Molecular basis for the deficiency in humans of gulonolactone
Transcription
Molecular basis for the deficiency in humans of gulonolactone
Molecular basis for the deficiency in humans of gulonolactone oxidase, a key enzyme for ascorbic acid biosynthesis13 Morimitsu Nishikimi ABSTRACT bic and The acid is known inability an enzyme that vitamin. Isolation allowed us to study ofa level to L-gulono-y-lactone covering lactone oxidase human the analysis oxidase ofmutations as a pseudogene gene on the genetic Enzymatic L-gulono-y- indicated has accumulated being active genome. zyme deficiency, nucleotide oxidase, sequence, aspects Before a and that Am J we will ascorbic acid, en- The dealing ent pseudogene in the as its essentiality as a nutrient number of exceptional species animals. In other vitamin C, or L-ascorbic demonstrated that as shown (from uronic acid and two steps tion. The oxidase the pathway acid. latter and thus This need trait has been most prevalent carried genetic prone carry We have deficiencies fication carried been enzyme (8). is missing intake arose through disorder, generations. because of the gluc- synthesis point from branches consists and of oxida- by L-gulono-’y-lactone of vitamin during guinea pigs L-ascorbic C to prevent the genetic scurvy-prone and this In a sense, all individuals this is the of scurvy- characterization of rat and studies 12035-85. defect animals. Printed underlying Starting goat liver on the enzyme in USA. with GLO GLO puri(3), from gene level biological studies in humans. we deficiencies © 1991 American basis of the GLO enzymological deficiency, aspects in vertebrates The resides from in the liver animals: and reptiles is located enzyme and in the kidney Asano from rat liver revealed the by which 000 is present to the N(l) position ofa histidyl group at the C(8) methyl (9, 1 1). During the electrons from the substrate reduced flavin is oxidized 2-oxo-L-gulono-y-lactone the products; the former L-ascorbic above. liver (3) the method the enzyme was successfully of by use of appropriate The enzyme weight residue by GLO, the di- being apoprotein of the linked through isoalloxazine flavin L-gulono-’y-lactone, by molecular adenine enzyme, ofthe position as de- gel electro- of flavin of the de- preparations molecular molecule molecule catalysis of birds goat sulfate-polyacrylamide (3, 9). One per in both the level of species rat and purified properties. dodecyl of improving microsomes nucleotide the by liver of primitive mentioned from of the following by sodium is -5O (9) (10), Characterization phoresis in certain a and/ it is pres- in the kidney mammals to homogeneity chicken and is missing scurvy-prone GLO of GLO. is interesting enzyme of amphibians tergents. to form evolution, genetic some enzyme purified termined trait. studying 199l;54: D-glucose in the above-mentioned deficiency out immunochemical Am J C/in Nutr is catalyzed molecular deficiency (7). The as in the solubilized it was starting lactonization a dietary this in these and step is part acid possible at the oft-ascorbic-acid-synthesizing The Nakagawa synthesize from the branch viz, and of the metabolic pathways defect in the liver of highly evolved species, and and liver of certain species at the intermediate We a limited higher l950s, eg, humans, other primates, and these animals cannot synthesize enzyme species acid) after reactions, oxidation This is synthesized metabolic part can In the first portion of L-ascorbic The of enzymatic (GLO). scurvy. of the animals body. to L-gulonic one exceptional