Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene

Braz J Med Biol Res, March 1998, Volume 31(3) 355-363

Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene

M.P. Nobrega¹, M.A.S. Graminha², E.N. Troitskaya² and F.G. Nobrega³

Departamentos de ¹Fisiologia and ²Biologia, Instituto de Biociências, and ³Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil

References

Abstract

Introduction

Material and Methods

Results

Discussion

Acknowledgments

^{Correspondence and Footnotes}

The mutants of Saccharomyces cerevisiae assigned to complementation group G199 are deficient in mitochondrial respiration and lack a functional cytochrome oxidase complex. Recombinant plasmids capable of restoring respiration were cloned by transformation of mutants of this group with a yeast genomic library. Sequencing indicated that a 2.1-kb subclone encompasses the very end (last 11 amino acids) of the PET111 gene, the COX7 gene and a new gene (YMR255W) of unknown function that potentially codes for a polypeptide of 188 amino acids (about 21.5 kDa) without significant homology to any known protein. We have shown that the respiratory defect corresponding to group G199 is complemented by plasmids carrying only the COX7 gene. The gene YMR255W was inactivated by one-step gene replacement and the disrupted strain was viable and unaffected in its ability to grow in a variety of different test media such as minimal or complete media using eight distinct carbon sources at three pH values and temperatures. Inactivation of this gene also did not affect mating or sporulation.

Key words:Saccharomyces cerevisiae, gene cloning, cytochrome oxidase, subunit VII, gene disruption

In the yeast Saccharomyces cerevisiae, cytochrome oxidase is composed of eleven different polypeptides (1) and the enzyme contains two spectrally distinct heme a groups and two copper atoms. The three large subunits (COX1, COX2, and COX3) which are generally believed to represent the major catalytic centers of cytochrome oxidase are encoded by mitochondrial DNA and possess significant similarity in their primary sequence to the three subunits of prokaryotic cytochrome oxidases (2). All additional subunits are encoded by the nuclear genome. These subunits are thought to function in the regulation of catalysis or in the assembly of the holoenzyme (3). The main function of cytochrome oxidase, the terminal enzyme in the respiratory chain, is energy conservation. This hetero-oligomeric lipoprotein complex of the mitochondrial inner membrane is a molecular pump that uses redox chemistry to drive protons from the mitochondrial matrix across the membrane (4). Besides the eleven enzyme subunits, about 60 more nuclear genes are known to be necessary for the activity of cytochrome oxidase (5,6), an unexpected and exciting problem for study.

In order to define the role of one more nuclear-encoded subunit, we have characterized a pet mutant assigned previously to complementation group G199. Pet mutants are yeast strains which, due to a recessive mutation in a nuclear gene affecting mitochondrial function, have lost the ability to grow on non-fermentable carbon sources, but grow normally on fermentable substrates (5).

The G199 gene was cloned by complementation of the inability to grow on glycerol/ethanol in mutant E880/U1, after transformation with a yeast genomic plasmid library. After subcloning, a 2.1-kb HindIII fragment still had the ability to restore respiration in the mutants. The sequence of this DNA segment contained the end of the PET111 gene (7), the COX7 gene (8,9) and a new ORF. A search at the Saccharomyces Genome Database (SGD), Stanford, CA, confirmed our results and showed that this region is located between coordinates 777570 and 779664 on yeast chromosome XIII. The function of this new ORF (YMR255W) was explored by phenotypic analysis after gene disruption.

Yeast strains and media

The genotypes and sources of yeast strains used in this study are described in Table 1. The nuclear pet mutants were isolated by mutagenesis of the respiratory competent haploid S. cerevisiae strain D273-10B/A1 with ethyl methanesulfonate as described previously (5).

Yeast growth media were YPD (2% glucose, 2% peptone, 1% yeast extract), YEPG (1% yeast extract, 2% peptone, 2% glycerol, 2% ethanol), WO (2% glucose, 0.67% yeast nitrogen base without amino acids (Difco), amino acids and other supplements were added as required at 20 µg/ml), and YPGal (2% galactose, 2% peptone, 1% yeast extract). All solid media contained 2% agar. Plasmid-containing Escherichia coli RR1 (D(gpt-proA)62 leuB6 thi-1 lacY1 hsdSB20 rpsL20 (Strr) ara-14 galK2 xyl-5 mtl-1 supE44 mcrBB) were grown in L-broth (0.5% yeast extract, 1.0% tryptone, 0.5% NaCl and 0.1% glucose) in the presence of 50 µg/ml ampicillin.

