The tubulin gene family of Paramecium: Characterization and

Bid
Cell (1996)
0 Elsevier,
83
87,83-93
Paris
Original
article
The tubulin gene family of Paramecium: Characterization
and
expression of the aPT1 and aPT2 genes which code for
a-tubulins with unusual C-terminal amino acids, GLY and ALA
Pascale Dupuis-Williams
a*, Catherine Klotz a, Honor6 Mazarguil b, Janine Beisson a
a Centre de Ghe’tique Moltkulaire,
Centre National de la Recherche Scientifique,
Associe’ h I’llniversite’ Pierre et Marie Curie, 91198 Gif-sur-Yvette;
b Institut de Pharmacologic
et de Biologie Structurale,
Centre National de la Recherche Scientijique, 31400 Toulouse, France
(Received 15 July 1996; accepted 26 September 1996)
- The ciliated protozoan Paramecium harbours a particularly large diversity of microtubule networks, ranging from the
elaborate and stable ciliary axonemes and basal bodies to very dynamic cytoplasmic, cortical or intranuclear arrays. Their organization
and individual cycle of assembly/disassembly
are well known and extensive immunocytochemical
studies of the post-translational
modifications in the various microtubule systems have been reported. However, in order to better understand the biogenesis of these
multiple and diverse microtubule arrays, it seemed necessary to characterize the tubulin gene family. We show that P tetraurelia possesses four a- and three Pgenes and we report the cloning and sequencing of two intronless a-genes, @Tl and clpT2, which code for
very similar polypeptides, differing only by their unusual C-terminal amino acids, respectively GLY and ALA. Partial sequencing
of
the two other a-genes suggests an absence of any further isotype diversity. In an attempt to study the expression of @Tl and cPT2,
polyclonal antibodies were raised against the twelve C-terminal amino acids corresponding to the deduced polypeptide sequences. The
reactivity of these anti-sequence antibodies was studied on blots of soluble tubulin and in situ and compared with that of other well
characterized anti-a-tubulin
antibodies. The molecular data show that in Paramecium, like in other ciliates, microtubule diversity does
not arise from tubulin isotype diversity. The immunocytological
data indicate that the native C-terminal sequences are predominantly
detected in transient or nascent microtubule arrays and lead us to propose: 1) that the C-terminal TYR, absent in Paramecium and in
most cilate species, has no intrinsic functional role; and 2) that post-translational
modifications do not seem directly instrumental in the
geometry and functions of microtubule arrays.
Summary
tubulin
multigene
family
/ cytoskeleton
/ tubulin
diversity
/ post-translational
Introduction
Microtubules are engaged in a variety of functions as
diverse as cell division, cell shapedetermination, intracellular transport and locomotion, related first to their intrinsic
dynamic properties [37], and to the great diversity in their
biochemical properties, organization, stability, and associated proteins (MAPS). The major componentsof microtubules, the a- and Ptubulins, are encoded by multigenic
families whose complexity has increased in the course of
evolution of eukaryotes [45, 481. The diversity resulting
from differences in primary sequenceis further increased
by post-translational modifications: acetylation, detyrosilation/tyrosylation of a-tubulins, phosphorylation of Ptubulins [30], glutamylation [21] and polyglycylation [61] of
both sub-units. Over the last 20 years, researchon a large
variety of organismshas aimed at sorting out the role of this
molecular microheterogeneity in the functional and structural differentiation of microtubule arrays.
The functional significance of tubulin sequencediversity, first postulated by Fulton and Simpson [25], has been
* Present &dress: Ldboratoire
lake,
Ecole
Vauquelin.
SupCrieure
75005
Paris,
de neurobiologie
de Chimie
et Physique
France
de la diversit
Industrielles,
cellu10, rue
modifications
challenged by several lines of evidence. Not only do tubulins of various origins coassemble into microtubules in
vitro, but gene disruption [33, 661 or transfection experiments [12], and microinjection experiments using exogenous tubulins [29, 741 all seemto show that an apparently
specialized isoform can be replaced by any exogenousisoform to form a functional microtubule. Furthermore, in
vivo, microtubules can be copolymers of the various available tubulin isoforms [46]. In several lower eukaryotes, all
the microtubule arrays share a single a- or Pgene product
[3, 52, 771. Then, to account for microtubule diversity,
many studieswere devoted to the role of post-translational
modifications, but have globally been elusive: in no case
did the modifications appear instrumental in determining
directly the properties of microtubule arrays and at any rate
acetylation of a-tubulin was proved to be totally dispensable [26, 381. Conversely, genetic analysis of tubulin
mutants in several specieshas more recently provided evidence that some microtubule systems specifically require,
for assemblyor function, a given a- or Ptubulin gene product; such is the case in Drosophila
(the @ gene is required
for spermatogenesis[34, 501 and one a-tubulin for the ovocyte meisosisand cleavage mitosis [50]), in Caenorhabditis
(the met-7 gene for touch sensing[65]), and in Aspergillus
(the tubB gene for ascoporogenesis[36]). These studiesalso
suggestedthat the functional specificity of microtubule net-
works
depends
on the ratio of the different
iaotyptta con>-
posing the tubulin pool. Furthermore, isotype specificit),
was shown to influence the self-assembly properties 01
tubulins [53. 721 and the biological properties of microtu
hules suchas their resistanceto cold [ 17. 5 I].
All in all it appearsthat different organismsmight adopt
different strategiesand that the organization and functions
of microtubule arrays can be controlled at diffe.rent levels.
isotype diversity, ratio of particular isotypes, post-trznsla~,
tional modifications, interactions with MAPS, and presunably properties of the MTOCs. This is why.a full understanding of the basis of microtubule diversity requires a
comprehensive description of all these parameters. which
have seldom been studied together in a single cell type.
Hence, unicellular organismslike Purarner.Gn
may be of
special interest.
Indeed, the ciliate P tetnrureliu
displays a particularly
high diversity of microtubule arrays. Cytological and
immunological data distinguish 16 microtubule networks.
which differ by their geometry, dynamic properties and
functions [ 14, 20, 231 and extensive analysis of their post-translational modifications have been carried out 12. 5, ‘,I.
As a necessary complement, we have undertaken a molecular study of the tubulin family in P tetruuretia
and we
show that it is composed of four a- and three Ptubulin
genes, significantly more than in the other ciliates that have
been studied: Tetrahymena
[3], Euplntes
[43] and Stylon~chia
[16. 321. We have cloned and sequencedtwo of the
a-genes, which encode proteins differing only in their C
terminal amino acids, respectively ALA and GLY. Partial
sequencedata on the two other a-genes seemto preclude
any further isotype diversity, We have raised antibodies
against the twelve C-terminal amino acids encoded by the
two genes.Their preferential reaction on the most dynamic
microtubule arrays and in the early stagesof assembly ot
more stable arrays indicates that in structurally and functionally diverse microtubules arrays, the C-terminal
sequenceof the a-tubulins, whether native oi weakly modified, remains accessible. These observations, along with
the molecular data, concur to the conclusion that, in Pntw
mccium. neither t,ubulin diversity nor post-translational
modifications suffice to account for the diversity of organization and functions of the microtubule networks.
Materials and methods
Cells
und odture
conditions
Wild type cells of stock d4-2 of P tt’tncureiia
(701 were grown at
ham) by rarttlum-priming
(Bocfrringer. <ierrrtany). t*i!Lzrs ii, \li.
autoradlographed
on Kodak X-Omat AK5 films at -70°C'
uGfJt
intensifying screens.