species, (2); as a consequence, acid acid D-glucose L-gulonic all higher in their 1 (1). The cycle, D-glucose; nearly L-ascorbic inasmuch is restricted to only among phylogenetically acid, in Figure pathway from words, discovered the of GLO kidney (8) as well the vitamins our GLO discuss standpoint evolution among with distribution mammals. Introduction C is unique genetic isolation it became of GLO first briefly species, kidney Vitamin of the accomplished that phylogenetic Gulonolactone of the we summarize basis We (5); as a result, clone 199 l;54:l203S-8S. WORDS (4). GLO nature article, or the kidney KEY monkey overlapping rat study since it stopped in the human the (6). In this deficiency genomic of the pig and for rat-liver to investigate oxidase as well as three guinea of a cDNA of this this human region Sequence L-gulono-’y-lactone Nuir underlying ofa coding eDNA. large number it now exists C/in defect oxidase entire biosynthesis for rat L-gulono-”y-lactone basic in the and oxygen. ring accepts two the resulting In this process, and hydrogen peroxide are formed product isomerizes nonenzymatically as acid. I From the Institute Mitake, Gifu, Japan. 2 Supported in part of Applied Biochemistry, by a Grant-in-Aid (01580156) Yagi Memorial Park, for Scientific Re- search from the Ministry of Education, Science, and Culture of Japan. 3 Address reprint requests to M Nishikimi, Institute of Applied Biochemistry, Yagi Memorial Park, Mitake, Gifu 505-01, Japan. Society for Clinical Nutrition 12035 Downloaded from ajcn.nutrition.org by guest on June 29, 2015 related L-ascor- of L-gulono--y-lactone for the led to isolation clones the is required the to synthesize to a lack cDNA and Yagi ofhumans to be due oxidase, at the gene Kunio 12045 NISHIKIMI AND YAGI COOH H-C-OH H-C-OH H-C-OH H-C-OH CH2OH FIG Cloning of a cDNA To elucidate GLO antibody used vector, amino amino the acid acid of rat GLO culated from the of the expression 1 cells with SV4O late the (M Nishikimi The that GLO, enzyme tide protein sequence ofthe of 1320 ATG co-ion protein. The GLO The adenine ular weight tail nor the genetic value region, the a polyadenylation Yet, it was defect utilized in GLO fixation antibody and latter signal, region the cDNA as a versatile deficiency, contains tool as will isolation RNA any preparation was detectable the conditions signal not Yet, humans region a have expressed and in the next question GLO occurs clone is not the use ofthe in our study of genomic of gene protein is not it is very from out Northern related RNA from liver poly(A) guinea no guinea RNA pigs mRNA human to that do not in their liver contains mRNA, in view gene that was discovered to a great extent (see products of the GLO indicated animals, to be addressed rat GLO if it or not scurvyin their liver showed that whether in the aberrant we have had no opportunity to of human liver poly(A) RNA. scurvy-prone DNAs present sequence analysis same material whether mRNA DNA (13) the of detection. us to carry blot levels in the genomes using limit no GLO-specific mRNA Udenfriend ofGLO-specific that the GLO has deteriorated measurement protein of that or that any yet determined should con- 1/600 < by the it is clear amount microsomes and with poly(A) where rat (6). Thus, detectable the observation human genome a 5’- be discussed and that monkey ofcross-reacting enabled Northern green or below GLO rat cDNA genome. We have The neither was animals ofthe anform cross-reactive microcomplement Sato the amount that and technique GLO blot analyses to examine