The RFC2 gene, encoding the third-largest subunit of the replication factor C complex, is required for an S-phase checkpoint in Saccharomyces cerevisiae.

Replication factor C (RF-C), an auxiliary factor for DNA polymerases delta and epsilon, is a multiprotein complex consisting of five different polypeptides. It recognizes a primer on a template DNA, binds to a primer terminus, and helps load proliferating cell nuclear antigen onto the DNA template. The RFC2 gene encodes the third-largest subunit of the RF-C complex. To elucidate the role of this subunit in DNA metabolism, we isolated a thermosensitive mutation (rfc2-1) in the RFC2 gene. It was shown that mutant cells having the rfc2-1 mutation exhibit (i) temperature-sensitive cell growth; (ii) defects in the integrity of chromosomal DNA at restrictive temperatures; (iii) progression through cell cycle without definitive terminal morphology and rapid loss of cell viability at restrictive temperatures; (iv) sensitivity to hydroxyurea, methyl methanesulfonate, and UV light; and (v) increased rate of spontaneous mitotic recombination and chromosome loss. These phenotypes of the mutant suggest that the RFC2 gene product is required not only for chromosomal DNA replication but also for a cell cycle checkpoint. It was also shown that the rfc2-1 mutation is synthetically lethal with either the cdc44-1 or rfc5-1 mutation and that the restrictive temperature of rfc2-1 mutant cells can be lowered by combining either with the cdc2-2 or pol2-11 mutation. Finally, it was shown that the temperature-sensitive cell growth phenotype and checkpoint defect of the rfc2-1 mutation can be suppressed by a multicopy plasmid containing the RFC5 gene. These results suggest that the RFC2 gene product interacts with the CDC44/RFC1 and RFC5 gene products in the RF-C complex and with both DNA polymerases delta and epsilon during chromosomal DNA replication.

Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning.

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was >/=60 bp were utilized, approximately 2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is approximately 60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

Base analog 6-N-hydroxylaminopurine mutagenesis in the yeast Saccharomyces cerevisiae is controlled by replicative DNA polymerases.

Genetic control of mutagenesis by the base analog 6-N-hydroxylaminopurine (HAP) was studied in a set of isogenic yeast strains carrying null or point mutations in DNA repair and replication genes. Null alleles of the PMS1, RAD6, REV3 and RAD52 genes did not affect HAP mutagenesis. Defects in 3'- > 5' exonucleases associated with DNA polymerases epsilon and delta led to 2- to 3-fold increases in HAP-induced forward Can(r) mutant frequency. A similar increase was observed for FOAr mutants but only in the strain with a defective exonuclease of the polymerase epsilon (mutation pol2-4). The polymerase epsilon mutations, pol2-9 and pol2-18, which lead to temperature-sensitivity, and pol2-1 (insertion of URA3 at the position coding for amino acid 1134 in the POL2 gene) substantially reduced HAP mutagenesis. The polymerase delta mutation, cdc2-2, slightly reduced HAP mutagenesis. Enhanced proofreading was not the cause of the antimutator effect in the pol2-18 bearing strain, inasmuch as antimutator effect was observed in the pol2-4,18 mutant strain lacking proofreading. From the data obtained, we conclude that both DNA polymerase epsilon and delta participate in mutation generation by HAP.

Mutagenic specificity of the base analog 6-N-hydroxylaminopurine in the LYS2 gene of yeast Saccharomyces cerevisiae.

We used the LYS2 gene mutational system to study mutation specificity of the base analog 6-N-hydroxylaminopurine (HAP) in yeast. We characterized phenotypes of mutations using codon-specific nonsense suppressors and the test employing inactivation of the release factor Sup35 due to overexpression and formation of prion-like derivative [PSI]. We have shown that HAP induces predominantly nonsense mutations. While the tests using codon-specific nonsense-suppressors allowed to identify only about 50% of nonsense-mutations, all the nonsense-mutations were identified in the test with defective Sup35. We determined and analyzed the spectrum of HAP-induced nucleotide changes in two regions of the gene. HAP induces predominantly GC-->AT transitions in a hotspots of a central position of trinucleotide GGA or AGG. Directionality of these transitions is consistent with the idea that initial dHAPMP incorporation in the leading strand is more genetically dangerous than in lagging DNA strand. We revealed a specific context inhibitory for HAP mutagenesis, a "T" in -1 position to mutation site.

The genetic activity of N6-hydroxyadenine and 2-amino-N6-hydroxyadenine in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae.

The genetic activity of 2-amino-N6-hydroxyadenine or 2-amino-N-hydroxylaminopurine (AHA) and N6-hydroxyadenine or 6-N-hydroxylaminopurine (HAP) was studied in S. typhimurium, E. coli and Saccharomyces cerevisiae strains. AHA was a more potent mutagen for bacteria and a less potent mutagen for yeast than HAP. The mutagenic activity of analogs was not influenced by excision, mutagenic or double-strand DNA repair mutations. On the other hand, the uvrBdel mutation has a drastic effect on the mutagenicity and toxicity of both analogs in the Salmonella strains studied. HAP was a very potent mutagen in yeast with a low capability of inducing mitotic recombination contrary to common mutagens, possessed unique intergenic specificity and was able to induce mutations in diploids at rather high frequency.