‘The genomlc library was obtained by Jean Cohen by Sa243A
tial digestion of Purumrcium
total DNA, phosphoryladon
pa
und
ligation with RumHI digestedlambda~vectorEMBL;4 [24]. Tt~Lx
ligated DNA was then packaged in vitro and recombinan;
phages, when plated, led to estimate the titer of this librarIG at
I.:! x IO5particles/ml.Accordingto [63], the whole non-ampIfied library. ic 4.8 x 101 recombinant phages, was therefore rcpresentativc of the Parutiecium
genome whose tiomplexity 1.~
estimated to 2 x 10s bp [47]. Phages containing
cl-tubuli;l
sequences were isolated.by pbque hybridization v&h the dI”f‘
gene
131.The cloneswerethenpurified to hornet
c$’ T p~rifirmis
geneity and anatysed by restriction mapping and Southern blot<.
The fragments hybridizing with dfT were subcloned in a pB+uc
Script plasmid (PBS) (Stratagene. USA).
‘Iwo genes, aPTI and CXPT~., were entirely cloned in PBS. ih::
I‘irst step led to the cloning of two fragments (a 2.7.kb EccrRf
insert and a 2.7-kb BgEII insert called respectively ~El4 and
aB23) from two independent lambda clones. However, sequence
analysis showed that the a-tubulin gene in each sub-clone was
incomplete,eachmissingapproximately100bp at one end.The
3’ end of &I4 (&‘Tl gene) was then subcloned from the s:~~mc
L-clone. in a 240 bp EcoRI fragment. The aPT2 gene was
recloned in a 85kh Hind111 fragment containing the whole cod-.
ing sequence, named &H.
Specific probes for each gene were subsequently obtained by
sequential deletion by exonuclease 111of the tiEI and the nB2?
clones until1 about -1000 bp from the initiation codon of eack
gene, as shown by size determination on agarose gels (not shown;.
Genomic DNA was cut by HindIII and lractionated own 1% agarose gels. The 2.5kh band.(containing the entire &‘I’3 gene) atid
the 7-8-kb band (containing the remaining three g&es) were cm.
their DNA purified and PCR amplified between the primer>
Avctl: (GATCTTATGTATGCCAAGAGAGCCTT) and Avu.!
(AAATGTTTTAATATTTGATTTGTATATATATTC).
designed to surround the C-termini of both the aPTI and the
uPT2 genes. Thirty cycles of 1 min at 90°C. 45 s at 55’C and
I min at 72°C were carried out. Each amplification product was
directly sequenced with each primer in turn, using the sequencmg kit from Pharmacia in the presence of 0.5% Nonidet P-40 and
after denaturation by incubation at 90°C and immediate cooling
in dry ice.
I)NA
.sryuenc~itr~
and
.srffuet2ce
unu1ysi.s
17°C in a buffered infusionof wheatgrasspowder (PinesInternational Co. USA), supplemented
bacterizedwith
Enterobactrr
with 0.4 pglml psitosterol
and
uerogmcs.
Exponentially growingparameciawerepelletedand taken up IO
50°C for at Ieast4 h, in 2 lysis buffer: 0.S M EDTA (pH 9). 1%
SDS, and 1 mglmfproteinaseK (Merck, USA). After thiee phz-
The inserts of the plasmids were gradually digested by cxonuclease 111in order to obtain overlappingclonesof 150to 300 hp
which were sequenced each at least twice. DNA sequence wah
determined according to the method described in.[64], using the
sequencing kit from LJtiited States Biochemicala
(USA). The
analysis and comparison of the sequences were carried out usink
the [JWGCG programs [ 181.
nol extractions, the mixture was first dialysed against 20% ethanol, and finally against 10 mM Tris-WC1 (pH 8);l mM EDTA.
Restricted Parumecium
DNA was fractionated on I % (w/vi agarose gelsand transferredontn Nylon N+ membranes
(Amersham,
UK) in a 0.4 N NaOH solution. Prehybridization
and hybridization were carried out at 62°C as described by Chilrch and Gilberi
( 1 I] with probes radiolabelled (QP dATP, A00 Cilmmol; Amers-
l’ubutin was prepared accordmg to Vallee and CoIlin> 1731, iram
the post-microsomal supernatant of an exponential culture. This
supematant, obtained according to 1621, was made SO mM Pipeb,
I mM MgCl,. 1 mM EGTA. Tubulin was induced EOpolymeri?c
by addition of 1 mM GTP and 40 ,uM taxol (generously provided
by-Dr Y GuCnard) for 15 min at 3S”, then pelleted 30 min at
5000 Lq on a 10% sucrose cushion containing I mM GTP and
10 ,uM taxol
a-Tubulin
Electrophoresis
and Western
genes
Antibodies
The following antibodies were used. The anti-Paramecium axonemal tubulin has been previously described [l, 131 and shown to
recognize a post-translationally
modified form of tubulin [2],
recently identified as a polyglycylation
[61]. The ‘universal’
monoclonal DMlA [4] was purchased from Amersham (UK) and
the monoclonal
YL l/2 [35, 751 from Interchim (Montlu9on,
France). The monoclonal anti-acetylated tubulin [56] was from
Sigma (St-Louis, Missouri). The monoclonal anti-ffiLU,
ID5,
raised against the 14 C-terminal aminoacids of detyrosinated pig
brain tubulin [76] was a gift from Dr Wehland. In Paramecium,
whose a-tubulins lack a C-terminal Glu, the selective decoration
of basal bodies and a limited subset of all microtubule arrays
suggest that this antibody recognizes poly-Glu motifs, arising
from post-translational
modifications [5].
The polyclonal antibodies R200 and R255 described here
were raised against the 12 C-terminal amino acids (439-450) of
the deduced polypeptide sequence corresponding respectively to
the two Parameciuma-tubulin genes, a-PTl an a-PT2 characterized in this study. The corresponding peptides were synthesized on an ABI 430A (Foster City, CA, USA) automated
synthesizer. The purity of the peptides was verified by reverse
phase chromatography
and FAB mass spectrometry. 5 mg of
each peptide was coupled with 5 mg keyhole limpet hemocyanin
(KLH, from Sigma) in 5 ml 0.15 M NaCl for 24 h. Rabbits were
immunized by four subcutaneous injections at 2-week intervals
of the conjugatedpeptide as an emulsion with complete Freund
adjuvant.
On immunoblots from both 1D and 2D SDS-PAGE gels, the
two antibodies, R200 and R255, were shown to recognize Parameciuma-tubulin. In particular on blots from 1D gels they specifically label a single band, which is that labeled by DMl A.
R255 but not R200, also labeledthe a: polypeptidesin purified
porcine brain tubulin samples. In all cases, the specificity of
these reactionswasascertained
by their extinction whenthe antibodies were pre-incubated
with the homologous
peptide. In
immunofluorescence,
extinction was obtained with 5 mM of the
peptide; on immunobfots, extinction was observed with 20 mM
of the peptide. On Paramecium,the two antibodiesdisplayed
some cross-reaction, as their preincubation with the heterologous
peptide, under the same conditions as above, led to a partial
extinction of the reaction, observed both on blots and in situ.
immunojluorescence
P tetruurelia cytoskeletons
85
blots
SDS-PAGE was carried out on 10% acrylamide gels with a bisacrylamide:total
acrylamide ratio of 0.135:10 using SDS from
BDH (Poole, UK) according to [ 11.
Two-dimensional
gel analysis was performed essentially
according to O’Farrel [53] on a mini-gel system from BioRad.
Isoelectric focalisations were made in 0.8 mm inner diameter capillaries, gels were 0.8% of 3-10 and 1.2% of 46.5 Pharmalytes
(Pharmacia-LKB
Biotechnology,
Uppsala, Sweden). Tubulin
samples were 0.2% SDS and treated with 10 mM iodoacetamide
before addition of urea and mercaptoethanol (buffer A) in order to
block SH groups and to decrease tubulin aggregation. Migration
time by voltage amounted to 12 000 V/h. The second dimension
was performed as above. Gels were either stained with Coomassie
blue (0.25% in 50% methanol, 10% acetic acid) or electrophoretitally transferred to nitrocellulose filters (Schleicher and Schtiell;
Dassel, Germany) on a semi-dried system (Phannacia-LKB).