possess GLO-specific a strong liver. to that a 3’-noncoding that Nishikimi pig liver for guinea scurvy-prone an aberrant African protein Later, GLO-specific mRNA, because test an undegraded preparation flavin of the guinea microsomes. indicated in their gave that microsomes and investigation the Ouchterlony and reactive showed studies possess a molec- contains pig indicated liver positive signal pig liver under sulfate-polyacryl- the cDNA and reading weight primates deficiency, a radioimmunoassay GLO bound this by using guinea in rat liver established poly(A) mi- of immunologically present acid in In their the question ofwhether pigs and monkeys, have found test also no nucleofirst other contains no detectable protein immunologically with antirat GLO rabbit antiserum. Their the of 22 nucleotides Because of the The to form is comparable dodecyl They liver and Southern prone animals (3). to the coding of full length. this of GLO. the like deficiency introduction. basis (4) raised as guinea liver of the scurvy-prone is present at all. (12). Thus acids of the molecular molecular These the consensus to have the Udenfriend imals, such to GLO in GLO in the of the the is removed lacking into as that an open are as mentioned L-Ascorbic approach pSVL amino acid by sodium a covalently Humans in human Moreover, surrounding presumed sodium the 1991). The a presumed is thus by poly-A below. (FAD) In fact, nucleotides. control vector to contain with by of COS- analysis. well with with enzyme determined of 776 au- described by Kozak encodes 440 amino polypeptide region cal- was the same methionine gel electrophoresis noncoding the blot sequence agreed of 5 1 267. In addition on The mature dinucleotide of rat GLO amide was found amino-terminal of 50 483. that The CH2OH in animals. genetic pigs, tam (5). observations, synthesized (PCCATGG) that the clone the mature under of corn- with gel electrophoresis. cDNA that acid presumed from the deduced that of rat GLO determined (ATCATGG) tiation sequence we concluded with amino expression by Western nucleotides. 33- confirmed unpublished as demonstrated The amino by transfection placed sulfate-polyacrylamide frame identical the further eukaryotic K Yagi, the molecular weight sequence agreed with dodecyl was the (5). its deduced sequence protein was in the and size ofthe ofrat cDNA GLO cDNA promoter acid in an encoding agreement amino cloned library amino-terminal was and a to us, isolated and in reasonable of functional was 2. The enzyme deduced acid biosynthesis GLO available a cDNA cDNA sequence, was the to obtain cDNA GLO in Figure of the amino position thenticity cloned shown sequence deduced of rat HO-C-H L-Gulono--y’iactone acid of L-ascorbic animal. was a rat-liver 1 1 . As a result, ofthis are underlying it is pivotal rat GLO to screen sequence sequence sequence pathway L-ascorbic-acid-synthesizing against Agt acid nucleotide acid ofan as a probe expression entire mechanism directed HO-C-H CH2OH Molecular humans animals, H-C that as discussed is whether ofthe cDNA various the animals gave answer to this question (6). In the hybridization only DNAs from L-ascorbic-acid-synthesizing The related blot an is not above. animals. Southern at both gene gene scurvy-prone as a probe, gene the of in the below). to With analysis unequivocal analysis, not animals (mouse, Downloaded from ajcn.nutrition.org by guest on June 29, 2015 encoding it was molecular in scurvy-prone Because 1 . The metabolic for rat GLO the deficiencies L-Gulonic 0 HO-C H-C CH2OH Acid II 0 HO-C-H