YEAST
VOL.
6: 353-361(1990)
Analysis of the THR4 Region on Chromosome I11 of the Yeast Saccharomyces cerevisiae GERTRUD MANNHAUPTt, GERARD VAN DER LINDENt, IRENE VETTER*, KICK MAURERt, URSULA PILZ*, RUDI PLANTAT AND HORST FELDMANN*
* Instirut fur Physiologische Chemie, Physikalische Biochemie und Zellhiologie der Universitur Miinchen, Schillei~sti-aJ.3e 44, D-8000 Miinchen 2 , F.R.G. t Biochemisch Lahorator-ium, Vr-ije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Nttherlands Received 27 November 1989; revised 14 March 1990
The gene encoding threonine synthase (THR4 ) from the yeast Saccharomyces cerevisiae was cloned by complementation of a thr.4 mutant. This gene was also found on a lambda clone (5239) consisting of a fragment of chromosome 111 inserted in the vector lambdaMG3. The THR4 gene encodes a protein of 514 amino acids (M.W. 58 kDa), which has extensive homologies with E.coli threonine synthase (rhrC) and B.suhtilis threonine synthase. The 5’ flanking region of the gene contains three regulatory sequences [TGACT(C)] for the general amino acid control (GCN). About 130 bp downstream of the THR4 gene another large open reading frame (563 amino acids) is found in the opposite orientation. This may imply that this open reading frame, called CTR86, shares a terminator region with l H R 4 . The function of the protein encoded by CTR86 is not yet clear, but the fact that the upstream region contains a GCN4 responsive site suggests that the gene product may also be involved in amino acid biosynthesis. KEY WORDS-Threonine
metabolism; amino acid biosynthesis; homologous domains: chromosome 111; gene organisation.
INTRODUCTION In the yeast, Saccharomyces cerevisiae , nearly 800 genes have been identified to date (Mortimer et ul., 1989). In most of the cases, the genes have been assigned to specific chromosomal locations through genetic analysis or by physical mapping. Furthermore, the great majority of these genes have been characterized phenotypically or functionally, or both. A large number of the genes have been analysed to the molecular level, and corresponding proteins have been isolated. One basic approach to isolate a gene is the use of a previously defined mutation in complementation analysis. a procedure that has been widely employed in studies of genes involved in amino acid metabolism. For instance, we have recently applied this approach in characterizing T Y R I , the gene encoding prephenate dehydrogenase (Mannhaupt
et al., 1989), and T H R l , the gene encoding homoserine kinase (Mannhaupt et al., 1990) from yeast. On the other hand, it is unlikely that all of the possible genes will be identified by mutation. For the identification and mapping of new, as yet undefined, genes current molecular approaches are very useful. A complete sequence analysis of a DNA fragment and the subsequent comparison of the encoded protein sequence with other protein sequences available in the data banks, and the presence of well-known transcriptional regulatory sites, may lead to a preliminary functional identification of the new gene, which can then be confirmed by biochemical analysis. In this study, we show that the above mentioned approaches complement each other and lead to the identification of new genes. In brief, we have identified THR4, the gene encoding threonine
Correspondence: Dr H. Feldmann, Institut fur Physiologische Chemie, der Universitat Miinchen. SchillerstraBe 44, D-8000 Miinchen 2. F.R.G. Tel. 49-89-59 96 45 1 Fax 49-89-59 96 3 16 0749-503 X/90/040353-09$05.00 @ I990 by John Wiley & Sons Ltd
354 synthase, genetically mapped to the right arm of chromosome I11 (Mortimer and Hawthorne, 1966), and a second open reading frame of unknown function (CTR86), closely linked to THR4. Furthermore, we were able to determine the physical location of these two genes on chromosome 111. MATERIALS AND METHODS Plasmids, strains and media The yeast recombinant DNA library constructed from strain AB320 (HO, ade2-1, lys2-I, tip5-2, leu2-I, canl-100, ura3-1 and/or ui-al-1, met4-1; M.V.Olson) by Nasmyth and Reed (1980) with YRp7 (Struhl et al., 1979) as a vector was used to isolate pYthr4. Yeast strain S2068B (Yeast Genetic Stock Centre) (n, thr4, a d e l , trpl ; RKM) was used as a recipient strain in protoplast transformation (Beggs, 1978; Hinnen