Large-scale phenotypic analysis—the pilot project on yeast chromosome III

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome. © 1997 John Wiley & Sons, Ltd.     

A Review of Phenotypes in Saccharomyces cerevisiae

A summary of previously defined phenotypes in the yeast Saccharomyces cerevisae is presented. The purpose of this review is to provide a compendium of phenotypes that can be readily screened to identify pleiotropic phenotypes associated with primary or suppressor mutations. Many of these phenotypes provide a convenient alternative to the primary phenotype for following a gene, or as a marker for cloning a gene by genetic complementation. In many cases a particular phenotype or set of phenotypes can suggest a function for the product of the mutated gene. © 1997 John Wiley & Sons, Ltd.     

Sequencing the yeast genome: An international achievement

The yeast genome is currently being sequenced by a Consortium of European laboratories, in collaboration with a wider international network of researchers. It is expected that within the next two years Saccharomyces cerevisiae will become the first eukaryotic organism to have been completely genetically mapped and sequenced. This article traces the sequencing enterprise from its beginnings, outlining the intentions, the organisation, and the achievements so far. The tasks which remain are discussed, emphasising the follow-on research into the evolution of primitive karyotypes, and, more particularly, into the nature of novel genes revealed during sequencing. The functional analysis of novel genes is attracting an ever wider community of yeast scientists, so that research which began with a decision to sequence a simple genome promises to remain a focus for international cooperation.     

A computer filtering method to drive out tiny genes from the yeast genome

The authors of the first yeast chromosome sequence defined a minimum threshold requirement of 100 codons, above which an open reading frame (ORF) is retained as a putative coding sequence. However, at least 58 yeast genes shorter than 100 codons have an assigned protein function. Therefore, the yeast genome may contain other tiny but functionally important genes that are discarded from analyses by this simple filtering rule. We have established discriminant functions from the in-phase hexamer frequencies of functional genes and of simulated ORFs derived from a stationary Markov chain model. Fifty-two out of the 58 genes were recognized as coding ORFs by our discriminating method. The test was also applied to all the small ORFs (36 to 100 codons) found in the intergenic regions of published chromosomes. It retained 140 new potential tiny coding sequences, among which we identified seven new genes by similarity searches. Our method, used conjointly with similarity searches, can also highlight sequencing errors resulting from the disruption of the coding frame of longer ORFs. This method, by its ability to detect potential coding ORFs, can be a very useful tool for functional analysis.     

Sequence and analysis of 24 kb on chromosome II of Saccharomyces cerevisiae

In the course of the European yeast genome sequencing project, we determined 23,920 bp of a continuous chromosome II right arm sequence. Analysis of data revealed 13 open reading frames (ORFs), three of which corresponded to previously identified genes; two tRNA genes and one repetitive element. One ORF showed considerable homology (46%) to a hypothetical chromosome III gene; another, putatively very hydrophobic gene product, was 30% identical to the heat-shock protein HSP30. Two ORFs were homologous to human genes. The complete sequence was submitted to the EMBL data bank under the Accession Number Z46260 Authorin submission ‘3’.     

Analysis of a 23 kb region on the left arm of yeast chromosome IV

We have determined the complete nucleotide sequence of a 23 kb segment from the left arm of chromosome IV, which is carried by the cosmid 1L10. This sequence contains the 3′ coding region of the STE7 and RET1 (COP1) genes, and 13 complete open reading frames longer than 300 bp, of which ten correspond to putative new genes and three (CLB3, MSH5 and RPC53) have been sequenced previously. The sequence from cosmid 1L10 was obtained entirely by a combined subcloning and walking primer strategy.     

Sequence and functional analysis of a 7·2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II

In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79·2 kDa (corresponding gene YBR156c), 12·1 kDa (YBR157c), 62·7 kDa (YBR158w) and 38·7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grr1 (Cat80) protein with respect to the leucine-rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains. The DNA sequence was deposited at the EMBL data base with accession numbers Z36025 (YBR156c), Z36026 (YBR157c), Z36027 (YBR158w) and Z36028 (YBR159w).     

Analysis of 21·7 kb DNA Sequence from the Left Arm of Chromosome VII Reveals 11 Open Reading Frames: Two Correspond to New Genes

The DNA sequence of a fragment of 21 731 bp (nucleotides 87408 to 109138) located on the left arm of chromosome VII from Saccharomyces cerevisiae S288C has been determined using a random cloning strategy followed by an oligonucleotide-directed sequencing. This fragment contains eight complete genes previously sequenced (CLG1, SKI8, VAM7, YPT32, MIG2, SIP2, SPT16 and CHC1), the 5′ part of POX1 and two other complete unidentified open reading frames of more than 100 amino acids. © 1997 John Wiley & Sons, Ltd.     

Suitability of replacement markers for functional analysis studies inSaccharomyces cerevisiae

The complete yeast sequence contains a large proportion of genes whose biological function is completely unknown. One approach to elucidating the function of these novel genes is by quantitative methods that exploit the concepts of metabolic control analysis. An important first step in such an analysis is to determine the effects of deleting individual genes on the growth rate (or fitness) of Saccharomyces cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, they are most readily determined by competition against a standard strain in chemostat cultures where the true steady state demanded by metabolic control analysis may be achieved. We have constructed two different standard strains in which the HO gene is replaced by either HIS3 or kanMX. We demonstrate that HO is a selectively neutral site for gene replacement. However, there is a significant marker effect associated with HIS3 which, moreover, is dependent on the physiological conditions used for the competition experiments. In contrast, the kanMX marker exhibited only a small effect on specific growth rate (≤±4%). These data suggest that nutritional markers should not be used to generate deletion mutants for the quantitative analysis of gene function in yeast but that kanMX replacements may be used, with confidence, for such studies. © 1997 John Wiley & Sons, Ltd.     

XI. Yeast sequencing reports. The yeast YKL741 gene situated on the left arm of chromosome XI codes for a homologue of the human ALD protein

The yeast gene YKL741 is situated on the left arm of chromosome XI, 12 kb closer to the centromere with respect to the previously localized PAS1 gene. The new yeast gene codes for a homologue of the human ALD protein (ALD: adrenoleukodystrophy). The similarity between the YKL741 protein and the ALD protein is very high in the C-terminal half, which contains an ATP-binding cassette characteristic of the ABC family of transporters. Additionally the YKL741 protein shows some similarity to the ALD protein in the N-terminal half in three putative transmembrane spanning domains. The sequence has been deposited in the EMBL data library under Accession Number X76133 SC YKL.     

Discrimination between fortuitous and biologically constrained open reading frames in DNA sequences of Saccharomyces cerevisiae

The systematic sequencing of the yeast genome has raised the problem of the biological significance of the open reading frames (ORFs) revealed: it is possible that some of these are fortuitous. To avoid the analysis of such fortuitous ORFs, a minimum length of 100 sense codons was adopted. Nevertheless, the presence of fortuitous ORFs of more than 100 codons cannot be excluded. Thus, in the context of functional analysis, a method for discrimination between fortuitous and biologically active ORFs may be useful. The discrimination method described here is based on multiple criteria: ORF length, codon bias, and both amino-acid and dipeptide composition of the corresponding polypeptide. The thresholds for each criterion are based on the comparison between two learning sets: one drawn from random DNA sequences and the second from known genes. The method was validated by two test sets (one random and one biological) and then applied to the ORFs of chromosomes I, II, III, V, VIII, IX and XI. This method predicts 123 fortuitous ORFs among the 1773 identified on these chromosomes.     

传统诱变与基因组重排技术相结合选育耐高温酿酒酵母菌株

以酿酒酵母AY12为出发菌株,采用传统人工诱变育种与基因组重排技术相结合的方法选育出耐高温且高产乙醇的酿酒酵母菌株.通过将出发菌株进行紫外诱变,获得酿酒酵母突变群体,并从中筛选出耐高温且高产乙醇的23株突变株作为正向突变文库;将该文库通过酵母高效产孢与杂交反应实现突变文库的全基因组重排,筛选得到的基因组重排最优菌LYQ-F1的高温耐受性较出发菌株AY12明显提高,且在高温模拟发酵工艺中其乙醇产量较出发菌株提高了22.1％.     

ARTP诱变以及基因组重排筛选具有耐高温性能的酿酒酵母

在工业生产中,酿酒酵母进行高温发酵具有发酵速度快、发酵周期短、发酵原料黏度低、生产成本少并且更适用于同步糖化发酵工艺等优点,因此,选育耐高温的酿酒酵母菌株在工业应用方面具有重要意义。本研究以从工业菌种中分离出来的酿酒酵母AY12作为出发菌株,对其进行常压室温等离子体诱变,在37℃下进行初筛,得到150株单菌落长势较好的菌株。将其进行产孢、杂交以实现基因组重排,在37℃下进行复筛,得到137株生长性能良好的菌株。在35℃玉米水解液中发酵,进行二次复筛,从而获得具有良好发酵性能的14株菌株。最后,进行35℃同步糖化发酵,并测定每菌株的乙醇产量和残糖含量。,最终筛选出7株具有良好耐高温发酵性能的菌株,其中发酵性能最好的菌株为L-38,其同步糖化发酵完成后,乙醇产量为16.20%(V/V),较出发菌株AY12提升了8.00%,残糖含量为35.50 g/L,较AY12降低了32.38%。     

The Characterization of Two New Clusters of Duplicated Genes Suggests a ‘Lego’ Organization of the Yeast Saccharomyces cerevisiae Chromosomes

The systematic sequencing of 42 485 bp of yeast chromosome VII (nucleotides 377948 to 420432) has revealed the presence of 27 putative open reading frames (ORFs) coding for proteins of at least 100 amino acids. The degree of redundancy observed is elevated since five of the 27 ORFs are duplications of a previously identified gene. These duplicated copies may be classified in two types of cluster organization. The first type includes genes sharing a significant level of identity in the amino acid sequences of their predicted protein product. They are recovered on two different chromosomes, transcribed in the same orientation and the distance between them is conserved. The second type of cluster is based on one gene unit tandemly repeated. This duplication is itself repeated elsewhere in the genome. The level of nucleic acid identity is high within the coding sequence and the non-coding region between the two repeats. In addition, the basic gene unit is recovered many times in the genome and is a component of a multigene family of unknown function. These organizations in clusters of genes suggest a ‘Lego organization’ of the yeast chromosomes, as recently proposed for the genome of plants (Moore, 1995). The sequence is deposited in the Yeast Genome Databank under Accession Number from Z72562 to Z72586. © 1997 John Wiley & Sons, Ltd.