HO-C-H D-Glucuronic D-Glucose I H-C-OH COOH HO-C HO-C-H HO-C-H HO-C-H H-C-OH cDNA HO-C-H H-C-OH HO-C-H CO GULONOLACTONE OXIDASE DEFICIENCY IN 12055 MAN -22 GATCCTCCTGATCACTGGAATC 1 ATGGTCCATGGGTACAAAGGGGTCCAGTTCCAAAATTGGGCAAAGACCTATGGTTGCAGTCCAGAGGTGTACTACCAGCCCACCTCCGTG M 91 V H C Y K C V Q F Q N W A K T Y G C S P E V I Y Q P T S V 30 K V V G C G H S P S D I A 60 V D K E K K Q I T V E A C 90 S N L G A V S D V T V A G 120 T Q V V A L T L M T A D G 150 R V H L G C L G I I L T V 180 T L K E V L 0 N L D S H L 210 S I I Y Q D H T N K A P S 240 F L L W T S T Y L P C L V 270 S S N L S H K I F T Y E C 300 E A L L E L K A M L E A H 330 D I L L S P C F Q R 0 5 C 360 0 Y W L A Y E T I N K K F 390 E E M Y P T F H K F C D I 420 V F Y GAGGAGGTCAGAGAGGTGCTGGCCCTGGCCCGGGAGCAGAAGAAGAAAGTGAAGGTGGTGGGTGGTGGCCACTCGCCTTCAGACATTGCC E 181 E V R E V L A L A R E Q K K K V TGCACTGACGGTTTCATGATCCACATGGGCAAGATGAACCGGGTTCTCCAGGTGGACAAGGAGAAGAAGCAGATAACAGTGGAAGCCGGT C 27 1 T D G F M I H M C K M N R V L Q ATCCTCCTGGCTGACCTGCACCCACAGCTGGATGAGCATGGCCTGGCCATGTCCAATCTGGGAGCAGTGTCTGATGTGACAGTTGCTGGT I 3 61 L L A D L H P Q L 0 5 H G L A M GTCATTGGATCCGGAACACATAACACAGGGATCAAGCACGGCATCCTGGCCACTCAGGTGGTGGCCCTGACCCTGATGACAGCTGATGGA V 45 1 I G S G T H N T G I K H G I L A GAAGTTCTGGAATGTTCTGAGTCAAGAkaTGCAGATGTGTTCCAGGCTGCACGGGTGCACCTGGGTTGCCTGGGCATCATCCTCACCGTC E 54 1 V L E C S E S R N A D V F Q A A ACCCTGCAGTGTGTGCCTCAGTTTCACCTTCAGGAGACATCCTTCCCTTCGACCCTCAAAGAGGTCCTTGACAACCTAGACAGCCACCTG T 63 1 L Q C V P Q F H L Q E T S F P S AAGAGGTCTGACTACTTCCGCTTCCTCTGGTTTCCTCACACTGAGAACGTCAGCATCATCTACCAAGACCACACCJJCAAGGCCCCCTCC K 721 R S E Y F R F L W F P H T E N V TCTGCATCTAACTGGTTTTGGGACTATGCCATCGGGTTCTACCTACTGGAGTTCTTGCTCTGGACCAGCACCTACCTGCCATGCCTCGTG 8 11 A S N N F N 0 Y A I G F Y L L E Downloaded from ajcn.nutrition.org by guest on June 29, 2015 S GGCTGGATCAACCGCTTCTTCTTCTGGATGCTGTTCAACTGCAAGAAGGAGAGCAGCAJCCTCAGTCACAAGATCTTCACCTACGAGTGT G 90 1 W I N R F F F N M L F N C K K E CGCTTCAAGCAGCATGTACAAGACTGGGCCATCCCTAGGGAGAAGACCAAGGAGGCCCTATGGAGCTAAAGGCCATGCTGGAGGCCCACC R 99 1 F K Q H V Q 0 W A I P R E K T K CCCAAAGTGGTAGCCCACTACCCCGTAGAGGTGCGCTTCACCCGAGGCGATGACATTCTGCTGAGCCCCTGCTTCCAGAGGGACAGCTGC P 10 81 K V V A H Y P V E V R F T R C D TACATGAACATCATTATGTACAGGCCCTATGGAAAGGACGTGCCTCGCCTAGACTACTGGCTGGCCTATGAGACCATCATGGAAGTTT Y 1 171 N N I I M Y R P Y G K D V P R L GGAGGAAGACCCCACTGGGCAAAGGCCCACAATTGCACCCAGAAGGACTTTGAGGAAATGTACCCCACCTTTCACpJGTTCTGTGACATC G 12 6 1 G R P H W A K A H N C T Q K 0 F CGTGAGAAGCTGGACCCCACTGGAATGTTCTTGAATTCGTACCTGGAGAAAGTCTTCTACTACCAGGAGTGGAACAAACCACCCTGA R E K L D P T C M F L N S Y L E K 1351 CCCCTCACACTTCTGCTGCCCCCGGGGGTCTGGGGAGCAGAGAAGTGCCTCACAAGCACAATGGGAACTGACCTCTCCTCCTGACCACAA 1441 AGAAAGGCTGGGCTCTGGGCGGGTCCTCTCTGCCTTCGGCATCATTTCCCTTACATCCAGGCGAAGAAGTGGCCTCTCACTCAAATTCCT 1531 GTTAGCATTTCCATGGGTCACACATA)ACTGCAATCCTCTCAGGAGAAGGGGATCCCTGATACATCATATCTATCCAGACTAAGGATGT 1621 GGTTCTTCCTAGATTCTATGGCTCCACCAGGTATAGAGAGATTCCTGGGGCCTGCAGTTCTCCATCCCTCTTCAGGGGAGGGATCCCT 1711 TGGCGAGAGTTTGGCTCAGAGGTGGCATGAAGCATGCTCTGCTCTCTCTTACCCTTGAAGGTCCTTCGGATGCCCAGAGATGTCTGCTGG 1801 TCCTGGGCAAGCCATCATTCAAACGTCCAJCCTGGCCTTCTGTCTGCCATGGCCTGACCCTCGCAGTGTCTCTTCCAGATGTTTAG 1891 AGTGGAACTCGCTTCAACCTCTTAACCAGTTGCTGATCCCTGTGTTTCTCTCCCTTCTCCTTAGACTACTCTTGGAGGGGGATCCCAC 1981 CATCTCCTTGGCTTTCCCTGGGTATTGTTCTCCTCTTCCTCTTCACAAATATGATTTCAGTTTGATTTGTGGCCTTTCTGGAGTGTTCCT 2071 TGGACAACCAAGATGTTCCAGCTAC FIG 2. The nucleotide sequence ofa cDNA encoding rat liver GLO and its deduced amino acid sequence. Revisions were made from our previous paper (5) as follows: a change