A new family of yeast vectors and S288C-derived strains for the systematic analysis of gene function.

The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2 Delta 0 ade8 Delta 0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.

Exploring redundancy in the yeast genome: an improved strategy for use of the cre-loxP system.

Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome. As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed. We constructed three replacement cassettes in which different selectable markers were placed between two loxP loci. Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers (kanMX, LYS2, KlURA3 and SpHIS5). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains.

Genes encoding three new members of the leukocyte antigen 6 superfamily and a novel member of Ig superfamily, together with genes encoding the regulatory nuclear chloride ion channel protein (hRNCC) and an N omega-N omega-dimethylarginine dimethylaminohydrolase homologue, are found in a 30-kb segment of the MHC class III region.

Many of the genes in the class III region of the human MHC encode proteins involved in the immune and inflammatory responses. We have sequenced a 30-kb segment of the MHC class III region lying between the heat shock protein 70 and TNF genes as part of a program aimed at identifying genes that could be involved in autoimmune disease susceptibility. The sequence analysis has revealed the localization of seven genes, whose precise position and order is cen-G7-G6-G6A-G6B-G6C-G6D-G6E-tel, five of which are fully encoded in the sequence, allowing their genomic structures to be defined. Three of them (G6C, G6D, and G6E) encode putative proteins that belong to the Ly-6 superfamily, known to be GPI-anchored proteins attached to the cell surface. Members of the family are specifically expressed and are important in leukocyte maturation. A fourth gene, G6B, encodes a novel member of the Ig superfamily containing a single Ig V-like domain and a cytoplasmic tail with several signal transduction features. The G6 gene encodes a regulatory nuclear chloride ion channel protein, while the G6A gene encodes a putative homologue of the enzyme N omega,N omega-dimethylarginine dimethylaminohydrolase, which is thought to be involved in regulating nitric oxide synthesis. In addition, three microsatellite markers, 9N-1, 82-2, and D6S273 are contained within the sequence, the last two of which have been reported to be strongly associated with the autoimmune disease ankylosing spondylitis.

An integrated map of human chromosome 6p23.

The human chromosomal band 6p23 is a Giemsa-negative (light) band that may be expected to be relatively gene rich. The genes for spinocerebellar ataxia type 1 (SCA1), guanosine monophosphate reductase (GMPR), DEK involved in a subtype of acute myeloid leukemia (AML), and the folate-sensitive fragile site FRA6A, have already been mapped to 6p23. Recent linkage data have suggested evidence for a susceptibility locus for schizophrenia in the region. We have constructed a single YAC contig of approximately 100 clones spanning the entire 6p23 band from 6p22.3 to 6p24.1 and covering 7.5-8.5 Mb of DNA. The YAC contig contains 55 markers including genetically mapped STSs, physically mapped STSs, anonymous STSs, anonymous ESTs, and ESTs from the genes mapped to the region. The order of the genetically mapped STSs is consistent with their order in the contig and some of the markers not resolved on the genetic map have been resolved by the YACs. Four of the YACs from 6p23 and covering approximately 3 Mb of DNA have been used to isolate approximately 300 cosmids from a flow-sorted human chromosome 6 cosmid library, which have been organized into pockets. The proposed susceptibility locus for schizophrenia is most closely linked to D6S260, which is located within the YAC contig along with genetic markers < or = 5 cM on either side. Therefore, the presented materials are valuable reagents for characterization of the genomic region implicated in schizophrenia.