Identification of a 450-bp region of human papillomavirus type 1 that promotes episomal replication in Saccharomyces cerevisiae

Human papillomaviruses (HPVs) replicate as nuclear plasmids in infected cells. Since the DNA replication machinery is generally conserved between humans and Saccharomyces cerevisiae, we studied whether HPV-1 DNA can replicate in yeast. Plasmids containing a selectable marker (with or without a yeast centromere) and either the full-length HPV-1 genome or various regions of the viral long control region (LCR) and the 3' end of the L1 gene were introduced into S. cerevisiae and their ability to replicate episomally was investigated. Our results show that HPV-1 sequences promote episomal replication of plasmids although the yeast centromere is required for plasmid retention. We have mapped the autonomously replicating sequence activity of HPV-1 DNA to a 450 base-pair sequence (HPV-1 nt 6783-7232) that includes 293 nucleotides from the 5' region of the viral LCR and 157 nucleotides from the 3' end of the L1 gene. The HPV-1 ARS does not include the binding sites for the viral E1 and E2 proteins, and these proteins are dispensable for replication in S. cerevisiae.     

Identification of extragenic suppressors of the cif1 mutation in Saccharomyces cerevisiae

The cif1 mutation of Saccharomyces cerevisiae causes inability to grow on glucose and related fermentable carbon sources. We have isolated two different suppressor mutations that allow growth on glucose of yeasts carrying the cif1 mutation. One of them, sci1-1, is recessive and caused inability to grow on non-fermentable carbon sources and to de-repress fructose-1,6-bisphosphatase. The other suppressor mutation, SCI2-1, is dominant and diminished the capacity to phosphorylate glucose or fructose. The SCI2-1 mutation decreased sporulation efficiency by 70% in heterozygosis and by more than 90% in homozygosis. In a CIF1 background, cells carrying the mutation SCI2-1 accumulated trehalose during the logarithmic phase of growth and hyperaccumulated it during the stationary phase. Genetic tests showed that SCI2 was either allelic, or else closely linked, to HXK2. The concentrations of the glycolytic metabolites measured during growth on glucose in cells carrying the cif1 mutation and any of the suppressor mutations were similar to those of a wild-type. Both types of suppressor mutations restored the transient cAMP response to glucose to cif1 mutants.This paper is dedicated to Prof. J. R. Villanueva on the occasion of his 65th birthday     

Identification and characterization of four new GCD genes in Saccharomyces cerevisiae

Summary Mutant strains, resistant against the amino acid analogues 5-methyltryptophan, 5-fluorotryptophan and canavanine were isolated, starting with a trp2 leaky auxotrophic strain. Of 10 such strains, only four turned out to be of the general control derepressed (gcd) mutant type. Three other isolates were shown to be defective in the general amino acid permease system, while the remaining three strains displayed low spore viability and were not further investigated. Complementation tests amongst the four new gcd-mutant strains, including strain RH558 gcd2-1 isolated earlier, yielded five complementation groups: GCD2, GCD3, GCD4, GCD5, and GCD6. All mutant strains showed a dual phenotype, which was not separable by wild type backcrosses: constitutive derepression and slow growth. Epistatis of all gcd mutations over gcn1-1, gcn2-1 and gcn3-1 was found with respect to both phenotypes, except for gcd5-1, which was lethal in these combinations. On the other hand gcn4-101 was found to be epistatic over all gcd mutations, but only with respect to the constitutive derepression phenotype, and not to slow growth; again the combination with gcd5-1 was lethal. Mutation gcd2-1 was mapped on chromosome VII, 50 cM from leu1 and 22 cM from ade6. A new model is discussed, in which GCD-genes are involved in the amino acid uptake into the vacuoles.     

Resistance to cadmium is under the control of the CAD2 gene in the yeast Saccharomyces cerevisiae

Summary A cadmium-resistant strain, X3382-3A, which is able to grow in a medium containing 0.2 mM cadmium sulfate, was picked out from our laboratory stock strains of Saccharomyces cerevisiae. The cadmium resistance of this strain is controlled by a single dominant nuclear gene, denoted as CAD2. The locus of CAD2 was mapped by gene linkage to a site 15.5 centimorgans to the right of the his7 locus on the right arm of chromosome II. The cadmium resistance of the strain carrying CAD2 was evaluated for its properties of cadmium uptake, cadmium distribution and cadmium-metallothionein formation, in comparison with those of some other strains. The results suggest that the novel type of cadmium resistance controlled by CAD2 does not involve production of a cadmiumm-metallothionein.     