et al., 1978) and complementation experiments. Yeast synthetic minimal medium plus dextrose supplemented with all but the nutrient selected for (SD medium) was used (Sherman et al., 1982). E.coli strain 490A was employed in transformation experiments, and strain JM 103 in combination with M13 derivatives for sequencing. Molecular cloning procedures, i-estriction analysis and sequencing Recombinant DNA methods followed standard protocols (Maniatis et al., 1989). Restriction endonucleases, T4 DNA ligase, E.coli DNA polymerase (Klenow fragment), and calf intestine alkaline phosphatase were obtained from Boehringer Mannheim GmbH and used according to the manufacturer's instructions. Sequenase (version 2.0) was from United States Biochem.Corp. For sequencing (method A), appropriate restriction fragments from pYthr4 (cf. Figure 1) were subcloned into M 13 derivatives. DNA sequences were determined by the dideoxy chain termination method (Sanger et al., 1977) using ['5S]dATP and Sequenase. All sequences were read from both strands in at least two independent sets of experiments. The phage lambda clone 5239 was obtained from S.G.Oliver (Manchester) and was derived initially from a yeast DNA bank of mapped clones, assigned to yeast chromosomes, constructed by M.V.Olson
G. MANNHAUFT ET AL.
(St.Louis). The sequence analysis of this clone was performed as part of the European Community's enterprise to sequence whole chromosome 111. Half of the insert of this clone, about 1 1 kb, was sequenced on both strands according to the method of 'gene hopping' using synthetic oligonucleotides as internal sequencing primers. The oligonucleotides were synthesized with the Applied Biosystems DNA synthesizer. The sequencing was performed according to the dideoxy chain termination method using [''SIdATP and Sequenase. Computer.-assisted compaii~ons Sequence comparisons were performed on a MicroVaxII workstation in conjunction with sequence data from EMBL Bank (release 58), the MIPS protein data library (release 20), and the PIR system. For sensitive amino acid sequence comparisons, the programme developed by R. Rechid, M. Vingron, and P. Argos (Argos, 1987) was employed. RESULTS AND DISCUSSION The characterization of the THR4 region of chromosome 111 was achieved by two independent approaches: (i) isolation of a plasmid clone complementing a thr4 mutation and sequence analysis of the yeast DNA insert of this clone, (ii) partial sequence analysis of lambda clone 5239. Analyses of the data obtained in the two approaches revealed one open reading frame, the deduced amino acid sequence of which conforms to the properties predicted for the THR4 gene product, yeast threonine synthase. A second open reading frame (designated CTR86) was found to be located closely downstream from THR4. Cloning of the THR4 gene region @om S.cerevisiae by complementation As a source to isolate a clone complementing the yeast thr4 mutation, we used the recombinant yeast DNA library constructed by Nasmyth and Reed (1980). Yeast strain S2086B served as the thr4mutant in protoplast transformation (Beggs, 1978; Hinnen et al., 1978); several transformants were obtained. On retransformation of E.coli A490 with total DNA of these single yeast transformants, we
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Figure I . Restriction map of the THH4 region of yeast chromosome 111 and complementation experiments. The upper bar shows the 4.6 kb insert of the recombinant pYthr4 (see Materials and Methods); vector sequences are hatched. The bars underneath delineate the inserts of subclones that were derived from pYthr4 by cloning the corresponding restriction fragments into appropriate sites of M 13 vectors for sequencing. The open reading frames within the 4.6 kb insert are indicated. Below. those inserts of rubclones in YRp7 are listed (4-5 through 4-10) which were employed in the complementation studies. Black bars, fragments complementing thr.4 : open bars. fragments not complementing r/l/'4.The following abbreviations for restriction enzymes are used: A, A K ~ :B, BuriiHl: C. Clrrl: E, EcoRI: G, B,qIII: H, Hiridlll: I, HincII; K , KpriI; P. PIwII; SI. SulI: Q. X h t r I : T. P.\/l: ( B ) indicate\ that the originill L i c i i i i H I \ites of the vcctoI bere abolished by cloning of. the Sorr3A fragments.