Transformation of S. cerevisiae

Mutant E880/U1 originated from a haploid spore derived from the sporulation of a diploid resulting from the mating of E880 and W303-1A. It was grown in 100 ml YPGal to an approximate density of 10⁶ cells/ml and the entire culture was transformed by the method of Schiestl and Gietz (10) with a yeast genomic library. The library (provided by Dr. Marion Carlson, Department of Human Genetics, Columbia University) consists of partial Sau3AI fragments (average length 12 kb) ligated to the BamHI site of YEp24 (11). Uracil-independent clones were selected on minimal glucose medium and were subsequently checked for respiratory competence by the ability to grow on glycerol-rich medium (YEPG). Plasmid DNA was isolated from independent uracil⁺ and glycerol⁺ transformants and amplified in Escherichia coli RR1. One plasmid (pG199/T1) that was checked as positive after transformation of the original recipient was used to subclone and characterize the nuclear DNA insert.

Subcloning and DNA sequencing

Restriction fragments from the plasmid pG199/T1 were subcloned into YEp352/351 (12) and pKS+ (Bluescript^® II KS (+) phagemid, Stratagene) and the DNA was sequenced by the method of Maxam and Gilbert (13). All restriction sites used for 5'-end labeling were crossed from neighboring sites and the sequence was confirmed from the complementary strands. The ends of the insert were sequenced by the method of Sanger et al. (14) using primers (New England Biolabs) adjacent to the BamHI cloning site in the YEp24 vector.

Construction of a deletion strain at ORF YMR255W

A 1.1-kb TaqI fragment (from pG199/T5) containing the entire ORF was treated with Pfu DNA polymerase (Stratagene) to generate blunt ends beginning 336 bp before the ORF's ATG and extending 211 bp after the termination codon. The fragment was ligated to the pKS+ vector previously digested with ApaI, BstXI and the Pfu DNA polymerase (for blunting), generating plasmid pKS/T8. To construct a disrupted allele of the YMR255W gene, plasmid pKS/T8 was digested with BstEII and EcoRV, a procedure that removes 137 bp internal to the ORF. The deleted plasmid was ligated to a 1.1-kb HindIII fragment containing the yeast URA3 gene. The disrupted orf::URA3 allele was recovered from this construction as a linear 2.0-kb PvuII fragment and was used to transform both the haploid W303-1A strain and the isogenic diploid strain W303 by the procedure of Rothstein (15).

DNA manipulation procedures and miscellaneous methods

Standard methods were used for the transformation of E. coli, the preparation of recombinant plasmid DNA, digestion of DNA with restriction endonucleases, isolation and ligation of DNA fragments, and nick translation (16). The isolation of yeast nuclear DNA and the conditions for Southern analysis have been described elsewhere (17).

Functional analysis

Media used to test the growth of gene disruptants included complete glucose medium (YPD) or synthetic medium with glucose (WO) and also both media supplemented with the following carbon sources replacing glucose: 2% fructose, 2% galactose, 2% maltose, 2% raffinose, 2% potassium acetate, 2% lactate, and 2% glycerol at three different pH values (pH 3.5, pH 6.6 and pH 8.5). Plates with the test media were incubated at three different temperatures (18, 30 and 37^oC). The proper haploid derivatives were mated to generate diploids homozygous for the disruption of YMR255W. The sporulation of these cells was induced and examined as described by Rockmill et al. (18).

Phenotype of COX7 mutants

E880 is the mutant from the pet mutant collection of Tzagoloff and Dieckmann (5) carrying the mutation allelic to the complementation group G199. The mutant is unable to grow on non-fermentable carbon sources and is complemented by mating to a rho^o tester strain, indicating that the respiratory deficiency is caused by a recessive mutation in a nuclear gene. The phenotype of this mutant suggests that the respiratory deficiency stems from a specific defect in cytochrome oxidase. Mitochondria of the mutants lack cytochromes a and a₃ but have the absorption bands corresponding to cytochromes b, c and c1 (data not shown).

Cloning and sequencing of COX7 and ORF YMR255W

To clone the gene carrying the mutation in E880, strain E880/U1 was transformed with a yeast genomic library. Five of the uracil-independent and respiratory-competent clones obtained from the transformation were found to have plasmids with related inserts of yeast nuclear DNA. The nuclear DNA insert of those plasmids was ascertained to be approximately 9 kb long. An XbaI fragment of 6.9 kb was obtained from the original recombinant plasmid YEp24 and ligated to the XbaI site in the polylinker of plasmid YEp352 originating recombinant plasmid pG199/T2 (Figure 1). Initially, a total of three subclones were generated from this XbaI fragment (T3, T4 and T5; Figure 1). T3 is a 2.9-kb EcoRV fragment, T4 is a 3.2-kb XbaI-HindIII fragment and T5 is a 2.1-kb HindIII fragment. The DNA fragments were cloned in YEp352 and their ability to complement the respiratory-deficient phenotype of G199 mutants was investigated. The 2.1-kb recombinant plasmid pG199/T5 was the smallest complementing clone and its sequence was determined. It was found that this region contained the C-terminal end of the PET111 gene, the COX7 gene and another open reading frame of sufficient length to qualify as a protein-coding gene. Three new subclones were generated from pG199/T5. Subclone pG199/T6 (EcoRV-HindIII, 1.3 kb; Figure 1), that contained the entire COX7 gene and part of gene YMR255W, complemented the respiratory defect in the mutants. Subclone pG199/T7 (HindIII-EcoRV, 0.7 kb; Figure 1) contained part of gene YMR255W and did not complement mutants. Subclone pG199/T8 was constructed (1.1 kb, TaqI fragment from pG199/T5; Figure 1) to contain the entire YMR255W gene including its upstream (promoter) region. The sequence of this region is shown in Figure 2 with the centromere proximal region located at the beginning and the telomere proximal region at the end of the sequence. The ORF of YMR255W encodes a polypeptide of unknown function with 188 amino acids and a calculated molecular mass of 21,584 Da (Figure 2) without significant homology to any known protein.

Figure 1
- Partial restriction maps of pG199/T2 and derivative plasmids. The larger insert (T2) is a 6.9-kb fragment of nuclear DNA ligated at the XbaI site of vector YEp352. The locations in YEp352 of the URA3 and the ß-lactamase genes are shown. The bars in the upper part of the figure represent the size and position of the indicated subclones relative to the larger (T2) insert. The arrows below the restriction map represent the main ORFs present in this segment of chromosome XIII. The two TaqI sites that define subclone T8 are indicated. The plus signs denote the recombinant plasmids that complement the respiratory deficiency of group G199 mutants.

Figure 2
- Nucleotide sequence of YMR255W and flanking regions. The sense DNA strand from the TaqI fragment (pG199/T8) and the translated reading frame corresponding to YMR 255W are shown (GenBank accession number AF007064). Restriction enzyme sites used for cloning, disruption and probe preparation are identified.

In situ disruption of the YMR255W gene

Plasmid pKS/T8 was constructed to explore the function of this new gene by one-step gene disruption. A null allele was created by insertion of the yeast URA3 gene replacing the BstEII-EcoRV fragment (137 bp) internal to the coding sequence (Figure 3). The disrupted allele was isolated on a linear (PvuII) fragment and used to transform the respiratory-competent haploid strain W303-1A and the isogenic diploid strain W303. Southern analysis showed that the uracil-independent clones obtained from the transformation had acquired the disrupted ymr255w::URA3 allele. Nuclear DNA from the parental strains, as well as from each transformant, was digested with HindIII and probed with a 833-bp KpnI-SacI fragment of pKS/T8. The probe recognizes a 2.1-kb fragment in the genomic DNA of the parental strain. The genomic DNA of the knockout mutant strain has a new species cross-hybridizing at about 3.1 kb, the size expected after the manipulations used in the construction and after the insertion of the 1166-bp fragment bearing the URA3 gene (Figure 3).

