Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.     

The effect ofrecA mutation on the expression ofEcoKI andEcoR124Ihsd genes cloned in a multicopy plasmid

Type I restriction-modification (R-M) endonucleases are composed of three subunits—HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of clonedEcoKI andEcoR124Ihsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wthsd genes in a multicopy plasmid inEscherichia coli recA ⁺ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the clonedhsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation inE. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.     

Repair of plasmid and genomic DNA in a rad7 delta mutant of yeast.

Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined in a yeast plasmid of known chromatin structure and in genomic DNA in a radiation-sensitive deletion mutant of yeast, rad7 delta, and its isogenic wild-type strain. A whole plasmid repair assay revealed that only approximately 50% of the CPDs in plasmid DNA are repaired after 6 h in this mutant, compared with almost 90% repaired in wild-type. Using a site-specific repair assay on 44 individual CPD sites within the plasmid we found that repair in the rad7 delta mutant occurred primarily in the transcribed regions of each strand of the plasmid, however, the rate of repair at nearly all sites measured was less than in the wild-type. There was no apparent correlation between repair rate and nucleosome position. In addition, approximately 55% of the CPDs in genomic DNA of the mutant are repaired during the 6 h period, compared with > 80% in the wild-type.     

The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene

Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA. We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism. Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially. The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity.     

A palindromic mutation of the linear killer plasmid k2 of yeast.

Production of the killer toxin in Kluyveromyces lactis is dependent on the presence of two linear DNA plasmids, k1 and k2. We isolated a non-killer mutant, VM5, with a modified plasmid composition. It had lost k1, but conserved k2, and acquired, in addition, three new DNA species. The new species were found to be rearranged derivatives of the k2 plasmid. One of them, pVM5-1, was made of the left terminal 4720 bp sequence of k2, including the inverted terminal repeat, and was organized as a large palindromic dimer molecule. The second, pVM5-2, was made of one strand of the pVM5-1 palindrome, folded into a hairpin structure. Like normal k2, pVM5-1 and 2 were present in a high copy number. The third species, pVM5-x, of variable size, was also a deletion product of k2, but not palindromic, and did not contain the terminal repeat. Genetic analysis showed that the presence of the palindromic derivatives appeared to destabilize the normal k2 genome, leading to gradual accumulation of plasmid-less cells.     

Isolation of yeast tRNALeu genes. DNA sequence of a cloned tRNALeu3 gene.

A library of cloned yeast DNA fragments generated by digestion of yeast DNA with the restriction endonuclease Bam HI has been screened by colony hybridization to total yeast [32P]tRNA. Four hundred colonies carrying yeast tRNA genes were isolated. By hybridization to 125I-tRNALeu3, we have isolated from this collection 14 colonies carrying fragments containing yeast tRNALeu genes. The size of the yeast Bam HI inserts ranged from 2.45 x 10(6) to 14 x 10(6) daltons. One of these fragments was mapped in detail by restriction endonuclease digestion and hybridization to 125I-tRNALeu3. The presence of a tRNALeu3 gene was confirmed by DNA sequence. The results indicate that the tRNALeu3 coding region is not co-linear with the tRNALeu3. An intervening tract of 33 base pairs interrupts the coding sequences 1 base pair past the anticodon coding region. The putative structure of a tRNALeu3 precursor is deduced in which the anticodon base pairs with residues from the intervening sequence.     

A map of the restriction targets in yeast 2 micron plasmid DNA cloned on bacteriophage lambda

Summary The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage . The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of the targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin sensitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.     

Mutagenesis by site-specific arylamine adducts in plasmid DNA: enhancing replication of the adducted strand alters mutation frequency

Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by greater than 7-fold to 2.9% and of the AAF adduct by greater than 12-fold to 0.75%. The mutation frequency of the AF adduct was greatly reduced in a uvrA- strain while no mutations occurred with the AAF adduct in this strain. The sequence changes resulting from these treatments were dependent on adduct structure and the presence or absence of uracil on the strand opposite the adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid.

By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block. In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain. The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.     

Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector.

Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts.     

The rad2 mutation affects the molecular nature of UV and acridine-mustard-induced mutations in the ADE2 gene of Saccharomyces cerevisiae

We have studied the molecular nature of ade2 mutations induced by UV light and bifunctional acridine-mustard (BAM) in wild-type (RAD) and in excision-deficient (rad2) strains of the yeast, Saccharomyces cerevisiae. In the RAD strain, UV causes 45% GC → AT transitions among all mutations; in the rad2 strain this value is 77%. BAM was shown to be highly specific for frameshift mutagenesis: 60% frameshifts in the RAD strain, and as many as 84% frameshifts in the rad2 strain were induced. Therefore, the rad2 mutation affects the specificity of UV- and BAM-induced mutagenesis in yeast. Experimental data agree with the view that the majority of mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the rad2 strain are predominantly postreplicative events. Our results also suggest that: (1) cytosine-containing photoproducts are the substances responsible for major premutational damage to DNA; (2) a fraction of the mutations may arise in the course of excision repair of UV photoproducts.     

