Vol 10 No 2 - Journal of Cell and Molecular Biology

Transcription

Journal of Cell and
Molecular Biology
Volume 10 · No 2 · December 2012
http://jcmb.halic.edu.tr
•Circadian rhythm genes in cancer
•Tunneling nanotubes
•Genetic screening of Turkish barley genotypes
•Strontium ranelate induces genotoxicity
Journal of Cell and
Molecular Biology
Volume 10 · Number 2
December 2012
İstanbul-TURKEY
Editor-in-Chief
Nagehan ERSOY TUNALI
Haliç University
Faculty of Arts and Sciences
Journal of Cell and Molecular Biology
Founder
Gündüz GEDİKOĞLU
Our Children Leukemia Foundation
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Rector
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Ferruh ÖZCAN, İstanbul, Turkey
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Gizem DİNLER DOĞANAY, İstanbul, Turkey
Hande ÇAĞLAYAN, İstanbul, Turkey
Hande GÜRER ORHAN, İzmir, Turkey
İlhan YAYLIM ERALTAN, İstanbul, Turkey
İsmail ÇAKMAK, İstanbul, Turkey
İsmail TÜRKAN, İzmir, Turkey
Kemal BÜYÜKGÜZEL, Zonguldak, Turkey
Maria V. KALEVITCH, Pennsylvania, USA
Mehmet TOPAKTAŞ, Adana, Turkey
Meral KENCE, Ankara, Turkey
Meral ÜNAL, İstanbul, Turkey
Müge TÜRET SAYAR, İstanbul, Turkey
Mustafa DJAMGÖZ, London, UK
Nermin GÖZÜKIRMIZI, İstanbul, Turkey
Nevin Gül KARAGÜLER, İstanbul, Turkey
Nihat BOZCUK, Ankara, Turkey
Pınar SAİP, Istanbul, TURKEY
Rezan FAHRİOĞLU YAMACI, Nicosia, Cyprus
Şehnaz BOLKENT, İstanbul, Turkey
Selma YILMAZER, İstanbul, Turkey
Sevtap SAVAŞ, Toronto, Canada
Uğur ÖZBEK, İstanbul, Turkey
Ünal EGELİ, Bursa, Turkey
Valentine KEFELİ, Pennsylvania, USA
Zihni DEMİRBAĞ, Trabzon, Turkey
Ziya ZİYLAN, İstanbul, Turkey
CONTENTS
Volume 10 · Number 2 · December 2012
Review Articles
Transforming acidic coiled-coil proteins and spindle assembly
S. TRIVEDI
1
Marker Systems and Applications in Genetic Characterization Studies
Y. ÖZŞENSOY, E. KURAR
11
Research Articles
COX5B and COX2 gene expressions in Multiple Sclerosis
N. SAFAVIZADEH, S. A. RAHMANI , M. ZAEFIZADEH
21
Curcumin rendered protection against cadmium chloride induced
testicular damage in Swiss albino mice
P. SINGH, K. DEORA, V. SANKHLA, P. MOGRA
31
Study of Klebsiella pneumoniae isolates with ESBL activity, from ICU
and Nurseries, on the island of Mauritius
S.K. MUNGLOO-RUJUBALI, M.I. ISSACK, Y. JAUFEERALLY-FAKIM
39
HIV-1 reverse transcriptase inhibition by Vitex negundo L. leaf extract
and quantification of flavonoids in relation to anti-HIV activity
M. KANNAN, P. RAJENDRAN, V. VEDHA, G. ASHOK, S. ANUSHKA,
P. CHANDRAN RAMACHANDRAN NAIR
53
Genetic characterization and bottleneck analysis of Korki Jonub
Khorasan goats by microsatellite markers
B. MAHMOUDI, O. ESTEGHAMAT, A. SHARIYAR. M.Sh. BABAYEV
Low-Stringency Single-Specific-Primer PCR as a tool for detection of
mutations in the matK gene of Phaseolus vulgaris exposed to
paranitrophenol
61
71
Mohamed R. ENAN
Short Communication
Characterization of Paenibacillus larvae isolates from Brazil
S.S. CHAGAS, R.A. VAUCHER, A. BRANDELLI
Guidelines for Authors
79
83
Front cover image: “Cell division”
Shutterstock image ID: 4099351
Journal of Cell and Molecular Biology 10(2):1-10, 2012
Haliç University, Printed in Turkey.
Review Article 1
Seema TRIVEDI*
(* author for correspondence; [email protected])
Received: 2 January 2012; Accepted: 5 November 2012
Abstract
Transforming acidic coiled-coil proteins (TACC) are essential for mitosis not only by their association
with the centrosome and assembly of spindle microtubules, but also by involvement in cell cycle
checkpoints. In order to stabilize spindle fibers, TACCs interact with other microtubule-associated
proteins (MAPs). Dysregulation of TACCs may lead to abnormal cell division that may result in
chromosomal abnormality or tumorigenesis. This review focuses on facts known so far regarding
TACC proteins, their interactions and their involvement in spindle microtubule stability in higher
eukaryotes.
Keywords: Cancer, microtubules, centrosome, spindle fibers, transforming acidic coiled-coil proteins
Özet
Dönüştürücü asidik sarmalanmış sarmal proteinleri ve iğ iplikçiğinin birleşmesi
Dönüştürücü asidik sarmalanmış sarmal proteinleri (TACC) sadece sentrozomlarla ve iğ iplikçiği
mikrotübülleriyle birleşmesiyle olan ilişkileri açısından değil, ayrıca hücre döngüsü kontrol
noktalarına dahil olmaları açısından da mitoz için gereklidir. İğ iplikçiklerini stabilize etmek amacıyla,
TACC’ler diğer mikrotübül ilişkili proteinlerle (MAP) etkileşime girer. TACC’lerin yanlış
düzenlenmesi kromozomal anormallikler ya da tümörigenez ile sonuçlanan anormal hücre
bölünmelerine neden olabilir. Bu derleme, bugüne kadar TACC proteinleri, etkileşimleri ve yüksek
ökaryotlarda iğ iplikçiği kararlılığına katkıları ile ilgili bilinen gerçekler üzerinde durmaktadır.
Anahtar Kelimeler: Kanser, Mikrotübüller, Sentrozom, İğ iplikçikleri, Dönüştürücü asidik
sarmalanmış sarmal proteinleri
Abbreviations
TACC = Transforming Acidic Coiled-coil, MAP = Microtubule Associated Protein, Msps = Mini
spindles, MT = Microtubule, ch-TOG = Colonic–hepatic Tumour-Overexpressed Gene, AAK =
Aurora A Kinase
Introduction
Transforming acidic coiled-coil protein (TACC) is
a family of the microtubule associated proteins
(MAPs) that is crucial for spindle assembly,
maintaining bipolarity and microtubule (MT)
stability during mitosis (Still et al., 2004; Brittle
and Ohkura, 2005; Pearson et al., 2005). TACCs
are even important in acentrosomal (acentriolar or
anastral) meiosis in females of many animal
species where spindle formation is
chromosome centric (Pearson et al., 2005).
TACCs do not have microtubule
stabilizing activity on their own. Instead,
they form complexes with other MAPs to
stabilize spindle fibers. TACC/MAP
complex provides stable association of MTs
with centrosomes at the minus end (Albee
and Wiese, 2008) possibly after release from
2 Seema TRIVEDI
the nucleation site from kinetochores (reviewed in
Raff, 2002). The complex associates at the plus
ends of the MTs growing out from the centrosome
during nucleation (reviewed in Raff, 2002).
However, the exact mechanism by which TACC
interacts with other MAPs and recruits them to the
centrosome is not clearly understood. In
Drosophila, D-TACC recruits Mini spindles protein
(Msps; a conserved family of MAP) (reviewed in
Raff, 2002) and in Xenopus, maskin (TACC3) form
a complex with XMAP215 (Albee and Wiese,
2008). However, the XMAP215 complex may
associate with both plus and minus ends of the MT.
Similarly, human TACCs and ch-TOG (colonic–
hepatic tumour-overexpressed gene) (homologue of
Msps) possibly form a structural lattice at
centrosomes to maintain the integrity of spindle
poles and to stabilize spindle MTs (reviewed in
Raff, 2002; Gergely et al., 2003). However, it is not
known why in cultured human cells TACC3 and, in
Drosophila D-TACC, these proteins are apparently
not essential for recruitment of XMAP215 analogue
(Brittle and Ohkura, 2005). This review focuses on
genomic locations, protein characteristics and
interactions of TACC proteins in humans and
general aspects of role of TACC proteins in spindle
dynamics in higher eukaryotes.
TACC genes and proteins
Three TACC proteins have been identified in
humans, namely TACC1, 2 and 3. The three human
TACCs are related by ~200-amino-acid C-terminal
region (the ‘TACC domain’), which is predicted to
form a coiled coil domain (reviewed in Raff, 2002).
Besides TACC domain, these proteins share
homology (except Drosophila D-TACC) in the
SDP domain as well (Lauffart et al., 2002; Still et
al., 2004). SDP domain is composed of functionally
conserved repeats of 33 amino acids, though the
numbers of repeats are different in different TACC
proteins. Interaction of SDP domain with
transcription factor GAS41/NuBI1 (also known as
YEATS4) has been established (Lauffart et al.,
2002) but this interaction possibly does not directly
affect spindle formation.
Genomic locations of human TACC
reference gene sequences as per NCBI and
details of proteins obtained from SwissProt
are given in Table 1.
Though implicated in several cancers, no
mutations have been reported in the TACC1
gene (Still et al., 2008). TACC1 mRNA
exon 3 contains a predicted nuclear
localization signal (Still et al., 2008), shows
ubiquitous expression and encodes a
cytoplasmic protein which is mainly
perinuclear protein (molecular mass of 125
kDa) (Conte et al., 2002) but different
isoforms in different cells may be localized
in the cytoplasm as well (Lauffart et al.,
2006).
TACC1 protein does not show
compositional bias whereas TACC2 has
poly-lysine and poly-proline rich regions
and TACC3 has poly-serine regions (Table
1). In addition, different sites in the TACC
proteins show post translational covalent
modifications (Table 2). Phosphotyrosine is
seen only in TACC1, while N6-acetyl lysine
is
seen
only
in
TACC3
and
phosphothreonine is not seen in TACC1. It
is also pertinent to note that TACC1
phosphorylation varies in different isoforms
(Still et al., 2008).
TACC2 concentration at the centrosome
has been observed even during interphase
unlike TACC1 and TACC3 proteins
(Gergley et al., 2000a). TACC3, also known
as ERIC1 (Still et al., 2004; Eslinger et al.,
2009) a non-motor MAP, was also identified
as an ARNT interacting protein (Aint1) in
mice (Aitola et al., 2003) and is expressed in
proliferative tissues (Aitola et al., 2003;
reviewed in Hood and Royle, 2011). During
mitosis, TACC3 is localized to centrosome
but during interphase, TACC3 is seen in
cytoplasmic or perinuclear regions (Piekorz
et al., 2002). The TACC3 gene has five
reported mutations: CAG to CGG, TCG to
TTG, CGT to TGT, CAG to TAG and TCA
to TTA (Eslinger et al., 2009).
TACC proteins in spindles 3
Table 1. Human TACC Genomic Location (as per NCBI+), Introns, mRNA And Splice Variants (as per NCBI AceView+)
and Protein Details (as per SwissProt*).
Official Symbol
Genomic Location
TACC1
8p11.22
TACC2
10q26
TACC3
4p16.3
Genomic Sequence
NC_000008.10
38585704..38710546
NC_000010.10
123748689..124014057
NC_000004.11
1723266..1746898
Number of Introns+
44
49
23
mRNA+
38 (7 unspliced)
35 (7 unspliced)
16 (3 unspliced)
Protein*
TACC1 (O75410
TACC1_HUMAN)
805
TACC2 (O95359
TACC2_HUMAN)
2948
TACC3 (Q9Y6A5
TACC3_HUMAN)
838
Length (number of amino acids)
Number of Isoforms+
27
Features
Description
Coiled coil
Potential
TACC1
610 - 805
26
15
Position
TACC2
TACC3
2675 - 2703
637 - 837
2746 - 2947
2420 - 2423
Poly-Lys
Compositional
bias
155 - 160
Poly-Ser
Pro-rich
1956 - 2016
SPAZ
2315 - 2403
482 - 549
Domain
Motif
SPAZ 1
215 - 297
SPAZ 2
Bipartite
nuclear
localization
signal 1
Potential
Bipartite
nuclear
localization
signal 2
Potential
359 - 507
Interaction
with CH-TOG
226 - 241
455 - 471
701 - 805
Interaction
with LSM7
and SNRPG
1 - 55
Interaction
with TDRD7
152 - 259
Interaction
with YEATS4
206 - 427
Region
+
Thierry-Mieg and Thierry-Mieg 2006, AceView-Dec 2009
4 Seema TRIVEDI
Table 2. Modification types and positions of modifications in TACC proteins as per SwissProt.*
N6-acetyl
Protein Position
Phosphoserine Phosphothreonine Phosphotyrosine
lysine
TACC1
TACC2
TACC3
44
50
52
54
55
57
228
276
406
533
197
201
493
571
575
758
962
1025
1267
1313
1426
1562
1946
1949
2072
2073
2226
2246
2256
2317
2321
2359
2389
2390
2392
2394
2403
2512
2884
25
59
71
175
317
434
558
*Grey cells indicate presence and blank cells indicate absence or not known.
Spindle assembly and TACC proteins
TACC proteins interact with MTs (particularly at
the minus end) and are mainly associated with
centrosome (Gergely et al., 2000a and 2000b). The
distribution and concentrations of TACC proteins
during
spindle
formation
vary.
TACC1
concentration at centrosome is weak and is seen
only during mitosis. TACC2 concentration is strong
at centrosome throughout the cell cycle. TACC3 is
strongly concentrated in a more diffused region
around
centrosomes (during G2 phase) at the minus
end of spindle MTs (Gergely et al., 2000a;
Gergely et al., 2000b; Barr et al., 2010).
Once the spindle has formed, TACC3
protein is not found at astral MTs (reviewed
in Raff, 2002; Hood and Royle, 2011).
TACC3 protein is localized at centrosomes
with γ-tubulin and spindle MTs with αtubulin (Piekorz et al., 2002, reviewed in
(Raff, 2002; Hood and Royle, 2011),
particularly during S, G2 and M phases of
cell cycle (Piekorz et al., 2002). TACC3/ch-
TOG/clathrin complex increases the stability of Kfibers during early mitosis. This is achieved by
reduction in MT catastrophe by anchoring to the
spindle and establishing short bridges. Involvement
of TACC proteins in formation of long bridges is
not known but possibly HURP and the kinesinrelated protein HSET/KIFC1 may be involved in
longer bridges (Booth et al., 2011).
TACC3 depleted cells do not show proper
metaphase alignment; therefore it is possible that
TACC3 protein may be essential for chromosome
alignment (Gregely et al., 2003). TACC3 may also
affect early mitotic checkpoint by associating with
pS939-TSC2 (tuberous sclerosis complex 2) and
regulating its localization at spindle poles and also
possibly affect nuclear envelope (Gomez-Baldo et
al., 2010). TACC3 affects spindle checkpoint by
affecting SAC (spindle assembly checkpoint
assembly) by stabilizing spindle and is important
for microtubule-kinetochore interaction. Depletion
of TACC3 results in activation of the SAC (spindle
assembly checkpoint protein), which prevents
degradation of cyclin B1 and anaphase transition.
Cyclin B1 is present in cells from late G2 to
metaphase and is degraded prior to anaphase.
TACC3 depletion increases the levels of cyclin B1
but not cyclin A (S/G2) (Schneider et al., 2007),
thus presence of TACC3 would affect degradation
of cyclin B1 and help in anaphase transition.
These observations indicate differences in roles
of different types of TACC proteins during mitosis.
For proper assembly and stability of spindle
fibers and minus end of centrosome associate MT,
phosphorylation of TACC is essential which is
mediated by mitotic kinases (e.g. Aurora A kinase
i.e. AAK) (Barros et al., 2005, Peset et al., 2005).
Absence of phosphorylated TACC may result in
either shorter centrosomal MT or absence of these
spindle fibers (Peset et al., 2005, Kinoshita et al.,
2005). Phosphorylation occurs at different sites in
different animals. In Xenopus TACC3/Maskin, at
least two residues (main site is Ser626) are
phosphorylated; in Drosophila D-TACC Ser863 is
phosphorylated exclusively at centrosomes and in
human Ser558 is phosphorylated in TACC3
(reviewed in Raff, 2002; Brittle and Ohkura, 2005;
LeRoy et al., 2007). Phosphorylation of TACCs
may also help G2/M checkpoint by proper
microtubule assembly, thus the control of mitosis
by affecting G2/M transition (Conte et al., 2002;
reviewed in Bettencourt-Dias and Glover, 2007),
particularly
via
spindle
checkpoint
(Schneider et al., 2007).
However, AAK itself must be activated
prior to being able to phosphorylate the
TACC proteins. In humans, AAK is
activated by binding with TPX2 [Targeting
Protein for Xklp2 (Xenopus plus enddirected kinesin-like protein)]. AAK-TPX2
binding is activated by HURP (Human
hepatoma up-regulated protein) which is a
MAP protein that can also bind directly to
MTs. However, HURP itself needs
activation by Ran (RAs-related Nuclear
protein) (Sato et al., 2009; Sato and Toda,
2010).
On the other hand, dephosphorylation of
TACC proteins may also affect spindle
stability. In this regard, Mars (a D.
melanogaster sequence homologue of
HURP) mediates spatially controlled
dephosphorylation of TACC for spindle
stability, perhaps only at the centrosome.
Dephosphorylated TACC establishes lateral
interactions with MT or with plus ends
which may be impaired due to TACC
phosphorylation at Ser863 (Tan et al.,
2008).
Spindle fibers and proteins interacting
with TACC proteins
Minimal interactions of TACCs with other
proteins or ligands were determined using
the STRING web interface (Jensen et al.,
2009), and are shown in Figure1. As
previously stated, TACC1 and TACC3 bind
with ch-TOG (clathrin, colonic and hepatic
tumor overexpressed gene) (Conte et al.,
2002; Lauffart et al., 2002); however, from
the interaction shown in Figure 1 it appears
that all three TACC proteins interact with
CKAP5 (homologue XMAP215 or chTOG). The other two common interactions
of the three TACC proteins are YEATS4
(also called YAF9; GAS41; NUBI-1)
(transcription factor, protein located in
nucleoli) and LSM7 (a conserved subfamily
of Sm-like small proteins). LSM7 associates
with U6 snRNPs and plays a role in several
aspects of mRNA processing (Conte et al.,
2002; Lauffart et al., 2002).
6 Seema TRIVEDI
TACC1 may also be involved in gene regulation by
affecting mRNA translation by interaction with
TDRD7 (tudor domain containing 7) (Figure 1).
TDRD7 protein is a part of cytoplasmic RNA
granules involved in mRNA regulation. It is not
known whether the involvement of TACCs in RNA
processing or interaction with transcription factors
has any role in MT dynamics during cell division.
As per Figure 1, TACC1 and 3 also interact
with AURKB and AURKA. These two proteins
also interact with Cyclin B1 (which controls entry
in mitosis) (Conte et al., 2002). Studies have shown
that TACC3 is involved in localization of the
mitotic kinase Aurora B and the checkpoint protein
BubR1 at kinetochores thus affecting MT
attachment (Schneider et al., 2007).
TACC2 protein also directly interacts with
SMYD2 (SET- and MYND-containing protein 2)
(Figure 1). SMYD2 activates TACC2 gene by
methylation of H3K4 in the promoter region of
TACC2 gene besides the involvement of SMYD2
in some protein-protein interactions. These protein
interactions may have roles in centrosomal MT
formation (Abu-Farha et al., 2008) but apparently
not by directly interacting with TACC2 protein.
Phosphorylated TACC3 also directly interacts
with clathrin heavy chain (CLTC) that promotes
accumulation of other complex members at the
mitotic spindle (reviewed in Hood and Royle,
2011). Another direct interaction of TACC3 protein
with septin-7 (SEPT7, CDC10 protein homolog) is
shown in Figure 1. SEPT7 associates with the
mitotic spindle and the kinetochore. It has also been
shown that SEPT7 is needed for stable localization
of CENP-E (centromere-associated protein E) at
kinetochore besides affecting spindle checkpoint
(Zhu et al., 2008). Association of SEPT7 and
MAPs, particularly MAP4, in MT dynamics both
during interphase and mitosis has been established
(Silverman-Gavrila et al., 2008), and it is possible
that similar interaction of SEPT7 with TACC
proteins may affect MT stability.
Expression of TACC3 is high in hematopoietic
tissue unlike TACC1 and TACC2. Apparently
higher levels of TACC3 proteins are not for
proliferation of tissue but for direct or indirect
regulation of p53 levels to prevent apoptosis
although not true for other tissue (Piekorz et al.,
2002). Other study confirmed direct interaction of
TACC3 with p53 (that is also concentrated at
centrosomes) possibly to keep it inactive during
mitosis (reviewed in Raff, 2002) (not seen in
Figure 1).
TACC3 is necessary for proper
localization of phosphorylated TSC2
(Tuberous sclerosis proteins, tuberin) to the
mitotic apparatus and cytokinetic structures.
This interaction may be through the TSC2HBD domain (TSC2 hamartin-binding
domain) and result in promotion of
cytoskeletal remodeling (Gómez-Baldó et
al., 2010).
Though little is known about the factors
that control or regulate length of spindle
fibers, TACC3 may be one of the proteins
that control length of MTs and time for
chromosome alignment at metaphase plate
(reviewed in Raff, 2002). AAK may also
regulate MT length, as seen in Drosophila
embryos, where AAK function disturbance
leads to abnormal shortening of centrosomal
MTs. This disturbance also leads to
inefficient concentration of D-TACC at
centrosomes (reviewed in Raff, 2002).
TACCs dysregulation and cancer
Human TACC 1, 2 and 3 are present in
genomic regions that are rearranged in
certain cancer cells (reviewed in Raff, 2002;
Stewart et al., 2004; Still et al., 1999).
Aberrations of TACC genes (TACC3 in
particular)
contribute
to
tumorigenesis/cancer (reviewed in Raff,
2002; Lauffart et al., 2005).
Since TACC proteins are also involved
in centrosomal dynamics, cell cycle
checkpoints (Schneider et al., 2007) and can
form multiple complexes, any dysregulation
in these proteins may be important during
tumorigenesis (Lauffart et al., 2002). It has
been noted that increase or decrease in
levels of the TACCs can lead to impairment
of spindle functions (reviewed in Raff,
2002). Disruption of spindle function can
then lead to misalignment of chromosomes
and or abnormalities in chromosome
separation. Altered levels of TACC proteins
can lead to abnormal mitosis although next
p53 dependent checkpoint should eliminate
such cells. However, if there is simultaneous
alteration in TACC levels and p53 is either
absent or depleted, then such cells would not
Figure 1. Predicted functional partnes (minimum interaction) of the three TACC proteins (as per
STRING Jensen et al., 1999)
undergo elimination, resulting in genetic instability
that could contribute to the development of cancer
(reviewed in Raff, 2002).
Some studies show that TACC3 may enhance
stability of tumors. This can be achieved by
TACC3 and ch-TOG in clustering of multipolar
spindles in tumor cells into two poles that may lead
to somewhat ‘normal’ division (reviewed in Hood
and Royle, 2011). However, TACC2 may be a
potential tumour suppressor (reviewed in Raff,
2002) contrary to studies that suggest no role of
TACC2 in tumor suppression (Schuendeln et al.,
2004).
Unanswered questions
Though much is known regarding TACC function
in spindle and microtubule dynamics, there are few
aspects that still remain unknown. These are
summarized below:
Roles of TACCs in regulation/control of
spindle fiber lengths are not fully
understood. Though the levels of TACC and
particularly roles of TACC3 and AAK are
indicated in control of MT length (reviewed
in Raff, 2002), the precise mechanism by
which this is achieved is not known.