from G to C at nucleotide position 567 accompanying a change from glutamine to histidine at amino acid residue I 89, and deletion of the G at nucleotide position I and the Cs at positions 1460 and 2097 of the previous sequence. The amino-terminal amino acid sequence determined by the sequence analysis of purified rat liven GLO is underlined. Adapted from Koshizaka et al (5). dog, pigs cow, gave and chicken) but also positive signals (Fig both humans and related to rat GLO. of Figure were 3 that far weaker synthesizing indicates than using were those studied a 5’-terminal do coding have for DNAs and the human extent The structures pigs pigs GLO a nucleotide sequence However, it should be noted from inspection the intensities of the signals for human DNA mammals that to a great guinea DNAs from humans and guinea 3). Accordingly, it is clear that even GLO gene since the ofthe GLO-related in some fragment gene from from sequence stopped detail the L-ascorbic-acid- chickens. genes and encoding the amino-terminal (nucleotides 947-1293) the amino The results contain acid sequence close demonstrated coding carboxyl-tei-minal that sequences parts amino acid sequence of the same cDNA enthe to the carboxyl human corresponding of GLO, and terminus and guinea to both hence pig amino- probably the finding entire has become altered To more quantitatively analyze the alteration of the human GLO gene, we attempted to isolate both the rat and human GLO genes. Screening of partial EcoRI- and HaeIII-cut rat ge- genes ofhumans (nucleotides (6). cDNA a fragment This functioning. by Southern and blot -22-367) and guinea analysis of the by rat coding sequence. nomic DNA libraries in Charon overlapping clones covering the 4A entire led to coding isolation region of three of the rat 12065 NISHIKIMI AND 0) YAGI carboxyl-terminal tyrosine. exon-related C C) C C) . .2 D) 0 0 . C) E 0 0 0 .E I The of the in Figure relatively - tified analysis c ization probe, even at lower weak hybridization resulting (! by Southern in the human of the four single 23.0 sequences. and two out human and When mology S was 4.4 insertion. It was junctions sequenced did not conform splice-donor site sequences of the words, the GLO found in the rule: the of the introns were GC human four exon-related were compared the overall ho- At the amino and, accumulated pressure gene what a large once now acid level, is more, number it ceased exists many of mutations to be active; as a pseudogene in other in the human exon junction coding region sequences was found The determined identical with three followed the to be divided nucleotide the sequence differences. Two and C to T at position codons for glutamine probably are due Screening may during homologous to several and unpublished K Yagi, encompasses that the encode to A at position polymorphism. 12 1 1), which ofa (G regions except 252 alters genomic from other the codon also be a polymorphism the cloning procedures. clone regions The that exons DNA ofthe observations, corresponding phenylalanine rat GLO 1991). gene (M Nishikimi Perhaps, this clone to the six rat GLO at position 202 gene to the and of amino site and (Ks) per acid sub- nonsynon- two genes. substitutions The obtained, ofthe K value estimated between The value of K calculated to this to the K value one, GLO neutral indicating genes been nonsynonymous by the selective restricted that occurred we can make theory ofevolution. to suppose that the substitution is far less in the rat GLO gene have the following rate It may at nonsynon- than substitutions pressure the at the in the human in the rat gene during evolution whereas the nonsynonymous have occurred as frequently substitutions in the human gene as the synonymous substitutions since active. this to the re- and of Miyata number ofthe (18). is comparable on the basis gene, because the sequences 4 by Nei method site (Ks) between for superimposed in the GLO or may in EMBL3 (silent) substitutions as in other genes. be reasonable ymous sites for glutamine nucleotide genes gene substitutions be an of Figure of the the synonymous genes is similar and an average human and rat genes is 0.823 GLO and genes. was nonsynonymous ancestor site million at neutral sites lineage years The gene the we can date ago, assuming estimated number occurred as the between as 2.3 X iO (19). the that obtained On this assumption, at ‘-70 time of mammalian at the synonymous Therefore, site the common of 0. 16, which per the primate being nonsynonymous gene value of GLO tutions stopped per human stitutions library contained difference data (15) K, respectively) ( 1 7) were 0.86 and 0. 1 6, respectively. synonymous substitutions are not subjected to selective during evolution, their number calculated for various and Because pressure (IC discussion (14). The by I 1 in- sequences ofthe exon of the rat GLO cDNA differences of a human in isolation GT/AG rule into 12 exons sequence version (amino-acid-altering) of K and K corrected In regard unpublished observathat all of the intron- 432) occur in the third position of the and threonine, respectively, and most to allelic (A to G at position for arginine, that arose K Yagi, showed genes ( 16) for calculating for the GLO (M Nishikimi and 199 1). Sequence analysis the per synonymous same rate cDNA of GLO modified stitutions FIG 3. Southern blot analysis ofgenomic DNAs from various animals. Genomic DNAs from the indicated animals were digested with EcoRI and analyzed by Southern blot hybridization. The rat GLO eDNA fragment covering the 5-noncoding region and most of the coding region (nucleotides -22- 1293) was used as a probe. Size markers are shown on the left. Adapted from Nishikimi et al (6). exons gene analyzed ymous values suIted also GT/AG in number, four rat exons, (“-‘70%); (a for one to the the species. yet lower evolution Yasunaga to that artifact between GLO Molecular 2.0 for deletions were not subtle and two stop codons were present sequences. These results led us to conclude that no selective We trons. were sequences three eight was under Gojobori’s tions, human were genome. S GLO as a hybrid- This may be due to too number of substitutions single-nucleotide intron-exon iden- substitution date radiation. By letting the sites be equal to that at the of between could be close number of sub- rat and human the primates’ the rate . site’ loss of substi. y corresponds substitution nonsynonymous for to the rate Downloaded from ajcn.nutrition.org by guest on June 29, 2015 the human rat cDNA cor- not nucleotides ‘-80% substitutions in the human 6.6 were three the the homology determined two regions determined regions, a total of 548 nucleotides with those of the corresponding 9.4 the and at the GG. using for two deletions gene dinucleotides other stringency. from a large of four were rat exons there ofthe GLO 4. The regions, one sequences ones short In the exon-related nucleotide deletion) that blot nucleotide six expected and the result is shown responding to the two c C) Cl) regions GULONOLACTONE OXIDASE DEFICIENCY IN MAN 12075 202 Human F LH S LD ? HWXKSED tCCtgCtgaggtCagTTCCTCCATAGCCTGGACAG-CATTGGAAGTCTGAGGACTTCTGCTTCCTCTGGTTCCCACA *** ** Rat t CCCt Ct *** **** * * *** ***** ** FC ***** ****** #{163}LWFPB ***** ************* ** ** tgtaCCagGTCCTTGACAACCTAGACAGCCACCTGAAGAGGTCTGAGTACTTCCGCTTCCTCTGGTTTCCTCA VLDNLDSHLKR$EYRFISWPPH E S t S V A I Q H 0. 