Dominant chromosomal mutation bypassing chromosomal genes needed for killer RNA plasmid replication in yeast

Yeast strains carrying a double-stranded RNA plasmid of 1.4–1.7 x 10⁶ daltons encapsulated in virus-like particles secrete a toxin that kills strains lacking this plasmid. The plasmid requires at least 24 chromosomal genes (pets, and mak1 through mak23) for its replication or maintenance. We have detected dominant Mendelian mutations (called KRB1 for killer replication bypass) that bypass two chromosomal genes, mak7 and pets, normally needed for plasmid replication. Strains mutant in mak7 and carrying the bypass mutation (mak7–1 KRB1) are isolated as frequent K⁺R⁺ sectors of predominantly K^-R ^- segregants from crosses of mak7–1 with a wild-type killer. All KRB1 mutations isolated in this way are inherited as single dominant centromere-linked chromosomal changes. They define a new centromere. KRB1 is not a translational suppressor. KRB1 strains contain a genetically normal killer plasmid and ds RNA species approximately the same in size and amount as do wild-type killers. Bypass of both mak7 and pets by one mutation suggests that these two genes are functionally related.

Two properties of the inheritance of KRB1 indicate an unusually high reversion frequency: (1) Heat or cycloheximide (treatments known to cure strains of the wild-type killer plasmid) readily induce conversion of mak7–1 KRB1 strains from killers to nonkillers with concomitant disappearance of KRB1 as judged by further crosses, and (2) mating two strains of the type mak7–1 KRB1 with each other yields mostly 2 K⁺R⁺: 2 K^-R^- segregation, although the same KRB1 mutation and the same killer plasmid are present in both parents.

     

基于基因突变频率识别低级别脑胶质瘤驱动基因

癌症通常由基因变异的累积所驱动,有效地识别癌症的驱动突变是一个巨大的挑战。目前已有方法更多是通过将基因组区域中观察到的突变率与背景突变率(BMR)预期的突变率进行比较或功能影响测试来识别驱动基因,该驱动基因本质上是存在统计异常的基因。而且并未对已有明确分类的癌症的子类之间驱动基因进行研究。本文引入关联规则算法,探寻发生该基因突变诱使病人患该子类低级别脑胶质瘤的有效规则,将突变数据与患癌结果通过算法建立关系,再通过支持度、置信度和提升度这三个指标对产生的规则进行筛选和评估,来预测候选驱动基因以及类间驱动基因差异。最后利用491例低级别脑胶质瘤体细胞突变数据,得到22个与结果存在关联的驱动基因及其所属的子类,敏感性和假阳性结果优于目前已有的单一算法,且22个基因均具有重要的生物学功能。同时建立了基于22个基因的低级别脑胶质瘤子类识别方法,模型总体准确率达98.99%,方法可有效区分三子类。     

Antimutagenic effect of alpha-tocopherol on the gene mutation frequency in Salmonella]

The work presents the data on the antimutagenic effect of alpha-tocopherol on the frequency of N-nitroso-N-methyl urea induced gene mutations in Salmonella in vitro and in vivo. In vitro tests have revealed the dependence of decreasing the rate of induced mutations both on combination of treatments with mutagen/antimutagen and on the dose of the mutagen. In vivo tests have demonstrated that the mutagenic effect of alpha-tocopherol depends on the duration of its action on organisms.     

Use of cloned htpR gene of Escherichia coli to introduce htpR mutation into the chromosome

A deletion htpR mutant of Escherichia coli has been constructed on the basis of site-directed mutagenesis. To this end, the chromosomal allele of htpR gene was substituted by a mutant allele introduced into the cell with a recombinant plasmid. The htpR mutant is characterized by a reduced level of proteolysis and therefore by a decreased rate of proteolytic degradation of RNA polymerase of bacteriophage T7. The mutation in htpR is linked with chloramphenicol resistance.     

The effects of three rad genes on UV induced mutation rates in haploid and diploid Saccharomyces cells

Effects of the rad 2-20, rad 9-4, r1s, and the corresponding wild type RAD alleles in haploid and homozygous diploid Saccharomyces strains on UV induced mutation rates from adenine, lysine and histidine dependence to independence are reported. The UV induced mutation rates were similar for the RAD, r1s, and rad 9-4 haploids, whereas the rad 2-20 mutation causes a marked increase in the UV induced mutation rates. The diploid rad 2-20 strain also exhibits a marked increase in the UV induced mutation rates, whereas the rad 9-4 diploid has reduced mutation rates when compared to the wildtype. The UV induced mutation rates of haploid and diploid RAD strains are almost identical. For the rad 2-20 and rad 9-4 diploids, however, these rates are smaller than in the corresponding haploid strains. Differential effects of the rad genes on the ratio of locus to suppressor mutations were found. The implications of these findings on possible repair processes in yeasts are discussed.     

Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain.

The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.     

The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast

During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression.