Download Molecular Cloning and Nucleotide Sequence of the 3

Journal of General Microbiology (1 987), 133, 1089-1 097. Printed in Great Britain
1089
Molecular Cloning and Nucleotide Sequence of the 3-Isopropylmalate
Dehydrogenase Gene of Cmdida utilis
By K A Z U H I R O HAMASAWA, Y U K U H A R U KOBAYASHI, S H I G E Y O S H I
H A R A D A , K O J I YODA, M A K A R I Y A M A S A K I * A N D G A K U Z O T A M U R A
Department of Agricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
(Received 2 July 1986; revised 27 October 1986)
A 34sopropylmalate dehydrogenase (3-IMDH, EC 1.1 . 1 .85) gene was cloned from a gene
library of Candida utilis. One of the plasmids, pYKL30, could complement Escherichiu coli leuB
and Saccharomyces cerevisiae leu2 auxotrophs; a 2.2 kb Hind111 fragment subcloned in pBR322
could still complement the IeuB mutation. Southern hybridization confirmed that this fragment
was derived from C. utilis. An open reading frame of 1089 bp that corresponded to a polypeptide
of 363 amino acids, one residue shorter than the 3-IMDH of S . cerevisiae, was found in the
cloned fragment. The homology between the 3-IMDHs of C. utilis and S . cerevisiae was 76.2% in
nucleotides and 85.4% in amino acids. In contrast, the homology between the 3-IMDHs of C.
utilis and Thermus thermophiluswas much smaller and was restricted to some regions of the gene.
INTRODUCTION
Cell mass of strains of Candida utilis has been recognized to be useful as a food resource since
the early studies of Fink et al. (1936) on the fermentative nature of the strains. As C.utilis
generally requires no growth factors such as vitamins and has a high growth rate, it has been
cultured on a large scale with sulphite waste liquor containing hemicellulose as the major carbon
source. Culture broth of some strains of C. utilis contains many medically useful materials such
as RNA, glutathione, NAD and coenzyme A. Although C. utilis is highly useful in the
fermentation industry, there is little genetic information on this organism mainly because of the
difficulty in obtaining auxotrophic mutants. In this report, a gene coding for 3-isopropylmalate
dehydrogenase (3-IMDH, EC 1.1 . 1 .85) was cloned from a strain of C. utilis and examined in
detail. This gene is homologous to LEU2 of Saccharomyces cerevisiae, which is frequently used in
genetic studies. It should be of help to construct Leu- mutants, by gene disruption, to be used as
recipient strains with a selectable marker in cloning based on C. utilis.
METHODS
Strains, media and reagents. Candida utilis ATCC 9226 (a food yeast) obtained from the American Type Culture
Collection, Saccharomyces cerevisiae AH22 (MATa leu2-3 leu2-112 his3-1 canl-100)obtained from Y. Fukuda, the
Mitsubishi-Kasei Institute of Life Science, and Escherichia coli K12 RRI (F- leuB6 proA2 thi-1 arall) obtained
from J. Lederberg, University of Stanford, were used in this study. The general growth medium was YPD medium
(yeast extract, 1 %, w/v, peptone, 2%, w/v, glucose, 2%, w/v) for yeasts or nutrient broth (peptone, 1 %, w/v, meat
extract, 0.3%, NaCI, 0.5%) for bacteria. Bacterial transformants were selected on broth agar plates containing
ampicillin (100 pg ml-*). Complementation tests were done on synthetic medium (Davis, 1948)for bacteria or on
Difco nitrogen base medium for yeast. Restriction enzymes were products of Takara Brewery Co. (Japan) or
Boehringer Mannheim (West Germany).
Cloning of Sau3AI fragments from C . utilis chromosomal DNA. Chromosomal DNA (50pg) of C . utilis was
partially digested with Sau3A 1. The digested DNA was electrophoresed in a low-melting-temperature agarose gel,
Abbreciations: 3-IMDH, 3-isopropylmalate dehydrogenase; ORF, open reading frame.
0001-3565 0 1987 SGM
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1090
K . HAMASAWA A N D OTHERS
C
EIH
[[
c,I J
BamH I
Pv
(6 9 kb)
a,
P
4
T4 ligase
s
EH
Hind[[]
Hind111 2.5 kb
fragment
C
T4 ligase
E
E
Suu3A I
partial
digest
".) LJj
pYKL45
EIH
Bg
A, I /BamH I
c
Pv
//
pYKL40
r v
EcoRI partial digest
T4 ligase
Suu3A I!