Saturation, immunological
labelling of blots and antibody dilutions
were made in TBST (10 mM Tris HCl, 150 mM NaCl, 0.1%
Tween-20) containing 5% non-fat dry milk. The secondary antibodies were labelled with alkaline phosphatase (Promega Biotec,
Madison, WI) and the enzymatic activity was localised by addition of the substrates: BCIP (5-bromo-4-chloro-3-indolyl
phosphate) and NBT (nitro blue tetrazolium).
fixation.
in Parnmecirrm
were prepared either with or without
Cells were first permeabilized
for 3 min in 1% Triton
Fig 1. Representation of plasmids aE14 and c~B23. The black
boxes representthe coding partsof the genes(rPT1 and aPT2,
the grey boxes the respective non-coding parts. The 5 ’ probe
used in Southernexperimentscorresponds
to the 1600-bprestriction fragmentdelimitedby the KpnI-EcoRI sites,which includes
430..bp of the 5’part of the crPT1 coding sequence.
X-100 in PHEM-buffer [67] thenfixed for 2&30 min in 2% paraformaldehyde
in PHEM,
washed twice in TBST,
then in TBST
containing 3% BSA (TBSTIBSA), then processedthrough primary antibodies and FITC or RITC secondary antibodies from
the Pasteur Institute in TBST/BSA.
Alternatively,
the fixation
step was omitted and the permeabilized
cells were briefly incu-
bated in TBSTIBSA then processedas above through primary
and secondary antibodies. The polyclonal antibodies R200 and
R255 and anti-Paramecium axonemal tubulin were diluted
11400, DMlA l/1000 and ID5, a culture supematant,l/10. The
secondary antibodies were used at a l/200 dilution.
Results
The multigenic tub&n family of Paramecium
Preliminary Southern blots of Paramecium genomic DNA,
probed with cloned a-tubulin genesfrom other ciliates, Z’etrahymena pyriformis [3] and Stylonychia lemnae [3 11,
revealed identical patterns composed of several bands of
high molecular massimplying the existence of more than
one a-tubulin gene (not shown).
A genomic lambda library of P tefraurelia ]19] was then
screenedwith the cST of T pyriformis. The positive clones
were classified by endonucleasemapping and blot hybridization, in order to identify restriction fragments presumed
to contain an entire tubulin gene, which would then be
cloned in a pBluescript plasmid. Two a-tubulin genes,
aPT1 and aPT2, were thus isolated (fig l), as describedin
Materials and methods.
Probing Southern blots with either aPT1 or aPT2 led to
identical results, showing for most restriction patterns four
high molecular mass bands of equal intensity (fig 2).
Washes following hybridization with aPTI, carried out
over a range of increasing stringencies, revealed only a
slight difference of intensity between the bands, suggesting
that the nucleotide sequencesof the four a-genes are nearly
identical.
To ensurea correct interpretation of the complex patterns
observed, the blots were screenedwith probes restricted to
the 5’ parts of the Paramecium genes (see Materials and
methods). Figure 2 shows the hybridizations carried out
with a 5’ restriction fragment from the aE14 plasmid,
including the first third of the coding sequenceof the aPT1
gene. Comparison of these data with those obtained using
the complete coding sequencessupportsthe conclusion that
four a-tubulin genes are present in the macronuclear
Xh
EcoRl
Hindlll
H~all
Bglll
Xholl
6glll
Haelll
Hindlll
Haelll
2.3
Fig 2. Identificationof a-tubulin genes in Parumeciurn. Southernblotsof total DNA here probedwith the wholesequence
of the ctoned
Paramecium
a-tubulin geneaPTI (A) or with a proberestrictedto the the 5’ part of the CZFTI gene (B) (see fig 1). The con~parism uf
the two patterns leads to the conclusion
that four oz-tubulingenesarepresentin the macronuclearDNA of P tetraureliu. The identifica
tionsof respectively@‘I’1 (*) and WI’2 (0) on the restrictionpatternswereobtainedby subsequent
hybridizationswith specific.probes
(asdescribed in Materials arm’methods).
genome of P tetraureliu. The same type of experiments,
carried out with ,@tubulin probes, revealed the existence of
three Ptubulin-genes (described elsewhere ]19]; and not
shown).
This result was somewhatunexpected, as similar studies
in other ciliates show that their tub&n family contains a
limited number of genes, one or two for each sub-unit [3.
16, 27, 31, 431 and that when two genesare present, they
encodeidentical or slightly divergent proteins.
The cloned genes were then individually identified on
the Southern blots (fig 2), by hybridization with probesspecific for each cloned gene and constructed as described in
Materials and methods.
particularly high proportion of A+T: 73% upstream from
the ATG, and almost 80% in the non-coding down-stream
region. Although no consensusemergedfrom the comparative analysis of the overall upstream sequencesknown in
Paramecium (not shown), the presencewas noted in pasition -29 from the ATG, of the motif AGAGAG~also found
In the upstream regions of several Tetruhymena genes 1.6.
27, 691 and therefore considered as a putative element of
transcription control. In addition, regarding the regulation
of the fubulin genes,the presenceof somerepeats-(fig 3jat
similar positions (towards the ATG) in the two cz- and tht:
Ptubulin genesmight indicate that the expression of these
genes is coordinately regulated as shown in Tetrahymena
[27,69].
Nucleotide sequenceanalysis
The sequences of the two complete genes aPT1 and
(xPT2 (fig 3), as well as their flanking regions, were
determined on sequentially deleted overlapping clones.
Their EMBL accession numbers are respectively X99489
and X99490.
The coding regions of clPT1 and aPT2 are both 1350
nucleotides long, including the stop codon TGA, and are
not interrupted by any introns. The two genes are highly
similar: their nucleotide sequencesexhibit 98.8% identity.
Only 18 substitutions were observed along the whole coding sequence, mostly T/C transitions, all situated in the third
position of the codon, except for a silent conversion of TAA
into CAA in position 396 of the clpT2 gene, and for the
final codon. Respectively ten and nine internal TAA codons
were found in CcpTl and czPT2,coding for Gln, as could be
deduced from comparison with tubulin sequencesfrom
other organisms.and aspreviously shown in surface antigen
genes[lo, 571.
In agreement with available data on non-coding ciiiate
DNA 1581.the flanking regions of the two genescontain a
The striking feature emerging from the comparison ot
the two genesis their very high degreeof identity. N.ot only
are the coding sequencesalmost identical, but more unusually, the non-coding sequencesare highly similar overtheir
entire length and absolutely identical over 60 bp upstream
from the ATG.
Based on this observation, primers were designed in
order to amplify the terminal parts of the two remaining
genes(seeMaterials cmdm&hods). Unfortunately, the cloning of these genes in E coli turned out to be impossible
because of the highly recombinogenic nature of these
sequences;all attempts,using different piasmid vectors and
a variety of host E coli strains, repeatedly led to partia)ly~
deleted clones. To avoid thesedifficulties - already encountered during the cloning of aPTI and. aPT2 or of other
genomic fragments from Puramecium or Tetrahymena -direct sequencing of the amplification products from the
terminal parts of the geneswas undertaken and preliminary
results indicate that the two other gene t.&nini are not different from the two already sequencedgenes. More prccisely,. the partial sequenceof the gene.designedas-crPT3
was identical to aPTI, including the ammo-terminal GLY.
a-Tubulin
genes
in Paramecium
87
-224
CcPTl AAATTAAGCTAGGGAGTTTATTGATGAGTTAMGAGATGAATGTCAGAATTAG
aPT2 AAATTAAGTTTGGTTGTTTCTTATCGAGCCAACGTAATAAAGGTGAGAATTAG
-231
-171
AAGATGAAGCATATACTTATTTTATAATTGATTGATAATT......