Figure 3
- Southern hybridization analysis of yeast genomic DNA from representative isolates of strain W303 after disruption of YMR255W. The location of the restriction sites is marked along the line that represents the DNA. The ORFs and respective orientations are shown below. A, Map of the YMR255W region indicating the BstEII and EcoRV sites used to delete a 137-bp internal fragment of the gene before the insertion of a 1166-bp DNA fragment carrying the URA3 gene. B, Map of the same chromosomal region after gene replacement with the construction explained above. C, Electrophoretogram of total yeast DNA prepared from the control strain W303-1A (lanes 1 and 5) or from individual transformants from experiments done with the W303-1A haploid strain (lanes 2, 3 and 4) or with the diploid W303 strain (lanes 6, 7 and 8). About 0.2 µg of DNA was digested with HindIII, fractionated by electrophoresis on 1% agarose gel and blotted onto Hybond N. The Southern blot was hybridized to the labeled TaqI-SacI fragment (probe) and exposed to X-ray film. The size of the fragments detected is indicated on the left-hand margin.

Phenotypic analysis of ORF YMR255W disruptants

The YMR255W gene was successfully disrupted in a haploid strain. Consequently the gene knockout is viable. Growth behavior of the disruptant in the different media and under the conditions listed in the Material and Methods section failed to disclose a behavior distinct from the wild-type strain. The disruptants also presented a normal frequency in the generation of spontaneous cytoplasmic petite mutants.

The HindIII fragment characterized by sequencing is located between coordinates 777570 and 779664 on the right arm of chromosome XIII. The gene order (Figure 1) is centromere proximal, YMR255W, YMR 256C (COX7), and YMR257C (PET111). Our sequence analysis placed these genes together for the first time. This has been fully confirmed by the results of the systematic yeast genome sequencing project. The phenotype of the mutant E880 that originated this study was respiratory incompetence. The yeast strains belonging to this group were unable to grow on non-fermentable substrates and upon spectral and enzymatic examination revealed the absence of cytochrome c oxidase activity and a lack of the spectral bands assigned to this respiratory complex (heme a-a₃). The activities of the other membrane enzymes were present (data not shown). Here we demonstrate that the mutant phenotype could be completely rescued by transformation of the mutant with plasmid pG199/T6 carrying a 1.4-kb fragment that contained only the COX7 gene and its promoter region. This gene has been sequenced previously (8,9) and the sequence of the fragments amplified by PCR using degenerate primers corresponded to the amino- and carboxyl-terminal regions of the sequenced subunit VII polypeptide. We now used a genetic method that independently led to the cloning of the same gene. Although cytochrome oxidase in yeast or mammals is a complex enzyme with 11 to 13 subunits, studies with the corresponding Paracoccus denitrificans enzyme showed that the mininal unit required for electron transfer-linked proton pumping consists of subunits I and II (4). Current research aims to understand the function of the other subunits. The recent elucidation of the three-dimensional structure of the mammalian enzyme (19) will certainly direct new attempts to define the role of the accessory polypeptides such as subunit VII (COX7). Another interesting problem is the detection and study of the many nuclear genes that affect cytochrome oxidase activity without being part of the purified active enzyme (6).

One of the genes adjacent to COX7 is PET111 (7). This is also a respiratory gene that functions as a translational factor for the mitochondrially encoded COX2 mRNA requiring the 5'-untranslated portion of the messenger (20). The presence of two neighboring respiratory genes made us wonder if the next gene YMR255W, of unknown function, had a mitochondrial role. To that effect a specific null mutant was generated. The disruption of this gene behaved as many other inactivated yeast genes (21), i.e., its knockout does not result in an observable phenotype as examined by growth in rich and minimal media with different carbon sources and at different temperatures. The stability of the mitochondrial genome was found to be normal and was estimated by measuring the spontaneous generation of petite mutants. Also, the null mutant did not affect mating or sporulation in tests carried out with the appropriate haploids or the homozygous diploid yeast. The usual assumption in those cases is that the genome harbors at least one functional equivalent of the disrupted gene or that the experimental conditions tested in the laboratory do not include the physiological demanding conditions that would require this gene product.

