TRP1 Locus Research Articles

A TRP1-marker-based system for gene complementation, overexpression, reporter gene expression and gene modification in Candida glabrata.

ABSTRACTAlthough less prevalent than its relative Candida albicans, the yeast Candida glabrata is a successful pathogen of humans, which causes life-threatening candidiasis. It is thus vital to understand the pathogenicity mechanisms and contributing genes in C. glabrata. However, gene complementation as a tool for restoring the function of a previously deleted gene is not standardized in C. glabrata, and it is less frequently used than in C. albicans.In this study, we established a gene complementation strategy using genomic integration at the TRP1 locus. We prove that our approach can not only be used for integration of complementation cassettes, but also for overexpression of markers like fluorescent proteins and the antigen ovalbumin, or of potential pathogenicity-related factors like the biotin transporter gene VHT1. With urea amidolyase Dur1,2 as an example, we demonstrate the application of the gene complementation approach for the expression of sequence-modified genes. With this approach, we found that a lysine-to-arginine mutation in the biotinylation motif of Dur1,2 impairs urea-dependent growth of C. glabrata and C. albicans. Taken together, the TRP1-based gene complementation approach is a valuable tool for investigating novel gene functions and for elucidating their role in the pathobiology of C. glabrata.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in <em>Saccharomyces cerevisiae</em>

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9. Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-Δ3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-Δ5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in <em>Saccharomyces cerevisiae</em>

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-Δ3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-Δ5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild-type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.

A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae

Protein decay rates are regulated by degradation machinery that clears unnecessary housekeeping proteins and maintains appropriate dynamic resolution for transcriptional regulators. Turnover rates are also crucial for fluorescence reporters that must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamic responses. Here, we use components of the Escherichia coli degradation machinery to construct a Saccharomyces cerevisiae strain that allows for tunable degradation of a tagged protein. Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter. We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes. Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis–Menten-type interactions. In addition to providing a reporter with tunable dynamic resolution, our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways.

Construction of a Trp- commercial baker's yeast strain by using food-safe-grade dominant drug resistance cassettes.

We have designed a food-safe-grade module for gene disruptions in commercial baker's yeast strains, which contains the G418 resistance cassette, KanMX4, flanked by direct repeats from the MEL1 gene of Saccharomyces cerevisiae. This module was used to obtain a Trp(-) auxotrophic mutant of the polyploid HY strain by successive targeting to the TRP1 locus and later in vivo excision of the kan(r) marker. Southern blot analysis indicated that HY contains five copies of the TRP1 gene. However, after four disruption rounds, a strain named HYtrpM(4), unable to grow in the absence of tryptophan, was selected. Southern and Northern analysis of HYtrpM(4) cells showed that a remaining functional wild-type copy was still present, suggesting that the level of phosphoribosylanthranylate isomerase activity, resulting from a single copy of TRP1, is too low to sustain growth. Accordingly, a high reversion frequency of the Trp(-) phenotype, through gene conversion, was found in cells of the mutant strain. Nevertheless, this was not a drawback for its use as a recipient strain of heterologous genes. Indeed, YEpACT-X24 transformants were stable after 25 generations and expressed and secreted high levels of active recombinant xylanase. These data indicate that the new Trp(-) strain can be used to generate a stable recombinant yeast that fulfils all the requirements and market criteria for commercial utilisation.

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface.

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of alpha-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence.

T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis.

The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used.

The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively.

The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.

The Neurospora aab-1 gene encodes a CCAAT binding protein homologous to yeast HAP5.

The expression of the am (glutamate dehydrogenase) gene is dependent upon two upstream activating sequences, designated URSam(alpha) and URSam(beta). A heteromeric nuclear protein Am Alpha Binding protein (AAB) binds specifically to a CCAAT box within the URSam(alpha) element. AAB appears to be composed of three components. We used polyclonal antiserum raised against the highly purified AAB1 subunit to isolate a partial aab-1 cDNA clone, which was then used to isolate a full-length cDNA and a genomic clone. The full-length cDNA has the potential to encode a 272 amino acid protein with a calculated molecular weight of 30 kD. Amino acid sequence obtained by Edman analysis of the AAB1 protein confirmed that the aab-1 gene had been cloned. AAB-1 shows similarity to the HAP5 protein of yeast and the CBF-C protein of rat. Each of these proteins is an essential subunit of their respective heteromeric CCAAT binding proteins. The aab1 gene maps on linkage group III of Neurospora crassa near the trp-1 locus. Disruption of the aab-1 gene results in pleiotropic effects on growth and development as well as a 50% reduction in glutamate dehydrogenase levels. Transformation of the aab-1 disruption mutant strain with the cloned genomic copy of the aab-1 gene rescued all of the phenotypic alterations associated with the aab-1 mutation.