Though TACC proteins are present in
cytoplasm during interphase, their fate at the
end of mitosis is not known with respect to
their redistribution to cytoplasm of the
daughter
cells.
If
there
is
exclusion/reduction in amount of TACC
proteins from nucleoplasm, the mechanism
remains elusive. Further, it is not known
whether destruction/recycling/decrease in
8 Seema TRIVEDI
expression of all TACCs (like cyclins) is important
for ending anaphase.
It is known that TACC3 associates with
kinetochore MTs (K fibers), but association with
interpolar MTs (reviewed in Hood and Royle,
2011) or astral MTs is not confirmed. It is also not
known whether all three TACCs associate with
kinetochore MTs (K fibers), interpolar MTs and
astral MTs or there are different TACC for each
fiber type.
If different TACCs are involved in K-fibers,
interpolar and astral MTs; it is not known how this
association difference is achieved.
Different interaction partners at different
subcellular locations of each splice variant of
TACC1 protein have been reported during different
stages of embryonic development. This may be
possible due to retention of coiled coil domain in
each splice variant but there are differences in
interaction motifs at N-terminus (Lauffart et al.,
2006). However, it is not known whether different
isoforms of TACC proteins have different roles in
spindle assembly during mitosis.
Conclusion
It is known that TACCs are important in
maintaining bipolarity and stability of spindle fibers
through
their
association
with
other
binding/interacting partners. TACCs also play a
role in cell cycle checkpoints due to their
association with centrosomes and p53. However,
there are many unresolved functional and structural
aspects of the three TACC proteins. Further
advancement in studies with respect to the unsolved
facets of these proteins may help in enhancing basic
understanding of spindle MT dynamics and related
disorders.
Acknowledgements
I am extremely indebted to Prof. S. D. Kapoor,
former Head, Department of English, JN Vyas
University, Jodhpur (Raj.) and Emeritus Fellow of
UGC (University Grants Commission) of India, for
correcting the language and expression in the
manuscript.
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Review Article 11
Markör Sistemleri ve Genetik Karakterizasyon Çalışmalarında
Kullanımları
Marker Systems and Applications in Genetic Characterization
Studies
Yusuf ÖZŞENSOY*1,2, Ercan KURAR2
1
2
Bitlis Eren Üniversitesi Sağlık Yüksekokulu, 13000, Bitlis
Selçuk Üniversitesi Veteriner Fakültesi Genetik Anabilim Dalı, 42031, Konya
Received: 18 September 2012; Accepted: 20 December 2012
Abstract
Nowadays, owing to the developments in molecular biology, genetic markers are generally used to
describe specific regions of the genome. Three different marker systems, namely, protein and DNA
markers, are used in genome analyses and in various genetic studies. Following the discovery of
polymerase chain reaction (PCR), PCR-based marker systems are widely preferred in genetic studies.
Genetic characterization studies are critically important to determine the level of genetic diversity
between and within populations, origin of domestication and migration and development of
conservation programs. Different biochemical marker systems, alloenzymes, mitochondrial DNA and
Y chromosome are used for genetic characterization studies. DNA markers, especially the
polymorphic microsatellite markers, are the most preferable marker systems in PCR applications.
Recent progresses in molecular biology techniques allow rapid and economical identification of single
nucleotide polymorphisms analyses and their applications along with microsatellites.
Keywords: Genetic Characterization; Marker systems, Microsatellite, SNP, RFLP
Özet
Moleküler biyoloji alanındaki gelişmeler sonucunda günümüzde markörler genel olarak genomun
özgün bir bölgesini tanımlamak amacıyla kullanılmaktadır. Genom analizleri ve genetik çalışmalarda
morfolojik, protein ve DNA markörleri olmak üzere üç tip markör kullanılmaktadır. Polimeraz zincir
reaksiyonunun (PZR) keşfinden sonra genetik çalışmalarda PZR-temelli markörler daha fazla tercih
edilmeye başlanmıştır. Genetik karakterizasyon çalışmaları, popülasyon içi ve popülasyonlar arası
genetik çeşitlilik seviyesinin belirlenmesi, koruma programlarının geliştirilmesi, evcilleştirilme ve göç
yollarının tespiti gibi çalışmalar için oldukça önemlidir. Genetik karakterizasyon çalışmalarında farklı
biyokimyasal markör sistemleri, alloenzimler, mitokondriyal DNA ve Y kromozomuna özgün
markörler kullanılmaktadır. DNA markörleri, özellikle polimorfik mikrosatellit markörleri, PZR
uygulamalarında en çok tercih edilen markör sistemini oluşturmaktadır. Son zamanlarda geliştirilen
yeni moleküler biyoloji teknikleri tek nükleotid polimorfizmleri analizinin daha hızlı ve ekonomik
olarak yapılabilmesine ve mikrosatellitler ile birlikte kullanılmasına olanak sağlamaktadır.
Anahtar Kelimeler: Genetik karakterizasyon, Markör Sistemleri, Mikrosatellit, SNP, RFLP
GİRİŞ
Genomun özgün bir bölgesini tanımlamak amacıyla
birçok markör sistemi kullanılmaktadır. Genom
analizleri ve genetik çalışmalar başta olmak
üzere moleküler çalışmalarda morfolojik,
12 Yusuf ÖZŞENSOY ve Ercan KURAR
protein ve DNA markörleri olmak üzere 3 tip
markör kullanılmaktadır (Liu, 1998).
Morfolojik Markörler
Çok sayıdaki morfolojik markör insan, hayvan ve
bitki genetik çalışmalarında kullanılmaktadır.
Genler ve kromozomlar hakkındaki bilgi
eksikliğinden dolayı ilk çalışmalar, göz rengi, kanat
yapısı, boynuzluluk, deri rengi gibi basit Mendel
kalıtımı gösteren özellikler üzerinde yapılmıştır. Bu
gibi morfolojik karakterler, özgün genler için
güvenilir indikatörler olarak kullanılabilirler ve bu
özellikleri kodlayan genlerin kromozom üzerinde
yerlerinin tanımlanmasında faydalı olmaktadırlar.
Morfolojik markörlerin gözlenmesi kolay olmasına
rağmen alel sayılarının nispeten az olmasından
dolayı kullanımı kısıtlı kalmaktadır (Liu, 1998).
Protein Markörleri
Amino asit bileşimi, moleküler ağırlıkları ve
antikor-antijen ilişkilerindeki farklılıklar nedeniyle
proteinler için farklı aleller bulunabilmektedir.
Moleküler büyüklük ve amino asit bileşimi
farklılıklardan dolayı proteinler, jel elektroforez
yöntemi kullanılarak kolaylıkla ortaya çıkarılabilir
ve genetik markör olarak kullanılabilirler. Genetik
çalışmalarda
ilk
zamanlarda
protein
polimorfizmlerinin araştırılması amacıyla kan
antijenleri ve izoenzim markörleri yaygın olarak
kullanılmıştır. İzoenzimler, bir enzimin alternatif
bir formudur ve aynı enzim aktivitesine sahip
olmalarına rağmen elektroforetik hareketleri
farklılık göstermektedir (Liu, 1998). Genetik
çalışmalarda kan ve doku proteinleri markör olarak
yaygın bir şekilde kullanılmaktadır. Fakat özellikle
kan grubu ve protein markör sistemlerinin genomun
bazı
bölgelerinde
toplanmış
bulunmaları,
polimorfizm değerlerinin nispeten düşük olması,
özgün olarak kan örneklerine gereksinim
duyulması, iş yükünün ağır olması ve analizlerin
uzun zaman alması nedeniyle ve moleküler
biyolojideki gelişmelere bağlı olarak yerini DNA
temelli markörlere bırakmıştır (Kurar, 2001).
DNA Temelli Markörler
DNA markörleri, bir tür içerisindeki farklı
bireylerde dizi polimorfizmi gösteren DNA
bölgeleridir ve varyasyonun belirlenmesinde
günümüzde en sık kullanılan yöntemdir (Liu,
1998). Polimeraz Zincir Reaksiyonunun (PZR)
keşfinden sonra genetik çalışmalarda PZR temelli
markörler daha çok tercih edilmeye
başlanmıştır. Karry Mullis tarafından 1985
yılında ilk kez ortaya konulan bu teknoloji
sayesinde genomun özgün bölgelerinin in
vitro
şartlarda
çoğaltılabilmesi
ve
elektroforez teknikleri ile görüntülenmesi
mümkün hale gelmiştir. DNA teknolojisi ve
moleküler biyolojideki hızlı gelişmeye
paralel olarak daha ekonomik, kolay ve
polimorfik olmalarından dolayı özellikle
PZR temelli DNA markör sistemleri (RFLP,
RAPD, EST, STS, SSCP, AFLP, STR ve
SNP) genetik çalışmalarda daha yaygın
olarak kullanılmaya başlanmıştır (Weber
and May, 1989; Liu, 1998).
Restriksiyon
polimorfizmleri
parça
uzunluk
Restriksiyon endonükleaz (RE) enzim
kesimleri ile oluşturulan farklı DNA parça
uzunlukları restriksiyon parça uzunluk
polimorfizmleri
(RFLP-“Restriction
fragment length polymorphism”) olarak
adlandırılmaktadır.
RE’leri,
DNA
diziliminde
belirli
sırayla
bulunan
nükleotidlerden kendisine özgü özel tanıma
dizilimi bölgelerini tanıyarak bu dizilimin
belli bir noktasından keserek DNA’yı ikiye
ayırmaktadır. Her bir RE’nin kendisine özel
kesim bölgeleri bulunmaktadır.
RFLP teknolojisi ile DNA dizisinde
bulunan dizilim farklılıkları kolayca tespit
edilebilmektedir.
RFLP’lerin
belirlenmesinde DNA öncelikle bir RE
enzimi ile kesilerek DNA parçacıkları
agaroz jel elektroforezinde ayrıştırılır. DNA
dizilim
farklılıklarına
göre
genom
bölgesinde farklı RE kesim alanları ve
dolayısıyla bireyler arasında farklı DNA
fragman profilleri oluşacaktır. Alkali jelde
denatüre olmuş DNA fragmanları Southern
blot yöntemi ile nitroselüloz kâğıdı üzerine
alınır. Radyoaktif işaretli bir DNA probu
kullanılarak özgün DNA fragmanları ve
profili tespit edilebilir (Botstein et al.,
1980).
RFLP markörlerin en önemli avantajı
özgün dizi bilgisine ihtiyaç bulunmamasıdır.
RFLP yöntemi, türler, cinsler hatta büyük
popülasyonların
analizinde
kullanılabilmektedir. Polimorfizm oranı çok
Markör Sistemleri 13
yüksek olmasından dolayı aile ağacı ve haritalama
analizlerinde tercih edilen markör sistemi olmuştur.
RFLP markör sisteminin dezavantajları ise; analizin
yapılabilmesi için yeterli miktarda DNA’ya ihtiyaç
duyulması ile birlikte teknolojik olarak pahalı, uzun
ve yorucu bir yöntem olmasıdır (Botstein et al.,
1980).
Tek zincir konformasyon polimorfizmleri
Tek zincir konformasyon polimorfizmi (SSCP“Single-strand conformational polymorphism”)
markörleri, bir DNA dizilim bölgesindeki (1000
baz çiftinden daha kısa) dizi varyantları ve
mutasyonları (özellikle nokta mutasyonları)
belirlemede kullanılan bir markör sistemidir (Orita
et al., 1989). PZR ile çoğaltılan bir genom bölgesi,
uygun ısıda denatürasyona tabi tutularak mutasyon
bölgesinde II. ve III. DNA konformasyonlarının
oluşturulması esasına dayanmaktadır. Varyasyona
bağlı olarak oluşan II. ve III. konformasyondaki
DNA molekülleri jel elektroforezinde farklı bant
profilleri oluşturarak varyantların tespitine olanak
sağlamaktadır. Teknoloji olarak basit olmasına
rağmen, her bir mutasyon için farklı ortamların
oluşturulması gerekliliği, bu tekniğin uygulamasını
kısıtlayan en önemli etkendir (Liu, 1998).
Rastgele çoğaltılmış polimorfik DNA
Rastgele çoğaltılmış polimorfik DNA (RAPD“Randomly amplified polymorphic DNA”)
markörleri, PZR tabanlı olup ilk kez Williams et
al., (1990) tarafından geliştirilmiştir. Rastgele
nükleotid dizilimine sahip olan tek bir primerin
kullanılmasıyla DNA parçaları çoğaltılmakta ve
oluşan farklı bant profiline göre DNA polimorfizmi
tespit edilebilmektedir. Bu markör kullanılarak
yapılan çalışmalarda, aynı lokustaki iki farklı alel
belirli büyüklükteki bantların varlığıyla ya da
yokluğuyla ayırt edilebilmektedir (Liu, 1998).
RAPD markörlerinin avantajları, DNA dizi
bilgisine ihtiyacın olmaması, diğer markörlere göre
ucuz ve daha az miktarda DNA ile kısa sürede
sonuçlar alınabilmesi olması rağmen en önemli
dezavantajı diğer markörlere göre özelliklede
RFLP’ye göre güvenilirliğinin düşük olmasıdır.
Bundan dolayı genellikle RFLP ile birlikte
değerlendirmeye alınırlar (Williams et al., 1990).
Çoğaltılmış parça uzunluk polimorfizmi
Çoğaltılmış parça uzunluk polimorfizmi (AFLP“Amplified fragment length polymorphism”)
tekniği, RE enzimleri ile kesilmiş genomik
DNA parçalarının seçici PZR ile
çoğaltılması temeline dayanmaktadır. Bu
teknik, DNA’nın enzimlerle kesilmesi ve
oligonükleotid adaptörlerin bağlanması,
kesilen bölgelerin seçici PZR yöntemiyle
çoğaltılması
ve
çoğalan
bölgenin
poliakrilamid jelde analiz edilmesi olmak
üzere 3 temel aşamadan meydana
gelmektedir. RE ile kesilen parça bölgeleri
nükleotid
dizilimi
bilinmeden
jel
elektroforez
yöntemi
ile
görüntülenebilmektedir.
Parmak
izi
analizlerinde ağırlıklı olarak kullanılan
AFLP markör sistemi, RFLP markörüne
benzer özelliklere sahip olmakla birlikte
RFLP’ye göre analizi daha kolaydır ve daha
az
miktarda
DNA’ya
gereksinim
duymaktadır (Vos et al., 1995).
Tek nükleotid polimorfizmleri
Tek nükleotid polimorfizmleri (SNP-“Single
nucleotide
polymorphism”)
genomun
herhangi bir bölgesindeki tek nükleotid
dizilim farklılıklarıdır. Genomda oldukça
yaygın bulunan bu markörlere intron ve
ekzon bölgelerinde, 500–1000 bç sıklıkta
rastlanılabilir (Wang et al., 1998).
Genellikle iki alele sahip olan SNP
markörlerinin polimorfizmleri daha düşük
kalmakta, veri tabanı katalog bilgisine ve
polimorfizm
dizi
bilgisine
ihtiyaç
duyulmaktadır (Smigielski et al., 2000).
SNP’ler araştırmacıların ihtiyaçlarına göre
çalışmaları kolaylaştırmak için dizi konumu,
fonksiyonu, türler arası homoloji ve
heterozigotluk derecesi olmak üzere 4 büyük
bilgi ekseni tek veya daha fazlası bir arada
olmak
üzere
düzenlenebilmektedir
(Smigielski et al., 2000). Genomda bilinen
1.42 milyon SNP’nin her 1.91 Kbç başına 1
SNP yoğunlukta bulunduğu bilinmekle
birlikte ekson gen bölgelerinde 60 000 SNP
bulunduğu ve eksonun %85’inin SNP’nin 5
Kbç yakınında yer aldığı belirlenmiştir (The
International SNP Map Working Group,
2001). SNP bilgilerine ulaşmak için;
GenBank, PubMed, LocusLink ve Genome
Sequence gibi kaynak bilgiler ile NCBI veri
tabanındaki
bilgiler
kullanılmaktadır
(Smigielski et al., 2000). Geliştirilen yeni
moleküler biyoloji teknikleri (mikrodizin, gerçek
zamanlı PCR) ile çok sayıda SNP’nin analizi daha
hızlı ve ekonomik olarak yapılabilmektedir.
SNP’ler genetik çeşitlilik, popülasyon yapısı,
kantitatif özellik lokusları (QTL), markör destekli
seleksiyon (MAS) çalışmalarında ve ailesel
ilişkilerin araştırılmasında yaygın kullanılmaktadır.
Mitokondriyal DNA
Maternal kalıtım gösteren mitokondrial DNA
(mtDNA), çift zincirli, halkasal yapıda ve aerobik
solunumu destekleyen genleri içermektedir.
mtDNA toplam genetik materyalin %0.3’ünü
oluşturmaktadır (Rokas et al., 2003; Başaran,
2004). Tipik bir somatik hücre 500-1000
mitokondri içermektedir (Rokas et al., 2003).
mtDNA’da gözlenen mutasyon oranı nükleer
DNA’ya (nDNA) göre daha hızlıdır. Bu durumun
sebebi olarak, mtDNA’da meydana gelen
mutasyonların
nDNA’da
meydana
gelen
mutasyonlardan yaklaşık 10–20 kat daha fazla
olması ve mtDNA’nın tamir mekanizmasının
bulunmaması olduğu gösterilmektedir. Sonuçta
oluşan mutasyon oranı, mtDNA’nın baz diziliminde
çok farklı varyasyonların oluşmasına yol
açmaktadır (Başaran, 2004). mtDNA, canlıların
orjinleri, göç haritalarının çıkarılması, adli tıp,
dejeneratif hastalıkların sebebinin araştırılmasında
ve kanser çalışmalarında kullanılmaktadır. mtDNA,
rekombinasyon eksikliğinin tespiti ve genetik
olarak klonlanan canlıların kalıtımının tespit
edilmesinde yaygın kullanılmaktadır (Rokas et al.,
2003).
mtDNA’nın
kodlanan
bölgesindeki
varyasyonun iyi anlaşılması popülasyonların
genetik sonucunun (filogenetik geçmişinin)
belirlenmesinde yararlı olacaktır (Finnilä et al.,
2001).
Mikrosatellitler
Genomda bir lokusta arka arkaya gelen rastgele
tekrar dizilerine kısa ardışık tekrarlar (STR-“Short
Tandem Repeat”) denilmektedir. STR’lerin 1–6 bç
tekrarlarından oluşmuş markörlere mikrosatellit
markörler veya basit dizi tekrarları (SSR-“Simple
Sequence Repeat”) olarak isimlendirilmektedir
(Weber and May, 1989; Liu, 1998). Genomda 9–
100 bç arasında değişen rastgele dizi tekrarları ise
minisatellit markörler veya değişken ardışık
nükleotid tekrarlar (VNTR-“Variable Number of
Tandem Repeats) olarak tanımlanmaktadır.
Mikrosatellitlerin tekrar sayısı genelde 100’den,
minisatellitlerin tekrar sayısı ise 1000’den
daha azdır (Liu, 1998). Mikrosatellitler
prokaryot ve ökaryot genomun herhangi bir
bölgesinde
bulunabilmektedir.
Prokaryotlarda birçok biyolojik fonksiyona
sahip olduğu halde ökaryot hücrelerde rolü
tam olarak bilinmemektedir (Bennett, 2000).
Mikrosatellit markörler, yaygın olarak 2
nükleotidli tekrarlardan [(CA)n] oluşmakla
birlikte farklı formlarda da (AC, AT, AAC,
AAT, CCG vb) bulunabilmektedir (Ellegren
et al., 1997; Orti et al., 1997; Bruford et al.,
2003).
STR markörleri, PZR teknolojisinin
yardımıyla genetik çalışmalarda en çok
tercih
edilen
markör
sistemini
oluşturmaktadır (Weber and May, 1989;
Liu, 1998). Mikrosatellitlerde tekrar
bölgelerini kuşatan DNA dizileri bir türün
bireylerinde aynı olmasına rağmen tekrar
dizilim sayıları bireyler hatta bireyin
homolog kromozomları arasında dahi
farklılık gösterebilmektedir. Üç nükleotid
tekrarlı mikrosatellit bölgelerinin %60
oranında polimorfik olduğu, 2 bç tekrarlı
mikrosatellitlerin ise %100 polimorfik
özelliğe sahip olduğu belirlenmiştir (Metta
et al., 2004). Mikrosatellitler, genomda
yaygın olarak bulunmaları, polimorfizm
oranının yüksek olması ve kullanımının
kolay olması nedeniyle birçok moleküler
biyoloji çalışmasında rahatlıkla kullanımı
tercih edilmektedir.
GENETİK ÇEŞİTLİLİK VE GENETİK
KARAKTERİZASYON
Hayvansal üretimde genetik çeşitlilik, ıslah
programlarının temelini oluşturmaktadır.
Genetik çeşitlilik, belli bir coğrafik bölgeye
uyum sağlamış, ilgili bölgede yaygın olarak
yetiştirilen canlı türlerinin, bu türlere ait
ırkların genetik niteliklerini (kalıtsal bilginin
zenginliğini)
ve
içinde
yaşadıkları
ekosistemde birbirleri ile ilişkilerinin
niteliğini ifade eder. Evcil hayvanlarda
genetik çeşitlilik, ırk içi ve ırklar arası olmak
üzere iki çeşittir.
Genetik karakterizasyon çalışmaları,
ırklar arası ve ırk içi genetik çeşitliliğin
belirlenmesi ve ırkların tanımlanması
amacıyla önemli bir yere sahiptir. Dünyada
ve Türkiye’de 1980’li yıllarda yerli ırkların genetik
yapıları ve bazı verim özellikleri ile olan ilişkileri
kan ve süt protein polimorfizmi (Ceriotti et al.,
2003; Ibeagha-Awemu and Erhardt, 2005)
kullanılarak incelenirken en son gelişmelerle
birlikte mikrosatellit markörler ve SNP’ler (Cañón
et al., 2001; Li et al., 2006; McKay et al., 2008;
Kang et al., 2009; Molaee et al., 2009) daha yaygın
olarak kullanılmaya başlanmıştır.
Irkların kökeni ve evcilleştirilme bölgelerinin
tespit edilmesi amacıyla genetik karakterizasyon ve
arkeolojik çalışmalar yapılmaktadır. Avrupa
ırklarının göç yollarının Tuna Nehri boyunca
Kuzeyden Merkezi Avrupa’ya ve Akdeniz kıyısı
boyunca olmak üzere iki farklı göç yolu izlediği,
sığır, koyun, keçi, domuz ve mandanın iki farklı
Asya Bölgesi’nde ilk evcilleştirilmeye başlandığı
bildirilmektedir (Bruford et al., 2003). Bu
merkezlerden en eski olanı Doğu-Güneydoğu
Anadolu bölgesini kapsamaktadır ve ırkların bu
bölgeden tüm Dünya’ya özellikle Anadolu’dan
Avrupa’ya yayıldığı belirtilmektedir (Loftus et al.,
1994; 1999; Luikart et al., 2001; Troy et al., 2001;
Hiendleder et al., 2002; Cymbron et al., 2005).
Genetik
karakterizasyon
çalışmalarında
mikrosatellitler, insan (Bowcock et al., 1994; Deka
et al., 1995), sığır (MacHugh et al., 1997; Loftus et
al., 1999; Edwards et al., 2000; Cañón et al., 2001),
keçi (Luikart et al., 2001; Maudet et al., 2002),
koyun (Mukesh et al., 2006; Lawson Handley et
al., 2007; Molaee et al., 2009), köpek (Boyko et al.,
2009; Kang et al., 2009), at (Luís et al., 2007), eşek
(Aranguren-Méndez et al., 2002), domuz (Behl et
al., 2006; Sollera et al., 2009), manda (Flamand et
al., 2003) ve diğer birçok hayvan türlerinde (Cosse
et al., 2007; Vijh et al., 2007; Li et al., 2009)
kullanılmaktadır.
Genetik
karakterizasyon
çalışmalarında
mikrosatellitler dışında farklı biyokimyasal markör
sistemleri de (Moazami-Goudarzi et al., 1997;
Kantanen et al., 1999; 2000; Ceriotti et al., 2003;
Ibeagha-Awemu and Erhardt, 2005) kullanılmıştır.
AFLP (Negrini et al., 2007), mtDNA (Loftus et al.,
1994; Bradley et al., 1996; Finnilä et al., 2001;
Mannen et al., 2004), Y kromozomuna özgün
mikrosatellitler (Edwards et al., 2000; Cai et al.,
2006; Li et al., 2007; Kantanen et al., 2009)
ağırlıklı olarak kullanılmakla birlikte SNP (Li et
al., 2006; McKay et al., 2008) markörleri de
kullanılmaya
başlanmıştır.
Ayrıca
genetik
karakterizasyon çalışmalarında markör sistemleri
dışında aile ağacı kayıtlarından da
yararlanılmaktadır (Trinderup et al., 1999;
Honda et al., 2006).
Sığır
ırkları
üzerinde
yapılan
mikrosatellit
markörler
ile
yapılan
analizlerinde Taurin ve Zebu karışımı
bulunmamışken diğer markör sistemlerinden
olan Y kromozomu ve mtDNA ile yapılan
çalışmalar birlikte değerlendirildiği zaman Y
kromozomu markörü verilerinde bu ırklarda
Zebu karışımının olduğu belirlenmiştir
(Lirón et al., 2006).
SNP’ler ise bitkiler, deniz ürünleri ve
birçok
hayvan
türünde
moleküler
çalışmalarda aynı anda sayıca fazla miktarda
kullanılmaktadır (Muir et al., 2008;
Matukumalli et al., 2009; Yan et al., 2009;
Zhu et al., 2012).
Sonuç olarak markör sistemlerinde PZR
teknolojisi öncesi birçok markör sistemi
kullanılırken, PZR teknolojisi ile birlikte
mikrosatellitler
ağırlıklı
kullanılmaya
başlanmıştır. Son yıllarda ise SNP
markörleri üzerinde çalışmalar artmış ve
SNP çipleri oluşturulmaya başlanmıştır.
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Research Article 21
Naeimeh SAFAVIZADEH1, Seyed Ali RAHMANI 1, Mohamad ZAEFIZADEH2*
1
Department of Science, Ahar Branch, Islamic Azad University- Ahar- Iran.
Department of Science, Ardabil Branch, Islamic Azad University- Ardabil- Iran.
Received: 8 June 2012; Accepted: 14 August 2012
2
Abstract
Multiple Sclerosis (MS) is an autoimmune inflammatory disease which affects the Central Nervous
System (CNS) and leads to the destruction of myelin and atrophy of the axons. Genetic factors, in
addition to environmental ones, seem to play a role in MS. Numerous studies have reported
mitochondrial defects including a reduction in COX complex function related to the decrease of
mitochondrial gene expression in the cortex tissue of MS patients. This study aimed to assess COX5B
and COX2 gene expression in MS patients and controls. By using Real-Time PCR method, expression
levels of the COX5B and COX2 were determined, with reference to ß-actin and GAPDH. The results
showed that COX5B gene expression is significantly reduced in MS patients compared to control
(P<0.05), whereas there was no significant difference in the COX2 gene expression. Thus, it can be
claimed that down-regulation of mitochondrial electron transport chain genes supported the
hypothesis that hypoxia-like tissue injury in MS may be due to mitochondrial gene expression
impairments.
Keywords: Multiple Sclerosis, Mitochondria, COX5B, COX2, Real-Time PCR
Özet
Multipl Sklerozda COX5B and COX2 gen ekspresyonu
Multipl Skleroz (MS) Merkezi Sinir Sistemini (MSS) etkileyen, akson atrofisi ve miyelin yıkılımına
neden olan otoimmün bir hastalıktır. Çevresel faktörlere ek olarak genetik faktörlerin MS’de rol
oynadığı görülmektedir. Pek çok çalışma MS’li hastaların korteks dokularındaki mitokondriyal gen
ekspresyonunun azalmasıyla ilişkili COX kompleks fonksiyonundaki azalmayı kapsayan
mitokondriyal defektleri bildirmiştir. Bu çalışma MS hastalarında COX5B ve COX2 gen
ekspresyonlarını değerlendirmeyi ve onları kontrollerle karşılaştırmayı amaçlamıştır. Real-Time PCR
metodu kullanılarak COX5B, COX2, genlerinin ve ß aktin ve GAPDH referans genlerinin ekspresyon
düzeylerini belirledik. Elde edilen veriler referans genlerle standardize edilmiş ve Excel ve SPSS
programlarıyla bağımsız örnek t-test kullanılarak analiz edilmiştir. Sonuç olarak kontrol ve hastalar
arasında COX2 gen ekspresyonunda anlamlı farklılık bulunmazken MS hastalarında kontrole kıyasla
anlamlı olarak azalmış COX5B gen ekspresyonunu gösterilmiştir (P<0,05). Böylece; mitokondriyel
elektron transport zincir genlerinin aşağı düzenlenmesinin, MS’de hipoksi benzeri doku hasarının
mitokondriyel genler ve farklı ekspresyon azalmalarındaki farklılıklar nedeniyle olabileceği hipotezini
desteklediği iddia edilmektedir.
Anahtar Kelimeler: Multipl Skleroz, Mitokondri, COX5B, COX2, Real-Time PCR.
22 Naeimeh SAFAVIZADEH et al.
Introduction
Multiple Sclerosis (MS) is an inflammatory
demyelinating disease of the central nervous system
with axonal degeneration (Frohman et al., 2006).
The loss of myelin in MS may be the result of
direct damage to myelin through immune mediated
processes and dysfunction of oligodendrocytes
(Vercellino et al., 2009). Approximately 15–20%
of MS patients have a family history of MS, but
large extended pedigrees are uncommon, with most
MS families having no more than two or three
affected individuals. Studies in twins (Calabresi,
2007) and conjugal pairs indicate that much of this
familial clustering was the result of shared genetic
risk factors, while studies of migrants
(Ramagopalan et al., 2010) and apparent epidemics
(Koch et al., 2008) indicated a clear role for
environmental factors. Mitochondrial defects are
known to occur during aging, cancer, heart disease,
and a wide variety of degenerative diseases, such as
Alzheimer’s disease (AD), Parkinson’s disease
(PD), Huntington’s Disease (HD) and MS
(Henchcliffe et al., 2008). Mitochondria contain the
respiratory chain where energy in the form of ATP
is most efficiently produced (Damiano et al., 2010).
The mitochondrial respiratory chain is located in
the inner mitochondrial membrane and consists of
four complexes (complexes I–IV), whilst complex
V is directly involved in ATP synthesis. The
complexes of the mitochondrial respiratory chain
include multiple subunits; all but complex II (which
is entirely encoded by nuclear DNA) contain
proteins encoded by nuclear and mitochondrial
DNA (mtDNA). The final respiratory chain
complex [complex IV or cytochrome-c oxidase
(COX)] is the site at which over 90% of oxygen is
consumed. This complex is also involved in proton
pumping, essential for ATP synthesis (Alston et al.,
2011). The mammalian COX is composed of 13
subunits, of which the three largest are encoded by
the mtDNA and form the catalytic core of the
enzyme. The remaining ten, evolutionary younger,
nuclear-encoded subunits are involved in assembly
and regulation of the enzyme (Zee et al., 2006).
The function of mammalian COX can be
physiologically modulated and the enzyme
represents one of the key regulatory sites of energy
metabolism (Fernandes-Vizarra et al., 2009). COX
transfers electrons from cytochrome-c to molecular
oxygen, which is reduced to water. The electrons
pass from cytochrome-c, binding at subunit II,
through Cu (A) and heme as cofactors, to
the binuclear center buried inside subunit I
and composed of heme a3 and Cu (B), where
the incoming four electrons together with
four protons from the matrix are sequentially
used for oxygen reduction. This exergonic
redox reaction is coupled with proton
pumping across the inner mitochondrial
membrane, but the coupling of the two
processes (H+/e- stoichiometry) can be
modulated. In addition to Mitchell’s
chemiosmotic theory, a “second mechanism
of respiratory control” has been proposed
that involves the binding of adenine
nucleotides to nuclear-encoded COX
subunits. The key event is the
phosphorylation of subunit IV. Activity of
phosphorylated COX is regulated by
ATP/ADP ratio and respiratory rate is
precisely controlled according to the ATP
utilization. The membrane potential is kept
low (100-150 mV) and COX works at high
efficiency of proton translocation (H+/e-= 1).
The COX biosynthesis and assembly is
timely and complicated process involving
several rate-limiting steps reflecting the
sequential incorporation of the subunits
from either the cytosol (nuclearly coded
subunits) or from the mitochondrial matrix
(subunits I, II and III). The majority of COX
defects thus originate from mutations in
nuclear genes (Acin-Perez et al., 2009).
Mutations in the genes encoding several
COX assembly factors have been described
as a frequent cause of mitochondrial
diseases and have been assigned with
specific clinical symptoms. The dysfunction
of COX in these cases is mostly caused by
structural changes rather than by the changes
in amount of the enzyme (Galati et al.,
2009). Cytochrome-c oxidase subunit II,
abbreviated as CoxII, is the second subunit
of cytochrome-c oxidase subunit 2 (CO II)
transfers the electrons from cytochrome-c to
the catalytic subunit 1. It contains two
adjacent transmembrane regions in its Nterminus and the major part of the protein is
exposed to the periplasmic or to the
mitochondrial
intermembrane
space,
respectively. CO II provides the substratebinding site and contains a Cu centre called
Cox Expression in MS 23
Cu(A), probably the primary acceptor in
cytochrome-c oxidase. An exception is the
corresponding subunit of the cbb3-type oxidase
which lacks the Cu(A) redox-centre. Several
bacterial CO II have a C-terminal extension that
contains a covalently bound heme c (Barrientos et
al., 2009). Subunit Vb of mammalian cytochrome c
oxidase is encoded by a nuclear gene and
assembled with the other 12 COX subunits encoded
in both mitochondrial and nuclear DNA. This gene
located on chromosome 2, region cen-q13
(Williams et al., 2005). There is a common
symptom of MS with mitochondrial diseases such
as AD and PD, and also the point mutations in
mtDNA can cause damage to the myelin (DiMauro
et al., 2008). In this study, we compared COX5B
and COX2 gene expression among MS patients
with controls. In regard to the importance of
mitochondria in MS, we mainly centralized our
study on quantifying the expressions of COX5B
and COX2 using Real-Time PCR.
Materials and Methods
RNA Extraction
Total RNA was isolated by using a
RNAeasy kit from Roche (Germany),
according
to
the
manufacturer’s
recommendations. The RNAsamples were
incubated with RNAse-free DNAse I at
37°C for 15 min. RNA samples were
purified with a RNAeasy kit. It is possible to
preserve extracted RNA for months under
−80 °C. Total RNA quality and quantity
were assessed in 2 ways. In the first method,
we estimated the RNA concentration by
ultraviolet absorbance at 260 nm (1
absorbance unit at 260 nm = 40 ng/μL RNA)
and the RNA purity by measuring the ratio
of absorbance at 260 nm and 280 nm (1.8 <
A260/A280<2.1 for pure RNA). Total RNA
was run on 1% agarose gels to check size
and integrity. The quality of RNA was
confirmed by the detection of 18S and 28S
bands after 1% agarose gel electrophoresis.
Primer synthesis
Subjects
Thirty-six patients (16 male, 20 female) and 30
controls (14 male, 16 female) took part in this
study. Written informed consent was obtained from
each individual. Peripheral blood samples (5ml)
were obtained from the cubital vein and collected in
cell preparation tubes containing an anticoagulant
(EDTA). Peripheral blood mononuclear cells
(PBMC) were isolated by EDTA density
centrifugation.
Primer sequences were designed using
Primer3
software
for
COX5B
(NM_001862.2), COX2 (NC_012920.1) and
two reference genes (ß-actin and GAPDH),
and then primer sequences edited with the
Bioedit software and confirmed in the NCBI
/ BLAST database (Table 1).
Table 1. Sequences of the primers used for RT-PCR
Gene name
Primer sequence
COX5B
Forward: 5cccaaagggagcttcagg3
Reverse: 5cgacgctggtattgtcctct3
COX2
Forward:5tatccctggtcggttgaatc3
Reverse:5ggcataaaaccgtgatttaca3
GAPDH
ß-actin
Forward:5ccactcctccacctttgac3
Reverse:5accctgttgctgtagcca3
Forward:5ggcatgggtcagaaggatt3
Reverse:5ccacacgcagctcattgta3
cDNA Synthesis
Total RNA from each sample was used to generate
cDNA with a Reverse Transcriptase cDNA
synthesis kit (Roche, Germany) with oligo (dT)
primers, according to the manufacturer’s protocol.
Briefly RNA and 1 μL oligo (dT) were mixed and
then heated at 70°C for 5 min. They were chilled on
ice until the other components were added. Then,
we added 2μL dNTP, 4 μL of buffer, and 1 μL of
ribolack (RNase inhibition). The samples were
mixed and incubated at 37°C for 5 min. Then 1 μL
of Reverse Transcriptase was added, and the
samples were mixed and incubated at 42°C for 60
min. The reaction was inactivated at 70°C for 10
min. Finally, cDNA was stored at -20°C.
Quantitative Real-Time PCR
Amplification was performed over 30 cycles on the
MJ Research PTC-200 Gradient Cycler (MJ
Research, Waltham Mass, USA). The annealing
temperature was 54°C, extension occurred at 72°C,
and denaturation occurred at 94°C. After
completion of PCR, the product was separated by
electrophoresis in 1% agarose gel and detected by
ethidium bromide staining. cDNAs testing positive
for ß-actin and GAPDH expression was used in the
Real-Time PCR. Relative expressions of COX2 and
COX5B in the blood samples was carried out by
Real-Time PCR analysis using the ABI PRISM
7700 Sequence Detection System (Applied
Biosystems, Darmastadt, Germany) and the SYBR
Green PCR Kit (Qiagene). The data were evaluated
by the CT method, which measures the
expression level of the target genes normalized to a
reference gene and relative to the expression of the
genes in the calibrator samples. After efficiency
testing, ß-actin and GAPDH were chosen as the
reference housekeeping genes and the same primer
pairs used for the standard PCR were utilized for
the Real-Time PCR. Amplification was performed
over 45 cycles. The annealing temperature was
54°C, extension occurred at 70°C, and denaturation
occurred at 94°C. Every cDNA samples were
measured in four separate preparations to correct
for minor variations. Melting curve analyses were
carried out after completion to confirm the presence
of single amplified species. The transcript levels of
COX2 and COX5B in the samples were normalized
to the transcriptional level of the housekeeping
genes ß-actin and GAPDH, and calculated relative
to the expression levels of the target genes in a
calibrator blood samples. The arithmetic
mean of the relative expression levels of
COX2 and COX5B for each group of
patients and controls were calculated.
Statistical analysis
Statistically significant differences were
calculated by the Student’s t-test, using the
SPSS 18.0 and Excel. Finally a Pvalue0.05
was accepted as the level of significance.
Results
To determine the role of mitochondriaencoded gene and nuclear-encoded gene in
MS pathogenesis, using quantitative RealTime PCR analysis with SYBR-Green
fluorescent dye, we calculated the mRNA
fold change for one mitochondria-encoded
gene in complex IV (COX2) and one
nuclear-encoded gene in complex IV
(COX5B) of oxidative phosphorylation.
Real-Time PCR data analysis for genes
expression
Quantitative measurement of (COX2,
COX5B) gene expression was investigated
in 36 MS patients. Five different standard
concentrations were used to draw a standard
curve (Figure 1). Quantitative measurement
of gene expressions, achieved in the PCR of
the cycles, was done in the progressive
phase. Real-Time PCR Software can
determine the threshold cycle automatically
(Figure 2). In addition, due to temperature
changes in the melting curve analysis, there
is only one peak associated with the COX5B
and COX2 genes, which is a symptom of
lack of cases such as nonspecific products
and primer dimer (Figure 3). Then PCR,
standard curve, as a template standard DNA
of successive dilutions, was plotted for the
COX5B and COX2 genes (Figure 4).
Standard curve for the COX5B gene stands
for index (R2)=0.98 and also standard curve
of obtained COX2 gene stands for index
(R2)=0.95.
Cox expression in MS 25
Figure 1. Amplification plots for MS COX5B
Figure 3. Melting curve for COX 5b gene
Figure 2. Ct values of COX5B in subjects
Figure 4. Standard curve for COX5B gene
(determination of index R2=0.98).
26 Naeimeeh SAFAVIZA
ADEH et al.
Standard
dized results Ct
Accordinng to the studiies that were done
d
after RealTime PCR
R test, the aveerage of standdardized Ct in
regard to the state of -actin

and GAPDH genes in
COX5B among controol and patientts affected with
MS are 6.21, 6.71 and
a
3.95, 5.446 respectiveely
(Figures 5).
5 In additionn, the average of standardizzed
Ct in regard to state of
o -actin andd GAPDH gennes
in COX2 among controls and patiennts affected with
MS are 4.86, 4.38 and 4.84, 4.12 resspectively.
Figure 5. The Ct CO
OX5B gene, staandardized wiith
ß-actin
Comp
parison betweeen the statisttical analysis
of CO
OX5B t-test annd the resultss showed that
there are meaningfful differencees in COX5B
gene (P0.05). Onn the other hannd, decreased
gene expression in
i COX5B w
was observed
amon
ng MS patiennts rather thhan controls,
howeever, the resullts showed noo meaningful
differrences for the COX2 gene. For equality
of vaariances, t-tesst and Levenee’s test were
perfo
ormed. The results showed that the data
relateed to Ct was not significanntly different
COX
X5B gene amoong patients and control.
Ct comparison
c
was performed with t-test in
patien
nts and contrrols. The ressults showed
that th
he measured t is significantt for COX5B
gene (Pvalue=0.0168) and (P
Pvalue=0.005),
whilee the data related too Ct has
signifficantly diffferent COX22 gene in
patien
nts and contrrol. Ct com
mparison was
perfo
ormed with t-teest in pateintss and control.
The results show
wed that meeasured t is
signifficant for CO
OX2 gene ((Pvalue=0.117)
and (P
( value=0.124) (Table 2, 3)). Generally,
the reesults showedd that actin aand GAPDH
geness are similaar among ccontrol and
patien
nts.
Table 2. COX55B and COX22 genes expresssion (Ct) on
n based of B-aactin referencee gene
G
Gene
C
Compare
Means (Ct)
Std. Errror mean
Patieent Vs Controll
6.71
0.1
19746
6.21
0.1314
4.38
1.6
4.86
0.88
0
CO
OX5B
Patieent vs Controll
C
COX2
d
df
Tvalue
Pvalue
6
64
2.69
00.0168
6
64
-1.67
0.117
Taable 3. COX5B
B and COX2 genes
g
expresssion (Ct) on based
b
of GAP
PDH referencee gene.
Gene
Compare
Means (Ct)
Std.Error
S
meean
Patiient vs Controol
5.46
0.89
3.95
0.78
4.12
1.44
4.84
0.69
COX5B
B
Patiient vs Controol
COX22
df
Tvaalue
Pvalue
64
2.991
0.005
64
-1.52
0.124
Determination of changes in gene expression
levels
To determine changes in the expressions COX5B
and COX2 genes Livak formula was used (Livak
and Schmittgen, 2001).
-actin gene: COX5B = 2-CT= 2-(6.21- 6.71)= 20.5 =
1.41
COX2 = 2-CT= 2-(4.86-4.38)= 2-0.48
GAPDH gene: COX5B = 2-CT= 2-(3.95-5.46)= 21.51=
2.84
COX2 = 2-CT= 2-(4.84-4.12)= 2-0.72
Total results of the Real-Time PCR
According to the results obtained, it seems that
COX2 gene has no effect on MS patients; but
COX5B gene expression was decreased in MS
patients and this gene could play an important role
in neurodegenerative diseases. Correlation between
COX5B and COX2 gene expression turned out to
be R=0.92 in control. This correlation was R=0.84
in patients, who, despite being significant, was
lower compare to control. This difference, if
verified with further subjects and studies, can be
considered as a hypothesis about this disease.
Discussion
In this study, the expression levels of COX5B and
COX2 genes in MS patients and controls were
compared. The results showed that COX5B gene
expression in MS patients was significantly lower
compared to controls (P=0.0168) and (P=0.005). In
the case of Cox5B gene expression, a highly
significant difference between controls and
patients, despite the low number of samples,
indicates the role of this gene in patients, but it is
not clear that reduced expression of this gene is one
of the causes of disease, or when the disease
induces, the gene expression will be reduced. While
there was no significant differences in the COX2
gene expression between controls and patients,
differences between P=0.117 and P=0.124 was not
significant. One of the reasons that Cox2 gene is
not significant, it can be due to the low number of
samples and low df, it is necessary to consider the
mentioned term to report the more accurate results.
Degeneration of chronically demyelinated axons is
a major cause of the continuous, irreversible
neurological disability that occurs in the chronic
stages of MS (Aschrafi et al., 2008). Microarray
studies from cortical and white matter tissue of
patients with progressive MS showed upregulation of genes involved in hypoxic
preconditioning and decreased expression of
mRNA for mitochondrial proteins (Lin et
al., 2006). The dominant loss of small axons
in MS lesions suggests energy deficiency as
a major mechanism included in axonal
degeneration (Stys, 2005). The newly
reported research provides evidence that
neurons in MS are respiratory-deficient due
to mtDNA deletions, which are extensive in
GM and may be induced by inflammation.
They propose induced multiple deletions of
mtDNA as an important contributor to
neurodegeneration in MS (Campbell et al.,
2010). This presents a unique challenge to
neurologists wanting to identify, diagnose,
and manage patients and families with
mitochondrial disease (Druzhyna et al.,
2008). Clonally expanded multiple deletions
of mtDNA causing respiratory deficiency
are well recognized in neurodegenerative
disorders and aging. Given the vulnerability
of mtDNA to oxidative damage and the
extent of inflammation in MS, often starting
with a preclinical phase and remaining
throughout the disease course, mtDNA
deletions might be expected in MS
(Nicholas et al., 2009). Repair of damaged
mtDNA rather than oxidative damage to
mtDNA per se is the most likely mechanism
by which mtDNA deletions are formed, and
clonal expansion is regarded as the
mechanism
responsible
for
causing
respiratory deficiency. Where respiratory
deficiency is caused by induced multiple
DNA deletions, cells will have initially
contained
deletions
with
different
breakpoints, one of which then clonally
expands to high levels over time (Krishnan
et al., 2008). Clonally expanded multiple
deletions of mtDNA are reported in
inclusion body myositis, a condition
associated with chronic inflammation.
Decrease in density of respiratory-deficient
neurons in lesions is a likely reflection of
mtDNA deletion-mediated cell loss as well
as increase in susceptibility to other insults
because of the clonally expanded mtDNA
deletions. The extent of mtDNA deletions
identified using long-range PCR on a global
scale regardless of cell type, and even in neurons
with intact complex IV activity, reflects the
potential of cells in MS to become respiratory
deficient through clonal expansion over the course
of the disease. By isolating individual neurons at a
single time point we identified high levels of
multiple mtDNA deletions within respiratorydeficient cells. In contrast, mtDNA deletions
detected by long range PCR within respiratoryefficient neurons in MS and controls were not
expanded to high levels (Micu et al., 2006).