5 t S CAGTGAGAACGTCAGTGCCATCCACCAGGACCACACCAGCAGgCagCtgC ** ************ **** **** ********** ***** CACTGAGAACGTCAGCATCATCTACCAAGACCACACCAACAAGgt T Et4V$ I Z YQP * **** aaCtgC HNK 264 I F I Q C I V G W I l G CCCCtgCagCATCTTCTTGCAGTGCCTTGTGGGCTGGATCAATGGCTTTTT’rTTCTGACTCCTGTTTGCCTGC aCCCtgt ** **** tgt t CCt ** ****** ** ***** ************** **** ** * ***** r L L * A C ***** **** YLpCLVGwiNRrrwMLrNc QEN SNL$ P KX FTHVCH ‘KQRVQHWA I -CAGGAGAACAGCAACCTCAGCCCCAAGATCTTCACCCACGTGTGCCACTTCAAGCAGCATGTCCAGCACTGGGCCAT - - *** FrE’ CaCCggCagCACCTACCTGCCATGCCTCGTGGGCTGGATCAACCGCTTCTTCTTCTGGATGCTGTTCMCTGC T 7 * F ****** ************ * ************* *** *** * *************** ** ********** K RE S SNLS H I( I FT Y Downloaded from ajcn.nutrition.org by guest on June 29, 2015 AAGAAGGAGAGCAGCAACCTCAGTCACAAGATCTTCACCTACGAGTGTCGCTTCAAGCAGCATGTACAAGACTGGGCCAT ECRFKQRVQDWAI 314 P 1k KXTTEALLE CCCCAGgggCtCt *** L1A tgggCtgag-tgCagAAAGAAGACCACGGAGGCCCTGCTGGAGCTGAAGGC *** ********* CCCTAGgtaggag **** ********* ********* ******** ***** tgggCtgaCgggCagGGAGAAGACCAAGGAGGCCCTACTGGAGCTAAAGGC PR EXTKEALYE VLARP E VV S MY LKA LVGVfl ? EDD I LJS CGTGCTGGAGGCCCACCCTGAGGTGGTGTCCCACTACCTGGTGGGGGTACGCTTCACCTG-GAGGATGACATCCTACTGA * **************** * ***** ********* ** * *** ********* * * ******** ** **** CATGCTGGAGGCCCACCCCAAAGTGGTAGCCCACTACCCCGTAGAGGTGCGCTTCACCCGAGGCGATGACATTCTGCTGA MLEAHP KVVAHYPVEVR$’TRGDDI PCrwDs Ry LLS I LN N LY GCCCCTGCTTCCAGTGGGACAGCCGCTACCTGCATCAACCTGTACAGgtgaCag ************** ******** ***** ********* ********* * GCCCCTGCTTCCAGAGGGACAGCTGCTACATGAACATCATTATGTACAGgt PCFQRDSC ** aaaag YMN1 I MY 399 V tggt ** tgCtt P CT H S KNFE KMHP AF P KC * ********** Al Ct ******* ***** * * ** V *** * ** **** *** tgattgCttCCaCagGCCCACATTGCACCCGGAGGACTTTGAGGATGTACCCCACCTTTCACGTTCTGTGACAT AHNCTQKD B * It L E P T FE G M F L N L Y EMYPT t E FM It V F Y KFCD I * CTGAGAAGCTAGAACCCACTGGGATGTTCCTATTTGTATTTGGAGAGGTGTTCTACTGGTCAGA * * RE ** ***** K ** IDP ******** ****** TGMF * LN **** *** S ******* YLEKVFY ** ******* *** ** * FIG 4. Comparison of four exons of the rat GLO gene with the human nucleotide sequences related to them. Exon sequences are shown by uppercase letters, and intron sequences by lowercase letters. Identical residues in the rat and human sequences are indicated by asterisks. The deduced amino acid sequences for humans and rats are shown above and below the respective sequences; identical amino acids between the two species are shaded. Numbers above the human amino acid sequences indicate the residue numbers of rat GLO shown in Figure 2. Question marks in the human amino acid sequences represent the positions where there is a deletion of nucleotide(s) in the human sequence: and asterisks indicate stop codons. In the genomic sequence of the rat GLO gene, the A at nucleotide position 12 1 1 ofthe eDNA shown in Figure 2 is G (arrowhead). l208S NISHIKIMI sites in the human onymous substitution tution ‘ with gene, in the . y ‘. calculated average times site . rates consistent the GLO substitution the than calculate the and rat presumption loss of the L-ascorbic-acid-synthesizing thought keys to have and monkeys Old World ofboth L-ascorbic able, been though acid before monkeys lineages (20). they Thus, are (35-45 very rough years to be