A physical assay for meiotic recombination in Coprinus cinereus.

We have used the polymerase chain reaction (PCR) method to monitor meiotic recombination in the basidiomycete Coprinus cinereus. We used DNA-mediated transformation to recover strains with modifications of the trp1 locus. The modifications were designed to introduce unique PCR priming sites separated by a homologous 2.4 kb region in which crossing over could occur. We showed that exchange occurred in this region at the frequency expected for a typical region of this genome (2.4 kb should correspond to a genetic length of 0.08 cM). We also detected products resulting from crossing over in DNAs extracted from cells in meiotic prophase. The assay should be useful for monitoring exchange in mutants that cannot complete meiosis.

P-445 A suicide gene therapy of metastasized liver tumors

The cell-division-cycle gene, CDC7, of Saccharomyces cerevisiae has been cloned in a large plasmid (pCM6) containing an insert of about 45 kb of yeast chromosomal DNA in the yeast-Escherichia coli shuttle vector, YRp7. A subclone (pCM39), having a 7-kb insert with a unique BamHI site, was capable of conferring the ts + phenotype on a cdc7ts− strain of S. cerevisiae. When this insert was placed in the vector Ylp28, and then cut with BamHI and integrated into the ts − strain, genetic analysis showed the CDC7-complementing activity and an associated vector marker to be linked to the TRP1 locus i.e., to chromosome IV. Analysis of subclones covering the pCM39 insert showed that pCM52, in which a 3.3-kb EcoRI-HpaI fragment with the unique BamHI site has been cloned, possessed the full complementing activity. Although the CDC7 gene lies across the BamHI site, the main part of the gene appears to be in the 2.2-kb BamHI-HpaI segment of the pCM52 insert. A Northern blot analysis showed this region to give rise to a 1.7-kb mRNA which we conclude is the transcript of CDC7.The large insert of pCM6 also contains a 1.5-kb XhoI fragment which several observations suggest is that containing the centromere (CEN4) of chromosome IV to which the CDC7 gene is closely linked. The 45-kb DNA fragment in pCM6 is believed to be an uninterrupted piece of chromosome IV.

Differential repair and recombination of psoralen damaged plasmid DNA in Saccharomyces cerevisiae.

Psoralen photoreaction with DNA produces interstrand crosslinks, which require the activity of excision and recombinational pathways for repair. Yeast replicating plasmids, carrying the HIS3, TRP1, and URA3 genes, were photoreacted with psoralen in vitro and transfected into Saccharomyces cerevisiae cells. Repair was assayed as the relative transformation efficiency. A recombination-deficient rad52 strain was the least efficient in the repair of psoralen-damaged plasmids; excision repair-deficient rad1 and rad3 strains had repair efficiencies intermediate between those of rad52 and RAD cells. The level of repair also depended on the conditions of transformant selection; repair was more efficient in medium lacking tryptophan than in medium from which either histidine or uracil was omitted. The plasmid repair differential between these selective media was greatest in rad1 cells, and depended on RAD52. Plasmid-chromosome recombination was stimulated by psoralen damage, and required RAD52 function. Chromosome to plasmid gene conversion was seen most frequently at the HIS3 locus. In RAD and rad3 cells, the majority of the conversions were associated with plasmid integration, while in rad1 cells most were non-crossover events. Plasmid to chromosome gene conversion was observed most frequently at the TRP1 locus, and was accompanied by plasmid loss.

Functional analysis of the human estrogen receptor using a phenotypic transactivation assay in yeast

We have constructed yeast strains in which the expression of the Saccharomyces cerevisiae URA3 gene is induced by the human estrogen receptor (hER). Promoter sequences required for both basal and activated transcription of URA3 were replaced with one or three estrogen-response elements ( EREs) positioned upstream of the native TATA box. These constructs were each integrated at the TRP1 locus of a yeast strain in which the natural URA3 gene had been deleted, and the integrants were transformed with low- or high-copy-number shuttle plasmids expressing wild-type or truncated derivatives of hER. Transformants were assayed for growth on uracil-deficient medium plus or minus estradiol (E 2), for resistance to 5-fluoroorotic acid (5-FOA) and for activity of OMPdecase (orotidine-5'-monophosphate decarboxylase), the product of the URA3 gene. We show that the growth and 5-FOA-resistance (5-FOA R) phenotypes of these strains are strictly dependent upon the function of the receptor derivatives. Induction of URA3, measured by OMPdecase activity, was observed over a 20- to 2500-fold range depending on the receptor derivative, its expression level and the number of EREs in the responsive promoter. Both one- and three- ERE reporter strains expressing the full-length receptor are completely E 2-dependent for growth, and display a 5-FOA R phenotype in the absence of the hormone. We demonstrate that the individual hER transactivation functions, TAF1 and TAF2, are both functional in yeast, and that the hormone-dependent TAF2 is the more potent activator on our reporters. We show that hER displays strong homosynergism in yeast, and discuss the contributions of the two TAFs in hER synergism. The reporter system described here provides a phenotypic transactivation assay and constitutes a versatile genetic screening and selection system, as both phenotypic assays are amenable to positive selection strategies. In addition, the system may be adapted for the study of other inducible transcription factors.

High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with a high mitotic stability under non-selective conditions. Since high stability can best be assured by integration of the vector into chromosomal DNA we have set out to design a vector that is able to integrate into the yeast genome in a large number of copies. The rDNA locus appeared to be an attractive target for such multiple integration since it encompasses 100–200 tandemly repeated units. Plasmids containing several kb of rDNA for targeted homologous recombination, as well as the deficient LEU2-d selection marker were constructed and, after transformation into yeast, tested for both copy number and stability. One of these plasmids, designated pMIRY2 (for multiple integration into ribosomal DNA in yeast), was found to be present in 100–200 copies per cell by restriction analysis. The pMIRY2 transformants retained 80–100 % of the plasmid copies over a period of 70 generations of growth in batch culture under non-selective conditions. To explore the potential of pMIRY2 as an expression vector we have inserted the homologous genes for phosphoglycerate kinase (PGK) and Mn 2+ -dependent Superoxide dismutase (SOD) as well as the heterologous genes for thaumatin from Thaumatococcus danielli (under the GAPDH promoter), into this plasmid and analyzed the yield of the various proteins. Under optimized conditions the level of PGK in cells transformed with pMIRY2-PGK was about 50% of total soluble protein. The yield of thaumatin in the pMIRY2-thaumatin transformants exceeded by about a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene integrated at the TRP1 locus in chromosome IV.

Cloning and analysis of the Kluyveromyces lactis TRP1 gene: A chromosomal locus flanked by genes encoding inorganic pyrophosphatase and histone H3

The TRP1 gene of the yeast Kluyveromyces lactis has been cloned from a genomic library by complementation of the Saccharomyces cerevisiae trp1-289 mutation. The gene was located within the clone by transposon mutagenesis and the coding region identified by DNA sequencing. This has indicated that K. lactis TRP1 encodes a 210-amino acid polypeptide which shows 53% identity to the homologous S. cerevisiae protein. The K. lactis TRP1 gene has been disrupted by substituting the S. cerevisiae URA3 gene for a large part of the TRP1 coding sequence. Replacement of the chromosomal TRP1 locus with this construction has enabled the production of non-reverting trp1- strains of K. lactis, while a genetic analysis of the disrupted allele confirmed that the TRP1 gene had been cloned. DNA sequencing has also shown that the K. lactis TRP1 sequence is flanked by genes encoding inorganic pyrophosphatase and histone H3, which we have designated IPP and HHT1 respectively. Hybridization studies have shown that in common with S. cerevisiae, K. lactis has two copies of the histone H3 gene. Each H3 gene is closely linked to a gene encoding histone H4 and in both yeast species the IPP gene is tightly linked to one of the histone gene pairs.

Role of cell wall in Saccharomyces cerevisiae mutants resistant to Hg2+.

Hg2+-resistant mutants were isolated from Saccharomyces cerevisiae. Although they were very much like the parental strains in terms of colony-forming ability, they grew faster than the parental strains in the presence of sublethal doses of Hg2+. The Hg2+-resistant mutations were dominant. They were centromere linked and were divided into two groups by means of recombination; one of the mutations, designated HGR1-1, was mapped on chromosome IV because of its linkage to the TRP1 locus. The Hg2+-resistant mutants took up Hg2+ as much as, or slightly more than, the parental strains did. The mutants and parental strains retained only about 5 and 15%, respectively, of the cell-associated Hg2+ after removal of the cell wall; therefore, the mutants had less spheroplast-associated Hg2+ than did the parental strains. These results indicate that the cell wall plays an important role in protection against Hg2+ by acting as an adsorption filter and that the mutations described confer Hg2+ resistance by increasing the Hg2+-binding capacity of the cell wall.