Mitochondrial defects are increasingly recognized
to play a role in the pathogenesis of MS. Energy in
the form of ATP is most efficiently produced by
mitochondria, which also play a role in calcium
handling, production of reactive oxygen species
(ROS), and apoptosis (Lin et al., 2006). It is known
that mitochondria are intrinsically involved in the
cellular production of oxygen radicals and are
believed to play an important part in oxygen
radical-mediated cell damage in neurodegenerative
diseases. The investigations are in complete
agreement with the occurrence of activity defects of
COX in single muscle fibres. The mitochondrial
impairment is not to age or denervation-associated
muscular changes since the functional impairment
of mitochondria (Dutta et al., 2006). While
numerous pathogenetic mutations are routinely
detected in isolated COX deficiencies, the protocols
for characterizing the functional impact of these
mutations are still in early stages of development.
The integrative approach combining multiple
bioenergetic analyses performed in whole cells is
particularly promising, as the best way to
investigate the resulting pathogenetic changes
occurring in situ. The general severity of the
functional changes in COX defects suggests that
the development of effective drugs is very unlikely,
and that only gene therapy might lead to efficient
treatment of these diseases in the future (Smith et
al., 2005). Measurement of the threshold for
specific mtDNA mutations has been performed in
cultured cells elsewhere. The microphotometric
enzyme–assay system for measurement of COX
activity within individual muscle fibers is used in
conjunction with demonstration of myofibrillar
ATPase in serial sections, so that a normal range of
COX activity in individual muscle fibers of each
fiber type is defined in control samples. In previous
studies,
a
good
correlation
between
microphotometric enzyme analysis and biochemical
studies has been shown in patients with
Leigh syndrome (Ogbi et al., 2006). The
newly reported four proteins in particular
were responsible for distinguishing diseased
from healthy. Peptide fingerprint mapping
unambiguously
identified
these
differentially expressed proteins. Three
proteins identified are involved in
respiration including cytochrome c oxidase
subunit 5b (COX5b), the brain specific
isozyme of creatine kinase and hemoglobin
β-chain (Horváth et al., 2005). In previous
studies six genes, involved in energy
metabolism pathways, also had increased
transcript levels in the skeletal muscle of CR
rats compared with muscle of control rats.
These include genes associated with
mitochondrial ATP production, such as six
subunits of cytochrome c oxidase (COX I,
II, III, IV, Va, and VIII) and NADH
dehydrogenase (Kalman et al., 2007). All
mitochondria of the progeny are inherited
from the mother; and all 13 polypeptides
encoded by the mitochondrial genome are
located in the respiratory chain (complexes
I, III, IV and V). These biological principles
are helpful in understanding the clinical
syndromes and patterns of inheritance
associated
with
the
mitochondrial
myopathies
and
encephalomyopathies
(Broadwater et al., 2011). Cellular injury
often has been associated with disturbances
in mitochondrial function. Interestingly, our
analysis indicated a pronounced increase in
the expression of mitochondrial cytochrome
c oxidase subunits IV and Vb, a finding that
was validated further by RT PCR.
Cytochrome c oxidase has been used
frequently as a marker for neuronal
metabolic activity, especially in pathological
conditions involving oxidative stress.
Neurons subject to oxidative damage show
abnormalities in mitochondrial dynamics
even in the absence of any apparent
indication of degeneration (Hamblet et al.,
2006). Elevations in cytochrome c oxidase
expression or function in vulnerable
neuronal subpopulations in AD, following
traumatic brain injury and preceding
apoptotic death of spinal cord motor neurons
after sciatic nerve avulsion, have been
reported. Thus, an increase in cytochrome c oxidase
expression may reflect aberrant energy metabolism
and oxidative damage in the spinal cord during
EAE (Fukui et al., 2007).
With due attention to the results of correlation
between COX2 and COX5B gene expressions with
nuclear DNA and mitochondrial DNA source, we
can claim that there is an interaction between two
COX gene expressions of genome and
mitochondria source and the effectiveness is of
COX enzymes in the MS patients. While the
number of selected samples were not more than 36,
the state of COX5B between patient and control
groups was meaningful. This means that in a group
of a number of members, there is no impact on its
meaninglessness. In COX2, the main reason for
meaninglessness may relate to the low number of
samples. Therefore, it would be better to reproduce
the experiments with an expanded sample size.
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Research Article 31
Curcumin rendered protection against cadmium chloride induced
testicular damage in Swiss albino mice
Preeti SINGH*, Kanchan DEORA, Vandana SANKHLA, Priya MOGRA
Department of Zoology, College of Science, M.L.S. University, Udaipur-Rajasthan, 313001 India.
(* author for correspondence; [email protected] )
Received: 04 August 2011; Accepted: 01 November 2012
Abstract
Fertility interference, regulation and control have become a matter of global concern in order to maintain adequate,
sustainable and healthy population. Cadmium is known to interfere with reproductive physiology and adversely
affects the process of spermatogenesis, whereas curcumin is known to be a potent, protective herbal derivative,
which renders protection at the physiological, metabolic and cellular levels against numerous toxicants. In the
present research, four groups of Swiss albino mice, each group consisting of six mice, were treated with cadmium
chloride and curcumin. Group 1 was the control group, where mice were administered only vehicle. In the second
group, mice were administered only curcumin. In the third group mice were administered single oral dose of CdCl2,
50mg/kg/animal/day, for a day and were left for 15 days. The fourth group was pre-treated with curcumin
(10mg/animal/day) for 15 days and on the 16th day they were administered with single oral dose of CdCl2
(50mg/kg/animal). In animals administered only cadmium, significant perturbations were observed in the process of
spermatogenesis. In the seminiferous tubules there is loss of cellular contact and associations, as manifested by loss
of germ cells. In organisms pre-treated with curcumin, marked decline in histopathological damage was observed,
where the loss of germ cells was not so pronounced. Hence, the present research categorically elucidates the
protective effect of curcumin against a single high dose of cadmium chloride induced perturbation in the process of
spermatogenesis .
Keywords: Curcumin, cadmium chloride, testicular damage, spermatogenesis, reproduction
Özet
Zerdeçal Swiss albino farelerde kadmiyum klorür ile indüklenmiş testiküler hasara karşı
koruma sağlar
Fertilitenin engellenmesi, düzenlenmesi ve kontrolü, syeterli, devamlı ve sağlıklı bir populasyonun sağlanması için
dünyanın ilgilendiği bir sorun haline gelmektedir. Kadmiyumun üreme fizyolojisini engellediği ve spermatogenez
sürecini ters olarak etkilediği bilinirken, fizyolojik, metabolik ve hücresel, seviyelerde pek çok toksik maddeye karşı
koruyucu olan zerdeçalın güçlü, koruyucu bir bitki türevi olduğu bilinmektedir. Bu araştırmada, her bir grupta altı
fare bulunan dört grup Swiss albino fareye kadmiyum klorür ve zerdeçal uygulanmıştır. Sadece distile su verilen
Grup1, kontrol grubunu oluşturmaktadır. İkinci grupta farelere sadece zerdeçal verilirken üçüncü gruptaki farelere
bir gün için 50mg/kg/hayvan/gün CdCl2 tek doz oral olarak uygulanmış ve 15 gün boyunca beklenmiştir. Dördüncü
gruba önceden 15 gün boyunca (10mg/hayvan/gün) zerdeçal uygulanmış ve 16. günde farelere tek doz oral olarak
50mg/kg/hayvan CdCl2 uygulanmıştır. Sadece kadmiyum uygulanan hayvanlarda spermatogenez sürecinde belirgin
bir düzensizlik gözlenmiştir ve germ hücre hasarıyla açıkça gösterildiği gibi seminifer tübüllerde hücresel iletişim ve
birliktelik kaybı gözlenmiştir. Önceden zerdeçal uygulanan organizmalarda germ hücre hasarının bildirilmediği
yerlerde histopatolojik hasarlarda belirgin bir düşüş görülmüştür. Bunun sonucu olarak bu araştırma çalışması
yüksek tek doz kadmiyum klorürün spermatogenez sürecindeki düzensizliği indüklemesine karşın, zerdeçalın
koruyucu etkisini kategorik olarak açığa kavuşturmaktadır.
Anahtar Kelimeler: Zerdeçal, kadmiyum klorür, testiküler hasar, spermatogenez, üreme
32 Preeti SINGH et al.
Introduction
In order to maintain adequate, sustainable and
healthy population; fertility interference, regulation
and control have become a matter of global
concern. Exposure to metals is a common
phenomenon due to their environmental
pervasiveness. Some metals are essential for life,
others have unknown biological functions, either
favorable or toxic, and some others have the
potential to cause toxicity (Col et al., 1999; Benoff
et al., 2000; Suwazono et al., 2000). Overexposure
of metals is in fact, one of the oldest environmental
problems since they are widely distributed in the
environmental workplace. Heavy metals are
persistent environmental contaminants since they
cannot be degraded or destroyed easily. These are
chemical elements capable of spreading in the
environmental compartments and circulating
between them. These metals are emitted in to the
atmosphere with the composition of fine particles
or in the gaseous form and are transported by
atmospheric fluxes to considerable distances where
they enter ecosystems of remote regions. However,
many heavy metals are urgently necessary for
functioning of the human body and other living
organisms in small amounts and belong to the range
of nutrients. Others, when passed on to the living
organisms cause poisoning or death (Danielyan,
2010). Some toxic heavy metals are cadmium (Cd),
arsenic (As), nickel (Ni), mercury (Hg), lead (Pb),
zinc (Zn), chromium (Cr) etc. A substantial number
of couples seek fertility treatment due to poor
semen quality and there is evidence in the literature
that male reproductive function seems to have
deteriorated considerably in the past four-five
decades due to multifaceted reasons being
psychological, physical , physiological, or due to
the effect of certain hazardous substances. One of
the hazardous substances is cadmium, the 48th
element in the periodic table with an atomic weight
of 112.4 and is present in all components of our
environment viz. air, water and soil. It is used in
various industrial processes as an anti-corrosive
agent stabilizer in PVC products, as a color
pigment, and in fabrication of nickel-cadmium
batteries (Friberg et al., 1974; Fox, 1983; Elinder,
1985; Morrow, 1990). It serves no physiological
function within the mammalian system. The
Agency for Toxic Substances and Disease Registry
(ATSDR) has listed cadmium as number 7 in its top
20 list of hazardous substances and International
Agency for Research on Cancer (IARC) has
classified cadmium as a group 1 carcinogen. It has
extremely long biological half-life in mammals,
which is estimated to be about 17 years or longer in
humans (Nordberg, 1972; IARC, 1992)
Cadmium is highly toxic to various biological systems,
e.g. kidney (Friberg et al., 1974; Buchet et al., 1980;
Goyer, 1991), male and female reproductive systems
(Ferm and Carpenter, 1967; Massanyi et al., 2007; Wu et
al., 2008; Monsefi et al., 2010), brain (Beton et al., 1966;
Taylor et al., 1984; Bernard and Lauwerys,1986),
gastrointestinal tract (Sugawara and Sugawara, 1974),
liver , circulatory system (Stowe et al., 1972) and skeletal
system (Kawamura et al., 1978; Blumenthal et al., 1995;
Staessen et al.,1999). Due to the rapid industrialization and
overgrowing urbanization, the toxic effects of cadmium on
male reproduction is to be assessed and monitored and an
effort has to be made to check, counter balance or nullify
its toxicity .
India has a rich history of using plants for
medicinal purposes. Turmeric derived from Curcuma as a
medicine is used as home remedy for various diseases
(Ammon and Wahl, 1991; Eigner and Scholz, 1999).
Curcumin has been considered as a potent healing herbal
formulation, a strong antioxidant, which has been
considered to be more than three hundred times more
potent than vitamin E (Rao et al., 1982; Dikshit et al.,
1995). Curcumin is also reported to have anti-bacterial,
anti-amoebic, antifungal, anti-viral and anti-HIV activities
(Ammon et al., 1992; Azuine and Bhide, 1992; Ruby and
Kuttan, 1995 and Mortellini et al., 2000).
Hence, in the present study an effort has been made to
assess and monitor cadmium-induced reproductive toxicity
as after one single chance exposure and to observe whether
curcumin, derived from Curcuma longa has the potential
to protect and prevent testes from such toxicity.
Materials and methods
32-50 days old adult Swiss albino mice, weighing around
30-40 g, were maintained in plastic cages under controlled
lighting conditions (12 h light/12 h dark regime), relative
humidity (50 ± 5%) and temperature (37 ± 2°C). The
animals were fed on standard mice feed. Food and water
were given ad libitum. Each batch comprised of 6 mice
and their dose protocol was as followsGroup 1 - Mice were administered the vehicle (distilled
water) for 16 days.
Group 2- Mice were administered curcumin 10mg/animal
for 16 days.
Group 3- Mice were administered single oral dose of
CdCl2 50mg/kg/animal/day for a day and left for 15 days.
Group 4- Mice were pretreated with curcumin
(10mg/animal/day) for 15 days and on the 16th day mice
were administered with single oral dose of CdCl2
(50mg/kg/animal).
Twenty-four hours after administration of the last dose,
control and experimental animals were sacrificed. Testes
were excised and subsequently fixed in Bouins solutions.
After fixation testes were processed, wax blocks were
made. Wax sections were cut and slides were prepared,
Curcumin protects against cadmium chloride 33
where ‘b’ the Observed Frequency for category ‘a’
‘d’ is the Expected Frequency in the corresponding
category ‘c’
This experimental study was done after taking approval
from the Institutional Animal Ethics Committee
(No.Cs\Res\07\759).
then stained in haematoxylin and eosin for
histopathological studies (Kiernan, 2008).
For statistical evaluation of significance, 100
seminiferous tubules (‘a’ and ‘c’), per group were
assessed and X2 (Chi Square) test was conducted
using the formula as per the two by two table.
a
b
a+b
c
d
c+d
a+c
b+d
N
;
X2 =
N (ad-bc)2
(a+c ) ( b+d) ( a+b) (c+d)
Results
In the present study the testes of cadmium and
curcumin treated animals were assessed using the
following parameters: Changes in morphology,
testicular pathology, and cytostatic and cytotoxic
changes in the germ and Leydig cells.
The testes of control group mice administered only
vehicle showed normal pathology with distinct
seminiferous tubules undergoing different stages of
spermatogenesis. The interstitium was compact
with distinct Leydig cells (Fig.1). Spermatogonial
mother cells, primary and secondary spermatocytes,
maturing spermatids and spermatozoons embedded
in Sertoli cells were clearly visible (Fig.2).
Histopathological evaluation of only curcumin
treated mice testes showed normal structures
similar to control group. Testes of the cadmium
treated animals exhibited hemorrhage of its
vasculature, but the overall shape was not altered.
Seminiferous tubules appeared to lose their typical
spherical shape and tunica propria manifested
thickening and was irregularly and randomly
broken at places. Leydig cells were entrapped in the
degenerated interstitium (Fig. 3). There was general
derangement of morphology of spermatogonia,
spermatocytes and differentiating spermatids in the
seminiferous tubules (Fig. 4). In most of the peripheral
seminiferous tubules, the germ cells were seen to break
free end appeared in the lumen as puffs (Fig. 5). The cell
types which appeared to be most affected were
spermatocytes and spermatids. Tubular lumens were filled
with degenerated germ cells and multinucleated spermatid
aggregates (Fig. 3). Vacuolization of the seminiferous
epithelium was also observed (Fig. 6). The results show
that a single dose of cadmium causes a sudden increase in
testicular damage, apparently overpowering this tissue’s
natural defenses. Also, most of the seminiferous tubules of
testes in this group showed complete absence of primary
spermatocytes, secondary spermatocytes, spermatids and
spermatozoa and loss of spermatogenesis process (Fig. 6)
in comparison with normal structure of seminiferous
tubules in control mice. The seminiferous tubules of
experimental group animals pre-treated with curcumin for
15 days and cadmium chloride on 16th day showed very
slight histopathological damage in the peripheral tubules
and the inner tubules appeared to be normal showing all
stages of spermatogenesis viz. spermatoginal mother cells,
primary and secondary spermatocytes, maturing
spermatids and spermatozoons embedded in Sertoli cells
(Fig.7 and 8).
Figure 1. 1) Photomicrograph of testis of control group mice administered only vehicle. Normal seminiferous
tubules (ST) and Leydig cell (LC) are clearly visible (20X). 2) Testis of control group mice administered only
vehicle. Seminiferous tubule with distinct spermatogonial mother cells (SMC), primary spermatocyte (PS),
secondary spermatocytes (SS), maturing spermatids (MS) and spermatozoa (S) are seen (40X). 3) Testis of cadmium
chloride (50mg/kg) treated group mice. Degenerate seminiferous tubules(ST) are clearly visible (20x). 4) Testis of
cadmium chloride(50mg/kg) treated group mice. Degenerated germ cells in lumen of seminiferous tubules are
clearly evident (40x). 5) Testis of cadmium chloride(50mg/kg) treated mice showing exfoliated germ cells(GC) in
tubular lumen (100x). 6) Testis of cadmium chloride (50mg/kg) treated mice. Seminiferous tubules are left with only
vacuolised spermatogonial mother cells (SMC) and Sertoli cell (SC) (100x). 7) Testis of group pre-treated with
curcumin(10mg/kg for 15 days) and then administered cadmium chloride(50mg/kg). Seminiferous tubules (ST)
showing stages of spermatogenesis and pyknotic nuclei in Leydig cells (LC) are clearly evident (40x). 8) Testis of
group pre-treated with curcumin (10mg/kg for 15 days) and then administered cadmium chloride(50mg/kg).
Maturing spermatids (MS) and adhered germ cells are visible (100x)
2011). These observations are similar to the results of the
present study, which has revealed that cadmium chloride
induced severe alterations in histopathological profile of
testes as manifested by disarrangement of morphology of
Leydig cells and 100% seminiferous tubular damage
within which spermatogonia, spermatocytes and
differentiating spermatids were severely affected and were
lost in the luminal space of the tubules culminating in total
suppression of spermatogenesis. There was induction of
azoospermia. The results of present experiment also
correlate well with other reports where cadmium has been
shown to induce testicular damage in rat and mice (Gunn
et al., 1970., Herranz et al., 2010; and Mathur et al.,
2010). Our observations are also similar to the
observations of Monsefi et al., 2008; Chowdhury, 2009
and Adamkovicova et al., 2010 where, similar to our
results cadmium chloride has shown to cause rapid
testicular edema, haemorrhage, necrosis and degeneration
of testicular membrane tissue. Adaikpoh and Obi (2009)
have reported that cadmium increased total cholesterol
levels in the testes and prostate of rats, which affects
Leydig cell function negatively. In the present
experimental design administration of curcumin protected
testis of mice exposed to cadmium as evidenced by
appearance of about 75% normal structures of
seminiferous tubule of testis showing the ongoing process
of spermatogenesis as evidenced by the presence of
spermatids and spermatozoon, and lack of exfoliated cells
in the luminal space. Additionally, the present study
indicated that the exposure to heavy metals produce
testicular damage, which leads to spermatogenic arrest
which is rectified and prevented by curcumin intake.
Discussion
The management of infertility problems is the need
of time. The importance of drugs from plant origin,
as fertility regulating agents for the males has long
been recognized. Medicinal plants present a
repertoire capable of providing varied constituents
which could be helpful in infertility management.
Curcumin, a potent antioxidant compound derived
from turmeric, has been used for centuries as a
natural dye, seasoning and medicine (Huang et al,
1988). In Ayurveda, a 5000 year old system of
medicine originating in India, curcumin in turmeric
has been used to treat dozens of common
conditions. Hence, in the present experiment an
effort has been made to observe ameliorative effect
of curcumin on testicular damage induced by
cadmium chloride. The evidence of the past twenty
years have shown a disturbing trend in male
reproductive health hazards due to careless use of
certain chemicals cadmium being one of them
,which causes detrimental effects on different
organs. Broad-spectrum irreversible toxic actions of
cadmium at the cellular and molecular levels have
been observed mainly on the reproductive system
of humans and experimental animals, by a number
of researchers (Batra et al., 2001; Chowdhury,
2004; Massanyi et al., 2007; Burukog and Bayc,
2008; Almansour, 2009; Obianime and Roberts,
2009). Cadmium has also been reported to cause
testicular damage in Leydig cells and seminiferous
tubules (Massanyi et al., 2007; Burukog and Bayc,
2008; Almansour, 2009; Obianime and Roberts,
2009; De Souza Predes et al., 2009 and Al attar
Table 1. Alterations in the seminiferous germ cells of Swiss albino mice challenged with cadmium chloride and
curcumin
S. No.
Experimental Protocol
Total
number of
germ cell
layers
Types Of Germ Cells Present (P) And Exfoliated (E)
SMC
SC
P Sp
S Sp
S
SZ
Se C
1.
Control (distilled water as a
vehicle for 16 days)
P
P
P
P
P
P
P
2.
Curcumin (10 mg/animal/day
for 16 days)
P
P
P
P
P
P
P
3.
Cadmium Chloride(50
mg/kg/animal/day for 15 days)
P
P
E
E
E
E
P
2-3*
Χ2 =4.241
P=0.04
4.
Curcumin (10 mg/animal/day
for 16 days) +Cadmium
Chloride(50 mg/kg/animal/day
for 15 days)
P
P
E
E
P
E
P
6-10
Χ2=0.204
P=0.65
9-11
9-11
SMC – Spermatogonial Mother Cells; SC – Spermatogonial cells ; PSp- Primary Spermatocytes; SSp- Secondary
Spermatocytes; S- Spermatids; SZ- Spermatozoa; SeC- Sertoli Cells *Χ2 significant at P ≤0.05
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1303-1312, 2000.
Huang M, Smart RC, Wong C, and Conney AH.
Inhibitory Effect of Curcumin, Chlorogenic
Acid, Caffeic Acid, and Ferulic Acid on Tumor
Promotion in Mouse Skin by 12-OTetradecanoylphorbol-13-acetate.
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Research Article 39
Study of Klebsiella pneumoniae isolates with ESBL activity,
from ICU and Nurseries, on the island of Mauritius
Salima Khurshid MUNGLOO-RUJUBALI1, Mohamed Iqbal ISSACK2, Yasmina
JAUFEERALLY-FAKIM1*
1
Department of Biotechnology, University of Mauritius, Reduit, Mauritius.
Victoria Hospital, Ministry of Health and Quality of Life, Mauritius.
2
Received: 11 August 2011, Accepted: 2 November 2012
Abstract
Klebsiella pneumoniae with extended spectrum beta lactamase activity circulate widely among
nosocomial environments. Isolates were collected from hospitalised patients in ICU and nursery units
on the island of Mauritius, over a two year period. They were tested for their resistance/susceptibility
to various antibiotics using the double disc diffusion assay. Following the API biochemical assay their
genus/species were confirmed. Their genetic diversity was determined using RAPD, REP and BOXPCR. Among fifty isolates, most were highly diverse by these methods with a clustering into three
groups. No correlation was found among isolates of the same hospital or unit or with dates of
collection. Sequence analysis of parts of the TEM, SHV and CTX-M beta lactamase genes confirmed
the beta lactamase domain and the presence of SHV-11 and SHV-28. CTX-M 15 was found in some
isolates. Furthermore, integrase was PCR-amplified from a few isolates, showing the presence of an
integron-borne gene cassette. This study shows diverse clones of Klebsiella pneumoniae circulating
with ESBL activity.