AATTGGAAGCATATACTTATTTTATAAAATATATAT
-178
.AATTAT&XTTCAAATTTGATGTACCCCCATATTAGGATATT
TTTAATGTACCCCGGTGCAAGGATATT
-78
TTGGAGATTTAAATCTGGGGGTAACTGCTGAAATTCAMTAATTTTTT
TTGGATATTCUTATGAAAGTAACTGCTG
AAATTCAAATAATTTTTT
-78
GAGA
8 GAGA
TTTCATCACATAGCACTTTAAA
TTTCATCACATAGCACTTTA
TIGGGDDAFNTFFSETGAGKHVPRAVFLDLEPTVIDEVRT
241
GGAACATACAGATAATTATTCCACCCAGAATAATTGATTTC
C
GTYRQLPHPEQLISGKEDAANNFARGHYTIGKEIVDLCLD
C
A
C
A
361
AGAATCAGAAAGTTAGCTGATAACTGCACAGGTCTC~T~~T~~~TC~CACTCAGTCGGA~AGGTACC~ATCAG~~GffiATCACTC~A~GG~GA~GTCAGTCGAT
C
RIRKLADNCTGLQGFLVF
HSVGGGTGSGLGSLLLERLSVD
481
TATGGTAAGAAATCTAAGTGGGmCACCACCA~TATCCATCACCAT~GT~C~CTGCAGTCG~G~CCATAC~C~CATC~GTC~CCCACTCA~G~GG~CACACTGATGTC
T
YGKKSKLGFTIYPSPQVSTAVVEPYNSILSTHS
LLEHTDV
601
TGTGTCATGTTGGATAACGAAGCCATCTATGATATITGCAG~G~~G~TA~~G~C~CATACACC~C~G~CAGA~GA~GC~~G~A~TC~A~ACAGCT
T
T
CVMLDNEAIYDICRRNLDIERPTYTN
LNRLIAQVIS
S
F
TA
S
A
E
721
TCATTGAGATTCGATGGTGCCTTGAATGTCGATATCACTGAG~CT~CC~C~TCCCATATCCACG~TCCAC~A~CT~~TCATATGCCCC~TCATCTCA~TG~G
T
SLRFDGALNVDITEFQTNLVPYPR
IHFMLCSYAPI
I
K
841
GCTTATCACGAATAATTGTCAGTTGCTGAAATCAC~C~A~CTTCG~CCA~~CATGATG~T~G~TGACCC~GACAT~T~TACAT~C~TCCAT~~TACAGA
T
A Y
H E '1 LSVAEITNSAFEPANMMAKC
DPRHGKYMACSMLYR
961
GGTGATGTCGTCCCCAAGGATGTCILACGCTGCTA~CACCATC~GAC~GAG~CCATCT~~CGTCGAC~T~CC~CAGGA~C~~TC~~TC~CTATC~CCACCA
G
D
VV
:?
KDVNAAIATIXTKRTIQ
FVDWCPTGFKVGINYQPP
1081
ACAGTCGTCCCA(~AGGTGATPTGGCCAAGGTCATGAGA~TG~GTATGATC~C~C~~CT~CA~~TG~GTCTTCTCCAGAC~ATCAT~TTCGA~TTATGTATGCC
C
T V V P G GDLAKVMRAVCMISNSTAIAEVFSRLDHKFDLMYA
C
C
1201
AAGAGAGCCTTC(:TCCACTCGTCGGTGAAGGTATGCGCT
G
K
RA
F
V
HWYVG
EGME
EG
E
F
S
E
ARE
DLAAL
E
K
DY
E
E
VG
I
T
ETA
1321
GAAGGAGAAGGTGAAGAAGGAGAAGGTTGA
CA
E GE
G E E G E G STOP
A
1441
AATGCTAATTAAGAAATGTTTTCT
~A&CCAAATAGGAAATACTGCTT
Fig 3. Comparison of the aPT1 and &‘T2 genes and their deduced amino acid sequences. The differences between the two genes, shown
in the second line, are all clustered in the third codon positions, except for the Gln at position 397 which is encoded by TAA in &‘Tl
and CAA in apT2. In the 5’ non-coding parts of both genes, the direct repeats ATTCAAA and the AGAGAG boxes, potentially involved in the regulation of transcription, are underlined.
As for the &T4, it could not be conveniently isolated from
the other genes for the PCR experiments,
but direct
sequencing of PCR amplification obtained from total DNA
never revealed other sequences than the two already determined.
Amino acid sequences
As expected, when aligned with other ciliate a-tubulins, the
deduced ammo acid sequences of ml
and apT2 appear to
be highly conserved, apart from their C-termini (fig 4). Like
xx
Schizosaccharomyces pombe
cl1
a2
GEGMEEGEFSEAREDLAALERDYEEVGQl%MB3BBfYJi3J3lZEY
GEGNEEGEFSEAREDLAALERDYEEVGQBSX&v%J%'l%i=Y
Euplotes vannus
GEGMEEGEFSEAREDLAALEKDYEEVGIETAEGl!X+It!iZDBfA
Euplotes octocatitus
GEGMEEGEFSEAREDLAALEKDYEEVGITAZGEGB#3%3%ft
Oxytricha granulifera
GEGMEEGEFSEAREDLAALEKDYEEVGIETAECX%GRiXf83G@JZ
Stylonychia lemnae
al
a2
GEGMEEGEFSEVREDLAALEKDYEEVGIEIVEiGBX%EBGME
GEGMEEGEFSEAREDLAALEKDYEEVGIETAE~G~Wi3!l~G?#E
Tetrahymena pyrifornais
GEGMEEGEFSEAREDLAALEKDYEEVGIET&EG~GlZE~GY
Tetrahymena thermophila
GEGMEEGEFSEAREDLAALEKDYEEVGIETAEGEOEPEGlZEGY
Paramecium tetraurelia
Pig
ai
ff2
96,4's
97.9%
GEGMEEGEFSEAREDLAALEKDYEEVGXETAEQBZGEEGEG
GEGMEEGEFSEAREDLAALEKDYEEVGIETAEG-EaEA
GEGMEEGEFSEAREDMAALEKDYEEVGVDSVZWZGBZXGEl%Y
9L.W
Fig 4. C-terminalregionsof a-tubulina.Purumrciumtubulinsarc tn keepingwith the closeresemblance
ot’ all rx-tubuli@s
-- thsy t.tr~i:,
differ by some15C-terminalaminoacids(bold characters).The isology of the different a-tubtilinswith respectto rxFT1is indicatedin
percentages.
The sequences
are referencedby their accession
numberin Cienbankon the right of the figure.
the other cw-tubulins,they show somecharacteristics related
to post-translational modifications. in particular the LYS in
position 40, which was described as a substratefor xetylation [39]. This observation is consistent with the immunocytochemical data showing decoration of several microtubule arrays in Paramecium [2, 9. 141 with the 6-1 1B-l
monoclonal antibody raised against the acetylated Lys 30
1561.As for the C-termini of the molecules, it is interesting
to note that the two proteins differ only in the last amino
acid which is GLY in aPT1 and ALA in &T2. This observation, coupled with results obtained from partial sequencing of CXPT~and ccPT4 indicate that, in Purumecium the
four a-tubulin genesencodehighly similar or identical proteins. Hence, in accordance with what was described in
other ciliates studied so far and despite its relatively high
number of tubulin genes,there is low diversity in terms of
tubulin isotypes in Paramecium.
