Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein.

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.

Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome.

We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.

The yeast UME5 gene regulates the stability of meiotic mRNAs in response to glucose.

We reported previously that early meiotic transcripts are highly unstable. These mRNAs exhibit half-lives of approximately 3 min when expressed during vegetative growth in glucose medium and are stabilized twofold during sporulation in acetate medium. Two genes, UME2 and UME5, that regulate the stability of meiosis-specific transcripts have been identified. The wild-type UME5 gene, which has been analyzed in detail, decreases the stability of all meiotic mRNAs tested approximately twofold when expressed during vegetative growth but has no effect on the half-lives of a number of vegetative mRNAs examined. The UME5 gene is dispensable for mitotic and meiotic development. Cells in which the entire UME5 gene has been deleted are viable, although the generation time is slightly longer and sporulation is less efficient. The UME5 transcript is constitutively expressed, and its stability is not autoregulated. The UME5 gene encodes a predicted 63-kDa protein with homology to the family of CDC28 serine/threonine-specific protein kinases. The kinase activity appears to be central to the function of the UME5 protein, since alteration of a highly conserved amino acid in the kinase domain results in a phenotype identical to that of a ume5 deletion. Genetic epistasis studies suggest that the UME2 and UME5 gene products act in the same pathway to regulate meiotic transcript stability. This pathway is independent of deadenylation and translation, two factors known to be important in regulating mRNA turnover. Significantly, the UME5-mediated destabilization of meiotic mRNAs occurs in glucose- but not in acetate-containing medium. Thus, the UME5 gene appears to participate in a glucose signal transduction pathway governing message stability.

UME6 is a key regulator of nitrogen repression and meiotic development.

This report describes the identification, cloning, and molecular analysis of UME6 (CAR80/CARGRI), a key transcriptional regulator of early meiotic gene expression. Loss of UME6 function results in the accumulation of fully derepressed levels (70- to 100-fold increase above basal level) of early meiotic transcripts during vegetative growth. In contrast, mutations in five previously identified UME loci (UME1 to UME5), result in low to moderate derepression (2- to 10-fold increase) of early meiotic genes. The behavior of insertion and deletion alleles indicates that UME6 is dispensable for mitotic division but is required for meiosis and spore germination. Despite the high level of meiotic gene expression during vegetative growth, the generation times of ume6 mutant haploid and diploid cells are only slightly reduced. However, both ascus formation and spore viability are affected more severely. The UME6 gene encodes a 91-kD protein that contains a C6 zinc cluster motif similar to the DNA-binding domain of GAL4. The integrity of this domain is required for UME6 function. It has been reported recently that a mutation in CAR80 fails to complement an insertion allele of UME6. CAR80 is a gene required for nitrogen repression of the arginine catabolic enzymes. Here, through sequence analysis, we demonstrate that UME6 and CAR80 are identical. Analyses of UME6 mRNA during both nitrogen starvation and meiotic development indicate that its transcription is constitutive, suggesting that regulation of UME6 activity occurs at a post-transcriptional level.

Early meiotic transcripts are highly unstable in Saccharomyces cerevisiae.

Meiosis in Saccharomyces cerevisiae requires the induction of a large number of genes whose mRNAs accumulate at specific times during meiotic development. This study addresses the role of mRNA stability in the regulation of meiosis-specific gene expression. Evidence is provided below demonstrating that the levels of meiotic mRNAs are exquisitely regulated by both transcriptional control and RNA turnover. The data show that (i) early meiotic transcripts are extremely unstable when expressed during either vegetative growth or sporulation, and (ii) transcriptional induction, rather than RNA turnover, is the predominant mechanism responsible for meiosis-specific transcript accumulation. When genes encoding the early meiotic mRNAs are fused to other promoters and expressed during vegetative growth, their mRNA half-lives, of under 3 min, are among the shortest known in S. cerevisiae. Since these mRNAs are only twofold more stable when expressed during sporulation, we conclude that developmental regulation of mRNA turnover can be eliminated as a major contributor to meiosis-specific mRNA accumulation. The rapid degradation of the early mRNAs at all stages of the yeast life cycle, however, suggests that a specific RNA degradation system operates to maintain very low basal levels of these transcripts during vegetative growth and after their transient transcriptional induction in meiosis. Studies to identify specific cis-acting elements required for the rapid degradation of early meiotic transcripts support this idea. A series of deletion derivatives of one early meiosis-specific gene, SPO13, indicate that its mRNA contains determinants, located within the coding region, which contribute to the high instability of this transcript. Translation is another component of the degradation mechanism since frameshift and nonsense mutations within the SPO13 mRNA stabilize the transcript.

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.

Meiotic segregation of normal and deletion chromosomes in Saccharomyces cerevisiae.

We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on recombination and disjunction. Chromosome III derivatives in which the entire left arm or the entire right arm was deleted (telocentric) segregated with fidelity from a normal chromosome III. Recombination between either of these two deletion chromosomes and the intact chromosome also appeared normal. In a strain containing a right arm telocentric, a left arm telocentric and one normal chromosome both telocentrics disjoined from the normal chromosome. Homology on one arm was sufficient for proper recombination and segregation of these chromosomes. In strains containing two normal chromosomes and one telocentric chromosome the two normal chromosomes preferentially disjoined. In a few cases however, the two normal chromosomes cosegregated. Recombination between the two normal chromosomes or between one normal chromosome and the deletion chromosome increased the probability that they would disjoin, although cosegregation of recombinants was observed. A chromosome III derivative which contained a large centromeric deletion and an insertion of the centromere from chromosome V into a nonhomologous position segregated with fidelity from a normal chromosome III. These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs that directs meiotic chromosome disjunction.