Keywords: Klebsiella pneumoniae, ESBL, TEM, SHV, genetic diversity, beta lactamase
Özet
Mauritius adasında kreş ve yoğun bakım ünitesinden elde edilen ESBL aktiviteli
Klebsiella pneumoniae izolatlarının çalışması
Geniş spektrumlu beta laktamaz aktivitesi olan Klebsiella pneumoniae nosokomiyal çevrelerde büyük
ölçüde bulunmaktadır. İzolatlar, Mauritius adasındaki yoğun bakım ünitesinde yatan hastalardan ve
kreşlerden iki yıl boyunca toplanmıştır. Çift disk difüzyon testi kullanılarak izolatların çeşitli
antibiyotikler için dirençleri/duyarlılıkları incelenmiştir. Sonrasında API biyokimyasal test ile cins/tür
teyidi yapılmıştır. Genetik farklılıkları RAPD, REP ve BOX-PCR kullanılarak belirlenmiştir. Test
edilen elli kadar izolatın çoğu, üç grupta sınıflandırılan bu metodlar tarafından oldukça farklı
bulunmuştur. Aynı hastane, birimler, ya da aynı tarihlerde toplanan izolatlar arasında herhangi bir
korelasyon bulunmamıştır. TEM, SHV ve CTX-M beta laktamaz genlerinin dizi analizleri beta
laktamaz domeynini ve SHV-11 ile SHV-28 varlığını onaylamıştır. Bazı izolatlarda CTX-M 15
bulunmuştur. Bundan başka, birkaç izolattan PCR-amplifikasyonu yapılmış olan integraz ile, integronkaynaklı gen kasetinin bulunduğu gösterilmiştir. Bu çalışma ESBL aktivitesiyle dolaşan Klebsiella
pneumoniae’nın farklı klonlarını göstermektedir.
Anahtar Kelimeler: Klebsiella pneumoniae, ESBL, TEM, SHV, genetik çeşitlilik, beta-laktamaz
40 Salima Khurshid MUNGLOO-RUJUBALI et al.
Introduction
Klebsiella pneumoniae is a very common source of
infections in hospital environments and are
particularly of threat to immune-compromised
patients. It is a gram-negative bacillus of the
Enterobacteriaceae family. It is mostly treated with
beta lactams and fluoroquinolones, but over the
years the bacteria have accumulated significant
resistance to many of those. Beta lactamases,
encoded in bacterial sequences, are able to
efficiently hydrolyse the beta lactams of many
antibiotics, thus rendering them inactive. A large
number of studies have demonstrated their wide
occurrence and rapid spread during the era of
widespread antibiotic use for medical applications
culminating in the appearance of extended
spectrum beta lactamases (ESBLs), which are
resistant to third generation cephalosporins
(Nteimam, 2005).
The mode of action of beta lactamases relies on
their ability to bind to enzymes of the bacterial cell
wall biosynthesis process, thereby killing the cells.
They have an active site serine residue. They act on
peptidases that cross-link the penultimate D-alanine
of one peptidoglycan unit to a free amino acid of
diamino pimelic acid (gram negative) or a lysine
residue (Gram positive). The D-alanine-D-alanine
substrate of the peptidoglycan unit has a similar
stereochemistry to the beta lactam moiety thus
competing for binding at the active site of the
peptidases. TEM and SHV beta lactamases were
characterised first, followed by CTX-M. They are
of the Class A enzymes (Ambler, 1980). According
to Hall and Barlow (2004), TEM enzymes have
experienced larger phenotypic evolution, than SHV
and CTX-M. CTX-M ESBLs provide resistance to
Cefotaxime, but usually not to Ceftazidime. The
double disc synergy method using Cefotaxime and
Ceftazidime for detection of ESBLs is the
recommended one by the British Society for Anti
Microbial Chemotherapy (BSAC). ESBLs can
hydrolyse the so-called modern β-lactams that are
cefotaxime,
cefuroxime,
aztreonam
and
ceftazidime.
The last two decades have seen the appearance
of a large number of mutant forms of beta
lactamase genes from diverse organisms. Most
importantly, there seems to be a positive selection
for non-synonymous point mutations, which alter
the binding affinity of the enzyme for its
substrate. Key amino acid substitutions
bring changes in the substrate and inhibitor
binding affinities. Sequence comparisons of
the genes have identified the homology
within the TEM or within the SHV groups
and revealed codon alterations which lead to
different phenotypic profiles. Structural
comparison is useful in understanding the
outcome of nucleotide changes on the 3dimensional features of the protein. Alleles
TEM 1A and 1F differ by silent mutations;
however derivative variants can arise by
further point mutations or by crossing-over
between two alleles. Examples of
substitutions can be found at www.lahey.org
which lists all the reported variants and their
codon differences. Recent work has
identified key amino acid replacements
which are responsible for phenotypic
changes. Mutations that cause the ESBL
phenotype are known to be R164H (Arg to
His), R164S (Arg to Ser) and G238S (Gly to
Ser). TEM-68, which has a decreased
sensitivity to inhibitors, has a R275L (Arg to
Leu) replacement compared to TEM-1.
Similarly other mutations lead to modulating
effects such as increasing or decreasing
MIC’s (minimum inhibitory concentrations).
β -lactam antibiotics penetrate the outer
membrane of many gram-negative bacteria
through porins, hence antibiotic resistance
can also result from porin loss or deficiency
(Nikaido, 1989). Several reports have
demonstrated that beta lactamase expression
is not the only form of resistance in ESBL
bacteria. Other mechanisms such as porin
loss have been described in many species
(Tsu-Lan et al., 2001).
An understanding of the bacterial
populations carrying ESBLs is essential to
characterise
the
molecular
features
associated with the genes for β-lactamases
and understand their evolution. This study
describes the characterisation of Klebsiella
pneumoniae isolates from hospital sources
to assess their ESBL phenotype and identify
the corresponding gene sequences. The
genetic diversity of the strains carrying this
phenotype was determined to gauge the
K. pneumonia isolates with ESBL activity 41
extent of occurrence of the ESBL feature.
Isolation and characterisation of K. pneumoniae
the Ministry of Health, Mauritius. They
were pre-selected for their ESBL phenotype
by double disk assay. An enlargement of the
inhibition zone of 5 mm in the presence of
clavulanic acid is a confirmation of an ESBL
presence. The isolates were characterised
biochemically using the Analytical Profile
Index (API) system.
About fifty isolates of K. pneumoniae were
obtained between March 2003 and January 2005
from six different hospitals (Table 1). These were
collected by the Central Analytical Laboratory of
Table 1. List of K.pneumoniae isolates
ISOLATE
DATE ISOLATED
SEX
AGE
HOSPITAL
WARD
SOURCE
SS01
2.06.04
M
36
A
ICU
BLOOD
SS02
28.08.04
M
37
A
ICU
BLOOD
SS03
11.11.04
F
5 DAYS
D
2-3
N/A
SS04
3.08.04
M
37
A
ICU
BLOOD
SS05
9.11.04
F
73
B
D4
N/A
SS06
5.12.04
M
NEWBORN
A
NURSERY
BLOOD
SS07
23.08.04
F
60
C
MICU
BLOOD
SS08
27.01.04
F
69
C
1-3
N/A
SS09
14.11.04
F
4 DAYS
A
NURSERY
N/A
SS10
26.03.03
NEONATES
E
NURSERY
N/A
SS11
9.11.04
M
15 DAYS
C
NICU
RECTALS
SS12
9.09.03
F
3 WEEKS
C
NICU
N/A
SS13
24.10.04
NEONATES
D
NURSERY
N/A
SS14
6.02.04
A
NURSERY
ENV(SINK)
SS15
29.11.04
F
6 DAYS
A
NURSERY
N/A
SS16
23.06.04
M
29
B
04
BLOOD
SS17
19.11.04
M
62
B
D4
N/A
SS18
20.10.04
M
11
B
RDU
VAS.CAT.FLUID
SS19
14.02.04
F
2
C
S21
N/A
SS20
28.02.03
7 DAYS
F
SS21
22.11.02
4 DAYS
B
NICU
N/A
SS22
3.06.04
M
40
A
ICU
URINE
SS23
3.06.04
M
40
A
ICU
URINE
N/A
SS24
4.06.04
M
40
SS25
29.10.03
M
59
SS26
19.09.04
M
53
SS27
11.11.04
M
5 DAYS
SS28
27.10.03
M
67
SS29
23.01.04
F
15
SS30
1.09.04
F
SS31
31.07.04
SS32
A
ICU
URINE
ICU
CSF
D
4
N/A
C
NICU
RECTALS
ICU
CSF
A
ICU
N/A
53
E
DIALYSIS
BLOOD
M
50
C
MICU
ET-SECRETION
26.11.04
F
11 DAYS
A
NURSERY
BLOOD
SS33
14.01.05
M
44
A
2-4
BLOOD
SS34
19.02.04
F
61
E
F69
BLOOD
SS35
10.12.04
F
5 WEEKS
D
NURSERY
N/A
SS36
27.09.04
M
37
A
2-4
BLOOD
SS37
26.11.04
F
2 DAYS
NURSERY
N/A
SS38
14.01.05
M
57
E
B1
BLOOD
SS39
20.09.04
M
53
D
4
BLOOD-DIALYSIS
SS40
22.10.03
M
50
C
ICU
BLOOD
SS41
2.10.03
M
42
C
SS42
21.06.04
F
58
A
1-2
BLOOD
SS 43
18.11.03
F
68
B
ICU BURN UNIT
N/A
SS44
13.03.03
F
12 DAYS
B
NURSERY
CSF
SS45
27.09.03
F
56
C
ICU
BLOOD
SS46
21.06.04
F
3 WEEKS
D
NURSERY
BLOOD
SS47
16.06.04
M
44
C
MICU
N/A
SS48
4.10.04
C
CARDIAC ICU
ET SECRETION
SS49
30.11.04
M
34
A
ICU
BLOOD
SS50
29.07.04
M
34
D
1-3
BLOOD
N/A
RDU: Renal Dialysis unit, NICU: Neurological intensive care unit, MICU: Medical Intensive care
unit CSF: Cerebro spinal fluid , ET: endotracheal secretion; N/A: Not available
Their sensitivity to the following antibiotics either
alone or in the presence of clavulanic acid was
assessed on Mueller-Hinton agar using the pairs of
Oxoid combination discs: (ceftazidime-30
µg and ceftazidime/clavulanate-30/10 µg;
cefotaxime-30
µg
and
cefotaxime/clavulanate - 30/10 µg; cefpodoxime-30
µg
and
cefpodoxime/clavulanate-30/10µg;
cefpirome-30 µg and cefpirome/clavulanate30/10µg).
BOX-PCR and RAPD PCR amplification
For
the
BOX-PCR,
the
primer
5’CTACGGCAAGGCGACGCTGACG-3’ (Martin et
al, 1992) was used. PCR was carried out in a total
reaction mixture of 25 µl, containing 50 mM buffer,
2 mM MgCl2, 200 µM each deoxynucleoside
triphosphate, 25 pmol of primer, 25 ng of template
DNA and 1 U of Taq DNA polymerase .
Amplifications were performed with a DNA
thermocycler (BIO-RAD) as follows: 1 cycle of 95º
C for 4 minutes, 30 cycles of 94º C for 30 s, 92º C
for 30 s, 50º C for 1 minute, 65º C for 8 minutes
and 1 cycle of 65º C for 8 minutes. The PCR
products were run in 1.5 % agarose gel
stained with EtBr and observed to score for
markers.
Similar reaction was set up for RAPD
except that the annealing temperature was
32º C.
The gel banding patterns were scored and
the data was analysed by NTSYS; and the
tree was generated with Darwin 5.0.
Amplification and sequence analysis of
TEM, SHV and CTX-M genes
Standard PCR reactions were done for the
amplification of beta-lactamase genes using
published primer sequences (Table 2).
Products were purified after amplification
and sequenced. Sequence results were
manually checked and edited.
Table 2. List of primer sequences
Primer Name
SHV A
SHV B
SHV A1
SHV A2
SHV A11
SHV A12
Primer Sequence
Reference
5- ACT GAA TGA GGC GCT TCC-3
5- ATC CCG CAG ATA AAT CAC C-3
Babini & Livermore, 2000
5- TCA GCG AA AAC ACC TTG-3
5 –TCC CGC AGA TAA ATC ACC A-3
5- ATG CGT TAT ATT CGC CTG TG-3
5- GTT AGC GTT GCC AGT GCT CG-3
5- TGG TTA TGC GTT ATA TTC GCCSHV S1
3
Gniadkowski et al., 1998
SHV S2
5- GGT TAG CGT TGC CAG TGC T-3
SHV B1
5-ATG CGT TAT ATT CGC CTG TG-3
SHV B2
5- GTT AGC GTT GCC AGT GCT CG-3
5- GGG AAT TCT CGG GGA AAT
TEM D1
GTG CGC GGA AC-3
5- GGG ATC CGA GTA AAC TTG GTC
TEM D2
Bou et al., 2000
TGA CAG-3
TEM A1
5- TAA AAT TCT TGA AGA CG-3
TEM A2
5- TTA CCA ATG CTT AAT CA-3
CTX-M1
5- CGC TTT GCG ATG TGC AG-3
Jungmin Kim et al., 2005
CTX-M2
5- ACC GCG ATA TCG TTG GT-3
INT 2F
5-TCTCGGGTAACATCAAGG -3
Mazel et al., 2000
INT 2R
5- AAGCAGACTTGACCTGA -3
the nucleotide sequence were converted into
Sequence Analysis
contigs using the following web page
SHV, TEM and CTX-M homologue DNA
http://pbil.univ-lyon1.fr/cap3.php. Tblastx
sequences
were
identified
with
tblastx
was carried out with the contigs in NCBI.
(www.ncbi.nlm.nih.gov/blast) search of the nonThe contigs were translated into amino
redundant National Center for Biotechnology
acids by using the following program:
Information (NCBI) sequence database. First of all
http://www.biochem.ucl.ac.uk/cgi-
44 Salima Khurshid MUN
NGLOO-RUJU
UBALI et al.
bin/mcdoonald/cgina2aaa.pl
The amino
a
acid seequence with no stop coddon
was seleected. This was
w
then aliigned with the
t
corresponnding sequence that blasteed well withh a
low E-value.
The amino
a
acid seequences of SHV,
S
TEM and
a
CTX-M and their hoomologs werre aligned with
MultAliggn.
Results
Biochem
mical characteerisation
The API 20E system indicated thaat 45g negatiive
w
identifieed as Klebsiellla pneumoniaae,
isolates were
however these isolates could be classsified into thrree
groups baased on the prrofile obtainedd:
Group I: Profile 52157773
Group II:: Profile 52153373
Group IIII: Profile 52077773
Group IV
V: Profile 52055773
Group V: Profile 52557773
Group VII: Profile 52255773
ESBL acctivity
Four out of fifty isolatees were ESBL
L negative. Thhey
were isolates SS22, SS42,
S
SS45 and SS47. The
T
differencee in the zone of inhibition was less thann 5
mm for each one of
o them. Thee remaining 46
isolates were
w confirmed as ESBL producers.
The double
d
disc asssay revealed, that
t except for a
few, mosst of the isolaates produced evidence of an
extended spectrum beta lacctamase; with
Cefottaxime, isolates SS 35 andd SS 42 were
susceeptible with a zone of iinhibition of
aboutt 20 mm and 25 mm, resppectively, and
an in
ncrease of thiss zone by > 5 mm in the
preseence of clavullanate. Three isolates, SS
17, SS
S 31 and SS
S 42 were suusceptible to
Ceftaazidime with no
n increase inn diameter in
the
presence
of
inhibiitor.
With
Cefpo
odoxime, SS 42,
4 SS 45 andd SS 47 were
susceeptible, whilee with Cefpirrome SS 42
and SS
S 09 showedd some susceeptibility and
just a slight inccrease of 4 mm in the
preseence of inhibittor clavulanatte. Isolate SS
42 waas considered a non-ESBL.
BOX
X and RAPD PCR
P
BOX
X-PCR is commonly used for
fingerprinting baacterial genoomes using
primeers targeting repetitive elements in
interg
genic regionns. The oveerall results
show
wed that isolaates SS49, S
SS42, SS45,
SS43, SS44, SS48,, SS46, SS47,, SS50 which
were isolated duriing the same year, but in
differrent hospitalss, were groupped into one
clusteer (Figures 1, 2, 3). Isoolates SS42,
SS44
4, SS45, SS477, SS48, SS449 and SS50
also shared
s
the sam
me API profilee i.e profile 1
(Figu
ures 2 and 5).
X-PCR. Lane 42: SS42, Lanne 43: SS43,
Figure 1. DNA fingerrprint analysiss of K pneumooniae by BOX
4 SS45, Lanne 46: SS46, Lane
L
47: SS47
7, Lane 48: SS48, Lane 49: SS49, Lane
Lane 44: SS44, Lane 45:
H Hyperladdeer (11) Biolinne.
50: SS50. C: Control, H:
K.
K pneumonia issolates with ESBL activity 45
Figure 2. Dendogram generated froom BOX-PCR
R based on Diice similarity coefficient annd neighbour
joining cllustering for the
t 50 isolatess of Klebsiellaa pneumoniaee with a copheenetic correlattion (r) value
of 0.95299.
Figure 3.. RAPD fingeerprint of K. pneumoniae with primer OP
PA12. Lane 1: SS01, Lane 22:SS02, Lane
3: SS03, Lane 4: SS004, Lane 5: SS05, Lane6: SS06, Lane 7:
7 SS07, Lanee 8: SS08, Laane 9: SS09,
Lane10: SS10, Lane 11: SS11, Laane12: SS12, Lane13: SS1
13, Lane14: SS14,
S
Lane 115: SS15. C:
control, H:Hyperladder
H
r(11) Bioline
46 Salima Khurshid MUN
NGLOO-RUJU
UBALI et al.
Figure 4. RAPD fingeerprint of K .ppneumoniae with
w primer OPB
O
01. Lane442: SS42, Lanne 43: SS43,
Lane 44: SS44, Lane455: SS45, Lanee 46: SS46, Laane47: SS47, Lane48: SS488, Lane 49: SS
S49, Lane50:
SS50.C: control,
c
H:Hyyperladder(1) Bioline.
B
Figure 5. Dendogram generated froom RAPD-PCR
R based on Dice
D similarity coefficient annd neighbour
joining cllustering of RAPD
R
for the 50
5 isolates off Klebsiella pn
neumonia. Forr both sets of data with the
BOX andd RAPDs, the clustering of isolates
i
was nearly
n
the sam
me, with three discernable
d
grroups.
K.
TEM, SH
HV and CTX-M amplificaation
Differentt size productss were obtaineed for each set of primers. The
T TEM A1//A2 gave prodducts slightly
longer thaan 1 kb. The CTX-M
C
primeers amplified a product of 585
5 bp. Both sets
s of SHV A
A11 and A12,
and SHV
V B1 and B2 gave
g
a producct of 865 bp. Amplification
A
n were detecteed for thirty seven isolates
for SHV A11 and SHV
V A12; twentyy two isolates for SHVB1 and
a SHVB2, reespectively. A
Amplification
was achieeved with twenty-one isolaates for TEM
M A1/A2 and twenty eight isolates for T
TEM D1/D2.
There weere only twelve isolates, whhich amplified with CTX-M
M to give a product of 585 bpp.
Figure 6.. Protein blastt of translated product from
m isolate SS11 with primer SHV
S
A11/A122
Figure 7.. Alignment of
o amino acid sequence from
m isolate SS11 with SHV-228. The same w
was obtained
with isolaate SS25. Amiino acid sequeence from isollate SS47 alig
gned well withh CTX-M 15.
For one isolate, the presence of integrons was assessed using integrase specific primers. It was found
that the INT amplicon from isolate SS02 had the sequence for integrase. The BLAST result returned a
match with Corynebacterium diphtheriae integrase.
The results for integrase from isolate SS02 are shown below.
>2R.INT
QAMKT
ATAPLPPLRS VKVLDQLRER IRYLHYSLRT EQAYVHWVRA FIRFHGVRHP
ATLGSSEVEA FLSWLANERK VSVSTHRQAL AALLFFYGKV LCTDLPWLQE
IGRPRPSRRL PVVLTPDEVV RILGFLEGEH RLFAQLLYGT GMRISEGLQL
RVKDLDFDHG TII
ref|NP_940279.1| integrase [Corynebacterium diphtheriae NCTC ... 331 2e-89
REFSEQ: accession NC_002935.2
KEYWORDS
complete genome.
SOURCE
Corynebacterium diphtheriae NCTC 13129
ORGANISM
PUBMED
Corynebacterium diphtheriae NCTC 13129
14602910
Amino acid sequence
>2int
mktataplpp lrsvkvldql rerirylhys lrteqayvhw vrafirfhgv rhpatlgsse
veaflswlan erkvsvsthr qalaallffy gkvlctdlpw lqeigrprps rrlpvvltpd
evvrilgfle gehrlfaqll ygtgmriseg lqlrvkdldf dhgtiivreg kgskdralml
peslapslre qlsrglcckd wrqsevgcrs apirrllrng g
K.
Figuree 8. Conserveed domain of integrases.
Discussioon
The Klebbsiella pneumooniae isolates were analyssed
to determ
mine their geenetic relateddness and moost
came from ICU (42%
%). These isolaates were testted
by classiical biochemiical (growth on media, API
A
identificaation and Oxoiid Combinatioon disk test) and
a
DNA- baased methods.. Biochemicall profiles by the
t
API 20 E system identiified two grouups.
The phenotypic
p
chaaracterisation of sensitivity to
the anti--microbials inndicated thatt most of the
t
isolates thhat were colleected and pree-screened, weere
in fact ESBLs.
E
API confirmed thheir identity as
Klebsiellaa pneumoniaee. There are no
n reports of the
t
recent sittuation regardding the extent of spread of
ESBL froom Klebsiellaa pneumoniaae or any othher
resistant bacterial
b
pathogens in Mauuritius.
OPB 01, OPL 07 and OPA12 were
w
the RAP
PD
primers (Fig
(
3&4), chhosen from thee twenty five or
so primeers that weree screened. Primer
P
OPA 12
producedd more polymoorphic profilees than the othher
two and all
a the isolates were differeent. With prim
mer
OPL 07 the
t same profiile was obtainned from isolattes
SS40, SS
S41, SS42, SS43, SS44, SS445, SS46, SS447,
SS48, SS
S49, and SS500. They all haad only 3 bannds
of sizes sizes
s
700bp, 1000 bp and 1200 bp. Theese
same isollates gave diffferent patternns with the othher
two prim
mers. The denndrograms obbtained with the
t
results of
o BOX-PCR
R and that of
o RAPD booth
producedd three groups. Several of thhem were in the
t
same grouup for both methods.
m
No coorrelation wass found betweeen hospitals and
a
dates off isolation suuggesting thaat the ESBL
L -
h
originatedd from very
carrying strains had
diverse sources. Siimilarly, theree was little or
no co
orrelation fouund among sam
mples of the
same hospitals, probably
p
inddicating that
there had not beeen significannt spread of
ESBL
L carrying strains
s
withinn the same
centree, although thhe sampling w
was primarily
done from patientss. RAPD andd PFGE have
been used for typing off Klebsiella
pneum
moniae (Thouuraya et al., 2003). Both
PFGE
E typing and RAPD
D produced
conco
ordant resullts and diiscrimination
betweeen groups off epidemiologiically related
strain
ns could be maade.
Th
he presence of
o the beta-lacttamase genes
were detected by PCR amplifiication using
specific primers for
fo TEM, SHV
V and CTXM. All
A fifty isolates amplifieed the TEM
and/o
or SHV gennes while oonly twelve
produ
uced a prodduct with tthe CTX-M
specific primerss (data noot shown).
Ampllicons were purified andd sequenced.
BLAST results inddicated that isolates SS11
and SS25 carried SHV 28 whhereas SS27
harbo
oured SHV 11. For isolatees S01, S02,
S25 and S41-49, CTX-M 15 was present.
This variant is known
k
to bee commonly
found
d worldwide. There are fivve groups of
CTX--M enzymes, namely CTX
X-M 1, 2,3, 4
and 25. ESBL
L carrying strains of
entero
obacteria arre resistant to third
such
as
generration
cephhalosporins
cefotaxime, cefdazidime and ceftriaxone; and are
important threats. These enzymes do not affect
cephamycins or imipenems. CTX-M is widely
found in E. coli and Klebsiella isolates worldwide.
