`Mass-murder' of ORFs from three regions of chromosome XI from Saccharomyces cerevisiae1
Introduction
The yeast genome contains roughly 6000 protein-coding genes, two-thirds of which were discovered by the systematic sequencing program (Goffeau et al., 1996). At present, 43% of yeast genes are functionnally assigned either experimentally or because of the highly significant structural homology of their product to proteins with known biochemical functions (Mewes et al., 1997). The challenge now is to characterize the remaining 3400 ORFs. Classical genetics methods centred on given cellular functions may identify some of these genes, but these methods have been extensively applied over the years and progress may be painstakingly slow. Therefore, new systematic approaches are needed (Dujon, 1996). One of these is the analysis of the transcriptome, such as transcript maps from whole chromosomes (Tanaka and Isono, 1993, Richard et al., 1997) and more recently from the whole genome by novel methods such as SAGE (Velculescu et al., 1997) and hybridization microarrays (DeRisi et al., 1997). The yeast proteome has begun to be characterized by two-dimensional gel electrophoresis (Shevchenko et al., 1996; Fey et al., 1997), and systematic mapping of yeast protein interactions has begun using the two-hybrid system (Fields, 1997; Fromont-Racine et al., 1997).
Gene inactivation and examination of resulting phenotypes have played a considerable role in yeast genetics. The availability of the sequence, the large but defined number of genes to study and the efficiency of integrative transformation and homologous recombination in yeast open the way to systematic directed gene inactivation. The largest such program is part of EUROFAN, and others include chromosome-by-chromosome analyses [for a review, see Dujon (1998)]. EUROFAN is putting an initial emphasis on c. 1000 unknown genes, and there is a project to delete all 6000 yeast genes, one by one. Deleted strains are submitted to standard phenotypic assays (e.g. Huang et al., 1997) and are amenable to more specialized assays, characteristic of specific functions.
Before systematic sequencing, the `gene-number paradox' was already apparent from the fact that the number of genes giving selectable phenotypes when mutated is only a fraction of the total (Kaback et al., 1984; Goebl and Petes, 1986). Subsequent gene disruptions and mutageneses consolidated this paradox (Diehl and Pringle, 1991; Hampsey, 1997). It can be postulated that many genes are not needed for life in laboratory conditions but may be needed in specific environments, and/or that they are functionnally redundant. Furthermore, essential genes are relatively rare in yeast, representing 12–18% of all genes (Goebl and Petes, 1986; Chun and Goebl, 1996). These facts prompted us to develop a strategy in which several adjacent genes are deleted simultaneously so as to decrease the number of mutant strains to pass through the specialized functional assays from EUROFAN. This is a hierarchical strategy in which the primary phenotypic screening is performed on groups of genes that are then reinvestigated down to the single gene level. This strategy has been tested on 214 ORFs from chromosome XI which were not previously characterized. We describe here the design of the overall strategy, and its application to three regions spanning c. 70 kb and including 22 new ORFs from chromosome XI, along with the phenotypic analysis of the resulting mutants.
Section snippets
Nomenclature
The definition and nomenclature of chromosome XI ORFs were as described in Dujon et al. (1994)and Goffeau et al. (1996). In this work, inter-ORF regions were numbered, starting from the centromere, with ykl indicating the left arm, and ykr indicating the right arm. Inter-ORF names appear in the oligo and plasmid nomenclature (Table 1Table 2Table 3Table 5).
Amplification and cloning of TSH in split-marker vectors
This is summarized in Fig. 1 and was carried out as in Fairhead et al. (1996). TSH, target sequence homologs, are the regions obtained by PCR
Rationale
In order to accelerate the mapping of mutant phenotypes, we have introduced a strategy based on partially overlapping deletions and on hierarchical transformations and phenotypic analyses. This strategy has been used on three regions of chromosome XI. Partially overlapping deletions of several genes allow faster mapping of phenotypes than single deletions as illustrated by a theoretical example in Fig. 2. Here, three deletions define five genes unambiguously. The hierarchical protocol followed
Discussion
We have developed a hierarchical strategy for functional analysis of the yeast genome, which we have applied to 22 genes from chromosome XI. Although this is a limited number, the results are illustrative of such analyses (Table 6). Sixteen out of 22 deleted ORFs (73%) exhibit no mutant phenotype, three are essential (13.5%), two confer a growth rate defect, and one suppresses the yeast's ability to grow on glycerol. All three ORFs shown by us to be essential have also been characterized by
Acknowledgements
We thank our colleagues of the Unité de Génétique Moléculaire des Levures for advice and discussions. B.D. is Professor of Molecular Genetics at the Univ. P. et M. Curie and member of the Institut Universitaire de France. This work was supported by grant BIO4-CT95-0080 from the DGXII of the European Commission.
References (34)
- et al.
The primary structure of rat ribosomal protein L14
Biochem. Biophys. Res. Commun.
(1996) - et al.
Cloning and characterization of LAG1, a longevity-assurance gene in yeast
J. Biol. Chem.
(1994) The yeast genome project: what did we learn?
Trends Genet.
(1996)- et al.
Most of the yeast genomic sequences are not essential for cell growth and division
Cell
(1986) - et al.
Complete transcriptional map of yeast chromosome XI in different life conditions
J. Mol. Biol.
(1997) - et al.
Characterization of the yeast transcriptome
Cell
(1997) - et al.
Screening and identification of yeast sequences that cause growth inhibition when overexpressed
Mol. Gen. Genet.
(1997) - et al.
Subunits of the S. cerevisiae signal recognition particle required for its functional expression
EMBO J.
(1994) - et al.
Two S. cerevisiae genes which control sensitivity to G1 arrest induced by K. lactis toxin
Mol. Cell. Biol.
(1994) - et al.
The identification of transposon-tagged mutations in essential genes that affect cell morphology in S. cerevisiae
Genetics
(1996)
Complete inventory of the yeast ABC proteins
Nature Genet.
Exploring the metabolic and genetic control of gene expression on a genomic scale
Science
Molecular analysis of S. cerevisiae chromosome I: identification of additional transcribed regions and demonstration that some encode essential functions
Genetics
The complete DNA sequence of chromosome XI of S. cerevisiae (666 kb)
Nature
EUROFAN and the functional analysis of the Saccharomyces cerevisiae genome
Electrophoresis
Consequences of unique double strand breaks in yeast chromosomes: death or homozygosis
Mol. Gen. Genet.
New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using `split-marker' recombination
Yeast
Cited by (0)
- 1
Published in conjunction with A Wisconsin Gathering Honoring Waclaw Szybalski on occasion of his 75th year and 20years of Editorship-in-Chief of Gene, 10–11 August 1997, University of Wisconsin, Madison, WI, USA.
- 2
Present address: Laboratoire d'Ecophysiologie et Biochimie Métabolique, Université du Maine, Avenue Olivier Messiaen, F-72085 Le Mans Cedex 9, France.
- 3
Present address: Institut für Biochemie der RWTH, Pauwelsstrasse 30 D-52057 Aachen, Germany.