Immunologicul characterizution I$ the a;PTl clnd ~1~72
Seneproducts
The organization and dynamics of the various microtubule
arrays of Paramecium [2, 141 were already well known
from studiesbasedon the use of several antibodies. in particular the ‘universal’ DMlA 141directed aga~insta conserved sub C-terminal part of mouse a-tubulin and an antiParumecium axonemal tubulin [ 1-31shown to recognize a
particular post-translational modification [2] most recently
characterized asa polyglycylation of both (x- and ,!%tubulins
[61]. Acetylation of the LYS 40 of a-tubulins had also been
demonstratedon several stable [2] or transient arrays such
as the micronuclear spindles[14] as well as the presenceof
polyglutamylation of some of the stable microtubule arrays
[S]. These data and the more recent studiesof various antitubulin monoclonals raised against Purumecium [9, 13 1
demonstrated that the various post-translational modifications characterized different but often overlapping subsets
of microtubule arrays, without any clear correlation with
their stability, function or complexity.
In order to get new insights into the distribution of tubulin isoforms among the different microtubule arrays, polyclonal antibodies were raised against the 12 C-terminal
amino acids-of the putative products of c$‘U and aPi’-?
respectively, as their unusual C-termini suggestedthe popsibility of obtaining anti-C-terminal sequenceantibodies trj
be compared with antibodies againstC-terminal post-trans.”
lational modifications such as the previously demonstrated
polyglutamylation and/or polyglycylation.
The reactivity and specificity of the antibodies were i’zr~
examined on blots. By two-dimensional eltictrophntitic
analysis, the several isoforms of both a- and @-tubulim.
present in the soluble fraction of cell homogenatescan bc
separated{fig 5A). As the sequencedifferences between the
a-PTl and a-PT2 gene products cannot~resultin different
electrophoretic mobilities, it can be concluded that~the different observed isoforms are generatedby post-translatlan;if
modifications occurring in the soluble pool or maintained
after disassemblyof the dynamic micro&bules and that in
each spot of the 2D electrophoretogram, both aPT1 and
aPT2 gene products are presumably present. All a:iscjforms are recognized by the R200 antibodies (fig 5H?. An
identical reaction pattern was observed with R255 and titth
DMlA (not shown). In situ, both R200 and R2SS have the
sameproperties. Their reactivity dependson the conditions
(with or without fixation) of preparation of the cytoskeictons. In unfixed cells, both antibodies -rate
selectively
the labile and transient microtubule arrays: the intracytoplasmic network readily disassembledby nocodazole [7 t 1,
the cortical cytospindle assembledduring division and cl+assembledsoon after, the micronucleusdivision spindle and
the separation spindle made of 15 protofilament microtu
bules 1221;they also decorate the post-oral fibers which arr
disassembledat division. In fixed cells, most other microttlbule arrays, in particular basalbodies and cil-iary axonemex
are also labelled although more weakly and inconsistently.
In all cases,both antibodies decorated the same structurcr;
and the labeling was totally extinguished when the antibod
ies were reacted in the presenceof the homologouspeptide.
suggestinga homogeneousdistribution of both gene products on all the labeled microtubule arrays. The differencch
observed between unfixed and fixed cytoskeletons sup@urt
the conclusion that these antibodiesrecognize the native (‘
termini, predominant in dynamic arrays. masked in stable
arrays by post-transtationat modifications and MAPS, ku:
a-Tubulin
genes in Paramecium
89
function (since as soon as postoral fibers are detected by
R200 or R255, they ensure the transit of food vacuoles) nor
for their stability (since the newly formed structures are not
depolymerized by nocodazole).
Discussion
Fig 5.2-D analysis of soluble Paramecium tubulin. A. Coomassie blue stained gel. The lane MW indicates the position of the
molecular mass markers (200, 116, 92, 65, 43, 31 kDa); 01and p
correspond to porcine brain tubulin. Significant values of the pH
gradient in the first dimension are noted on the lower edge of the
gel. B. Corresponding blot reacted with the R25.5 antibodies. The
arrow points to the position of the basic edge of the IEF gel.
Identical patterns were obtained with DMlA or R200.
partly unmasked by fixation, as is the case for DMlA
whose reactivity requires fixation.
Figures 6 and 7 illustrate the reactivity of R200 during
the assembly of two complex microtubule arrays which are
neoformed at each division: the postoral fibers (a massive
microtubule bundle which sustains the transit of newly
formed food vacuoles) [15] and the contractile vacuole
system which form branched microtubule bundles originating from a microtubule-bound
cortical pore. During division, the postoral fibers disassemble and regrow within
minutes after division while the two contractile vacuole
systems are retained during division and ‘duplicate’, each of
the two new ones developing anterior to the old ones. In
double staining experiments, both the neoformed postoral
fibers (fig 6) and contractile vacuole microtubule arrays
(fig 7) were labeled by R200 or R255 as soon as they
developped and 15-20 min before being reactive with ID5.
Converse experiments (not shown) using ID5 and the polyclonal anti Paramecium axonemal tubulin, known to recognize the C-terminal
polyglycylation
of tubulins [42],
showed that labeling by the latter antibody also was late on
the neoformed structure, in agreement with other data [2],
just preceding the decoration by ID5.
These cytological observations show that in the cr-tubulin molecules present in labile, transient or newly formed
arrays of different geometries, the C-terminal epitope(s)
remain accessible and may be unmodified in arrays such as
the micronucleus spindles which are the most brightly decorated structures. Even if the microtubules
decorated by
R200 and R255 carry a low level of modifications,
as
revealed by TAP952 [23] but undetectable by R200 or
R255, this property is shared by most microtubule networks
[23] and therefore cannot be responsible for the diversity of
their organization. Our observations also indicate that, at
least for some microtubule networks, the C-terminal posttranslational modifications are neither necessary for their
The multigenic family of Paramecium tetraurelia is composed of four a- and three ptubulin genes with closely
related sequences, as shown by Southern blot experiments.
With the aim of understanding the part of genetic determinism in the assembly and dynamics of the different microtubule networks previously described in Paramecium, we
have undertaken the molecular characterization
of these
genes. Here, we report the cloning and sequencing of two
a-tubulin genes, aPT1 and aPT2. The deduced proteins are
both 450 amino acids long, identical except for their C-terminal residue, respectively
GLY and ALA, and highly
homologous to most a-tubulins.
Isoelectric focusing analysis of isoforms present in the
soluble intracellular tubulin pool revealed at least six a- and
five Ptubulin isoforms, considerably more than expected
from genetic data, suggesting than post-translational modifications are responsible for this diversity.
Polyclonal antibodies raised against the 12 C-terminal
amino acids of the deduced c@Tl and @I’2 gene products,
first characterized on Western blots of soluble cell extracts,
were then used in situ to localize primary gene products
which were shown to be associated with the most dynamic
or transient microtubule arrays in the cell.
A first implication of our results concerns tubulin evolution. The relatively high number of tubulin genes in Parumecium, as compared to other ciliates, may be interpreted
either as plurigenicity, or simply as the result of the macronuclear differentiation process by which a single gene of the
micronucleus (zygotic diploid nucleus) can be shared by
different chromosomes of the macronucleus (highly polyplaid, transcriptionally
active nucleus) [58]. However, the
sequencing data show that at least two different micronuclear a-tubulin genes coexist in the Paramecium genome.
The striking similarity between the cwPT1 and aPT2
genes, which extends outside the coding sequences, suggests a recent common origin for the two genes by duplication or homogenization by conversion. As regards the two
remaining genes, they are presumed to be very similar if not
identical, as attested to by the partial sequencing of their Ctermini. Moreover, mass spectrometry of the C-terminal
peptides obtained by proteolysis of a-isoforms of Purumecium axonemal tubulin distinguished only two different
types of gene products, with the corresponding
masses
expected for aPT1 and apT2 C-termini [61].
Considering that the three Ptubulins are homologous
(P Dupuis-Williams
et al, in preparation), we may conclude
that the complexity of the tubulin gene family in Parumecium conforms to the general case in ciliates, ie that a limited number of genes for each sub-unit encode identical or
slightly divergent proteins. Moreover, the comparison of
tubulin sequences in ciliates reveals that the different a- or
p-genes are more similar within a species than they are
between species, suggesting that they were homogenized
either by intraspecies duplication or by conversion processes. This situation is in opposition to what occurs in vertebrates where the different a- or Pgenes can be distributed
in common isotypic classes according to their functions or
tissue-specificities and suggests that the evolution of tubulin
sequences in ciliates is driven by the necessity of homoge-
Fig 6, immunodecaration of neoformed PUSI,
k. ~&-al fibers: double labeling by the mono&o@
i anti-cxCilu ID.5 and the polyclona~llR2OO. .The
$ ..post-oral fibers @of) form a bundle of micmlw
; bufes along which food vacuoles transit from
i: -the oral apparatus (oa) into the -cytoplasm.
$ Duting division, the pofs arc disa~emble&
:; -Within minutes after separation of the two
in daughter cells, their progressive regrdyth can
[ be monitored by immunoIat&ng with the R.200
1, antibodies. A, 3. The same ccl!, 1.5 &in-after
t -completion of cytolcinesis, respectively photographed for rhodamine fluorescence correspon: ding to ID5 labeling and-FITC corresponding to
; RZOO. C, D. The same double labeling of~anI other cell 30 min after cempletion of cytokine~
sis. At 15 min, the pofs, already well developed.
arebrightly labeled~by R200 but not by IDS. At
i 310tin, the fulty developed pofs are now st&i1 gly decorated by lD5 while labeling by R200
\ -is less inte~nse. The contractile vacuole pore
;, r&er&.ttbu~e bun&es~(cv) are no longer decorated by R2OQ.Bar, IO pm.
neity. This characteristic, shared by flagellate protists such
as Naegleria [40] could reflect the fact that homogeneity of
the tubulin pool is required to form such stable microtubule
structures as axonemes or flagella, as first hypothesized by
Little [44] and Silflow [68] and recently discussed by Gaertig ef al [27] and Lai et al [41]. Our data support this
assumption which appears especially relevant for Paramecium where molecular genetic studies seem to show a particular and intriguing tendency of its genome to tolerate and
maintain numerous multigenic families [ 19,28,49].
The second type of implications of our results bears on
the role of post-translational modifications. a-tubulins generally contain a C-terminal TYR, susceptible to removal by
a carboxypeptidase,
thus exposing the penultimate amino
acid (in almost all cases a GLU), and then repositioned by a
specific ‘tubulin tyrosine ligase’, in successive tyrosylation/detyrosylation
cycles [30]. Despite the considerable
amount of work devoted to the analysis of the functional
significance of the detyrosination/re-tyrosination
cycle of
a-tubulin [7], no clear functional role could be attributed to
this post-translational modification of m-tubulin, although a
correlation exists between detyrosylation
and ageing of
microtubules.
In Paramecium, the two sequenced~genes
lack a C-terminal encoded TYR. This appears to be the case
also for the two remaining genes,as evidenced by prelimrnary sequencing and supported by the fact that the antiTYR antibody YL1/2 [3S, 751 does not recognize farumecium tubulin on blots either of whole cell extracts or of
purified fractions (axonemes,basalbodies, soluble tub&n).
Furthermore, the study of non-tyrosinable tubulin in calf
brain [55] indicates that tyrosination requires a terminal
GLU-GLU. a situation not likely to occur in Paramecium.
Indeed, in ciliates (fig 3), the absenceof C-terminal TYR
seemsto be the norm, while in Tetruhymena, where it 15
present, biochemical studies could not reveal any tyrotinc
ligase activity ]59, 601. The universal function of the
TYR/GLU cycle has already beenchallengedby the discov ery of a-tubulins with unorthodox C-termini, not only lacking the terminal TYR but most likely unable to be a substrate for the tyrosine ligase, as discussedby Burns 181.
Moreover, the absenceof C-terminal TYR of both a- and
@.tbulin in Paramecium, as in Stylonychiu [31] or Euptolex
[43], negates the premiss of Lai et al’s proposal 141]
according to which, since an aromatic amino acid is evolutionarily maintained at the end of a- or @.&t&r; the-tyrosination/detyrosination cycle must play a fundamental role.
On the contrary, the ciliate situation would suggest~thatthc~
TyriGlu cycle has in itself no universal physiological signif-
a-Tubulin genes in Parumecium
91
Fig 7. Immunodecoration of the neoformed microtubules of the contractile vacuole system: double labeling by the monoclona1 anti&XI IDS and the polyclonal R200. During division, the contractile vacuole microtubule system is duplicated. In each presumptive
daughter cell, the neoformed contractile vacuole system (ncv) develops anterior to the old one (ocv). A, B. The same dividing cell respectively photographed by rhodamine fluorescence (ID5) and FITC (R200). Both ncv are brightly labeled by R200 (B), while only the
old contractile vacuole systems are labeled by ID5 (A). Note also (see text) the strong labeling of the two micronucleus separation
spindles (ss) by R200. ooa, old oral apparatus, retained in the anterior division product; noa, newly formed oa in the posterior division
product. Bar, 10 pm.
icance for microtubule dynamic properties, and at least
demonstrates that it does not participate in the morphogenesis of the highly elaborate microtubule edifices which characterize the ciliates.
Moreover, according to our immunocytochemical
data,
the sequential immunodecoration
of some neoformed
microtubule arrays by the antibodies directed against C-terminal polyglycylation
or polyglutamylation,
concomitant
with the masking of the epitope corresponding to the C-terminal region, confirms previous results showing that these
post-translational
modifications
arise on microtubules
assembled from unmodified or weakly modified isoforms.
This shows that the establishment of the highly specific
functions or geometries of these microtubule arrays are
acquired before the appearence of the post-translational
modifications,
or at least, before an immunologically
detectable level of modification is reached. These results,
together with the demonstration by Gaertig et al [26], in
Tetruhymena of the dispensability of acetylation seem to
show that post-translational
modifications
are neither
responsible for the geometry of microtubule networks nor
necessary for their function.
In conclusion, our analysis shows that the structural and
functional diversity of microtubule networks in Parumecium cannot result from genetic diversity nor, at least for
some networks, from subsequent post-translational modifications. In view of the homogeneity of isotypes, we can rule
out that ciliates use strategies based on isotype selection for
a given function, as occurs in metazoa. The question of
what generates structural and functional diversity in such a
protist is still open and further work will have to investigate
the role of MAPS which are yet poorly known in ciliates or
of the role of MTOCs which are especially diverse in Purumecium and other ciliates.
Acknowledgments
We gratefully acknowledge J Wehland for the generous gift of
the monoclonal lD5. We are grateful to C Rodrigues-Pousada,
I Barahona and H Helftenbein for the generous gift of their piasmids. We thank M Wright and A Adoutte for helpful discussions
and L Sperling, J Cohen and D Kerboeuf for critical reading of
the manuscript. This work was partly supported by grants from
the Fondation pour la Recherche Mtiicale and by a grant (70194)
from the Groupement d’Etudes et de Recherches sur le GCnome
and by the CNRS.