CTX-M 15 in Salmonella enterica has also been
shown to be present in stool samples of patients
suffering from acute diarrhoea (Rotimi et al.,
2008). The CTX-M 15 is encoded on large
incompatibility plasmids of sizes varying between
145,5 and 242,5 kb. Many are found as IncFII
together with IncFIA and IncFIB, with one report
showing IncFI not associated with any IncFII
(Mshana et al., 2009). This suggests that the
plasmids contribute to the lateral transfer of the
resistance genes. CTX-M ESBL hydrolyse
cefepime more efficiently than other ESBLs
(Mendonca et al., 2007).
TEM and SHV enzymes have evolved
significantly over the last decade or so and there is
some evidence of positive selection. A list of over
hundred different combinations of amino acid
substitutions have been reported for the 278 long
polypeptide. It is likely that the evolution of the
protein is directed towards finding the mutants that
have enhanced catalytic efficiency and can better
compete with others. Experimental prediction of
how resistance genes evolve in response to
selection pressure has shown that alleles of the
genes will mutate in a similar way as in nature.
Several in vitro experiments using gene shuffling,
or mutagenesis with Taq Polymerase under errorprone or the use of nucleoside analogs have led to
the appearance of mutants with extreme resistance
(> 64X or to beta-lactamase inhibitor). Interestingly
these in vitro experiments have recovered amino
acid substitutions that are naturally found among
the TEM beta-lactamases. Hall and Barlow (2002)
have used error-prone PCR for assessing the
evolution of TEM-1 under selection pressure.
Mutations were introduced into the genes and the
mutated genes cloned into E.coli. Growth in
increasing concentrations of antibiotic led to the
identification of mutations in the resistance genes
which allowed survival in the highest antibiotic
concentrations. The cycles of mutations and
selection are repeated until there is no further
increase in resistance.
The Barlow-Hall in vitro method recovered
seven out of the nine amino acid substitutions that
had arisen in nature. The same approach has been
used to isolate an allele giving a resistance with a
MIC of 256 µg/ml compared to 0.5 µg ml
for reported, natural form of TEM. Those
resistant alleles had between 2 to 6 amino
acid substitutions. Three of these were
identified that increased the MIC from 0.5 to
2 µg /ml then to 32 µg /ml and finally from
32 µg /ml to 256µg /ml. This would strongly
suggest that there is a “natural evolutionary”
series of replacement leading to the most
resistant allele (Ford and Avison, 2004).
Analysis of a K. pneumoniae genome
sequence (strainMGH78578) reported two
chromosomal
blaSHV
genes.
Closer
comparison of the surrounding sequence
(GC content and other genes) of the two
genes indicated that one had evolved from
within that strain itself. On the other hand
the second one was accompanied by
insertion sequences IS26 suggesting that it
was mobilised most likely from a different
K. pneumoniae strain. Nucleotide sequence
comparison and amino acid replacement at
the active site have pointed to convergent
evolution of ESBLs during the period of
intense cefotaxime use and later that of
ceftazidime.
Beta lactamase genes have been shown
to be vehicled as gene cassettes within
integron sequences. VEB-1 (Vietnamese
Extended Spectrum) and GES-1 (Guyanese
Extended Spectrum) were first described
associated with the intI1 integrase (Poirel et
al 1999., 2000). Being part of the integron
ensures that the resistance genes are rapidly
mobilised for lateral transfer intra- and interspecies. In this study, one CTX-M 15 from
isolate SS02, was found together with an
integrase (Figure 3c). This could explain its
rapid spread since it was first described.
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Research Article 53
HIV-1 reverse transcriptase inhibition by Vitex negundo L. leaf
extract and quantification of flavonoids in relation to anti-HIV
activity
Mohan KANNAN1*, Paramasivam RAJENDRAN2,
Gnanasekaran ASHOK3, Shanmugam ANUSHKA3,
RAMACHANDRAN NAIR1
Veerasami VEDHA3,
Pratap CHANDRAN
1
Department of Biotechnology and Research, K.V.M. College of Engineering and Information
Technology, Kokkothamangalam, Cherthala, Kerala, India.
2
Department of Microbiology, Sri Ramachandra Medical College & Research Institute, Sri
Ramachandra University, Porur, Chennai – 600 116, Tamilnadu, India.
3
Department of Microbiology, Post Graduate Institute of Basic Medical Sciences, University of
Madras, Taramani Campus, Chennai – 600 113, Tamilnadu, India.
(*author for correspondence; [email protected])
Received: 22 September 2011, Accepted: 08 November 2012
Abstract
This study aimed to determine the activity of ethanolic leaf extract of Vitex negundo L. against HIV-1
Reverse Transcriptase (RT) and to identify and quantify the flavonoids present. The effects of
ethanolic (85%) leaf extract of Vitex negundo L. on RT activity in vitro were evaluated with
recombinant HIV-1 enzyme, using a non-radioactive HIV-RT colorimetric ELISA kit. In addition,
identification and quantification of flavonoids such as Rutin, Luteolin, Myricetin, Quercetin,
Kaempherol, Isorhamnetin and Quercetagetin were analysed using HPLC. The plant Vitex negundo L.
ethanolic leaf extract exhibited the most notable activity of 92.8% against HIV-1 RT at 200 µg/ml
concentration. Phytochemical analysis revealed the presence of steroids, triterpenes, alkaloids,
flavanoids, antroquinone glycosides and amino acids. Among 7 flavonoids tested, 6 were identified in
the decreasing order of quantity as Kaempherol, Myricetin, Quercetin, Quercetagetin, Isorhamnetin
and Luteolin. The study revealed that the plant Vitex negundo L. leaf possess anti-RT substances and
probably the flavonoids act as anti-virus agents.
Keywords: Vitex negundo, flavonoids, HIV-1 reverse transcriptase, phytochemical, anti-HIV activity.
Özet
Vitex negundo L. yaprak özütüyle HIV-1 ters transkriptaz inhibisyonu ve antiHIV aktivitesiyle ilişkili flavonoidlerin kantifikasyonu üzerine bir çalışma
Bu çalışmada HIV-1 ters transkriptaza karşı Vitex negundo L. etanolik yaprak özütünün aktivitesini
tespit etmek ve flavonoidlerin varlığını ölçmek amaçlanmıştır. Vitex negundo L. etanolik (%85)
yaprak özütünün in vitro RT aktivitesi üzerinde etkileri, rekombinant HIV-1 enzimi ile radyoaktif
olmayan HIV-RT kolorimetrik ELISA kiti kullanarak ölçülmüştür. Ayrıca, Rutin, Luteolin, Myricetin,
Quercetin, Kaempherol, Isorhamnetin ve Quercetagetin gibi flavonoidlerin tanımlanması ve
kantifikasyonu HPLC kullanılarak analiz edilmiştir. Vitex negundo etanolik yaprak özütü 200 µg/ml
konsantrasyonda HIV-1 RT’e karşı %92,8 aktivite göstermiştir. Fitokimyasal analizler steroidlerin,
triterpenlerin, alkoloidlerin, flavonoidlerin, antrokinon glikoitlerin ve aminoasitlerin varlığını açığa
54 Mohan KANNAN et al.
çıkartmaktadır. Test edilen 7 flavonoidden altısı, azalan miktarlarına göre Kaempherol, Myricetin,
Quercetin, Quercetagetin, Isorhamnetin ve Luteolin olacak şekilde belirlenmiştir. Bu çalışma, Vitex
negundo bitki yaprağının anti-ters transkriptaz özelliği bulunduğunu ve flavonoidlerin retrovirüslere
karşı muhtemelen antivirüs ajan olarak rol oynadıkları ortaya çıkarmaktadır.
Anahtar kelimeler: Vitex negundo, flavonoidler, HIV-1 ters transkriptaz, fitokimyasal, anti-HIV
aktivitesi. Introduction
Acquired immunodeficiency syndrome (AIDS),
caused by the human immunodeficiency virus
(HIV), results in life-threatening opportunistic
infections and malignancies. HIV leads to the
destruction and functional impairment of the
immune system, subsequently destroying the
body’s ability to fight against infections (Kanazawa
and Matija, 2001). Moreover, the standard antiviral
therapies are too expensive for a common man. In
order to manage this condition alternative
treatments are explored.
Vitex negundo L., a member of Verbenaceae
family, an important medicinal plant is found
throughout India. Though almost all plant parts are
used, the extract from leaves and the roots is the
most important in the field of medicine and is sold
as drugs. The leaf extract is used in Ayurvedic and
Unani systems of medicine for treatment of various
ailments (Kapur et al., 1994). It also has mosquito
repellent activity (Hebbalkar et al., 1992), antiarthritic effect on rats (Tamhankar et al., 1994),
analgesic activity on mice (Gupta et al., 1999),
hepatoprotective activity (Kapur et al., 1994), antiinflammatory and anti-allergic activity (Chawla et
al., 1992; Jana et al., 1999). Besides being used as
a traditional medicine, its antiviral property,
especially against HIV, has not yet been explored
much.
Flavonoids have been proven to display a wide
range of biochemical and pharmacological actions
such as anti-carcinogenic, anti-viral, anti-microbial,
anti-thrombotic, anti-inflammatory, and antimutagenic activities. In addition, flavonoids can act
as free radical scavengers and terminate the radical
chains reaction that occurs during the oxidation of
triglycerides in food system (Turkoglu et al., 2007).
Moreover flavonoid compounds represent an
important natural source of anti-retrovirals for
AIDS therapy due to their significant anti-HIV-1
activity and low toxicity.
One of the possible approaches is the
screening of plants based on their
ethnomedicinal data for inhibition (Vlietinck
et al., 1998). Current strategies for anti-HIV
chemotherapy involve inhibition of virus
adsorption, virus-cell fusion, reverse
transcription,
integration,
translation,
proteolytic
cleavage,
glycosylation,
assembly, or release (Moore and Stevenson,
2000; Miller and Hazuda, 2001). Reverse
transcriptase is an enzyme that reads the
sequence of HIV RNA that has entered the
host cell and transcribes the sequence into
complementary DNA. Without reverse
transcriptase, the viral genome cannot be
incorporated into the host cell and as a result
a virus will not replicate. Reverse
transcriptase is therefore the principal target
enzyme of antiretroviral drugs such as
Nevarapine and Delavirpine that are used to
treat HIV infected patients (De Clercq,
2007; Woradulayapinji et al., 2005).
Therefore this study has been designed to
explore the possible anti-HIV activity by RT
enzyme inhibition assay and to quantify the
flavonoids from the leaves of Vitex negundo.
Plant material and extraction
The leaves of Vitex negundo L. were
collected from Kolli hills adjoining
downstream areas of Namakkal district,
Tamil Nadu, India and authenticated
(PARC/2010/587) by Dr. Jayaraman, Plant
Anatomy Research Centre, National
Institute of Herbal Science, Chennai, India.
The plant samples were washed, shadedried, powdered and extracted in 85%
ethanol and filtered. The extracts were then
concentrated to dryness under reduced
pressure and the residue was freshly
dissolved in appropriate buffer on each day
Anti-HIV property of Vitex negundo L 55
of experiment for the assays. Depending on the
assay, extract that could not dissolve in appropriate
buffer were dissolved in DMSO and later diluted to
different concentration needed for a particular
assay.
HIV-1 RT assay
The effect of the plant extract on RT activity in
vitro was evaluated with recombinant HIV-1
enzyme, using a non-radioactive HIV-1 RT
colorimetric ELISA kit (Roche) (Ayisi, 2003;
Harnett et al., 2005). The concentration of extract
used was 200µg/ml. The extracts, which reduced
activity by at least 50%, were considered as active
Percentage of Inhibition = 100 -
(Woradulayapinji
et
al.,
2005).
Azidothymidine (AZT) was used as a
positive control at 100 µg/ml. The control
(1) only contained the buffer and reaction
mixture (no enzyme and extracts were
added). For the control (2) the enzyme and
reaction mixture were added for the reaction
to take place. The absorbance was read on a
microtitre plate reader at 405 nm with a
reference wavelength of 490 nm. The mean
of the triplicate absorbance were analysed
using the formula:
Mean Sample absorbance X 100
Mean Control-2 absorbance
Preliminary phytochemical analysis
The ethanolic leaf extract was subjected to
preliminary phytochemical screening as per the
procedures of Harborne, 1998 and Kokate, 2003.
Quantitative analysis of flavonoids using HPLC
The procedure as described by Lawrence Evans
(2007) was used for the determination of flavonoids
in the plant extracts. The flavonoid standard used in
the study includes Rutin, Luteolin, Myricetin,
Quercetin,
Kaempferol,
Isorhamnetin
and
Quercetagetin (Sigma Chemicals, USA) and were
prepared at 1 mg/ml in methanol. A total of 1g of
plant extract was extracted with 78 ml of extraction
solvent (methanol, water and hydrochloric acid;
50:20:8). Extract was then refluxed at 90◦C for 2 h.
Then extract was cooled and latter 20 ml of
methanol was added and sonicated for 30 minutes.
All solutions were filtered through a 0.45µM
cellulose acetate membrane filter (Paul, USA)
before being injected into the HPLC. Aliquots of
the filtrate (20µl) were injected on to an HPLC
(Lachrom L-7000) column using C18 (Merck) (25
X 0.4 cm, 5µm) separately and eluted with mobile
phase solvent mixture comprising Water:
Methanol: Phosphoric acid (100:100:1, v:v:v) with
a flow rate at 1.5 ml/min. The UV detection was
carried out at 270 nm. The chromatograms
were recorded and the areas measured for
the major peak to quantify the flavonoids in
the tested plant sample.
Results
The results shown in Table 1 indicates the
inhibition percentage of ethanolic leaf
extract of Vitex negundo L. against the
reverse transcriptase (RT) enzyme. The most
notable activity of about 92.8% was detected
against RT at 200µg/ml. The phytochemical
analysis of the plant extract revealed the
presence of steroids, triterpenes, alkaloids,
flavanoids, antroquinone glycosides and
aminoacids. In this study, flavonoids content
of the ethanolic extract of Vitex negundo L.
leaves were evaluated. The HPLC
chromatogram of the flavonoid standard
used and the chromatogram of the tested
plant extract is shown in Figure 1 & 2.
Results revealed that the extract consisted of
different amount of various flavonoid types.
As shown in Figure 1, the retention times of
Rutin, Quercetin, Kaempherol Luteolin,
Isorhamnetin , Myricetin, and Quercetagetin
were at 19.40, 24.80 29.70, 33.80, 38.70,
41.26 and 43.8 min, respectively.
Table 1. Effect of ethanolic leaf extract of Vitex negundo L. on the activity of recombinant HIV-1
reverse transcriptase
Extract/Control Mean absorbance ± SD Percentage of Inhibition
Control 1
0.003 ± 0.01
Control 2
1.32 ± 0.01
AZT
0.193 ± 0.00
V. negundo L.
0.094 ± 0.01
100
0
85.37
92.8
Control 1- buffer and reaction mixture (no enzyme and extract); Control 2- enzyme and reaction
mixture (no extract); AZT – Azidothymidine (Positive control). The plant extract showing percentage
of inhibition greater than 50% has been considered as positive in inhibiting recombinant HIV-1
reverse transcriptase enzyme.
Figure 1. HPLC Chromatogram of the flavonoid standards used in the study. 1. Rutin 2. Quercetin,
3.Kaempherol, 4. Luteolin, 5.Isorhamnetin, 6.Myricetin, 7.Quercetagetin.
Figure 2 exhibits the presence of Quercetin, Kaempherol, Luteolin, Isorhamnetin, Myricetin and
Quercetagetin in Vitex negundo leaf extract as per the retention time. The flavonoid Rutin was not
identified in the chromatogram of the plant sample showing its absence in the plant extract. Amount
of tested flavonoid compounds in the extract were calculated by measuring the area obtained for the
peaks and in the order as Kaempherol (20.61 mg/g) > Myricetin (18.75 mg/g) > Quercetin (14.73
mg/g) > Quercetagetin (12.13 mg/g) > Isorhamnetin (11.01 mg/g) > Luteolin (6.40 mg/g).
Figure 2. HPLC chromatogram of Vitex negundo leaves. Probable flavonoids quantity as per area and
Retention time compared with the standard HPLC chromatogram: 1. Kaempherol ; 2. Myricetin ; 3.
Quercetin ; 4. Quercetagetin ; 5. Isorhamnetin ; 6. Luteolin.
Discussion
For centuries water extract of fresh mature leaves
are used in Ayurveda medicine as antiinflammatory, analgesic and anti-itching agents
internally and externally. However the ethanolic
extract of V. negundo leaves resulted in the
isolation of a new flavones glycoside along with
five known compounds which were evaluated for
their antimicrobial activities by Sathyamoorthy et
al., (2007). However studies on anti-HIV activity of
V.negundo are few. For example the water extracts
of Vitex negundo (aerial part) was shown to have
HIV-1 RT inhibition ratio (% IR) higher than 90%
at a 200µg/ml concentration (Woradulayapinij et
al., 2005). Similarly the present study also showed
that the polar solvent extract of ethanolic leaf
extract of Vitex negundo L. had 92.8% inhibition of
recombinant HIV-1 reverse transcriptase enzyme at
200µg/ml. Previous phytochemical studies on V.
negundo L. had revealed the presence of volatile
oil, triterpenes, diterpenes, sesquiterpenes, lignan,
flavonoids, flavones glycosides, iridoid glycosides,
and steroids as physiologically active compounds
(Azhar and Abdul, 2004; Mukherjee et al., 1981).
For centuries, preparations that contain
flavonoids as the principal physiologically active
constituents have been used by physicians and lay
healers in attempts to treat human diseases
(Havsteen, 1983). Flavonoids are the largest
classes of naturally-occurring polyphenolic
compounds (Geissman and crout, 1969).
Evidence has been presented that substances
closely related to flavonoids inhibit the
fusion of the viral membrane with that of the
lysosome (Miller and Lenard, 1981).
Therefore the many claims from lay medical
practitioners of the prophylactic effects of
flavonoids against viral attack have
substantial support (Beladi et al., 1977).
Although the mechanism of the inhibition
remains unclear, it seems that prostaglandins
participate in the fusion of cell membranes.
Since flavonoids inhibit their formation, a
rationale can be constructed for the
protective effect of flavonoids against viral
diseases (Nagai et al., 1995a, 1995b;
Carpenedo et al., 1969). Moreover from the
previous reports it is clear that certain
naturally occurring flavonoids can inhibit
reverse transcriptases of different origins
(Spedding et al., 1989) From the above
reviews it is clear that the flavonoids have
anti-microbial activity, particularly the
antiviral. Therefore the present study is
focused particularly on flavonoids among
other phytochemicals. Moreover the choice
of flavonoid standards used in this study was based
on those commonly found in herbs and vegetables
which have been studied earlier and evidenced to
possess anti-HIV activity.
Schinazi et al. (1997) showed that the flavonols
such as quercetin, myricetin, and quercetagetin,
which was used as standard control in this study,
have earlier been reported to inhibit certain viruses
in vitro, including the Rauscher murine leukemia
virus and the HIV virus. Among the 17 flavonols
tested by Schinazi et al. (1997) only 3-O-glucosides
of kaempherol, quercetin, and myricetin caused
significant inhibition of HIV-1 at nontoxic
concentrations. At the same time other comparative
studies with other flavonoids revealed that the
presence of both the double bond between positions
2 and 3 of the flavonoids pyrone ring, and the three
hydroxyl groups introduced on positions 5,6 and 7
(ie, baicalein) were a prerequisite for the inhibition
of RT-activity. Removal of the 6-hydroxyl group of
bacalein required the introduction of three
additional hydroxyl groups at position 3,3’ and 4’
(quercetin) to afford a compound still capable of
inhibiting the RT-activity. Quercetagetin which
contains the structures of both baicalein and
quercetin with an additional hydroxyl group on the
5’ position also proved strong inhibitors of RT
activity (Ono et al., 1990). Thus the activity of
Vitex negundo leaf extract against HIV-1 RT in the
present study might be due to the presence of above
mentioned flavonoids particularly due to the
presence of high quantity of Kaempherol, myricetin
and quercetin. However this needs to be explored
and confirmed. Probably this is the first report from
India confirming the possible anti-HIV activity of
Vitex negundo L.
Acknowledgement
We would like to thank Dr. Hannah Raichel
Vasanthi, Former in-charge of Herbal Indian
Medicinal Research laboratory, Sri Ramachandra
Medical College & Research Institute for providing
sophisticated lab facility for successful completion
of this work.
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Research Article 61
Genetic characterization and bottleneck analysis of Korki
Jonub Khorasan goats by microsatellite markers
Bizhan MAHMOUDI*1, Orang ESTEGHAMAT2, Ahmad SHAHRIYAR1 and
Majnun Sh. BABAYEV3
1
Islamic Azad University, Meshkinshahr Branch, Meshkinshahr, Ardabil, Iran
Department of Animal Sciences, Islamic Azad University, Astara Branch, Astara, Gilan, Iran
3
Department of Genetic, Faculty of Biology, Baku State University, Baku, Azerbaijan
2
(*author for correspondence: [email protected])
Received: 19 March 2012, Accepted: 14 November 2012
Abstract
The present study was undertaken for population genetic analysis at molecular level to exploit the
breed for planning sustainable improvement, conservation and utilization, which subsequently can
improve the livelihood of its stake holders. Iranian goat populations are recognized as an invaluable
component of the world’s goat genetic resources. The genetic characterization and bottleneck analysis
in Korki Jonub Khorasan (KJK) was analyzed using 13 microsatellite markers. The observed number
of alleles ranged from 3 (OarAE133) to 11 (TGLA122) with a total of 98 alleles and mean of 7.54
alleles across loci. The overall heterozygosity, PIC and Shannon index values were 0.845, 0.76 and
1.759 indicating high genetic diversity. Only 4 out of 13 loci were in Hardy-Weinberg equilibrium.
The mean Fis was -0.059.Only 3 loci had positive Fis values and 10 loci had negative values. Genetic
bottleneck hypotheses were also explored. Our data suggest that the KJK goats have not experienced a
genetic bottleneck in the recent past.
Keywords: Bottleneck, Genetic Diversity, Microsatellites, Korki Jonub Khorasan, Fis.
Özet
Korki Jonub Horasan keçisinin mikrosatellit markörleriyle darboğaz analizi ve
genetik tanımlanması
Bu çalışmada sahiplerinin geçimini arttıracak soyların sürdürülebilir gelişiminin planlanması,
korunması ve faydalanılması için moleküler düzeyde popülasyon genetik analizi yapılması
üstlenilmiştir. İran keçisi popülasyonu dünyanın keçi genetik kaynaklarının çok değerli bir parçası
olarak kabul edilmektedir. Korki Jonub Horasan (KJK)’da genetik tanımlama ve darboğaz analizi 13
mikrosatellit markör kullanılarak yapılmıştır. Gözlemlenen allel sayısı toplam 98 allelden
3(OarAE133) -11(TGLA122) arasında değişmektedir ve lokuslar genelinde ortalama 7,54 allel vardır.
Heterozigotluk, PIC ve Shannon indeks değerleri 0,845, 0,76 ve 1,759 olup yüksek genetik çeşitliliği
işaret etmektedir. 13 lokustan 4’ü Hardy-Weinberg dengesindedir. Ortalama Fis -0,059’dur. Sadece 3
lokusun pozitif ve 10 lokusun negatif Fis değeri vardır. Genetik darboyun analizi ayrıca incelenmiştir.
Verilerimiz KJK keçilerinin yakın geçmişte genetik bir darboğaz ile karşılaşılmadığını öne
sürmektedir.
Anahtar Kelimeler: Darboğaz, Genetik çeşitlilik, Mikrosatellitler, Horasan keçisi, Fis. 62 Bizhan MAHMOUDI et al.
Introduction
Genetic diversity, the primary component of
adaptive evolution, is essential for the long-term
survival probability of a population (Avise, 1995;
Coltman et al., 1998; Harley et al., 2002). Genetic
diversity within domesticated species depends on
several factors such as changing agricultural
practices, breed replacement and cross breeding.