Multiple antimutagenesis mechanisms affect mutagenic activity and specificity of the base analog 6-N-hydroxylaminopurine in bacteria and yeast.

Base analog 6-N-hydroxylaminopurine is a potent mutagen in variety of prokaryotic and eukaryotic organisms. In the review, we discuss recent results of the studies of HAP mutagenic activity, genetic control and specificity in bacteria and yeast with the emphasis to the mechanisms protecting living cells from mutagenic and toxic effects of this base analog.

Transformation-associated recombination (TAR) cloning of tumor-inducing Xmrk2 gene from Xiphophorus maculatus.

We modified the TAR methodology of YAC clone construction for application to fish genomic DNA isolated from Xiphophorus maculatus. YAC libraries were developed using the XIR1 repeat sequence as the recombinational hook. Construction of these libraries demonstrates that Xiphophorus DNA sequences can function as hooks in the yeast recombination system and that X. maculatus genomic DNA contains sequences that provide origin of replication function in yeast. By screening a subset of Xiphophorus YAC clones, we isolated a clone harboring the Xmrk2 locus that is associated with spontaneous and induced melanomagenesis. Modifications of the TAR technique allowed the targeted cloning of specific genes from genomic regions ranging in size from cDNAs to several hundred kilobases. Specific genomic regions can be isolated in a directional manner from fixed map locations to saturate these areas with physical markers. We discuss the applications of these and other yeast recombinational processes to fish genetics.

[Genetic analysis of spontaneous and 6-N-hydroxylaminopurine and propiolactone induced Adp+ mutants in Saccharomyces yeasts]. / Geneticheskii analiz spontannykh i indutsirovannykh 6-N-gidroksilaminpurinom i propiolaktonom mutantov Adp+ u drozhzhei sakharomitsetov.

652 spontaneous and 6-N-hydroxylaminopurine and propiolactone-induced mutants were obtained in yeast. 598 of them were LYS2 mutants. Detailed genetic analysis of the mutants was performed, including analysis of growth pattern on lysineless medium, suppressibility by nonsense suppressors of three types and localization on the recombination map of the LYS2 gene. Mutants induced by different agents were different for all these criteria, except for distribution among the map regions.

[Mutability of LYS2 gene in diploid Saccharomyces yeasts. II. Frequency of mutants induced by 6-N-hydroxylaminopurine and propiolactone]. / Izuchenie mutabil'nosti gena LES2 u diploidov drozhzhei sakharomitsetov. Soobshchenie II. Vozniknovenie mutantov, indutsirovannykh 6-N-gidroksilaminopurinom i propiolaktonom.

Chemical mutagens 6-N-hydroxylaminopurine (HAP) and propiolactone (PRO) induce Lys2 mutants with high frequency in diploid yeast Saccharomyces cerevisiae. HAP induces such mutants even in tetraploid strains. The genetic analysis of mutants was performed. It is shown that PRO induces mutants by means of "mutation-mitotic segregation" mechanism, while HAP induces mutants through novel mechanism "both allele mutation". Manifestation of such mechanism is the null fertility after meiosis of diploid mutants induced by HAP.

[Development of a system of intragenic mapping for molecular genetic analysis of mutations in the gene LYS2 of Saccharomyces yeasts]. / Razrabotka sistemy vnutrigennogo kartirovaniia dlia molekuliarno- geneticheskogo analiza mutatsii v gene LYS2 u drozhzhei sakharomitsetov.

The collection of overlapping lys2 deletions (five in the chromosomal and seven in the plasmid LYS2 gene) is constructed in this work. The deletions overlap the whole coding region of the gene and provide the system for intragenic recombinational mapping of lys2 mutations in one of 14 controlled regions. A portion of these regions can be correlated with the regions on the physical map of LYS2. Mutations in two regions can be easily cloned. The system constructed gives the possibility for the study of intragenic and molecular specificity of mutagenesis.

Transformation in the methylotrophic yeast Pichia methanolica utilizing homologous ADE1 and heterologous Saccharomyces cerevisiae ADE2 and LEU2 genes as genetic markers.

Development of transformation systems for methylotrophic yeasts is the starting point for research aimed at developing molecular genetics of these genera and will be the key to their further successful use in biotechnology. We transformed Pichia methanolica using selector genes ADE2 and LEU2 from Saccharomyces cerevisiae and ADE1 (homologue of S. cerevisiae ADE2 gene) from P. methanolica which was cloned and sequenced in our laboratory (Hiep et al., 1991). Lithium transformation of P. methanolica strains was inefficient with intact plasmids. Linearization of plasmids at a unique restriction site within the ADE1 gene prior to transformation substantially increased its frequency. Transformation with linear ADE1, ADE2 or LEU2 gene fragments was even more effective. Introduced DNA fragments either circularized in vivo, irrespective of the structures of their ends, giving unstable transformants; or integrated at different sites of the host genome. Using this transformation system, we obtained a disruption of the ADE1 gene on the chromosome by inserting the S. cerevisiae LEU2 gene. The disruption mutation ade1::LEU2 was used to study the mechanism of intragenic recombination in P. methanolica.