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G. MANNHAUFT ET AL.
were able to isolate the YRp7 recombinant plasmid pYthr4 carrying a 4.6 kb yeast DNA insert and complementing the thr4 mutation. The restriction map of this plasmid (Figure 1) was used as a basis to subclone various fragments of the yeast DNA insert into appropriate sites of the vector YRp7 (designated 4- 1 through 4- 10; Figure I , lower section). Each of these recombinants was tested for its ability to complement the thr-4 mutation in strain S2086B. As inferred from these experiments (Figure I), the 2,5 kb HiridllI/ClaI fragment of pYthr4 (designated 4-5a) was sufficient for complementation. Nucleotide sequence
of the THR4 region
Seqence analysis of the THR4 region was achieved by two different approaches. (i) Based on the restriction map, the 4.6 kb insert of pYthr4 was dissected into a number of appropriate fragments which were subcloned into M13 vectors (see Figure 1). The sequences of the inserts were determined by the dideoxy chain termination method (Sanger et a1 ., 1977). (ii) Based on the restriction map, appropriate fragments of the insert of the lambda clone 5239 were subcloned into pUC 18 vectors, followed by double strand DNA sequencing by the method of Sanger et al. (1977). The nucleotide sequence obtained by the two approaches is depicted in Figure 2. The sequence of a 4 kb segment (positions 480 to 4501) has been determined in duplicate; no ambiguities were found. The identification of the THR4 coding region is based on the presence of an open reading frame of 1542 bp (positions 720 through 2261 in Figure 2) entirely included in the 2.5 kb fragment which was shown to complement the yeast thr4 mutation (4-5a, Figure 1). The THR4 gene could thus code for a protein of 5 14 amino acids, with a calculated molecular weight of ca 58 kDa. The codon usage of THR4 corresponds to that observed for yeast genes expressed at low levels (Sharp et al., 1986). The open reading frame for THR4 (Figure 2) starts with an ATG and ends in a TAA stop codon; two further in-frame stop-codons follow. Several regulatory sites are present in the flanking regions of the coding part of THR4. Possible TATA boxes occur at positions 629 and 653, respectively; canonical transcription initiation signals (Guarente, 1987) are located at positions 668, 679, and 697,
respectively. By primer extension analysis a major transcription start site was found at position 679 (results not shown). The 5'-flanking region of the gene contains three canonical GCN4 regulatory sequences [TGACT(C), or inverse] suggesting that THR4 is under the general amino acid control (for review, see: Hinnebusch, 1988); these signals are located ca 410, 180, and 120 bp, respectively, upstream of the translation start point. A second large open reading frame (CTR86) is located 134 bp downstream of the translational stop of THR4, in opposite orientation (Figure 2). CTR86 starts with an ATG at position 4090 and ends in a TAA stop codon at position 2399. Thus, the open reading frame contains 1689 bp, and the putative protein is 563 amino acids in length (ca 63 kDa). A putative 'TATA box' is located at positions 4028/4034; putative transcription initiation sites are found at positions 41 13/41 16 and 4101/4105, respectively. A GCN4 box [TGACT] is located some 200 bp upstream from the translational start point, suggesting that this gene might also be under the general amino acid control. Interestingly, three of such boxes occur within the coding region of CTR86 . In addition, a stretch rich in purine residues, mainly A, is located between the canonical GCN4 box and the TATA box (positions 4218 to 4250). A canonical transcription termination signal (Zaret and Sherman, 1982) for CTR86 is located within the intergenic region (positions 2303 through 23 16). The codon usage of CTR86 corresponds to that observed for yeast genes expressed at low levels (Sharp el al., 1986). Matching ofthe genetic and the physical maps around THR4 THR4 has been genetically mapped on the right arm of chromosome 111, centromere distal from the MAT locus (Mortimer and Hawthorne, 1966). From the known orientation of lambda clone 5239 Figure 2. Nucleotide sequence of THR4 and flanking regions. Certain features of the sequence are high-lighted: GCN4 recognition signals are boxed. Putative TATA boxes and the putative transcription termination signal are underlined; putative transcription initiation sites are marked with arrows. The deduced amino acid sequences for THR4 and CTR86 are written underneath the nucleotide sequence in the one-letter code. The stop codons are marked.
ANALYSIS OF THE THR4 REGION ON CHROMOSOME 111 OF THE YEAST SACCHAROMYCES CEREVZSZAE
351
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VOL.