UV-inducible proteins in Saccharomyces cerevisiae

Summary Two UV-inducible proteins have been detected in the yeast, Saccharomyces cerevisiae. The proteins have molecular weights of 78,000 Daltons and 23,000 Daltons. This induction is specific for UV-irradiation as exposure to X-rays, mitomycin C and heat shock does not result in the synthesis of the proteins. The larger (78 kD) protein is induced in various rad strains and in a ° cir° strain. Attempts are being made to isolate the genes coding for these inducible proteins.     

Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae

Summary A pattern of active accumulation of trehalose during growth on glucose medium, TAC(+) phenotype, is controlled by a polymeric series of maltose fermentation (MAL) genes. An essential requirement for expression of the TAC(+) phenotype is that the MAL gene be in the constitutive state, MAL ^c. Mutation of a constitutive MAL allele to a maltose- inducible or nonfermenting (mal) state, alters the pattern of trehalose metabolism so that little or no trehalose accumulation occurs during growth on glucose medium. The TAC(+) phenotype is obtained in MAL ^c strains whether or not -glucosidase formation is sensitive or resistant to carbon catabolite repression. However, trehalose accumulation is sensitive to glucose levels even in MAL ^c strains in which -glucosidase formation is insensitive to catabolite repression. The effects of constitutive MAL genes on trehalose accumulation cannot be accounted for by an increase in trehalose-6 phosphate synthase or a decrease in trehalase as determined in vitro. A mechanism is proposed in which the gene-product of a MAL gene serves as a common positive regulator for expression of four genes coding respectively for maltose permease, maltase, -methylglucosidase and a component of the trehalose accumulation system.Paper I appeared in Cell. and Molec. Biology 25: 345–354, 1979     

Use of reporter genes for the isolation and characterisation of different classes of sporulation mutants in the yeast Saccharomyces cerevisiae

Reporter genes consisting of sporulation-specific promoters fused to lacZ were used as markers to monitor the sporulation pathway of the yeast Saccharomyces cerevisiae. Strains transformed with these lacZ gene fusions expressed -galactosidase (assayable on plates using the substrate 5-bromo-4-chloro-3-indolyl--D-galactopyranoside, X-gal) in a sporulation-dependent manner. Mutagenesis experiments performed on transformed strains resulted in the recovery of a number of novel sporulation mutants. Three classes of mutants were obtained: those which overexpressed the reporter gene under sporulation conditions, those which did not express the gene under any conditions, and those which expressed the gene in vegetative cells not undergoing sporulation. On the basis of the blue colony-colour produced in the presence of X-gal these have been described as superblue, white, and blue vegetative mutants, respectively. These were further characterised using earlier reporter genes and other marker systems. This study established that the multicopy reporter plasmids chosen do not interfere with sporulation; they are valid tools for monitoring the pathway and they provide a way to isolate mutations not readily selected by other markers.     

Chromium resistant mutants of the yeast Saccharomyces cerevisiae

Summary Many strains of Saccharomyces cerevisiae do not grow on YPD agar containing 750 g/ml CrO₃. Mutants able to grow in the presence of 850 g/ml CrO₃ were obtained from such strains after UV mutagenesis. All of the mutants grew even in the presence of 1,000 /ml CrO₃. Chromium resistance was dominant or partial dominant over normal response, therefore it was impossible to determine the number of genetic loci by complementation analysis. However, the segregation of representative mutants strongly indicated that resistance was determined by single mutations. In addition, a limited analysis of recombination suggested that the chromium resistant mutations were located on a certain region of the yeast genome. Although it was determined that the mutants had slightly reduced rates of Cr⁶⁺ uptake, the exact mechanism of resistance was not discovered. According to the studies of interactions between resistant mutations and sensitive mutations, however, we have proposed a preliminary pathway of Cr⁶⁺ detoxification.     

Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae

Summary We have isolated and characterized three conditional hyporecombination mutants, rec1-1, rec3-1 and rec4-1, that define three REC genes of Saccharomyces cerevisiae required for spontaneous general mitotic interchromosomal recombination. Each MATa/MAT rec/rec diploid is deficient in mitotic single site gene conversion, intragenic recombination, intergenic recombination and sporulation at the restrictive temperature (36°C). The rec1-1 mutation also confers conditional enhanced sensitivity to the killing effects of X-rays. The rec1-1 and rec3-1 mutations have been mapped to chromosome VII. The rec1-1, rec3-1 and rec4-1 mutations exhibit complementation at 36°C for both mitotic recombination and sporulation.     