Examination of the protein sequence encoded by YMR255W as analyzed by the Yeast Proteome Database shows that the 188-amino acid basic polypeptide (calculated pI of 9.95) is probably a gene with low abundance mRNA as suggested by the codon adaptation index of 0.138 (22) and low codon bias (0.039) (23). The sequence does not show any transmembrane domains but there are regions with the potential to generate coiled-coil domains (24) and potential phosphorylating sites for Cdc28 kinase and cAMP-dependent protein kinase (Yeast Proteome Database) as well as putative N-glycosylation sites (Figure 4). There is no extensive homology to any known protein. A similarity search within the yeast genome using the FASTA algorithm (25) through the SGD revealed 13 ORFs, 8 belonging to genes of unknown function and 5 to genes already named, all with 20 to 29% identity in overlaps that range from 120 to 188 amino acids. Another search using the National Center for Biotechnology Information (NCBI) BLAST program (26) against all non-redundant sequences deposited showed two or three regions (10 to 38 amino acids) of similarity in three additional yeast genes: TAF61, a subunit of transcription factor TFIID (27), ROD1, resistance to O-dinitrobenzene and ions such as zinc and calcium (28), and MLP1, a probable coiled-coil protein with myosin-like motifs and possible involvement in DNA repair (29). These proteins have similarity to YMR255W in its central region (residues 66 to 104 approximately) and also in the C-terminal region. Such similarities could eventually resolve the problem of finding a function for this gene. Further phenotypic testing (30), over-expression of the gene and a search for a synthetic lethal mutant (31) could provide important new clues.

Figure 4
- Derived amino acid sequence corresponding to the coding region of gene YMR 255W. Sequence motifs identified by the Yeast Proteome Database as putative targets for N-glycosylation (A), protein kinase A (B) or Cdc28 kinase (C) are underlined. Heptad sequences that represent potential coiled-coil structures have been identified (24) and are displayed in bold characters.

We thank Susy Coelho and Sandra M.M. Nascimento for technical help.

Address for correspondence: M.P. Nobrega, Departamento de Microbiologia, ICB, USP, Av. Prof. Lineu Prestes, 1374, 05508-900 São Paulo, SP, Brasil. E-mail: [e-mail]

Research supported by FAPESP (No. 96/0665-5), FINEP (No. 66.93.0618.00) and CNPq (No. 523941/94-3). M.A.S. Graminha is the recipient of a CAPES fellowship and E.N. Troitskaya was the recipient of a visiting fellowship from FAPESP (No. 93/4301-0). The present address of E.N. Troitskaya is A.N. Bach Institute of Biochemistry, Leninsky Prospect 33, Moscow, 117 071 Russia. Received June 25, 1997. Accepted October 28, 1997.