6: 353-361(1990)
Analysis of the THR4 Region on Chromosome I11 of the Yeast Saccharomyces cerevisiae GERTRUD MANNHAUPTt, GERARD VAN DER LINDENt, IRENE VETTER*, KICK MAURERt, URSULA PILZ*, RUDI PLANTAT AND HORST FELDMANN*
* Instirut fur Physiologische Chemie, Physikalische Biochemie und Zellhiologie der Universitur Miinchen, Schillei~sti-aJ.3e 44, D-8000 Miinchen 2 , F.R.G. t Biochemisch Lahorator-ium, Vr-ije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Nttherlands Received 27 November 1989; revised 14 March 1990
The gene encoding threonine synthase (THR4 ) from the yeast Saccharomyces cerevisiae was cloned by complementation of a thr.4 mutant. This gene was also found on a lambda clone (5239) consisting of a fragment of chromosome 111 inserted in the vector lambdaMG3. The THR4 gene encodes a protein of 514 amino acids (M.W. 58 kDa), which has extensive homologies with E.coli threonine synthase (rhrC) and B.suhtilis threonine synthase. The 5’ flanking region of the gene contains three regulatory sequences [TGACT(C)] for the general amino acid control (GCN). About 130 bp downstream of the THR4 gene another large open reading frame (563 amino acids) is found in the opposite orientation. This may imply that this open reading frame, called CTR86, shares a terminator region with l H R 4 . The function of the protein encoded by CTR86 is not yet clear, but the fact that the upstream region contains a GCN4 responsive site suggests that the gene product may also be involved in amino acid biosynthesis. KEY WORDS-Threonine
metabolism; amino acid biosynthesis; homologous domains: chromosome 111; gene organisation.
INTRODUCTION In the yeast, Saccharomyces cerevisiae , nearly 800 genes have been identified to date (Mortimer et ul., 1989). In most of the cases, the genes have been assigned to specific chromosomal locations through genetic analysis or by physical mapping. Furthermore, the great majority of these genes have been characterized phenotypically or functionally, or both. A large number of the genes have been analysed to the molecular level, and corresponding proteins have been isolated. One basic approach to isolate a gene is the use of a previously defined mutation in complementation analysis. a procedure that has been widely employed in studies of genes involved in amino acid metabolism. For instance, we have recently applied this approach in characterizing T Y R I , the gene encoding prephenate dehydrogenase (Mannhaupt
et al., 1989), and T H R l , the gene encoding homoserine kinase (Mannhaupt et al., 1990) from yeast. On the other hand, it is unlikely that all of the possible genes will be identified by mutation. For the identification and mapping of new, as yet undefined, genes current molecular approaches are very useful. A complete sequence analysis of a DNA fragment and the subsequent comparison of the encoded protein sequence with other protein sequences available in the data banks, and the presence of well-known transcriptional regulatory sites, may lead to a preliminary functional identification of the new gene, which can then be confirmed by biochemical analysis. In this study, we show that the above mentioned approaches complement each other and lead to the identification of new genes. In brief, we have identified THR4, the gene encoding threonine
Correspondence: Dr H. Feldmann, Institut fur Physiologische Chemie, der Universitat Miinchen. SchillerstraBe 44, D-8000 Miinchen 2. F.R.G. Tel. 49-89-59 96 45 1 Fax 49-89-59 96 3 16 0749-503 X/90/040353-09$05.00 @ I990 by John Wiley & Sons Ltd
354 synthase, genetically mapped to the right arm of chromosome I11 (Mortimer and Hawthorne, 1966), and a second open reading frame of unknown function (CTR86), closely linked to THR4. Furthermore, we were able to determine the physical location of these two genes on chromosome 111. MATERIALS AND METHODS Plasmids, strains and media The yeast recombinant DNA library constructed from strain AB320 (HO, ade2-1, lys2-I, tip5-2, leu2-I, canl-100, ura3-1 and/or ui-al-1, met4-1; M.V.Olson) by Nasmyth and Reed (1980) with YRp7 (Struhl et al., 1979) as a vector was used to isolate pYthr4. Yeast strain S2068B (Yeast Genetic Stock Centre) (n, thr4, a d e l , trpl ; RKM) was used as a recipient strain in protoplast transformation (Beggs, 1978; Hinnen et al., 1978) and complementation experiments. Yeast synthetic minimal medium plus dextrose supplemented with all but the nutrient selected for (SD medium) was used (Sherman et al., 1982). E.coli strain 490A was employed in transformation experiments, and strain JM 103 in combination with M13 derivatives for sequencing. Molecular cloning procedures, i-estriction analysis and sequencing Recombinant DNA methods followed standard protocols (Maniatis et al., 1989). Restriction endonucleases, T4 DNA ligase, E.coli DNA polymerase (Klenow fragment), and calf intestine alkaline phosphatase were obtained from Boehringer Mannheim GmbH and used according to the manufacturer's instructions. Sequenase (version 2.0) was from United States Biochem.Corp. For sequencing (method A), appropriate restriction fragments from pYthr4 (cf. Figure 1) were subcloned into M 13 derivatives. DNA sequences were determined by the dideoxy chain termination method (Sanger et al., 1977) using ['5S]dATP and Sequenase. All sequences were read from both strands in at least two independent sets of experiments. The phage lambda clone 5239 was obtained from S.G.Oliver (Manchester) and was derived initially from a yeast DNA bank of mapped clones, assigned to yeast chromosomes, constructed by M.V.Olson
G. MANNHAUFT ET AL.
(St.Louis). The sequence analysis of this clone was performed as part of the European Community's enterprise to sequence whole chromosome 111. Half of the insert of this clone, about 1 1 kb, was sequenced on both strands according to the method of 'gene hopping' using synthetic oligonucleotides as internal sequencing primers. The oligonucleotides were synthesized with the Applied Biosystems DNA synthesizer. The sequencing was performed according to the dideoxy chain termination method using [''SIdATP and Sequenase. Computer.-assisted compaii~ons Sequence comparisons were performed on a MicroVaxII workstation in conjunction with sequence data from EMBL Bank (release 58), the MIPS protein data library (release 20), and the PIR system. For sensitive amino acid sequence comparisons, the programme developed by R. Rechid, M. Vingron, and P. Argos (Argos, 1987) was employed. RESULTS AND DISCUSSION The characterization of the THR4 region of chromosome 111 was achieved by two independent approaches: (i) isolation of a plasmid clone complementing a thr4 mutation and sequence analysis of the yeast DNA insert of this clone, (ii) partial sequence analysis of lambda clone 5239. Analyses of the data obtained in the two approaches revealed one open reading frame, the deduced amino acid sequence of which conforms to the properties predicted for the THR4 gene product, yeast threonine synthase. A second open reading frame (designated CTR86) was found to be located closely downstream from THR4. Cloning of the THR4 gene region @om S.cerevisiae by complementation As a source to isolate a clone complementing the yeast thr4 mutation, we used the recombinant yeast DNA library constructed by Nasmyth and Reed (1980). Yeast strain S2086B served as the thr4mutant in protoplast transformation (Beggs, 1978; Hinnen et al., 1978); several transformants were obtained. On retransformation of E.coli A490 with total DNA of these single yeast transformants, we
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Figure I . Restriction map of the THH4 region of yeast chromosome 111 and complementation experiments. The upper bar shows the 4.6 kb insert of the recombinant pYthr4 (see Materials and Methods); vector sequences are hatched. The bars underneath delineate the inserts of subclones that were derived from pYthr4 by cloning the corresponding restriction fragments into appropriate sites of M 13 vectors for sequencing. The open reading frames within the 4.6 kb insert are indicated. Below. those inserts of rubclones in YRp7 are listed (4-5 through 4-10) which were employed in the complementation studies. Black bars, fragments complementing thr.4 : open bars. fragments not complementing r/l/'4.The following abbreviations for restriction enzymes are used: A, A K ~ :B, BuriiHl: C. Clrrl: E, EcoRI: G, B,qIII: H, Hiridlll: I, HincII; K , KpriI; P. PIwII; SI. SulI: Q. X h t r I : T. P.\/l: ( B ) indicate\ that the originill L i c i i i i H I \ites of the vcctoI bere abolished by cloning of. the Sorr3A fragments.