unable calculations and (19) based are that is 4-10 date of the in primates, ofNew million the above genes in rodents ability are reported substi- GLO As to the the divergence of syn- 10 X l0 Tanimura rate primates. ‘ synonymous by Li and in higher rate to be human substitution that the gene Interestingly, for the synonymous higher we can rat GLO it is World ago) monbecause to synthesize seem on AND reason- a number of a assumptions. We thank the Japanese Cancer Resources Bank for donating an EMBL3 human genomic DNA library that was constructed by Yoshiyuki Sakaki. References Biol Chem l988;263:l6l9-2l. 6. Nishikimi M, Koshizaka T, Ozawa and guinea pigs ofthe gene related ‘y-lactone oxidase. Arch Biochem T, Yagi K. Occurrence in humans to their missing enzyme L-gulonoBiophys 1988;267:842-6. 7. Chatteijee lB. Evolution and the biosynthesis l973;182: 127 1-2. 8. Chaudhuri CR, Chatterjee lB. i-Ascorbic phylogenetic trend. Science 1969;l64:435-6. ofascorbic acid acid. Science synthesis in birds: 9. Kiuchi K, Nishikimi M, Yagi K. Purification and characterization of i-gulonolactone oxidase from chicken kidney microsomes. Biochemistry l982;2 1:5076-82. 10. Nakagawa H, Asano A. Ascorbate synthesizing system in rat liver microsomes I. Gulonolactone-reducible pigment as a prosthetic group ofgulonolactone oxidase. J Biochem (Tokyo) 1970;68:737-46. 1 1. Kenney WC, Edmondson DE, Singer TP, Nakagawa H, Asano A, Sato R. Identification ofthe covalently bound flavin of i-gulono--ylactone oxidase. Biochem Biophys Res Commun l976;7l:1 194-200. 12. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNA. Nucleic Acids Res l984;l2:857-72. 13. Sato P, Udenfriend S. Scurvy-prone animals, including man, monkey, and guinea pig, do not express the gene for gulonolactone oxidase. Arch Biochem Biophys l978;l87:l58-62. 14. Mount SM. A catalogue ofsplicejunction sequences. Nucleic Acids Res 1982; 10:459-72. 15. Nei G, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 1986;3:418-26. 16. Miyata T, Yasunaga T. Molecular evolution of mRNA: a method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application. J Mol Evol l980;16:23-6. 17. iukes T, Canton CR. Evolution of protein molecules. In: Munro HN, ed. Mammalian protein metabolism III. New York: Academic Press, 1969:21-123. 18. Hayashida H, Miyata T. Unusual evolutionary conservation and frequent DNA segment exchange in class I gene of the major histocompatibility complex. Proc Natl Acad Sci USA 1983:80:2671-5. 19. Li W-H, Tanimura M. The molecular clock runs more slowly in man than in apes and monkeys. Nature l987;326:93-6. 20. Stone I. Studies ofa mammalian enzyme system for producing evolutionary evidence on man. Am J Phys Anthropol l965;23:83-6. Downloaded from ajcn.nutrition.org by guest on June 29, 2015 1 . Burns ii. Biosynthesis ofL-ascorbic acid: basic defect in scurvy. Am I Med l959;26:740-8. 2. Burns ii. Missing step in man, monkey and guinea pig required for the biosynthesis of L-asconbic acid. Nature 1957; 180:553. 3. Nishikimi M, Tolbert B, Udenfriend S. Purification and characterization of i-gulono-’y-lactone oxidase from rat and goat liven. Arch Biochem Biophys 1976;l75:427-35. 4. Nishikimi M, Udenfriend S. Immunologic evidence that the gene for L-gulono-’y-lactone oxidase is not expressed in animals subject to scurvy. Proc Natl Acad Sci USA l976;73:2066-8. 5. Koshizaka T, Nishikimi M, Ozawa T, Yagi K. Isolation and sequence analysis of a complementary DNA encoding rat liven i-gulono--ylactone oxidase, a key enzyme for L-ascorbic acid biosynthesis. J YAGI