References
1 Adoutte A, Claisse M, Maunoury R, Beisson J (1985) Tubulin evolution: ciliate-specific epitopes are conserved in the
ciliary tubulin of metazoa. J Mel Evol22, 220-229
2 Adoutte A, Delgado P, Fleury A, Levilliers N, Lain@ MC.
Marty MC, Boisvieux-Uhich
E, Sandoz D (1991) Microtubule diversity in ciliated cells: evidence for its genemtion by
post-translational
modification in the axonemes of Paramecium and quail oviduct cells. Rio1 Cell 7 1, 227-245
3 Barahona I, Soares H, Cyrne PD, Denoulet P. Rodrigue+
Pousada C (1988) Sequence of one (x and two P-tubuiin
genes of Tetrahymena pyrifnt-mix J Mel Biol202. 365-382
4 Blase SH, Meltzer DI, Feramisco JR (1984) 10 nm filaments
are induced to coll%pse in living cells microinjected
with
monoclonal and polyclonal antibodies against tubutin. .I Cell
Biol98,847-858
5 Br& MH, De NCchaud B, Wolff A, Fleury A ( 1994) Glutam
ylated tubulin probed in ciliates with the monoclonal antibody GT335. Cell Motil Cytoskel27, 337-349
6 Brunk CF, Sadler LA (1990) Characterization
of the pmmoter region of Tetrahymena genes. Nucleic Acid Res 18,
323-329
7 Bulinski JC, Gundersen GG (1987) A function for tub&n
tyrosination? Nature 328,676
8 Burns R (1987) Tubulin’s terminal tyrosine. Nature 327,
103-104
9 Callen AM, Adoutte A, Andrew JM, Baroin-‘l‘ourancheau
A.
Bre MH, Ruiz PC, Cl&rot JC, Delgado P, Fleury A, Jeailmaire-Wolf R, Viklicky V, Villalobo E, Levilliers N (1994)
Isolation and characterization
of libraries of monoclonal
antibodies directed against various forms of tubulin in Ptirczmecium. Biol Cell 8 1, 95 119
i 0 Caron F, Meyer E (1985) Dots Puramechm yrimuurelia USC
a different genetic code in its macronucleus? Nature 314.
185-188
11 Church GM, Gilbert W (1984) Genomic sequencing. .F-‘r-cii
NatlAc,adSci USA81, 1991-1995
12 Cleveland DW (1989) Use of DNA transfection to analyre
gene regulation and function in animal cells: dissection of the
tubulin multigene family. CeiE M&l Cytoskel 14. 147--l%
13 Cohen J, Adoutte A, Grandchamp S, Houdebine LM, Bctsson J ( 1982) Immunocytochemical
study of microtubulal
structures throughout the cell cycle ol’ PurcLmccium. /Iim/
Cell 44, 35-44
14 Cohen J, Be&on J (1988) ‘I‘ht: cytoskcleton. III: ~‘(IT(IIN<.
cium (G6rtz HD, ed) Springer-Verlag,
Berlin. 363-392
I.5
Cohen J: Garredu
de Loubrcsse N, Beisson J (1984) Actin
microfilaments
in Paramecium:
localization
and role in
intracellular movements. Cell Motil Cytoskel4, 443-468
16 Conzelmann KK, Helftenbein E (1987) Nucleotide sequence
and expression of two Ptubulin genes in Stylonychia Irw
me. J Mel Rio/ 198, 643-653
17 Detrich HW, Parker SK (1993) Divergent neural /I-tuhulm
from the antarctic fish Notothenia coriiceps neglecta: poten
tial sequence contributions to cold adapatation of microttlbule assembly. Cell Motil Cytoskcl24, 156-166
18 Devereux J. Haeberli P, Smithies 0 (1984) A comprehensive set of sequence analysis programs for the VAX. Nl&ii
Acids Res 12, 387-395
19 Dupuis P ( 1992) The fl-tubulin genes of Ptlrw~?eciunt xiiinterrupted by two 27 bp introns. 1%4R0 .I 11. 37 13537 lo
20 Dupuis P ( 1992) Structure. or,q~misatiori er r.Vpres.siorr tL,,<
g?rle.s tlr tuhuline chez la puramPcir. Thttse de Doctorat dc
1’UniversitC Paris XI
11 Eddr B, Rossier J, Le Caer JP, Drsbruyere,
I-;, Groa il.
Denoulet P (1990) Posttranslational
glutamylation
of (Itubulin. St+nce 247, 83-85
73 Eichenlaub-Ritter
U, Tucker JB (1984) Microtubules
with
more thdn 13 protofilaments in the dividing nuclei of ciliates. Nature 307, 60-62
73 Fleury A, Callen AM, Bre MH, lftode I;, Jeanmam-Wolf
K,
tevilliers N, ClCrot JC (1995) Where and when is microtubule diversity generated in Paramecium?
Immunological
24
25
76
27
28
29
30
31
32
i?
34
35
3h
37
.78
39
40
-I1
,12
3i
43
J;5
.$h
propertIes ot microtubular
networks in the interphase ankd
dividing ceHs. Protoplas&a 189, 37-60
Frischauf AM, Lehrach H, Poustka A, Murray N (.1983i
Lambda replacement vectors carrying polylinker sequence...
J Mel Biol 170, 827-842
Fulton C, Simpson PA (1976) Selective synthesis and mti
ization of flageflar tubulin. The multi-tub&n
hypothesis. In
Cell Motility (Goldmann R, Pollard T, Rosetibaum J, edh)
Cold Spring Harbor Laboratory Press, Cold Spring Harbour.
987-1005
Gaertig J, Cruz MA, Bowen J, Gu L, Pennock DG, Gorol
sky MA (1995) Acetylation of tysine 40 in a-tubulin is nc)!
essential in Tetrahymena thermophila.
J Cell Bioi $Zcr
1301-1310
Gaertig J, Thatcher TH, McGrath KE, Callahan KC, @)ru\-ski MA (1993) Perspectives on tubulin isotype function anti
evolution based on the observation that Tetmhyrnena tier
mophila microtubules contain a single u- and ptubulin. C<ii
Motil Cytoskel25, 243-253
Gautier MC, Sperling L, Madeddu L (1996) Clomng and
sequencing of genes coding for Parame&m
secretory gran
ule ftrichocyst) proteins. J Bin1 Chem 27 1, 1-9
Geuens G, Hilt AM, LevilIiers N, Adoutte A, Dcbrabandct
M (1989) Microtubule dynamics investigated by microinjertion of Parumecium axonemal tubulin: lack of nucleation
but proximal assembly of microtubules at the kinetochorc
during prometaphase. J Cdf Biol 108.939-953
Greer K, Rosenbaum JL (1989) Post-translational modifica
tions of tubulin.-ia: Cc12 movement. Volume 2 (Warner FD
McIntosh JR, eds) Alan R Liss inc, New York, 47-66
Helftenbein E (I 985) Nucleotide sequence of a macronu
clear DNA molecule coding for a-tubulin from the ciiiati
Stylonychiu lemnae. Special codon usage: TAA is not x
translation termination codon. Nucleic Acid RPS 13.4 15-1~ 1
Helftenbein E, Miitler E (1988) Both a-tubulin genes arc
transcriptionally
active in Stylonichia Zemnae. Curr Grnn?
I3,425--132
Katz WS, Solomon F ( 1988) Diversity among /&tuhuhn~. :t
carboxy-terminal
domain of yeast Ptubulin is not essential
in viva Mel Cell Biol8. 2730.-2736
Kemphues KJ; Raff EC, Kaufman TC ( 1983) G.enehc anaiy
sis of BZt, the structural gene for a testis specific ptubulin
subunit in Drosophila melanogaster. Genetics 105. 34553.56
Kilmartin JV, Wright B, Milstein C (1982) Rat monoclonal
antitubulin antibodies derived by using a new nonseeretmg
rat cell line. J Cell Biol93.