Fig. 1. Construction and restriction maps of subclones. Partial digests of the chromosomal DNA of C .
utilis with Sau3A1 were ligated with YRp7 at the BumHI site to make pYKL30. The fragment
containing TRPl and ARSI was removed from pYKL30 by HindIII digestion and recircularized to
make pYKL40. The 2.55 kb HindIII fragment of pYKL40 was inserted in pBR322 at the HindIII site to
make pYKL45. B, BumHI; Bg, BglII; C, CluI; E, EcoRI; H, HindIII; P, PstI; Pv, PvuII; S, SulI.
and the part of the gel containing fragments larger than 1 kb was cut out. The DNA was extracted by freezing and
thawing. The cloning vector YRp7 (4 pg) was digested with BumHI. Then the two types of fragment were ligated
with T4 DNA ligase and the ligation products were used to transform E. coli RR1. Ampicillin-resistant and
tetracycline-sensitive transformants were selected, and tested for complementation of leuB auxotrophy on
minimal medium.
Southern hybridization. Chromosomal DNAs of E. coli, S. cerevisiae and C. utilis were prepared as described
previously (Struhl et al., 1979)and digested with the restriction enzymes EcoRI and BamHI. The fragments were
transferred from agarose gel to a nitrocellulose filter (Southern, 1975).The hybridization probe DNA was labelled
by using a nick-translation kit (Amersham).
Sequencing. DNA was sequenced by the dideoxy chain termination method of Sanger et al. (1977). The sequence
kit of Takara Brewery Co. was used. All of the sequences were determined at least twice on the same strand. The
sequences were analysed with a PC9801F2 personal computer (Nippon Electronic Co., Japan) and the software
Genetyx-I11 (Software Development Co., Japan).
RESULTS
Cloning of the 3-IMDH gene of C . utilis
The E. coli RRl transformants containing C . utilis chromosomal DNA were screened for
complementation of the leuB6 auxotrophy. Fortunately, among the 492 transformants first
obtained, we found two that grew well in minimal medium lacking leucine, one of which had a
10.7 kbp plasmid. This plasmid, pYKL30, also complemented the leucine deficiency of the S .
cerevisiae leu2-3 leu2-112 auxotroph. The restriction map of plasmid pYKL30 is shown in Fig. 1.
Localization of the 3-IMDH coding region in pYKL30 was examined as follows. The fragment
containing TRPZ and ARSI was removed by HindIII digestion followed by recircularization to
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1091
Candida utilis 3-IMDH gene
0
1
1
I
l
C
ECHSaB
3
1
l
l
E BBe
H
4
1
H
5
1
P
1
BnC
1
1
S
1
1
Pv
I
~
k1b
;
P
1
I
40-E
9
Leu-
40-HI
1 Leu-
40-HlI
Leu'
40-Pv
] Leu+
] Leu+/Leu
I Leu-
&B- Bg
40-c
0
-
Leu-
3-IMDH
Fig. 2. Determination of the coding region of the C. utilis 3-IMDH gene. Bold lines represent the
nucleotide sequence derived from C. utilis.Open lines represent the DNA of pBR322. Light lines show
the deletion region in each derivative of pYKL40. The activity of complementation of the E. coli leuB6
auxotroph is indicated by Leu+ or Leu-. The line with arrowheads shows the coding region of the
3-IMDH.
c/o I
Suu3Al/BomHI
I
1
1
I
I
I
I
Hind I I I
Bgl I I
EroRI
I
I
I
I
I
I
I
1
3-IMDH
100 bp
Fig. 3. Strategy for sequencing the 3-IMDH coding and flanking regions. Fragments from pYKL45
were inserted in the multiple cloning site of pUC 18 vector. Arrows beneath the bold lines represent the
direction and extent of sequences determined. The arrow at the bottom shows the 3-IMDH coding
region. Vertical lines represent the positions of restriction sites.