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G. MANNHAUFT ET AL.
were able to isolate the YRp7 recombinant plasmid pYthr4 carrying a 4.6 kb yeast DNA insert and complementing the thr4 mutation. The restriction map of this plasmid (Figure 1) was used as a basis to subclone various fragments of the yeast DNA insert into appropriate sites of the vector YRp7 (designated 4- 1 through 4- 10; Figure I , lower section). Each of these recombinants was tested for its ability to complement the thr-4 mutation in strain S2086B. As inferred from these experiments (Figure I), the 2,5 kb HiridllI/ClaI fragment of pYthr4 (designated 4-5a) was sufficient for complementation. Nucleotide sequence
of the THR4 region
Seqence analysis of the THR4 region was achieved by two different approaches. (i) Based on the restriction map, the 4.6 kb insert of pYthr4 was dissected into a number of appropriate fragments which were subcloned into M13 vectors (see Figure 1). The sequences of the inserts were determined by the dideoxy chain termination method (Sanger et a1 ., 1977). (ii) Based on the restriction map, appropriate fragments of the insert of the lambda clone 5239 were subcloned into pUC 18 vectors, followed by double strand DNA sequencing by the method of Sanger et al. (1977). The nucleotide sequence obtained by the two approaches is depicted in Figure 2. The sequence of a 4 kb segment (positions 480 to 4501) has been determined in duplicate; no ambiguities were found. The identification of the THR4 coding region is based on the presence of an open reading frame of 1542 bp (positions 720 through 2261 in Figure 2) entirely included in the 2.5 kb fragment which was shown to complement the yeast thr4 mutation (4-5a, Figure 1). The THR4 gene could thus code for a protein of 5 14 amino acids, with a calculated molecular weight of ca 58 kDa. The codon usage of THR4 corresponds to that observed for yeast genes expressed at low levels (Sharp et al., 1986). The open reading frame for THR4 (Figure 2) starts with an ATG and ends in a TAA stop codon; two further in-frame stop-codons follow. Several regulatory sites are present in the flanking regions of the coding part of THR4. Possible TATA boxes occur at positions 629 and 653, respectively; canonical transcription initiation signals (Guarente, 1987) are located at positions 668, 679, and 697,
respectively. By primer extension analysis a major transcription start site was found at position 679 (results not shown). The 5'-flanking region of the gene contains three canonical GCN4 regulatory sequences [TGACT(C), or inverse] suggesting that THR4 is under the general amino acid control (for review, see: Hinnebusch, 1988); these signals are located ca 410, 180, and 120 bp, respectively, upstream of the translation start point. A second large open reading frame (CTR86) is located 134 bp downstream of the translational stop of THR4, in opposite orientation (Figure 2). CTR86 starts with an ATG at position 4090 and ends in a TAA stop codon at position 2399. Thus, the open reading frame contains 1689 bp, and the putative protein is 563 amino acids in length (ca 63 kDa). A putative 'TATA box' is located at positions 4028/4034; putative transcription initiation sites are found at positions 41 13/41 16 and 4101/4105, respectively. A GCN4 box [TGACT] is located some 200 bp upstream from the translational start point, suggesting that this gene might also be under the general amino acid control. Interestingly, three of such boxes occur within the coding region of CTR86 . In addition, a stretch rich in purine residues, mainly A, is located between the canonical GCN4 box and the TATA box (positions 4218 to 4250). A canonical transcription termination signal (Zaret and Sherman, 1982) for CTR86 is located within the intergenic region (positions 2303 through 23 16). The codon usage of CTR86 corresponds to that observed for yeast genes expressed at low levels (Sharp el al., 1986). Matching ofthe genetic and the physical maps around THR4 THR4 has been genetically mapped on the right arm of chromosome 111, centromere distal from the MAT locus (Mortimer and Hawthorne, 1966). From the known orientation of lambda clone 5239 Figure 2. Nucleotide sequence of THR4 and flanking regions. Certain features of the sequence are high-lighted: GCN4 recognition signals are boxed. Putative TATA boxes and the putative transcription termination signal are underlined; putative transcription initiation sites are marked with arrows. The deduced amino acid sequences for THR4 and CTR86 are written underneath the nucleotide sequence in the one-letter code. The stop codons are marked.
ANALYSIS OF THE THR4 REGION ON CHROMOSOME 111 OF THE YEAST SACCHAROMYCES CEREVZSZAE
351
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