Genetic diversity has been analyzed by using
protein polymorphism, mitochondrial diversity and
microsatellite marker in both domestic and wild
species (Harley et al., 2002;Li and Valenti, 2004;
Tapiio et al., 2006; Pastor et al., 2004; Barker et
al., 2001; Li et al., 2002; Joshi et al., 2004, Rout et
al., 2008). The domestic goat (Capra hircus) is
known for its ability to thrive on paltry fodder and
to with stand harsh environments. From an
agricultural standpoint, the world’s 700 million
goats provide reliable access to meat, milk, skin,
and fiber for small farmers particularly in some
countries like Iran. Iran is bestowed with 8% of
total world’s goat population comprised of 8
recognized and many non-descript populations.
Among them, Korki Jonub Khorasan (KJK) is the
major goat population of Khorasane Jonobi
province and is known for fiber quality, meat and
milk production.
There are several studies on genetic diversity
of goats, based on microsatellite markers, such as
Swiss breeds (Saitbekova et al., 1999), Chinese
indigenous populations (Li et al., 2002), goats from
Europe (includingalso the breeds represented here)
and Middle East (Canon et al., 2006), Mehsana
goat (Aggarwal et al., 2007), Indian domestic goats
(Rout et al., 2008), Barbari goats (Ramamoorthi et
al., 2009) and Iranian native goats (Mahmoudi et
al., 2011).
Microsatellites have been used successfully to
define genetic relationships among different breeds
in Iran. Microsatellites display higher levels of
variation, and consequently, enable population
differentiation to be found more efficiently, so as to
help breeders to implement rational decisions for
conservation and improvement of valuable
germplasm (Mahmoudi et al., 2011).
Bottleneck analysis of KJK population has not been
carried out. Hence, it is essential to genetically
characterize and unfold the genetic diversity of
indigenous breeds.
The aim of our pervious study was to analyze
the genetic diversity and calculation of genetic
distance of three Iranian native goat
populations (Raeini, Korki Jonub Khorasan
and Lori) through the use of microsatellite
markers. Our pervious study demonstrates
that the closest distance was observed
between Raeini and KJK (D = 0.4891) and
the largest between Raeini and Lori (D =
0.6298). The UPGMA tree shows that two
goat populations (KJK and Raeini) are
distinct from the other goat population
(Lori) (Mahmoudi et al., 2011).
But in this study, Microsatellite analysis
was carried out to test for signatures of
recent population bottlenecks in Korki
Jonub Khorasan goat. This analysis was
carried out on 51 DNA samples with 13
microsatellite markers. For these loci,
genetic variation was quantified using
measures of the total number of alleles,
number of polymorphic loci, observed and
expected heterozygosity per locus and allelic
richness. We tested the Hardy-Weinberg
(HW) equilibrium and also calculated the
values of Fis (inbreeding coefficient).
51 blood samples were collected in 12
villages in which the population has a major
concentration. Samples were collected from
the individuals exhibiting typical population
characteristics and at least two samples were
collected from each village of Khorasane
Jonobi province in Iran. An effort was made
to collect samples from unrelated
individuals based on information provided
by farmers. Blood samples were collected
from each animal using EDTA vacutainer
and stored at –20 ºC till further use.
Blood samples (5–6 ml) were collected
from the jugular vein of the animal in
vacutainers
containing
EDTA
as
anticoagulant. DNA was extracted from
whole blood using standard protocols
(Sambrook et al., 1989). The DNA isolation
procedure involved lysis of RBCs, digestion
of protein using proteinase K, and
precipitation of protein using phenol:
chloroform: isoamyl alcohol with 25: 24: 1
ratio.
In this study, 13 microsatellite primer
pairs were used, including MAF64,
Bottleneck analysis of KJH goats 63
BM4621,
BM121,
LSCV36,
TGLA122,
OarJMP23, OarFCB304, OarAE133, ILSTS005,
ILSTS022, ILSTS029, ILSTS033 and ILSTS034.
Most of primers used were independent and
belonged to different chromosomes. Typical
polymerase chain reaction (PCR) testing was
carried out under these conditions: 60 ng of target
DNA was used in 25-μl PCR reaction containing
1×PCR buffer, 50 ng of each primer, 200 μM of
dNTPs, 0.5 units of Taq DNA Polymerase and 1.5
mMMgCl2. A Common “Touchdown” PCR profile
included 3 cycles of 45 sec at 95°C, 1 min at 60°C;
3 cycles of 45 sec at 95°C, 1 min at 57°C; 3 cycles
of 45 sec at 95°C, 1 min at 54°C; 3 cycles of 45 sec
at 95°C, 1 min at 51°C and 20 cycles of 45 sec at
92°C, 1 min at 48°C. In all cycles elongation
temperature and time were 72°C and 1 min,
respectively. Alleles were scored using unlabeled
primers with products visualized by silver staining
(Bassam et al., 1991). Genotype of individual
animal at 13 microsatellite loci was recorded by
direct counting.Genotypic data were analyzed using
POPGENE (Yeh and Boyle, 1997) and GenAlex
(Peakall and Smouse, 2006) to calculate the
observed number of alleles, effective number of
alleles,
observed
heterozygosity,
expected
heterozygosity, and to test for Hardy–Weinberg
equilibrium (HWE).Allelic frequencies were
utilized for assessing polymorphic information
content (PIC), a measure of informativeness of a
marker, calculated according to Botstein et al.
(1980) using the given formula,
Where k is the number of alleles and xi and xj are
the frequencies of the ith and jth alleles
respectively.
Heterozygote deficiencies were estimated as Fis =
(Ho −He)/He, where Ho and He are the observed
and expected frequency of heterozygotes
respectively.
The bottlenecks program (Piry et al.,
1999) was used as an alternative measure of genetic
bottlenecks to test for excess gene diversity relative
to that expected under mutation-drift equilibrium.
The heterozygosity excess method exploits the fact
that allele diversity is reduced faster than
heterozygosity during a bottleneck, because rare
alleles are lost rapidly and have little effect on
heterozygosity, thus producing a transient
excess in heterozygosity relative to that
expected in a population of constant size
with the same number of alleles (Cornuet
and Luikart, 1996; Piry et al., 1999). To
determine the population ‘‘genetic reduction
signatures’’
characteristic
of
recent
reductions in effective population size (Ne),
the Wilcoxon’s heterozygosity excess test
(Piry et al., 1999) , standard differential test,
sign test and the allele frequency
distribution mode shift analysis(Luikart et
al.,
1998)
were
performed
using
BOTTLENECK (Piry et al., 1999). The
heterozygosity excess method was used to
analyzed the population and the data for the
heterozygosity excess test were examined
under the two-phased model (TPM;
95%stepwise mutation model with 5%
multi-step mutations and a variance among
multiple steps of 12), which is considered
best for microsatellite data (Piry et al., 1999;
Di Rienzo et al., 1994). We also analyzed
the allele frequency distribution for gaps.
Aqualitative descriptor of allele frequency
distribution (the mode-shift indicator),is
reported
to
discriminate
between
bottlenecked and stable population (Luikart
et al.,1998).
Results
In order to maintain genetic diversity,
breeding strategies that increase effective
population size minimizing genetic drift
effect should be implemented. Microsatellite
markers in combination with recent
statistical methodologies represent a useful
tool for the conservation and management of
endangered breeds. In the present work, the
actual situation concerning genetic diversity
and population structure of this breed has
been evaluated using the molecular
information derived from 13 microsatellites
loci and the use of clustering methods.
13 pairs of highly polymorphic
microsatellite markers were chosen based on
their genomic location (Table 1).Various
measures of genetic variation are presented
in the Table 2. The F-statistics estimates are
presented in Table3. The number of alleles
observed across the microsatellite loci
64 Bizhan MAHMOUDI et al.
studied varied from 3(OarAE133) to 11
(TGLA122) with an overall mean of 7.54 (Table 2).
The observed number of alleles across the loci was
more than the effective number of alleles (2.355 to
6.973). The Shannon information index (I) and
polymorphic Information Content (PIC) showed
that most of the loci were highly informative
indicating the high polymorphism across the loci
with an overall mean of 1.759 and 0.76
respectively.
The
average
observed
heterozygosity was more than the expected
(Table 3). The average expected gene
diversity (Nei, 1973) ranged from 0.581
(OarAE133) to 0.865 (TGLA122) with an
overall mean of 0.798. Nine out of total 13
loci studied showed significant deviations
from Hardy Weinberg Equilibrium.
Table 1. Details of the microsatellite used in the study
Locus
BM121
BM4621
ILSTS005
ILSTS022
ILSTS029
ILSTS033
ILSTS034
LSCV36
MAF64
OarAE133
OarFCB304
OarJMP23
TGLA122
Primer sequence
TGGCATTGTGAAAAGAAGTAAAA
CTAGCACTATCTGGCAAGCA
CAAATTGACTTATCCTTGGCTG
TGTAACATATGGGCTGCATC
GGAAGCAATGAAATCTATAGCC
TGTTCTGTGAGTTTGTAAGC
AGTCTGAAGGCCTGAGAACC
CTTACAGTCCTTGGGGTTGC
TGTTTTGATGGAACACAGCC
TGGATTTAGACCAGGGTTGG
TATTAGAGTGGCTCAGTGCC
ATGCAGACAGTTTTAGAGGG
AAGGGTCTAAGTCCACTGGC
GACCTGGTTTAGCAGAGAGC
GCACACACATACACAGAGATGCG
AAAGAGGAAAGGGTTATGTCTGGA
AATAGACCATTCAGAGAAACGTTGAC
CTCATCGAATCAGACAAAAGGTAGG
AGCCAGTAGGCCCTCACCAGG
CCAACCATTGGCAGCGGGAGTGTGG
CCCTAGGAGCTTTCAATAAAGAATCGG
CGCTGCTGTCAACTGGGTCAGGG
GTATCTTGGGAGCCTGTGGTTTATC
GTCCCAGATGGGAATTGTCTCCAC
AATCACATGGCAAATAAGTACATAC
CCCTCCTCCAGGTAAATCAGC
Type of
repeat
Chromosome
No.
(TC)18
16
(CA)14
6
(nn)39
10
(GT)21
3
(CA)19
3
(CA)12
12
(GT)29
5
(CA)16
19
(TG)13
1
(TG)24
Ann
(CT)11(CA)15
19
-
27
(CA)21
21
Within population inbreeding estimate (Fis) for
the investigated loci was -0.059. The estimates for
each locus are presented in Table 3. The values
ranged from –0.158 (TGLA122) to 0.047
(ILSTS05). Ten loci revealed negative Fis values.
The heterozygote deficiency may be a result of
inbreeding. The high genetic diversity observed in a
breed could be explained by overlapping
generations, mixing of populations from different
geographical locations, natural selection favoring
heterozygosity or subdivision accompanied by
genetic drift. Isolation, founder effects, genetic drift
and different selection pressures realized by
farmers in each population may have played major
role in differentiation of Iranian goats.
Any population that experienced a recent
bottleneck will show higher than expected
(equilibrium) heterozgosity for a large number of
loci. Microsatellite data were also subjected to
statistical analysis to test whether the populations
have undergone recent genetic bottleneck. Because
historical population sizes and levels of genetic
variation are seldom known, methods for detecting
bottlenecks in the absence of historical data would
be useful. Cornuet and Luikart, (1996) described
the quantitative methods suitable for analysis of
microsatellite data for detection of recent
bottlenecks in (100-200) generations.
Table 2. Number of alleles (Observed and
effective), Shannon's Information index and
Polymorphic Information Content for KJK goats
Locus name
BM121
BM4621
ILSTS005
ILSTS022
LSTS029
ILSTS033
ILSTS034
LSCV36
MAF64
OarAE133
OarFCB304
OarJMP23
TGLA122
Na
8
9
8
7
7
8
7
9
4
3
9
8
11
Ne
6.375
6.007
4.42
4.321
4.579
5.742
6.267
6.367
3.272
2.355
6.391
4.751
6.973
I
1.944
1.969
1.729
1.606
1.702
1.9
1.89
1.988
1.262
0.929
2.014
1.775
2.164
PIC
0.82
0.81
0.75
0.73
0.75
0.8
0.82
0.82
0.64
0.48
0.83
0.76
0.84
0.76
7.538
5.217
1.759
Mean
Na: Observed number of alleles; Ne: Effective number of
alleles; I: Shannon's Information index; PIC:
Polymorphic Information Content.
To determine whether a population exhibits
a significant number of loci with gene
diversity excess, there are three tests,
namely a "sign test", a "standardized
differences test" (Cornuet and Luikart,
1996) and a "Wilcoxon sign-rank test"
(Luikart et al., 1997).
Observed
and
expected
Table
3.
heterozygosity with p-value, Fis value for
each microsatellite locus and mean estimate
of different parameters for KJK goats
Locus
name
BM121
BM4621
ILSTS005
ILSTS022
ILSTS029
ILSTS033
ILSTS034
LSCV36
MAF64
OarAE133
FCB304
OarJMP23
TGLA122
Ho
He
Fis
HWE
0.902
0.804
0.745
0.863
0.902
0.902
0.941
0.843
0.725
0.667
0.882
0.804
1
0.852
0.842
0.781
0.776
0.789
0.834
0.849
0.851
0.701
0.581
0.852
0.797
0.865
-0.06
0.045
0.047
-0.113
-0.144
-0.082
-0.11
0.01
-0.035
-0.149
-0.036
-0.008
***
***
NS
NS
***
***
***
***
NS
NS
***
***
***
-0.158
Mean
0.845
0.798
-0.059
NS: Not Significant; ***: Significant at the
0.1% level
All the three models of microsatellite
evaluation Infinite Allele Model (IAM),
Stepwise Mutation Model (SPM) and Two
Phase Model (TPM) were utilized for the
purpose. In a population at mutation-drift
equilibrium (i.e., the effective size of which
has remained constant in the recent past),
there is approximately an equal probability
that a locus shows gene diversity excess or a
gene diversity deficit. The first test suffers
from low statistical power. The second test
is not very useful since it requires at least 20
polymorphic loci. The Wilcoxon test
provides relatively high power and it can be
used with as few as four polymorphic loci
and any number of individuals (15-40
individuals and 10-15 polymorphic loci is
recommend to achieve high power. So, the
null hypothesis was again rejected under
IAM for the sign test. Standard difference
test (T2 statistics) in this population provided the
significant (p<0.01) gene diversity excess under
TPM (4.019) and IAM (4.803) (Table 4).
In KJK goat, under Sign test, the expected
numbers of loci with heterozygosity excess were
7.70 (TPM) and 7.65 (SMM) which were
substantially lower than the observed numbers of
loci 13 (TPM) and 12 (SMM) with heterozygosity
excess (Table 4). So the null hypothesis that as the
population is under Mutation-drift equilibrium was
rejected. The expected number of loci (7.63) with
heterozygosity excess was significantly (p<0.05)
lower than the observed numbers of loci (13) with
heterozygosity excess under IAM.
In SMM there was heterozygosity excess
(2.167) and significant (p<0.05). Positive values of
the Bottleneck statistic T2 are indicative of gene
diversity excess caused by a recent reduction in
effective population size, while negative
value are consistent with a recent population
expansion
without
immigration
or
immigration of some private (unique) alleles
in population.
Under Wilcoxon rank test, probability
values of 0.00006 (IAM), 0.00006 (TPM)
and 0.00043 (SMM) were significant
(p<0.001). So, null hypothesis of mutation
drift equilibrium was rejected in all tests
under all the three models. Tantia et al.,
(2004) reported heterozygosity excess and
genetic bottleneck in the Indian goat breeds
(Chegu and Black Bengal). Sign test,
standardized difference test and Wilcoxon
rank test indicated heterozygosity excess in
KJK population.
Table 4. Mutation-drift equilibrium, heterozygosity excess/deficiency under different mutation
models in KJK population
Models Sign Test Standardized
Wilcoxon test
IAM
Hee= 7.63
T2= 4.803
P (One tail for H deficiency)= 1
Hd= 0
P= 0.00000
P (One tail for H excess)= 0.00006
He= 13
P (Two tail for H excess and deficiency)= 0.00012
P= 0.00006
TPM
Hee= 7.70
Hd= 0
He= 13
P= 0.00110
T2= 4.019
P= 0.00003
P (One tail for H deficiency)= 1
SMM
Hee= 7.65
Hd= 1
He= 12
P= 0.01017
T2= 2.167
P= 0.01513
P (One tail for H deficiency)= 0.99969
Parameters for T.P.M: Variance = 30.00 Proportion of SMM in TPM = 70.00%; Estimation based on
1,000 replications; Hee: Heterozygosity excess expected; Hd: Heterozygosity deficiency; He:
Heterozygosity excess; P: Probability; IAM: Infinite allele model, TPM: Two phase model, SMM:
Stepwise mutation model.
Under Wilcoxon rank test, probability
In SMM there was heterozygosity excess
values of 0.00006 (IAM), 0.00006 (TPM)
(2.167) and significant (p<0.05). Positive values of
and 0.00043 (SMM) were significant
the Bottleneck statistic T2 are indicative of gene
(p<0.001). So, null hypothesis of mutation
diversity excess caused by a recent reduction in
drift equilibrium was rejected in all tests
effective population size, while negative value are
under all the three models. Tantia et al.,
consistent with a recent population expansion
(2004) reported heterozygosity excess and
without immigration or immigration of some
genetic bottleneck in the Indian goat breeds
private (unique) alleles in population.
(Chegu and Black Bengal). Sign test,
standardized difference test and Wilcoxon rank test
indicated heterozygosity excess in KJK population.
The Mode-shift indicator test was also utilized as a
second method to detect potential bottlenecks, as
the non-bottleneck populations that are near
mutation-drift equilibrium are expected to have a
large proportion of alleles with low frequency. This
test discriminates many bottlenecked populations
from stable populations (Luikart et al., 1998;
Luikart and Cornuet, 1997). The distribution
followed the normal L-shaped form. The alleles
with low frequencies (0.01–0.1) are the most
numerous and proportion of alleles showed a
normal ‘L’ shaped distribution (figure 1) This
distribution clearly show that the studied population
has not experienced a recent bottleneck.
Ruhnu population showed a slight distortion
in distribution of allelic frequency
(Grigaliunaite et al., 2003).
Discussion
In conclusion, there was substantial genetic
variation and polymorphism across studied
loci in the KJK goat. And this population
was not in Hardy-Weinberg equilibrium at
most of the studied loci. The strong
inference that the KJK of goat has not
undergone bottleneck, as it suggests that any
unique alleles present in this breed may not
have been lost. Therefore, it can be
recommended that within breed diversity is
actively maintained to enable these
extensively unmanaged stocks to adapt to
future demands and conditions and there is
ample scope for further improvement in its
productivity through appropriate breeding
strategies.
Acknowledgments
This work was support by the Islamic Azad
University, Meshkinshahr Branch, Iran.
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Research Article 71
Low-Stringency Single-Specific-Primer PCR as a tool for
detection of mutations in the matK gene of Phaseolus vulgaris
exposed to paranitrophenol
Mohamed R. ENAN
Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC),
Giza, Egypt
(author for correspondence; [email protected]) Received: 26 April 2012; Accepted: 08 December 2012
Abstract
Low-stringency single specific primer polymerase chain reaction (LSSP)-PCR was assessed for its
suitability in detecting the genotoxic effect of paranitrophenol (PNP) in the dwarf bean (Phaseolus
vulgaris) exposed to different concentrations of PNP. DNA was extracted from both PNP-treated and
non-treated shoots that was amplified by specific PCR, using universal primers of maturase K
chloroplast DNA. PCR products of approximately 776 bp were subsequently used as a template for
LSSP-PCR analysis. We detected the genotoxic effect based on LSSP-PCR profiles of the DNA
generated in PNP-treated over the non-treated control of bean shoots. A complex electrophoretic
pattern consisting of many bands was obtained from control and treated samples. Surprisingly, DNA
sequencing data revealed that the homology among the maturase gene amplified from PNP-treated vs.
non-treated samples of dwarf beans are comparable. These results showed that the use of LSSP-PCR
analysis is not a proper tool to detect genotoxic effect in bean, at least in bean shoots that were
exposed to PNP.
Keywords: Genotoxicity, LSSP-PCR, Paranitrophenol, Phaseolus vulgaris, maturase K.
Özet
Paranitrofenole maruz kalan Phaseolus vulgaris’te matK geni mutasyonlarının
tespitinde bir araç olarak Düşük Kesinlikte Tek-Özgün Primerli PCR kullanımı
Düşük kesinlikte tek özgün primer lipolimeraz zincir reaksiyonunun (LSSP-PCR), paranitrofenolün
(PNP) sebep olduğu genotoksik etki tespitindeki uygunluğu farklı konsantrasyonlarda PNP’ye maruz
bırakılan bodur fasülyede (Phaseolus vulgaris) değerlendirildi. PNP ile muamele edilmiş veya
edilmemiş filizlerden izole edilen DNA, maturaz K kloroplast DNA’sının evrensel primerleri
kullanılarak özgün PCR ile çoğaltıldı. Hemen akabinde, 776 bç’lık PZR ürünleri LSSP-PCR analizi
için kalıp DNA olarak kullanıldı. PNP ile işlenmiş fasülye filizinden elde edilen DNA üzerindeki
genotoksik etkilerin PNP ile işlenmemiş kontrollere olan kıyasını LSSP-PCR profiline dayanarak
tespit ettik. Kontrol ve işlenmiş örneklerde birçok banttan oluşan karmaşık elektroforetik motifler elde
edildi. Şaşırtıcı bir şekilde DNA dizi analizi verileri PNP ile işlenmiş ve işlenmemiş bodur fasülye
örneklerinde çoğaltılan maturaz geni homolojisinin kıyaslanabilir olduğunu gösterdi. Bu sonuçlar,
LSSP-PCR analizinin fasülyede, en azından PNP ile işlenmiş filizlerde, genotoksik etkinin tespitinde
uygun bir araç olmadığını gösterdi.
Anahtar Kelimeler: Genotoksisite, LSSP-PCR, Paranitrofenol, Phaseolus vulgaris, maturaz K. 72 Mohamed R. ENAN
Introduction
Maturase K (matK) is a chloroplast-encoded gene
which is nested between the 5’ and 3’ exons of
trnK, tRNA-lysine (Sugita et al., 1985). Sequence
analysis indicated that this region displayed
homology to domain X of mitochondrial group II
intron maturases (Sugita et al., 1985; Neuhaus and
Link, 1987). Although maturase K gene (matK)
contains many indels (insertions and deletions)
throughout its reading frame, yet domain X lacks
any of these indels (Hilu and Liang, 1997; Hilu and
Alice, 1999; Hilu et al., 2003). Maturase K is the
only gene found in the chloroplast genome of
higher plants that contains this putative maturase
domain X in its protein (Neuhaus and Link, 1987).
Maturases are considered as splicing factors
because of their ability to splice and fold group II
introns. The coding region of matK is generally
located within intron of the chloroplast trnK gene
(Vogel et al., 1997). matK is very useful in DNA
barcoding to genetically identify plant families (Qiu
et al., 1999; Li and Zhou, 2007).
Genotoxic compounds are those which cause
damage to DNA. Para-nitrophenol is a synthetic
chemical that is used to manufacture drugs,
fungicides, insecticides (Yang et al., 2010).
Pesticides, such as parathion and methyl parathion,
are hydrolyzed and transformed to PNP; which in
turn these pesticides are considered as the main
source of PNP that is released to the environment
(Kitagawa et al., 2004). In vitro assay using CHO
cell PNP was positive for chromosome aberration
at levels of 100 µg/ml (Ohno et al., 2005), proving
the hypothesis that PNP induces chromosomal
aberrations.