make it easy to examine the region. This plasmid was named pYKL40. It should have no
autonomously replicating sequence (ARS) that works in S . cerevisiae, because when S . cerevisiae
AH22 was transformed with 1 pg pYKL40, less than Leu+ prototrophs appeared. Plasmid
pYKL40 was further digested with various restriction enzymes and recircularized to delete nonessential parts. The 3-IMDH activity of the deletion plasmids was examined by complementaDownloaded from www.microbiologyresearch.org by
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~
~
1092
K . H A M A S A W A A N D OTHERS
-300
Sau3AI/BamHI
~~TCAAGGGAGCAGAATC~TAAGAGAGC~AGAAATA~CCATGAAGGTTCAACA~TTGTACGATCAGACCAGTC~GTTCATTCA~CGTTGTTTAAT
-200
~ C A T A A T C ~ T T ~ A T C ~ C T T ~ ~ A A A G C A T C C C A C C A C C G C C ~ ~ C C T C T G T ~ T
-b
TCCACCTC~GTCATTCTC~GC~TGTTTC~CICCATT~~~AT~GCTACTAGC~CA~ACAACCATGCCAGAGAAAACAATTGT~GTTCTTCCAGG
YetProGluLYsThrlleValValLeuProGlv
I00
TGACCACG~CGGTACTGAGATCACTGCTGAGGCCATCAAGGTCCTCAAGGCCATCGAGGAGGTGAAGC~GGAGATCAAGTTCAA~TC~AACACCACC~G
AspHlsVaIGIyThrGIuIleThrAlaGIuAlalleLy~ValLeuLysAlalleGIuGIuVaILYsProGIuIleL~SPheASnPhetlnHlSHlsLeu
Clal
200
ATTGGAGGAGCTGC~ATC~ATGCCACCG;;TGTTCCATTGCCAGACGATGCCTTGGAGG~C~CCAAGAAGGCTGATGCTGTTCT~CTCGGTGCTGTTGGTG
IleGlYGIYAIaAlalleAspAlaThrGlyVa1ProLeuProAspAspAlaLeuGluAlaSerLysLYsAlaAsPAlaValLeuLe~GlYAlaValGlyGlY
300
GTCCAAAA~GGGGTACCGGTGCCGTCAGACCAGAGCAAGGTCTTCTGAAGATCAGAAAAGAGCTGAAC~TTTACGCCAACTTGAGACC~TGT
AACTTTGC
PrOLYsTrPGlYThrGlYAlaValArgProGluGlnGlyLeuLeuLyslleArgLysGluLeuAsnLeuTyrAlaAsnLeuAr9ProCYsAsnPheAla
400
TTCTGAGT~CTTGTTGGA~TTGTCTCCAATCAAGGCCGAGGTCGTCAAGGGCACAGAC~TTGTCGTTG~CAGAGAGCT~GTCGGTGGTATCTACTTTGGT
SerGluSerLeuLeuAsPLeuSerProlleLysAlaGluValValLysGlyThrAspPheValValValArgGluLeuValGlYGlYllelYrPheGly
500
GA A A G A A
A ~ G A G G A T G A C G G T T C A G G C G ~ T G C G T C C G A ~
GluAr9LYSGluAsPAsPGIYSerGlyValAlaSerAspThrGluThrTyrSerValProGluValGlnArglleThrAr~MetAlaAlaPherletAlaLeu
600
TGCAACACAACCCACCAT~GCCAATC~CI;TCTTTGGACAAGGCCAACG~CTTGGCCTC~TCCAGATTG~GGAGAAAGG~TGTCACTG
GlnHlsAsnProProLeuProlleTrPSerLeuAspLysAlaAsnValLeuAlaSerSerArgLeuTrpArgLysValValThrGluThrlleGluLys
700
EcoRl
GGAATTCCtACAATTGAC~GTCAA~~ACCAGTTGATCGACTCCGCTGC~ATGATCCTCATCAAGTACC~AA~ACAGCT~AACGGTATAGTCATCA~~T~C
GluPheProClnLeuThr~alGlnlilsGlnLeulle~sPSerAlaAlaMetlleLeulleLyslyrProThrC,lnLeuAsnGlylleValIleThrSer
BOO
AACATGTTTGGTGATATCATCTCTGATGAGGCATCTGTCATTCCAGGTTCTCTGGGTTTGTTGCCATCTGCCTCTTTGGCAT~CTGCCAGA~CGAACA
AsnMetPheGlYAspllelleSerAspGluAlaSerValIleProGlySerLeuGlyLeuLeuProSerAlaSerLeuAlaSerLeuProAsPSerA~nL~s
BamHl
900
,
AGGCCTTTGGTTTGTACGAGCCATGCCATGGATCCG~CCAGACTTGCCAGCCAATAAGGTGAACCCAATT~CCACTATCTTGTCTG~~~AATGATGTT