576-W
Kirk KE. Morris NR (1991 ) The tubs cr-tuhulin gcnc ;*
essential for sexual development in Aspcrgillus
nidulsts
Gcncs f)et, 5, 2014-7021
Kirschner-MW,
Mitchison TJ ( 1986) Beyond sclt”asscnrbi>
from microtubules to morphogenesis. Cell 45, 329-342
Kozminski
KG, Diener DR. Rosenbaum JL (1993) I-ii@
level expression of nonacetylatahle rx-tubulin irr Chlurr?$rmouas reinhardtii. Cell Motil Cytoskel 25, 158-i 70
L’flernauit
S, Rosenbaum JL f 1985) Chlamydomorw.~ trrubutin is post-translationally
modified by acetylation on the
c-amino group of a lysine. Biochemistry 24,473.-47Y
Lai EY, Remillard SP, Fulton C (1988) The e-tubulin gent:
family expressed during cell differentiation
in Nctegicrilz
,~rrthcri. J Cell Biol 106, 2035-2046
Lai EY, Remillard SP, Futton C ( 1994) A ,%tubulm gene tii
Naegleria encodes a carboxy-terminal
tyrosine. J Mel Bioi
235, 377.-388
Levilliers N, Fleury A, Hill AM ( lY95) Monoclonal and poi~‘clonal antibodies detect a new type of post-translationa! OWLSification of axonemal tubulin. J Call Sci 108, 30 1J-3028
Liang A, Schmidt HJ, Heckmann K (1994) The Q- amf &
tubulin genes of I:‘uplotes oc,tocclriPlcrtus. J Euk Micxc&o1
II. 163-169
Little M (1985) An evaluahon <)t’ tubuiirr as .t molecuiar
clock. Diosystems 18, 24 l-247
LiltJe M. Seehaus
T (1988) C’omparallve analysis oi’ twhuiirt
sequences. Camp Biochem Physiot 903. 655%670
Lopata MA, Cleveland DW (i987) In vi110 microtuhuiu, ajar:
a-Tubulin
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
genes in Paramecium
copolymers of available Ptubulin isotypes: localization of
each of six vertebrate Ptubulin
isotypes using polyclonal
antibodies elicited by synthetic peptide antigens. J Cell Biol
105, 1707-1720
Mac Tavish C, Sommerville J (1980) Macronuclear
DNA
organization and transcription in Paramecium primaurelia.
Chromosoma 78, 147-164
MacRae TH, Langdon CM (1989) Tubulin synthesis, structure, and function: what are the relationships? Biochem Cell
Biol67,770-790
Madeddu L, Klotz C, Le Caer JP, Beisson J (1996) Characterization of centrin genes in Paramecium. Eur J Biochem
238,121-128
Matthews KA, Rees D, Kaufman TC (1993) A functionally
specialized
a*-tubulin is required for oocyte meiosis and
cleavage mitoses in Drosophila. Development 117,977-991
Micelli C, Ballarini P, Di Giuseppe G, Valbonesi A, Luporini P (1994) Identification
of the tubulin gene family and
sequence determination of one Ptubulin gene in a cold poikilotherm protozoan, the antartic ciliate Euplotes focardii.
J Euk Microbial 4 1,42@-427
Neff NF, Thomas JH, Grisali P, Botstein D (1983) Isolation
of the Ptubulin
gene from yeast and demonstration of its
essential function in vivo. Cell 33, 21 l-219
O’Farrell PH (1975) High resolution two dimensional electrophoresis of proteins. J Biol Chem 250,4007-4020
Panda D, Miller HP, Banerjee A, Luduena RF, Wilson L
(1994) Microtubule
dynamics in vitro are regulated by the
tubulin isotype composition. Proc Nat1 Acad Sci USA 91,
11358-11362
Paturle-Lafanechere
L, EddC B, Denoulet P, Van Dorsselaer
A, Mazarguil H, Le Caer JP, Wehland J, Job D (1991) Characterization of a major brain tubulin variant which cannot be
tyrosinated. Biochemistry 30, 10523-10528
Piperno G, Fuller MT (1985) Monoclonal
antibodies specific for an acetylated form of a-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell
Biol 101, 208552094
Preer JR, Preer LB, Rudman BM, Barnett AJ (1985) Deviation from the universal code shown by the gene for surface
protein 51A in Paramecium. Nature 314, 188-190
Prescott DM (1994) The DNA of ciliated protozoa Microbiol Rev 58,233-267
Preston SF, Deanin GG, Hanson RK, Gordon MW (1979)
The phylogenetic
distribution
of tubulin: tyrosine ligase.
J Mol Evol 13, 233-244
Raybin D, Flavin M (1977) Enzyme which specifically adds
tyrosine to the a-chain
of tubulin.
Biochemistry
16,
2189-2194
Redeker V, Levilliers N, Schmitter JM, Le Caer JP, Rossier
J, Adoutte A, Bre MH (1994) Polyglycylation of tubulin: a
post-translational
modification
in axonemal microtubules.
Science 266, 1688-1691
93
62 Ruiz F, Garreau de Loubresse N, Beisson J (1987) A mutation affecting basal body duplication and cell shape in Paramecium. J Cell Biol 104,417-430
63 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. CSHL Press, New York, Cold
Spring Harbor
64 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing
with chain-terminating
inhibitors. Proc Nat1 Acad Sci USA
74,5463-5467
65 Savage C, Hamelin M, Culotti JG, Coulson A, Albertson
DG, Chalfie M (1989) met-7 is a tubulin gene required for
the production of 15protoIilament
microtubules in Caenorhabditis elegans. Genes Dev 3,870-88 1
66 Schatz PJ, Solomon F, Georges GE, Botstein D (1987)
Insertions of 17 amino acids into a region of a-tubulin do
not disrupt function in vivo. Mol Cell Bio17, 3799-3805
67 Schliwa M, Van Blerkom J (1981) Structural interaction of
cytoskeletal components. J Cell Biol90, 222-235
68 Silflow CD (1991) Why do tubulin genes family lack diversity in flagellate/ciliate protists? Protoplasma 164,9-l 1
69 Soares H, Cyrne L, Barahona I, Rodrigues-Pousada
C
(199 1) Different patterns of expression of beta-tubulin genes
in Tetrahymena pyriformis during reciliation. Eur J Biochem
197,291-299
70 Sonnebom TM (1974) Paramecium aurelia. In: Handbook
of Genetics (King R, ed) Plenum Publishing
Carp, New
York, 469-594
71 Torres A, Delgado P (1989) Effects of cold and nocodazole
treatements on the microtubular systems of Paramecium in
interphase. J Protozool36,
113-l 19
72 Trinczeck B, Marx A, Mandelkow EM, Murphy DB, Mandelkow E (1993) Dynamics of microtubules from erythrocyte marginal bands. Mol Biol Cell 4,323-335
73 Vallee RB, Collins CA (1986) Purification of microtubules
and microtubule-associated
proteins from sea urchin eggs
and cultured mammalian cells using taxol, and use of exogenous taxol-stabilized brain microtubules for purifying microtubule-associated proteins. Methods Enzymol 134, 116-127
74 Vantard M, Levilliers N, Hill AM, Adoutte A, Lambert AM
(1990) Incorporation of Paramecium axonemal tubulin into
higher plant cells reveals functional sites of microtubules
assembly. Proc Nat1 Acad Sci USA 87, 8825-8829
75 Wehland J, Schroder HC, Weber K (1984) Amino acid
sequence requirements in the epitope recognized by the atubulin-specific rat monoclonal antibody YL 112. EMBO J 3,
1295-1300
76 Wehland J, Weber K (1987) Turnover of the carboxy-terminal tyrosine of a-tubulin and means of reaching elevated
levels of detyrosination
in living cells. J Cell Sci 88,
185-203
77 Youngblom J, Schloss JA, Silflow CD (1984) The two p
tubulin genes of Chamydomonas reinhardtii code for identical proteins. Mol Cell Biol4, 2686-2696