The LSSP-PCR is a simple technique that
permits detection of single or multiple mutations in
gene-sized fragments (Pena et al., 1994). This
sensitive and rapid method uses PCR amplification
of a single oligonucleotide primer "driver" that is
specific to one of the extremities of the fragment,
under very low stringency conditions (Pena et al.,
1994). In a sequence-dependent manner, the driver
hybridizes both to the highly specific
complementary extremity, and to the low
specificity of multiple sites within the fragment.
The reaction thus yields a large number of products
that can be resolved by polyacrylamide gel
electrophoresis to give rise to a multiband DNA
fragment "signature" that reflects the underlying
sequence. Changes as small as single base
mutations can drastically alter the multiband
pattern, which ultimately produce new
signatures. LSSP-PCR has been broadly
used for the detection of mutations in human
genetic diseases (Pena et al., 1994),
sequence variations in human mitochondrial
DNA (Barreto et al., 1996) and for genetic
typing of infectious agents such as
papillomavirus (HPV; Villa et al., 1995),
Trypanosoma cruzi (Vago et al., 2006),
Trypanosoma rangeli (Marquez et al.,
2007), and Leishmania infantum (Alvarenga
et al., 2012). The objective of this study was
to describe the potential use of LSSP-PCR
as a molecular biomarker to detect DNA
mutation in maturase K gene in dwarf bean
tissues exposed to paranitrophenol.
Materials and methods
Plant growth and treatment conditions
The dwarf bean (Phaseolus vulgaris) was
used as the plant material in this study. The
selected seeds were sterilized with 75%
(v/v) ethanol for 2 min, followed by 20%
(v/v) sodium hypochlorite for 10 min and
were washed five times in sterile distilled
water. Uniformly three plant seedlings were
transferred to a Magenta box containing MS
(Murashige and Skoog, 1962) liquid
medium (control) or supplemented with
different concentrations of PNP (20, 40, 80,
160, 320, and 640 µg/ml). PNP-treated
seedlings were grown for 10 days in the
growth chamber. Plant growth conditions
was previously described (Enan, 2006).
DNA isolation
DNA was extracted from fresh plant shoots
using DNeasy plant minikit (Qiagen, USA),
following
the
instruction
of
the
manufacturer. The final DNA concentration
was
determined
by
agarose
gel
electrophoresis against known standards
(Invitrogen, USA).
Specific PCR amplification of matK
fragments
To eliminate any possibility of bacterial
contamination due to the very low-
matK mutations in Phaselous vulgaris 73 stringency conditions of the LSSP-PCR reaction,
all experiments were carried out with extreme
precautions. PCR reactions were performed with
specific
primers
matK472F
(5′CCCRTYCATCTGGAAATCTTGGTTC-3′) and
matK1248R
(5′GCTRTRATAATGAGAAAGATTTCTGC-3′) as
described by Yu et al. (2011). Amplification of
specific PCR products was carried out in a volume
of 25 μl containing 30 ng of genomic DNA and a
master mix containing 1.5 mM MgCl2, 200 μM of
each deoxynucleotide (dNPTs), 20 pmol of each
primer, 1.0 U Taq DNA polymerase (InvitrogenBRL), in 10 mM Tris–HCl [pH 8.0] and 50 mM
KCl. After an initial denaturation step of 94 °C for
5 min, the specific PCR program consisted of 35
cycles of 94 °C for 30 s, 56 °C for 1 min and 72 °C
for 1 min. The last cycle consisted of an extension
step at 72 °C for 5 min. The PCR products were run
on ethidium bromide-stained gel and the bands
corresponding to the specific fragment (876bp)
generated by universal specific primers were
purified using Purelink PCR purification Kit
(Invitrogen, USA).
LSSP-PCR analysis
For the production of LSSP-PCR signatures,
previously amplified matk fragments were purified
used as a template in the LSSP-PCR (Pena et al.,
1994). LSSP-PCR was also carried out in a 25μl
volume containing 5ng of DNA template, 1.5 mM
MgCl2, 200 μM of the four deoxynucleotide
triphosphates, 120 pmol of matK472F or
matK1248R primer 4.0 U Taq DNA polymerase in
10 mM Tris–HCl [pH 8.0] and 50 mM KCl. After a
denaturation step at 94 °C for 5 min the LSSP-PCR
program consisted of 35 cycles of denaturation at
94 °C for 1 min, annealing at 30 °C for 1 min and
extension at 72 °C for 1 min. Ten microliters of
LSSP-PCR
products
were
analyzed
by
electrophoresis on 8% (w/v) polyacrylamide gels
followed by ethidium bromide. The similarity
among the LSSP-PCR profiles of control and those
obtained with the DNA of PNP-treated samples
was analyzed accordingly.
DNA sequencing of PCR products
In order to determine the nucleotide sequence of the
776 fragments generated with universal specific
primers, PCR products of control and PNP-treated
samples were purified and sequenced by Source
BioScience (Nottingham, UK) according to Sanger
et al. (1977). The sequence was analyzed for
homology with database sequences with
Multiple Sequence Alignment by MultiAlin
(Corpet,
1988).
Results
We used the LSSP-PCR method to detect
mutations in matK gene of dwarf bean
tissues. DNA was amplified (first step)
using universal primers to produce 776 bp
fragments containing the maturase K region
(Figure 1).
Each fragment was isolated by
electroelution and subjected to a second
PCR amplification (second step) using a
single primer annealed under low-stringency
conditions. The generated profiles of the
PCR products of each sample were resolved
and
analyzed
by
non-denaturing
polyacrylamide gel. A complex pattern
consisting of many bands was obtained
which was different depending on the
concentration of PNP. We showed the
LSSP-PCR profiles of DNA obtained from
PNP-treated or PNP-untreated samples
amplified with either matk742 forward
primer (Figure 2) or matk1248R reverse
primer matK1248R primer (Figure 3).
Figure 1. Agarose gel electrophoresis of
PCR amplification of matk fragment with
776 bp obtained in control and treated
samples. Lane M: 100 bp DNA ladder; Lane
C: untreated sample (control); lanes 1-6:
plant samples treated with 20, 40, 80, 160,
320 and 640 µg/ml PNP, respectively.
74 Mohamed R. ENAN
(accession numbers JQ403111). We also
determined
whether
these
sequence
identities showed similarities between the
different samples treated with PNP (Figure
4). Our sequence alignment data obtained by
MultiAlin indicated that the sequence
identities of all treated samples shared 100%
homology with sequences of untreated
samples (Figure 4). This suggests that a
mutation in the matK gene as a hotspot gene
is not induced by treatment with PNP.
Figure
2.
Ethidium
bromide-stained
polyacrylamide gel electrophoresis showing gene
signatures obtained by LSSP-PCR with matk742
forward primer. Lanes M: 100 bp DNA ladder;
Lane C: untreated sample; lanes 1-6: plant samples
treated with 20, 40, 80, 160, 320 and 640 µg/ml
PNP, respectively.
Figure
3.
Ethidium
bromide-stained
polyacrylamide gel showing gene signatures
obtained by LSSP-PCR with matk1248 reverse
primer. Lane M: 100 bp DNA ladder; Lane C:
untreated sample; lanes 1-6: plant samples treated
with 20, 40 80 160, 320 and 640 µg/ml PNP,
respectively.
The LSSP-PCR profiles were unique for each
treatment, suggesting that this technique may be
applicable for the detection of genotoxic impact of
environmental contaminants. The sequence of the
maturase K gene was deposited in Genbank
Discussion
To our knowledge, this is the first study to
employ
LSSP-PCR
for
monitoring
biological effects of pollution. Molecular
biomarkers are effective early warning
signals of adverse biological effects. The
purpose of this study was to evaluate the
performance of LSSP-PCR method in the
detection
of
genotoxic
effect
of
paranitrophenol (PNP) on dwarf beans
(Phaseolis vulgaris). In the past 25 years,
numerous biomarkers have been developed
with the objective to apply them for
environmental biomonitoring (Sanchez and
Porcher,
2009).
Molecular
marker
techniques have provided new tools of
detection of mutations in DNA in response
to chemical pollution using DNA sequence
and structure. The alterations in genomic
DNA induced by genotoxic pollutants can
be monitored using different biomarkers’
assays both at the biochemical and the
molecular levels. In the past few years,
several of techniques revealed that
mutations in DNA could be generated and
identified mostly by the polymerase chain
reaction (PCR). Some of the examples of
PCR assays were utilized to detect genotoxic
effects of environmental pollutants arbitraryprimed PCR (AP-PCR; Welsh and
McClelland, 1990) and randomly amplified
polymorphic DNA (RAPD) (Williams et al.,
1990). One of the main advantages of using
LSSP-PCR
for
studies
related
to
genotoxicity is that the signatures were not
unduly sensitive to the concentration of
DNA template.
matK mutations in Phaselous vulgaris 75 Figure 4. DNA sequences of the matK genes from untreated and PNP-treated plant samples aligned
by using MultiAlin.
76 Mohamed R. ENAN
In the present study, genotoxic effect of PNP
was performed using LSSP-PCR that can detect
single or multiple mutations in gene-size DNA
fragments. The chloroplast maturase K gene
(matK) is one of the most variable coding genes of
angiosperms, which has been suggested to be a
“barcode” for land plants (Yu et al., 2011). Good
reproducibility as a solution in the LSSP-PCR
profiles using both forward and reverse primers
was obtained. However, we observed that many of
these bands are larger than the template. Our results
confirm the data obtained from other studies that
PCR products of the first few cycles may
themselves act as primers in further rounds of
amplification (Barreto et al., 1996). In the current
study, the sequenced PCR products of matK
fragment confirmed the results of the specific PCR
(Figure 1). On the other hand, unexpectedly, the
data of sequence alignment quite contradicts that of
the LSSP-PCR signatures. Sequence alignment of
all PNP-treated samples of dwarf bean with the
untreated control samples indicated that there is no
any nucleotide substitution in the matK sequence.
In previous study, Oliveira et al. (2003) described
that, very similar signatures were obtained with
specific primers (G1 and G2) for identification of
Leptospira interrogans serovars. Although the
sequence data of the 285 bp fragments of the three
serovars of L. interrogans indicated the presence of
three nucleotide alterations in these fragments, they
found that identical LSSP-PCR profiles were
obtained for the three serovars with individual
primers of G1 and G2. Barreto et al. (1996)
reported that the variations observed in LSSP-PCR
are attributed to several variables: (i ) the number
of cycles has a marked effect on the signature up to
35 cycles (ii) the ramping speed of thermocyclers (
type of thermo cyclers) had marked effect on the
LSSP-PCR signatures and (iii) changes in the
annealing temperature a range between 25-35◦C
had no marked effect but Ta > 40◦C showed a
deterioration of the signature.
In conclusion, the chloroplast matk used in this
study as a molecular biomarker gene to measure
genotoxicity of PNP using LSSS-PCR, is not
affected by PNP at DNA level but may be down
regulated at transcriptional or post-transcriptional
levels, which should be confirmed in further
studies.
Acknowledgment
The author would like to thank a lot Dr
Synan Abu Qamar, Ph.D, Purdue University
for his critical revising and constructive
comments on the manuscript.
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Short Communication 79
Sérgio Salla CHAGAS1, Rodrigo Almeida VAUCHER2, Adriano BRANDELLI2,*
1
Laboratório Nacional Agropecuário (LANAGRO/RS), Estrada da Ponta Grossa 3036, Porto Alegre,
Brazil.
2
Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Ciência e Tecnologia de
Alimentos, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, Brazil.
(*author for correspondence: [email protected])
Received: 24 February 2012; Accepted:11 November 2012
Abstract
Paenibacillus larvae is the agent of American Foulbrood disease (AFB), causing the death of the hive
and greatly affecting beekeeping. In Brazil, this bacterium was only isolated in the states of Rio
Grande do Sul and Paraná. The present study aimed to characterize the strains isolated in Brazil,
confirming its identification by molecular diagnosis. Eighty isolates from samples of honey,
honeycomb and pollen collected between 2002 and 2007 from different regions were selected for
analysis. Phenotypical characterization indicates that 77 strains were P. larvae but 3 strains showed
inconclusive results. PCR protocols based on detection of 16S rDNA and metalloproteinase gene
confirmed all strains being P. larvae. The PCR amplicon of 16S rDNA was sequenced, and
phylogenetic analysis was performed. The results indicated that there is high homology among the
strains isolated in Brazil.
Keywords: Paenibacillus larvae, 16S rDNA, metalloproteinase, American foulbrood disease,
phylogenetic analysis
Özet
Brezilya’dan Paenibacillus larvae izolatlarının karakterizasyonu
Paenibacillus larvae kovanın ölümüne sebep olan ve büyük oranda arıcılığı etkileyen Amerikan yavru
çürüklüğü hastalığının (AFB) etkenidir. Brezilya’da bu bakteri sadece Rio Grande do Sul ve Paraná
eyaletlerinde izole edilmiştir. Bu çalışma moleküler tanı ile yapılan identifikasyonu doğrulayarak
Brezilya’da izole edilen suşları karakterize etmeyi amaçlamıştır. Farklı bölgelerden 2002 ve 2007
yılları arasında toplanan bal, petek ve polen örneklerinden elde edilen seksen izolat analiz için
seçilmiştir. Fenotipik karakterizasyon 77 suşun P. larvae olduğunu, fakat 3 suşun yetersiz sonuç
gösterdiğini belirtmektedir. Metalloproteinaz geni ve 16SrDNA’nın saptanmasına dayanan PCR
protokolleri tüm suşların P. larvae olduğunu doğrulamaktadır. 16SrDNA PCR amplikonu
dizilenmiştir ve filogenetik analiz yapılmıştır. Sonuçlar Brezilya’da izole edilen suşlar arasında
yüksek homoloji olduğunu göstermektedir.
Anahtar Kelimeler: Paenibacillus larvae, 16S rDNA; metalloproteinaz; Amerikan yavru çürüklüğü
hastalığı, filogenetik analiz
80 Sérgio Salla CHAGAS et al.
Introduction
The American foulbrood (AFB) is a notifiable
disease of high economic importance and
significant international trade (OIE, 2009). Its
causative agent, Paenibacillus larvae, attacks only
the larval stage of the bee Apis mellifera and other
Apis spp. (Genersch, 2010). P. larvae are only
infective in the form of spores, which are extremely
tenacious and can remain viable for many years
(Genersch, 2008). Infection occurs by ingestion of
spores that germinate and spread after 24 hours,
producing septicemia and death (Allipi, 1992). The
clinical symptoms are typical, and the larvae have
affected to a dark and viscous form. Besides food
with honey containing spores, the introduction of
bees from hives infected and even beekeepers could
help its spread (Genersh, 2010).
In South America, the first isolation of P. larvae
occurred in 1989 in Argentina (Allipi, 1992), and
subsequently in Uruguay (Antunez et al., 2004). In
Brazil, despite efforts to prevent its introduction,
honey contaminated with P. larvae was found in
the state of Rio Grande do Sul in 2002 (Schuch et
al., 2003). More recently, the occurrence of P.
larvae was reported in hives in the state of Paraná
(MAPA, 2006). The aim of this work was to
confirm the presence of P. larvae by molecular
methods and to characterize the strains isolated in
Brazil.
This study used 80 isolates of P. larvae, originated
from samples of honey, honeycomb and pollen
collected from different Brazilian regions between
May 2002 and June 2007 by Laboratório Nacional
Agropecuário (LANAGRO/RS, Ministry of
Agriculture, Brazil), for the microbiological
analysis for the detection of spores (Schuch et al.,
2002). Of these 80 isolates, 30 were from suspected
samples and 50 originated from the process of
importation. Aliquots of 20 g of the various
products were diluted to 40 ml of distilled water
and centrifuged at 6000 g for 10 min. The pellet
obtained was resuspended in 1 ml of distilled water,
heated to 80°C for 10 min and seeded on selective
P. larvae agar plates (Schuch et al., 2002). These
plates were incubated at 35°C, and monitored for 5
days.
Isolated colonies were suspended in distilled
water and subjected to bacterial DNA extraction
using phenol-chloroform. P. larvae ATCC 9545
was used as a positive control. Bacillus
cereus ATCC 11778 and Paenibacillus alvei
isolated by LANAGRO/RS (Porto Alegre,
Brazil), were used as negative controls. The
amplification of the 16S rDNA was through
a nested PCR protocol, conducted in the
GeneAmp PCR System 2400 equipment
(Applied Biosystems, Foster City, CA,
USA). The amplification was initially
performed with external primers Ple(F)
TCGAGCGGACCTTGTGT and Ple(R)
CTATCTCAAAACCGCTCAGAG,
and
then with the external primers Pli (F)
CTTCGCATGAAGAAGTCATC
and
Pli(R) TCAGTTATAGGCCAGAAAGC.
The final concentrations of reagents were
0.2 mM of each primer, 1.25 U Taq DNA
polymerase, 0.2 mM dNTPs, 1.5 mM
MgCl 2, 5 μl of DNA (20 ng/μl) in a reaction
volume of 25 μl (Lauro et al. 2003). The
primers
MpPl(F)
CGGGCAGCAAATCGTATTCAG
and
MpPl(R)
CCATAAAGTGTTGGGTCCTCTAAG
were
used
for
amplification
of
metalloproteinase
gene.
The
final
concentrations of reagents was 1 mM of
each primer, 1 U Taq DNA polymerase, 0.2
mM of dNTPs , 2.0 mM MgCl 2, 5 μl of
DNA (20 ng/μl) in a reaction volume of 25
μl (Kilwinski et al., 2004). The amplified
DNA was subjected to electrophoresis in 1%
agarose gel stained with ethidium bromide.
The PCR products obtained by
amplification with primers Pli were purified
using the kit PureLink Quick Gel Extraction
(Invitrogen, Carlsbad, CA, USA) and
sequenced. The reaction was carried out
using the sequencing kit Big Dye
Terminator (Applied Biosystems), according
to the manufacturer’s instructions. The
analysis was performed on a 16 capillary
sequencer, model ABI 3130xl (Applied
Biosystems). The sequences of the 16S
rDNA were established, and the BLAST
algorithm was used to find homologous
sequences. The calculation of the distance
and the construction of the phylogenetic tree
were carried out by the neighbor-joining
method, with the help of the software
Characterization of Paenibacillus larvae 81
MegaBACE version 3.1 (Kumar et al., 2004).
Results
When microbial growth was observed on selective
medium, three colonies of each plate were selected
on the basis of colony morphology (hyaline aspect,
flat edges and flat center) and presence of a zone of
proteolysis for confirmatory tests (reaction
to catalase and Gram stain). Most of the
isolates were positive for P. larvae. Two
isolates from honeycomb showed atypical
colony morphology and one isolate from
pollen showed positive catalase reaction and
atypical morphology (Table 1).
Table 1. Evaluation of Paenibacillus larvae isolated from honey, honeycomb and pollen samples
(2002-2007).
Sample
n
Typical
colony Negative catalase Positive
PCR
morphology
reaction
results
Honey
37
37
37
37
Honeycomb
35
33
35
35
Pollen a
8
7
7
8
Total
80
77
79
80
a
The same pollen sample showed atypical colony morphology and positive catalase reaction.
The PCR protocols allowed definitive identification
of P. larvae by observing the specific amplification
of the 16S rDNA and metalloproteinase genes (Fig.
1). All the 80 isolates responded to this approach,
showing the presence of expected PCR fragments
with primers Ple (969 bp), Pli (572 bp) and
MpPl (271 bp). The specificity of amplicons
was checked by sequencing. Amplification
was not observed in the samples of negative
controls.
Figure 1. Agarose gel electrophoresis of PCR products. Bacillus cereus ATCC 11778 (B,C, 16S
rDNA; D, metalloproteinase); Paenibacillus larvae ATCC 9545 (E,F, 16S rDNA; G,
metalloproteinase); Paenibacillus alvei (H, I, 16S rDNA; J, metalloproteinase); Paenibacillus larvae
(K,L, 16S rDNA; M, metalloproteinase); A,N = 100 bp ladder.
The sequences were aligned with sequences
obtained from the GenBank database (accession no.
in parentheses) of the following strains:
Paenibacillus brasiliensis (D78476), Paenibacillus
glucanolyticus (D885140), Paenibacillus alvei
(X60604), Paenibacillus koreensis (AF130254),
Paenibacillus larvae (AY030079), Paenibacillus
alginolyticus (D78465), Paenibacillus azotofixans
(AJ 251192) and Paenibacillus polymyxa (AY
3596370). All P. larvae isolates investigated in this
study had >99% identity with the 16S rDNA
sequence of P. larvae (AY030079). These results
are the first sequencing of strains of P. larvae
isolated in Brazil and show that these strains are
highly correlated.
Discussion
Strains of P. larvae isolated from samples of honey,
honeycomb and pollen in Brazil were
characterized. Results from the microbiological
investigation often confirmed the presence of P.
larvae observed by growing in selective medium.
The most common discrepancy was on colony
morphology. Some degree of inconclusive results
from phenotypical characteristics could be expected
since different P. larvae genotypes may present
differences in morphological and physiological
characteristics (Genersch, 2010).
Although the isolation of the microorganism of
interest is often the gold standard for microbial
identification, molecular diagnosis may permit an
early detection of P. larvae, before the clinical
signs of the disease, in time to implement proper
control measures. In addition, some samples
showed inconclusive results in microbiological
testing, thus molecular verification may provide
security to the diagnosis. As an example, PCR
allowed to identify P. larvae in 91% of honey
samples, against 57% observed by cultural methods
(Lauro et al., 2003).
The high similarity among isolates of P. larvae,
together with the fact that until now there was only
one record on the presence of P. larvae in the state
of Rio Grande do Sul and one outbreak notification
in the state of Paraná, may indicate that this
pathogen was recently introduced in Brazil.
Phenotypic and genotypic characterization of P.
larvae isolated in the neighbor country Uruguay
revealed high strain similarity (Antunez et al.,
2007), while samples from Austria and Germany
show higher genetic diversity (Peters et al.,
2006; Loncaric et al., 2009).
The evolution of the disease in South
America (Allipi, 1992; Antunez et al., 2004)
direct to the strong control health deployed
to prevent the introduction of this pathogen
and its spread in the Brazilian territory. The
need for continuous surveillance indicates
that PCR-based methods for rapid detection
of P. larvae may be useful tools to be
adopted by regulatory agencies.
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83
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the paper has not already been published elsewhere;
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REVISED
December, 2011
Journal of Cell and
Molecular Biology
Volume 10 · No 2 · December 2012
Review Articles
S. TRIVEDI
Marker Systems and Applications in Genetic Characterization Studies
Y. ÖZŞENSOY, E. KURAR
1
11
Research Articles
21
N. SAFAVIZADEH, S. A. RAHMANI , M. ZAEFIZADEH
Curcumin rendered protection against cadmium chloride induced testicular damage in
Swiss albino mice
31
P. SINGH, K. DEORA, V. SANKHLA, P. MOGRA
Study of Klebsiella pneumoniae isolates with ESBL activity, from ICU and Nurseries, on the
island of Mauritius
39
S.K. Mungloo-RUJUBALI, M.I. ISSACK, Y. JAUFEERALLY-FAKIM
HIV-1 reverse transcriptase inhibition by Vitex negundo L. leaf extract and quantification of
flavonoids in relation to anti-HIV activity
53
M. KANNAN, P. RAJENDRAN, V. VEDHA, G. ASHOK, S. ANUSHKA, P. CHANDRAN
RAMACHANDRAN NAİR
Genetic characterization and bottleneck analysis of Korki Jonub Khorasan goats by
microsatellite markers
61
B. MAHMOUDI, O. ESTEGHAMAT, A. SHARIYAR. M. Sh. BABAYEV
Low-Stringency Single-Specific-Primer PCR as a tool for detection of mutations in the matK
gene of Phaseolus vulgaris exposed to paranitrophenol
71
Mohamed R. ENAN
Short Communication
79
S.S. CHAGAS, R.A. VAUCHER, A. BRANDELLI
Guidelines for Authors
83

Vol 10 No 2 - Journal of Cell and Molecular Biology

Transcription

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