AlaPheGlYLeuTyrGluProCYsHlsGlYSerAlaProAsPLeuProAlaAsnLYsValAsnProlleAlaThrlleLeuSerAlaAlaMetMetLeu
I000
GAAGTTCTCTTTGGACTTCTACGA~GAA~GTGTTGCGC~TGAGACTGC~GTTAAGCAAGTTCTCGATG~TGGTATCAGAACTG~TGAC~TGAAGGG,~A~A
LYsL~uSerLeuAsPLeuTYrGluGluGlYValAlaValGluThrAla~~alLYsGlnValLeuAsPAlaGlYlleAr9ThrGlYAspLeuLYsGlYThr
Bg/ 11
1100
AACTCAAC~ACCGAGGTTGGTGATGCTG;T~CTGAAGC~GTCAAGAAGATCTTGGCTJACT~-~~~~A~~~TA~TTT_AA_~
AsnSerThrThrGluValGlyAspAlaValAlaGluAlaValLysLyslleLeuAlaTRM
I200
-_----_--ACGAAACGAAG#~TR~CTATTATCATATCTTATGCAGA~GTAGTGATG~GACTTGAAC~GGCTTTGAC~CTAATTCTG~TGAGCAACAAAGTCATCAATG
I300
TCAATCTC~AAGATTCGAGCAGAACCAG;\GTTCGAAGTCTTTCTCTGC~TCT~TGCCTCCTGCTTCTCCAGTTTGCGTAACTTGGCTAATGGGCCATAGC
I400
CT A C A A C G ~ C C T T G T T C A ~ C T AATGGATCGAGGAA
CA
A ~
I500
ACCTTCGI CTCC~AACTTCTCC~CAACCAAGT~GTTGTCGTT~AACTTAACC~GTCTTGGCT~CTCGTCGTTGGCGTGTGTCACTGTGACGG~T
A ATGAT
1700
CTTCACAA~GACACCAGACTTGATCTTC~TCAATCTAAGCAGTTGAAGAACTTTCTGGACTTTGGAAGGGATCTTTGCACC~AC~GGG~C~GGGGTTC~G
Hind111
TCACAAGCT
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Cundidu utilis 3-IMDH gene
1093
tion tests of the E. colileuB6 auxotroph (Fig. 2). It was concluded that the 3-IMDH coding region
was situated in a BumHI/Suu3Al-HindIII fragment of 2.2 kbp. Therefore, this fragment was
recloned in pBR322 at the Hind111 site to form pYKL45 (Fig. 1.). Southern hybridization
experiments were done to test whether the sequence complementing the leuB6 mutation of E.
coli corresponded to C. utilis DNA. HindIII-digested chromosomal DNAs from C. utilis clearly
hybridized with pYKL40, while those from S . cerevisiue and E. colidid not (data not shown), so it
was confirmed that the 2.2 kbp fragment originated from C. utilis.
D N A sequence of the 3-IMDH gene of C. utilis
To analyse the DNA sequence of the 3-IMDH gene of C. utilis, we sequenced the 2.2 kbp
fragment of pYKL45 by the dideoxy chain termination method. Fig. 3 shows the sequence
strategy. Since the fragment bears three unique cleavage sites of CluI, EcoRI and BglII, it was
divided into four pieces by the enzymes. They were subcloned in the multiple cloning site of
pUCl8 vector (Yanisch-Perron et ul., 1985). Sequencing of each fragment was repeated two or
three times. The DNA sequence of the fragment (Fig. 4) consisted of 2209 bp and contained an
open reading frame (ORF) of 1089 bp encoding 363 amino acids. A translatable amino acid
sequence is shown under the nucleotide sequence in Fig. 4. The M , of the corresponding
polypeptide was calculated to be 38 700. A TATA-box-like sequence, the consensus sequence of
the RNA polymerase I1 recognition site, could also be found at two places, namely at positions
-258 and -30 (Sentenac & Hall, 1982).
DISCUSSION
The 3-IMDH gene of C. utilis was cloned and its nucleotide sequence was determined. This is
the first report of the nucleotide sequence of a functional gene of C. utilis as far as we know. The
sequence of 2209 bp was an alignment of four restriction fragments determined separately. The
sequences of HindIII-CluI, CluI-EcoRI, EcoRI-BglII and BglII-Hind111 fragments could be
clearly read from one end to the other. However, if there were two proximal CluI sites or an
EcoRI or BglII site which could not be detected by mapping with restriction endonucleases, they
would be missed. To test this possibility we cleaved pYKL45 with one of ClaI, EcoRI and BglII
completely, recircularized with ligase and used this DNA to transform E. coli R R l . As all the
ampicillin-resistant transformants were also Leu+, there should be no missing fragments.
Nucleotide sequences of the 3-IMDH genes of S . cerevisiue (LEU2)and T. thermophilus (IeuB)
have already been determined. When the DNA sequence of the 3-IMDH gene of C. utilis was
compared with that of S . cerecisiue, a considerable homology (76.2%) was found. Moreover,
since only the third letter of the corresponding codon differs in many cases, the homology of
amino acid sequences is as high as 85.4%. Thus the two sequences could be aligned in spite of the
difference in species (Fig. 5). The 3-IMDH gene from Cundidu multosu has been cloned
(Kawamura et ul., 1983), and its recently determined nucleotide sequence also has a high
homology (76%) with the 3-IMDH gene of S . cerevisiue (M. Takagi, personal communication).
The C. utilis 3-IMDH was one amino acid shorter than the S . cerevisiue 3-IMDH. In Fig. 5 , the
underlined sequence of 14 amino acids (288-301) in S . cerevisiue was considered by Andreadis et
ul. (1984) to be the substrate-binding site of the LEU1 and LEU2 gene products. The homology
was 85.7% in this region.
The nucleotide sequence of the leuB gene of Thermus thermophilus (Kagawa et ul., 1984) was
compared with that of the 3-IMDH gene of C. utilis. There is only 12.9% homology in the ORFs,
but in some parts more homologous stretches were found (Fig. 6). It is unknown what these
Fig. 4. Nucleotide and amino acid sequence of the 3-IMDH gene and its flanking regions. The amino
acid sequence of the ORF was translated from the nucleotide sequence. Numbering of both nucleotides
and amino acids start from the beginning of the coding sequence. TATA box-like sequences were
boxed. Underlines in the 5' non-coding region indicate a G-C-rich palindromic sequence. Broken lines
and boxes at the 3' non-coding region represents the presumptive consensus sequence of transcription
termination.
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1094
K . HAMASAWA A N D OTHERS
C. urilis
-MPEKT IVVL PGDHVGTEI T AEA IK V L I d EEVKPEI KFN FQHHLI GGAA IDATGVPLPii
S.cerevisiae
MSAPKK 1 V V L PGDHVGQE1 T AEA I KVLKA I SDVRSNVKFD FENHL I GGAA IDATGVPLI'E
* **** ****** *** **********
*
** * ******* **********
30
1
89
1I9
DALEASKKAD AVLLGAVGGP KWGTGAVRPE QGLLKIRKEL NLYANLRPCN FASESLLDLS
******* * ********** ***** **** ********** ********* *** ******
EALEASKKVD AVLLGAVGGP KWGTGSVRPE QGLLK I l?KEL QLYANLRPCN FASDSLLDLS
I20
I79
PI KAEVVKGT DFVVVRELVG GI YFGERK~; DGSGVASDTE TYSVPEVQRI TRMAAFMALQ
***
*** ********** ***** **** ** *** * * * ******* **********
PIKPQFAKGT DFVVVRELVG GIYFGKRKED DGDGVAWDSE QYTVPEVQRI TRMAAFMALQ
I50
I80
209
239
HNPPLPIWSL DKANVLASSR LWRKVVTETI EKEFPQLTVQ HQLIDSAAMI LIKYPTQLNG
* ******** ********** **** * ***
*** * ** ********** * * ** ***
HEPPLPIWSL DKANVLASSR LWRKTVEEI'I
KNEFPTLKVQ HQLIDSAAMI LVKNPTHLNG
Iv I TSNMFGD I I SDEASVI P GSLGLLPS~!!
LASLPDSNKA FGLYEPCHGS APDLPANK;~
210
240
* ******** ********** ********** ****** * * ********** ***** ***
IIITSNMFGD I I S D E A S V I P GSLGLLPSAS LASIJ'DKNTA
270
FGLYEPCHGS APDLPKNKVD
329
300
359
-P I AT 1LSAAM MLKLSLDLYE EGVAVETAVK QVLDAGI RTG DLKGTNSTTE VGDAVAEAVK
********** ****** * * ** * * *** ********* ** * ***** ******* **
P I AT I LSAAM MLKLSLNLPE EGKA I EDAYE KVLDAG I RTG DLGGSNSTTE VGDAVAEEV3i
363
KILA
****
KILA
364
Fig. 5. Comparisons of the putative amino acid sequences of the ORFs between C. utilis and S .
cerevisiae.The upper sequence represents C. utilis 3-IMDH and the lower one the S. cerevisiae 3-IMDH.
Asterisks indicate homologous amino acids. The underlined region represents a possible substrate
(3-isopropylmalate) binding site proposed by Andreadis et al. (1984).
partial homologies mean. Table 1 shows a comparison of codon usage among C. utilis, S .
cerevisiue and 7'. thermophilus in the 3-IMDH coding region. C. utilis and S . cerevisiue are
eukaryotes, and the codon usages are quite different from that of the prokaryote T. thermophilus,
especially in the codons of leucine, glycine, proline and arginine. In the third letter of the codons,
C. utilis preferred GC as compared to an AT choice in S. cerevisiue. As expected there is a high
occurrence of GC residues in the third letter of the codons in the 3-IMDH of 7'. thermophilus
(Table 1). It has been found that in the 5' non-coding region of the 3-IMDH gene (LEUZ)of S .
cerevisiue a leader peptide rich in leucine precedes the translation initiation site, and a stem and
loop structure can be formed at the end of the leader peptide (Yanofsky, 1983). This region
resembles the attenuator structure which regulates some E. coli genes concerning amino acid
synthesis. However, Erhart & Hollenberg (1983) later reported that this region is not relevant,
because deletions in it did not affect the regulation of 3-IMDH gene expression. We searched the
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Candida utilis 3-IMDH gene
l - G l v - G l ~ - A la-A1 a- I le-Asp-Al a-1
-Giy-Gii-Ala-Ala-I le-Asp-Ala-Gly-Gly-Ala-Ala-lle-Asp-Ala-
1095
(52)
-Ala-Asp-Ala-Val-Leu-Leu-Gly-Ala-Val-Gly-GlY-Pro-LYs-TrP-GlyLa-Val-Gly-GI Y-Pro-Lys-Trp-G1y-
‘-Val~Asp-Ala-Val-Leu-Leu-Gly-A
-Pro-Gly-Ser-Leu-GlY-Leu-Leu-Pro-Ser-Ala-Ser-Leu-Pro-Gly-Ser-Leu-Gly-Leu-Leu-Pro-Ser-Ala-Ser-Leu-
-Pro-Gly-Ser-Leu-Gly-Leu-Leu-Pro-Ser-Ala-Ser-Leu-G I u- Pro- C y s - H 1 s -GI y - S e r -A 1 a - Pro- As p-GIu - p r 0 - C ~- H~i s-GIy-Ser -A1 a-Pro- As P-Glu-ProfVa L f H I s-GI y - S e r - A 1 a-Pro-Asp--
(82)
(8.1)
(270)
(271)
(260)
(292)
(293)
(276)
-Pro-lle-Ala-Thr-lle-Leu-Ser-Ala-Ala-MET-MET-Leu-Pro-lle-Ala-Tlir-Ile-Leu-Ser-Ala-Ala-MET-MET-Leu-
(311)
(312)
- P r o ~ A l a ~ A l a ~ I l e - L e u - S e r - A l a - A l a - M E T - M E T - L e u -(295)
Fig. 6. Homologous sequences in the ORFs of C . utilis (a), S . cerevisiae (b) and T. thermophilus (c),
Identical amino acid sequences are boxed.