• 1. Taanman J & Capaldi RA (1992). Purification of yeast cytochrome c oxidase with a subunit composition resembling the mammalian enzyme. Journal of Biological Chemistry, 267: 22481-22485.
• 2. Raitio M, Salli T & Saraste M (1987). Isolation and analysis of the genes of cytochrome c oxidase in Paracoccus denitrificans EMBO Journal, 6: 2825-2833.
• 3. Buchwald P, Krummeck G & Rödel G (1991). Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane. Molecular and General Genetics, 229: 413-420.
• 4. Trumpower BL & Gennis RB (1994). Energy transduction by cytochrome complexes in mitochondrial and bacterial respiration: the enzymology of coupling electron transfer reactions to transmembrane proton translocation. Annual Review of Biochemistry, 63: 675-716.
• 5. Tzagoloff A & Dieckmann CL (1990). Pet genes of Saccharomyces cerevisiae Microbiological Reviews, 54: 211-225.
• 6. Poyton RO & McEwen JE (1996). Crosstalk between nuclear and mitochondrial genomes. Annual Review of Biochemistry, 65: 563-607.
• 7. Strick CA & Fox TD (1987). Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5'-leader. Molecular and Cellular Biology, 7: 2728-2734.
• 8. Calder KM & McEwen JE (1990). Nucleotide sequence of the gene encoding cytochrome c oxidase subunit VII from Saccharomyces cerevisiae Nucleic Acids Research, 18: 1632.
• 9. Aggeler R & Capaldi RA (1990). Yeast cytochrome c oxidase subunit VII is essential for assembly of an active enzyme: cloning, sequencing and characterization of the nuclear-encoded gene. Journal of Biological Chemistry, 265: 16389-16393.
• 10. Schiestl RH & Gietz RD (1989). High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Current Genetics, 16: 339-346.
• 11. Rose M, Grisafi P & Botstein D (1984). Structure and function of the yeast URA3 gene: expression in Escherichia coli Gene, 29: 113-124.
• 12. Hill JE, Myers AM, Koerner TJ & Tzagoloff A (1986). Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast, 2: 163-167.
• 13. Maxam AM & Gilbert W (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology, 65 (Part I): 449-560.
• 14. Sanger F, Niklen S & Coulson AR (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, USA, 74: 5463-5467.
• 15. Rothstein RJ (1983). One step gene disruption in yeast. Methods in Enzymology, 101: 202-211.
• 16. Sambrook J, Fritsch EF & Maniatis T (1989). Molecular Cloning - A Laboratory Manual 2nd edn. Cold Spring Harbor Laboratory Press, New York.
• 17. Holm C, Meeks-Wagner DW, Fangman W & Botstein D (1986). A rapid, efficient method for isolating DNA from yeast. Gene, 42: 169-173.
• 18. Rockmill B, Lambie EJ & Shirleen Roeder G (1991). Spore enrichment. Methods in Enzymology, 194: 146-149.
• 19. Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-Itoh K, Nakashima R, Yaono R & Yoshikawa S (1996). The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 Ĺ. Science, 272: 1136-1144.
• 20. Mulero JJ & Fox TD (1993). PET111 acts in the 5'-leader of the Saccharomyces cerevisiae mitochondrial COX2 mRNA to promote its translation. Genetics, 133: 509-516.
• 21. Skala J, Purnelle B, Crouzet M, Aigle M & Gouffeau A (1991). The open reading frame YCR101 located on chromosome III from Saccharomyces cerevisiae is a putative protein kinase. Yeast, 7: 651-655.
• 22. Sharp PM & Cowe E (1991). Synonymous codon usage in Saccharomyces cerevisiae Yeast, 7: 657-678.
• 23. Bennetzen JL & Hall BD (1982). Codon selection in yeast. Journal of Biological Chemistry, 257: 3026-3031.
• 24. Fischetti VA, Landau GM, Schmidt JP & Sellers P (1993). Identifying periodic occurrences of a template with applications to protein structure. Information Processing Letters, 45: 11-18.
• 25. Pearson WR & Lipman DJ (1988). Improved tools for biological sequence comparison. Proceedings of the National Academy of Sciences, USA, 85: 2444-2448.
• 26. Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ (1990). Basic local alignment tool. Journal of Molecular Biology, 215: 403-410.
• 27. Moqtaderi Z, Yale JD, Struhl K & Buratowski S (1996). Yeast homologues of higher eukaryotic TFIID subunits. Proceedings of the National Academy of Sciences, USA, 93: 14654-14658.
• 28. Wu AL, Hallstrom TC & Moye-Rowley WS (1996). ROD1, a novel gene conferring multiple resistance phenotypes in Saccharomyces cerevisiae Journal of Biological Chemistry, 271: 2914-2920.
• 29. Kolling R, Nguyen T, Chen EY & Botstein D (1993). A new yeast gene with a myosin-like heptad repeat structure. Molecular and General Genetics, 237: 359-369.
• 30. Smith V, Chou KN, Lashkari D, Botstein D & Brown PO (1996). Functional analysis of the genes of yeast chromosome V by genetic footprinting. Science, 274: 2069-2074.
• 31. Bender A & Pringle JR (1991). Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae Molecular and Cellular Biology, 11: 1295-1305.

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  07 Oct 1998
• Date of issue
  Mar 1998

History

• Accepted
  28 Oct 1997
• Received
  25 June 1997