Meiotic disjunction of homologs in Saccharomyces cerevisiae is directed by pairing and recombination of the chromosome arms but not by pairing of the centromeres.

We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on the pattern of disjunction and recombination of chromosome III during meiosis. The segregation of deletion chromosomes lacking part or all (telocentric) of one arm was analyzed in the presence of one or two copies of a normal chromosome III. In strains containing one normal and any one deletion chromosome, the two chromosomes disjoined in most meioses. In strains with one normal chromosome and both a left and right arm telocentric chromosome, the two telocentrics preferentially disjoined from the normal chromosome. Homology on one arm was sufficient to direct chromosome disjunction, and two chromosomes could be directed to disjoin from a third. In strains containing one deletion chromosome and two normal chromosomes, the two normal chromosomes preferentially disjoined, but in 4-7% of the tetrads the normal chromosomes cosegregated, disjoining from the deletion chromosome. Recombination between the two normal chromosomes or between the deletion chromosome and a normal chromosome increased the probability that these chromosomes would disjoin, although cosegregation of recombinants was observed. Finally, we observed that a derivative of chromosome III in which the centromeric region was deleted and CEN5 was integrated at another site on the chromosome disjoined from a normal chromosome III with fidelity. These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs, that directs meiotic chromosome segregation.

The mitotic stability of deletion derivatives of chromosome III in yeast.

We have constructed a series of deletion derivatives of chromosome III in yeast. Two telocentric chromosomes, one with a deletion of about 100 kilobases (kb) from the left arm and another with a deletion of about 240 kb from the right arm, are mitotically stable, showing only a 2- to 3-fold decrease in stability compared to a normal chromosome III. Chromosomes as small as 100 kb with deletions on both the left and right arms show only slight decreases in mitotic stability. Slight decreases in size in chromosomes smaller than 100 kb produce dramatic decreases in mitotic stability. In general, deletion chromosomes of similar size but different structure display similar stabilities. We find no evidence for the existence of any new cis-acting elements [besides the centromere, autonomously replicating sequences (ARS elements) and telomeres] essential for the stabilization of chromosome III.

Resolution of dicentric chromosomes by Ty-mediated recombination in yeast.

We have integrated a plasmid containing a yeast centromere, CEN5, into the HIS4 region of chromosome III by transformation. Of the three transformant colonies examined, none contained a dicentric chromosome, but all contained a rearranged chromosome III. In one transformant, rearrangement occurred by homologous recombination between two Ty elements; one on the left arm and the other on the right arm of chromosome III. This event produced a ring chromosome (ring chromosome III) of about 60 kb consisting of CEN3 and all other sequences between the two Ty elements. In addition, a linear chromosome (chromosome IIIA) consisting of sequences distal to the two Ty elements including CEN5, but lacking 60 kb of sequences from the centromeric region, was produced. Two other transformants also contain a similarly altered linear chromosome III as well as an apparently normal copy of chromosome III. These results suggest that dicentric chromosomes cannot be maintained in yeast and that dicentric structures must be resolved for the cell to survive.--The meiotic segregation properties of ring chromosome III and linear chromosome IIIA were examined in diploid cells which also contained a normal chromosome III. Chromosome IIIA and normal chromosome III disjoined normally, indicating that homology or parallel location of the centromeric regions of these chromosomes are not essential for proper meiotic segregation. In contrast, the 60-kb ring chromosome III, which is homologous to the centromeric region of the normal chromosome III, did not appear to pair with fidelity with chromosome III.

Construction of telocentric chromosomes in Saccharomyces cerevisiae.

We describe a simple method for the construction of large chromosomal deletions in yeast. Diploid yeast cells were transformed with DNA fragments that replace large regions of the chromosomes by homologous recombination. Using this method, we have constructed a telocentric chromosome III in which approximately equal to 100 kilobases (kb) of DNA has been removed from the left arm of the chromosome, so that the centromere is 12 kb from the left telomere. This telocentric chromosome is mitotically stable. Its rate of loss in a diploid strain is 2.5-7.4 X 10(-4) per cell division compared to a rate of loss of 0.36-1.8 X 10(-4) per cell division for a normal chromosome III. It also segregates 2+:2- with fidelity during meiosis. The construction of systematic deletions in a chromosome should be useful in determining the essential features for proper chromosomal segregation and replication.

Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae.

We have cloned a functional centromeric DNA sequence from Saccharomyces cerevisiae. Using the 2 mu chromosome-loss mapping technique and meiotic tetrad analysis, we have identified this DNA sequence as the centromere of chromosome V (CEN5). The CEN5 sequence has been localized on an 1,100-base-pair BamHI-BglII restriction fragment. Plasmids containing CEN5 and an autonomously replicating sequence are mitotically stable in S. cerevisiae and segregate in a Mendelian fashion during meiosis.