Table 1. Comparison of codon usage in the 3-IMDH gene of C . utilis, S . cerevisiae and
T. thermophilus
Frequency
Frequency
Frequency
Frequency
Amino
h Amino
r-h-h
acid
Codon C S T acid
Codon C S T
Amino
acid
Codon C S T
Amino
Codon C S T
acid
Leu
Tyr
TAT
TAC
0 0 1
6 4 5
Cys
TGT
TGC
1
1
His
CAT
CAC
1 3 0
5 3 5
Trp
TGG
3 4
Gln
CAA
CAG
7 9
2 0
Arg
Asn
AAT
AAC
CGT 0 2
CGC 0 0
CGA 0 0
CGG 0 0
AGA 10 9
AGG 0 0
Lys
AAA
3 10 0
AAG 21 18 15
Ala
GCT 16 13 2
GCC 17 16 28
GCA 4 5 1
GCG 2 2 1 1
TTA
TTG
CTT
CTC
CTA
CTG
0 9 0
25 21 4
4 5 4
5 0 15
0 4 1
6 1 10
Phe
TTT
TTC
Ile
ATT
ATC
ATA
4 8 0
20 16 7
1 1 1
Met
ATG
7 7 6
Val
GTT 16 18 0
GTC 14 7 12
GTA 0 0 0
GTG 2 4 20
5
4
Gly
Pro
GGT 22 28 1
GGC 2 0 1 0
GGA 4 1 7
GGG 0 0 19
CCT
ccc
CCA
CCG
5 3
5 10
Thr
ACT
ACC
ACA
ACG
0
0
21
1
7
0
16
0
4
18
1
7
7 4 0
8 8 6
5 4 0
0 0 6
Asp
GAT 9 17 1
GAC 1 1 7 I5
Glu
GAA 5 20 3
GAG 21 3 28
1
2
1 5 0
11 8 6
Ser
TCT
TCC
TCA
TCG
AGT
AGC
1 0
1 0
10
9
2
0
0 3
0 1
12
8
2
1
1
1
7
2
11
0
6
2
8
0
1
0
4
C, C.utilis; S , S . cerevisiae; T, T . thermophilus.
5’ non-coding region of the C. utilis 3-IMDH gene for similar structures, but could not find them.
Martinez-Arias et al. (1983, 1984) suggested on the basis of experiments on deletion mutants,
that a palindromic sequence upstream of the TATA box, 5’-TGAGAGGCCGGAACCGGCTTTTCA-3’, participates in the regulation of the expression of the S . cerevisiue
LEU2 gene. In the 5’ non-coding region of C . utilis similar sequences were located at -204 to
- 181, - 164 to - 15 1 and - 143to - 129, but they were downstream of the TATA box at - 256.
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1096
K . HAMASAWA AND OTHERS
A tRNALeugene containing an intervening sequence has been reported in the 5’ non-coding
region of the S. cerevisiue LEU2 gene (Andreadis et ul., 1982): we searched for a homologue of
the C. utilis tRNALeusequence described by Murasugi & Takemura (1978) but failed to find a
corresponding sequence. Zaret & Sherman (1982) suggested that the 3’ non-coding regions of
several genes contain transcription termination sequences. S . cerevisiue also has such a sequence
in the 3’ non-coding region of the LEU2 gene. In C . utilis, the consensus signal sequence for
termination and poly(A) addition, TAA/TAG/TGA***1-140***(T-rich)****TAGT/
TATGT***(A-T-rich)*****TTT*****poly(A)
was also found in this region (Fig. 4). Many
kinds of dehydrogenases have a structure which is thought to be part of the coenzyme-binding
domains. Recently it was proposed that ADP, a component of NAD or flavin-adenine
dinucleotide (FAD), would bind a structure of fl-strand-a- helix-fl-strand which was termed
an ADP-binding flap-fold (Froman et ul., 1984). The authors compared many ADP-binding
proteins and found two common characteristics of all complexes: (i) the occurrence of glycine
residues at the N-terminus of the helix (e.g. Gly*Gly**Gly), which results in a favourable
interaction between the a-helix dipole and the negatively charged pyrophosphate moieties, and
(ii) the occurrence of the eight amino acids at a specific position in a peptide fragment. The total
length of this amino acid sequence varies between 29 and 31 residues. We searched the C. utilis
3-IMDH gene for a sequence with these characteristics, but failed to find it, so the amino acid
sequence of the ADP-binding flap-fold may be different from the others reported (Wierenga et
ul., 1985, 1986).
There are many homologous sequences among the 3-IMDHs of C. utilis, S . cerevisiue and T.
thermophifus (Fig. 6). It is unknown what these homologies mean, but they may in some way
concern essential sequences such as the catalytic domain or the nucleotide binding site.
As we now have the nucleotide sequence of the 3-IMDH gene of C. utilis, it may be possible to
get Leu- auxotrophic mutants of C. utilis by the gene disruption technique (Rothstein, 1983).
Then, the 3-IMDH gene will be useful as a selective marker in gene manipulation of C. utilis.
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