The 5-aminoimidazole ribonucleotide-carboxylase structural gene of the methylotrophic yeast Pichia methanolica: cloning, sequencing and homology analysis.

The ADE1 gene of the yeast Pichia methanolica encodes phosphoribosyl-5-aminoimidazole-carboxylase (AIRC, EC 4.1.1.21), which is involved in purine biosynthesis. The gene was cloned by complementation of an ade2 mutation in Saccharomyces cerevisiae and a 3077 nucleotide DNA fragment was sequenced. The sequence possessed a single open reading frame, corresponding to a 543 amino acid sequence. The sequence of this putative protein has been compared to the proteins of homologous genes from S. cerevisiae, Schizosaccharomyces pombe, Escherichia coli, chicken and man. The analysis revealed remarkable homology between yeast AIRCs, while for other proteins homology was limited to defined regions.

Discovery of a novel, paternally expressed ubiquitin-specific processing protease gene through comparative analysis of an imprinted region of mouse chromosome 7 and human chromosome 19q13.4.

Using mouse BAC clones spanning an imprinted interval of proximal mouse chromosome 7 and the genomic sequence of the related interval of human chromosome 19q13.4, we have identified a novel mouse gene, Usp29 (ubiquitin-specific processing protease 29), near two known imprinted genes, Peg3 and Zim1. Gene Usp29 is located directly adjacent to Peg3 in a "head-to-head" orientation, and comprises exons distributed over a genomic distance of at least 400 kb. A similar human gene is also found in the homologous location in human chromosome 19q13.4. The mouse Usp29 gene is also imprinted and is transcribed mainly from the paternal allele with highest expression levels in adult brain, especially in the cerebral cortex and hippocampus, and in the forebrain, face, and limb buds of midgestation mouse embryos. Analysis of a full-length 7.6-kb cDNA clone revealed that Usp29 encodes an 869-amino-acid protein that displays significant homology with yeast and nematode ubiquitin carboxyl-terminal hydrolases. These data suggest that, like the candidate Angelman syndrome gene Ube3a (ubiquitin ligase), Usp29 may represent another imprinted gene involved in the ubiquitination pathway. This identification of a third imprinted gene, Usp29, from the Peg3/Zim1-region confirms the presence of a conserved imprinted domain spanning at least 500 kb in the proximal portion of mouse chromosome 7 (Mmu7).

Radial transformation-associated recombination cloning from the mouse genome: isolation of Tg.AC transgene with flanking DNAs.

Transformation-associated recombination (TAR) cloning allows entire genes and large chromosomal regions to be specifically, accurately, and quickly isolated from total genomic DNA. We report the first example of radial TAR cloning from the mouse genome. Tg.AC mice carry a zeta-globin promoter/v-Ha-ras transgene. Fluorescence in situ hybridization localized the transgene integrant as a single site proximal to the centromere of chromosome 11. Radial TAR cloning in yeast was utilized to create orientation-specific yeast artificial chromosomes (YACs) to explore the possibility that cis-flanking regions were involved in transgene expression. YACs containing variable lengths of 5' or 3' flanking chromosome 11 DNA and the Tg.AC transgene were specifically chosen, converted to bacterial artificial chromosomes (BACs), and assayed for their ability to promote transcription of the transgene following transfection into an FVB/N carcinoma cell line. A transgene-specific reverse transcription-polymerase chain reaction assay was utilized to examine RNA transcripts from stably transfected clones. All Tg.AC BACs expressed the transgene in this in vitro system. This report describes the cloning of the v-Ha-ras transgene and suggests that transcriptional activity may not require cis elements flanking the transgene's integration site.

HAM1, the gene controlling 6-N-hydroxylaminopurine sensitivity and mutagenesis in the yeast Saccharomyces cerevisiae.

The ham1 mutant of yeast Saccharomyces cerevisiae is sensitive to the mutagenic and lethal effects of the base analog, 6-N-hydroxylaminopurine (HAP). We have isolated a clone from a centromere-plasmid-based genomic library complementing HAP sensitivity of the ham1 strain. After subcloning, a 3.4 kb functional fragment was sequenced. It contained three open reading frames (ORFs) corresponding to proteins 353, 197 and 184 amino acids long. LEU2+ disruptions of the promoter and N-terminal part of the gene coding 197 amino acids long protein led to moderate and strong sensitivity to HAP, respectively, and were allelic to the original ham1-1 mutation. Thus this ORF represents the HAM1 gene. The deduced amino acid sequence of HAM1 protein was not similar to any protein sequence of the SwissProt database. The HAM1 gene was localized on the right arm of chromosome X between cdc8 and cdc11. Spontaneous mutagenesis was not affected by the ham1::LEU2 disruption mutation.