Phenotype traits associated with different alleles at the RPS5 locus in Saccharomyces cerevisiae

Summary The RPS5 gene has been characterised through its ability to reduce invertase production by the SUC5 gene. In this paper we show that RPS5 acts by maintaining low levels of SUC5 mRNA. We also show that RPS5 acts on the SUC1 and SUC4 genes but not on SUC2 and SUC3, which are members of the SUC family. RPS5 also shows a pleiotropic effect on the amount of mitochondrial cytochromes.     

Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae

Summary A specific deficiency in UDPG-linked trehalose-6-phosphate synthase in the yeast, Saccharomyces cerevisiae has been associated with a single nuclear gene, sst1. Strains bearing this abnormal allele lacked the capacity to accumulate trehalose during growth on glucose or galactose medium or when incubated with glucose in nonproliferating conditions. However, sst1 strains still exhibited trehalose accumulation during growth on maltose medium, provided they contained a gene for maltose fermentation (MAL gene). Introduction of a constitutive MAL ^c gene into an sst1 strain rendered the strain capable of accumulating trehalose during growth on glucose medium, but did not restore the normal capacity to convert glucose to trehalose in nonproliferating conditions. Different systems, I and II, of trehalose accumulation are proposed. System I would require the UPDG-linked synthase, whereas system II, which is normally specific for maltose, would utilize a different enzyme. It is unlikely that system II produces trehalose by trans-glucosylation, since it converted glucose to trehalose in MAL ^c sst1 strains. The results indicate that maltose specifically induces the production of the MAL gene-product, which, in turn, would stimulate the formation (or activation) of system II.     

A novel mutation occurring in the PHO80 gene suppresses the PHO4 c mutations of Saccharomyces cerevisiae

Summary We have isolated suppressors of a PHO4 ^c (a positive regulator) mutant which normally confers weak constitutivity for acid phosphatase production on the Saccharomyces cell. One dominant suppressor (PHO80-2) was found to be an allele of PHO80 (a negative regulator) that changes G to A, resulting in substitution of isoleucine for methionine 42 of the Pho80 protein. Substitution of valine (PHO80-3) or leucine (PHO80-4) for the same methionine by site-directed mutagenesis also suppressed PHO ^c. Suppression by PHO80-2) did show some allele specificity. From these results we were able to delimit the region of PHo80 which may interact with the Pho4 protein.     

Palindrome content of the yeast Saccharomyces cerevisiae genome

Palindromic sequences are important DNA motifs involved in the regulation of different cellular processes, but are also a potential source of genetic instability. In order to initiate a systematic study of palindromes at the whole genome level, we developed a computer program that can identify, locate and count palindromes in a given sequence in a strictly defined way. All palindromes, defined as identical inverted repeats without spacer DNA, can be analyzed and sorted according to their size, frequency, GC content or alphabetically. This program was then used to prepare a catalog of all palindromes present in the chromosomal DNA of the yeast Saccharomyces cerevisiae. For each palindrome size, the observed palindrome counts were significantly different from those in the randomly generated equivalents of the yeast genome. However, while the short palindromes (2–12 bp) were under-represented, the palindromes longer than 12 bp were over-represented, AT-rich and preferentially located in the intergenic regions. The 44-bp palindrome found between the genes CDC53 and LYS21 on chromosome IV was the longest palindrome identified and contained only two C-G base pairs. Avoidance of coding regions was also observed for palindromes of 4–12 bp, but was less pronounced. Dinucleotide analysis indicated a strong bias against palindromic dinucleotides that could explain the observed short palindrome avoidance. We discuss some possible mechanisms that may influence the evolutionary dynamics of palindromic sequences in the yeast genome.     

FLP recombinase induction of the breakage-fusion-bridge cycle and gene conversion in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SM^R) allele of ILV2] as selectable markers, and the 2 m site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir ⁺] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir ⁺] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA^– cells continued to segregate for loss of SMR1 until stable URA^– SM^R or URA^–SM^S cells were produced. Gene conversion was identified in stable URA^–SM^R cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 m DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.