First identification of an amber nonsense mutation in Schizosaccharomyces pombe: major differences in the efficiency of homologous versus heterologous yeast suppressor tRNA genes

In Saccharomyces cerevisiae ochre and opal, as well as amber mutations are known, whereas in the fission yeast Schizosaccharomyces pombe no amber alleles have been described. We have characterized trp1-566, an amber allele in the trp1 locus of S. pombe. The identification of trp1-566 as an amber allele is based on the following results: (a) The nonsense allele can be converted to an ochre allele by nitrosoguanidine mutagenesis. (b) trp1-566 is suppressed by a bona fide S. pombe amber suppressor tRNA, supSI. The supSI gene was obtained by primer-directed in vitro mutagenesis of a tRNASer from S. pombe. Unexpectedly, an S. cerevisiae amber suppressor tRNASer, supR21, transformed into S. pombe, failed to suppress trp1-566. Northern analysis of S. pombe transformants, containing supRL1 or S. cerevisiae tRNALeu or tRNATyr genes reveals that these genes are not transcribed in the fission yeast. As an additional tool for the analysis of nonsense mutations in S. pombe, we obtained by nitrosoguanidine mutagenesis two unlinked amber suppressor alleles, sup13 and sup14, which act on trp1-566.

Cloning of the CDC7 gene of Saccharomyces cerevisiae in association with centromeric DNA

The cell-division-cycle gene, CDC7, of Saccharomyces cerevisiae has been cloned in a large plasmid (pCM6) containing an insert of about 45 kb of yeast chromosomal DNA in the yeast- Escherichia coli shuttle vector, YRp7. A subclone (pCM39), having a 7-kb insert with a unique BamHI site, was capable of conferring the ts + phenotype on a cdc7ts − strain of S. cerevisiae. When this insert was placed in the vector Ylp28, and then cut with BamHI and integrated into the ts − strain, genetic analysis showed the CDC7-complementing activity and an associated vector marker to be linked to the TRP1 locus i.e., to chromosome IV. Analysis of subclones covering the pCM39 insert showed that pCM52, in which a 3.3-kb EcoRI- HpaI fragment with the unique BamHI site has been cloned, possessed the full complementing activity. Although the CDC7 gene lies across the BamHI site, the main part of the gene appears to be in the 2.2-kb BamHI- HpaI segment of the pCM52 insert. A Northern blot analysis showed this region to give rise to a 1.7-kb mRNA which we conclude is the transcript of CDC7. The large insert of pCM6 also contains a 1.5-kb XhoI fragment which several observations suggest is that containing the centromere ( CEN4) of chromosome IV to which the CDC7 gene is closely linked. The 45-kb DNA fragment in pCM6 is believed to be an uninterrupted piece of chromosome IV.

Organisation of the complex locus trp1 in the fission yeast Schizosaccharomyces pombe

65 trp1(-) alleles of Schizosaccharomyces pombe have been analysed for their interallelic complementation pattern, suppressibility by nonsense suppressors and position of the corresponding mutation site on an intragenic map of the trp1 locus. In addition to the three complementation classes previously described (Schweingruher and Dietrich 1973) as defective in phosphoribosylanthranilate isomerase (trp1A), indole glycerolphosphate synthetase (trp1B) and anthranilate synthetase (trp1C), two new complementation classes, trp1BC and trp1ABC, were found. The former is represented by a single allele which can only complement trp1A mutants. The latter is represented by six alleles which fail to complement tester mutants of the trp1A, trp1B or trp1C class. Classes trp1A, trp1B and trp1C correspond to mutations in three nonoverlapping regions mapping in the order trp1A, trp1B and trp1C All the alleles of the trp1BC and trp1ABC classes correspond to mutations in the trp1C region. Nonsense alleles of the opal (UGA) or ochre (UAA) type were found in the trp1A (2 alleles out of 17) and trp1ABC (5 alleles out of 6) classes only. These data indicate that the trp1 locus is transcribed as a single messenger RNA with transcription starting from the trp1C region. This messenger is probably translated in a single, multifunctional polypeptide, or at most in two polypeptides coded for by the trp1B-trp1C and by the trp1A regions. In addition, the polar effect of nonsense mutations in the trp1C region is cancelled by rare spontaneous mutations occuring at or very near the trp1 locus, which may act by creating an internal single for the initiation of transcription and/or translation.