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J J Miret

Publications and source records attributed to J J Miret.

12 recordsLinked to original sources
PubMed2001

Efficient repair of large DNA loops in Saccharomyces cerevisiae.

Small looped mispairs are efficiently corrected by mismatch repair. The situation with larger loops is less clear. Repair activity on large loops has been reported as anywhere from very low to quite efficient. There is also uncertainty about how many loop repair activities exist and whether any are conserved. To help address these issues, we studied large loop repair in Saccharomyces cerevisiae using in vivo and in vitro assays. Transformation of heteroduplexes containing 1, 16 or 38 nt loops led to >90% repair for all three substrates. Repair of the 38 base loop occurred independently of mutations in key genes for mismatch repair (MR) and nucleotide excision repair (NER), unlike other reported loop repair functions in yeast. Correction of the 16 base loop was mostly independent of MR, indicating that large loop repair predominates for this size heterology. Similarities between mammalian and yeast large loop repair were suggested by the inhibitory effects of loop secondary structure and by the role of defined nicks on the relative proportions of loop removal and loop retention products. These observations indicate a robust large loop repair pathway in yeast, distinct from MR and NER, and conserved in mammals.

Base Pair Mismatch↗
PubMed2001

Cis-elements governing trinucleotide repeat instability in Saccharomyces cerevisiae.

Trinucleotide repeat (TNR) instability in humans is governed by unique cis-elements. One element is a threshold, or minimal repeat length, conferring frequent mutations. Since thresholds have not been directly demonstrated in model systems, their molecular nature remains uncertain. Another element is sequence specificity. Unstable TNR sequences are almost always CNG, whose hairpin-forming ability is thought to promote instability by inhibiting DNA repair. To understand these cis-elements further, TNR expansions and contractions were monitored by yeast genetic assays. A threshold of approximately 15--17 repeats was observed for CTG expansions and contractions, indicating that thresholds function in organisms besides humans. Mutants lacking the flap endonuclease Rad27p showed little change in the expansion threshold, suggesting that this element is not altered by the presence or absence of flap processing. CNG or GNC sequences yielded frequent mutations, whereas A-T rich sequences were substantially more stable. This sequence analysis further supports a hairpin-mediated mechanism of TNR instability. Expansions and contractions occurred at comparable rates for CTG tract lengths between 15 and 25 repeats, indicating that expansions can comprise a significant fraction of mutations in yeast. These results indicate that several unique cis-elements of human TNR instability are functional in yeast.

Endodeoxyribonucleases↗
PubMed1998

Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae.

A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast. A promoter containing 25 repeats allows expression of a URA3 reporter gene and yields sensitivity to the drug 5-fluoroorotic acid. Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance. When integrated at either of two chromosomal loci, expansion rates were 1 x 10(-5) to 4 x 10(-5) per generation if CTG repeats were replicated on the lagging daughter strand. PCR analysis indicated that 5-28 additional repeats were present in 95% of the expanded alleles. No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52. The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system. In contrast, expansions of the complementary repeat CAG occurred at 500- to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence. When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand. Among the rare CAG expansions, the largest gain in tract size was 38 repeats. The control repeats CTA and TAG showed no detectable rate of expansions. The orientation-dependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments.

Base Sequence↗
PubMed1997

Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pair recognition.

Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6p form a complex that specifically binds to DNA containing base pair mismatches. In this study, we performed a genetic and biochemical analysis of the Msh2p-Msh6p complex by introducing point mutations in the ATP binding and putative helix-turn-helix domains of MSH2. The effects of these mutations were analyzed genetically by measuring mutation frequency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes. A mutation in the ATP binding domain of MSH2 did not affect the mismatch binding specificity of the msh2p-Msh6p complex; however, this mutation conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochemical defects consistent with defects in mismatch repair downstream of mismatch recognition. Helix-turn-helix domain mutant proteins displayed two different properties. One class of mutant proteins was defective in forming complexes with Msh6p and also failed to recognize base pair mismatches. A second class of mutant proteins displayed properties similar to those observed for the ATP binding domain mutant protein. Taken together, these data suggested that the proposed helix-turn-helix domain of Msh2p was unlikely to be involved in mismatch recognition. We propose that the MSH2 helix-turn-helix domain mediates changes in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.

Adenosine Triphosphatases↗
PubMed1997

Instability of CAG and CTG trinucleotide repeats in Saccharomyces cerevisiae.

A quantitative genetic assay was developed to monitor alterations in tract lengths of trinucleotide repeat sequences in Saccharomyces cerevisiae. Insertion of (CAG)50 or (CTG)50 repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expression and white colony color. Contractions within the trinucleotide sequences to repeat lengths of 8 to 38 restore functional expression of the reporter, leading to red colony color. Reporter constructs including (CAG)50 or (CTG)50 repeat sequences were integrated into the yeast genome, and the rate of red colony formation was measured. Both orientations yielded high rates of instability (4 x 10(-4) to 18 x 10(-4) per cell generation). Instability depended on repeat sequences, as a control harboring a randomized (C,A,G)50 sequence was at least 100-fold more stable. PCR analysis of the trinucleotide repeat region indicated an excellent correlation between change in color phenotype and reduction in length of the repeat tracts. No preferential product sizes were observed. Strains containing disruptions of the mismatch repair gene MSH2, MSH3, or PMS1 or the recombination gene RAD52 showed little or no difference in rates of instability or distributions of products, suggesting that neither mismatch repair nor recombination plays an important role in large contractions of trinucleotide repeats in yeast.

DNA Repair↗
PubMed1996

Recognition of DNA insertion/deletion mismatches by an activity in Saccharomyces cerevisiae.

An activity in nuclear extracts of S.cerevisiae binds specifically to heteroduplexes containing four to nine extra bases in one strand. The specificity of this activity (IMR, for insertion mismatch recognition) in band shift assays was confirmed by competition experiments. IMR is biochemically and genetically distinct from the MSH2 dependent, single base mismatch binding activity. The two activities migrate differently during electrophoresis, they are differentially competable and their spectra of mispair binding are distinct. Furthermore, IMR activity is observed in extracts from an msh2- msh3- msh4- strain. IMR exhibits specificity for insertion mispairs in two different sequence contexts. Binding is influenced by the structure of the mismatch since an insertion with a hairpin configuration is not recognized by this activity. IMR does not result from single-strand binding because single-stranded probes to not yield IMR complex and single-stranded competitors are unable to displace insertion heteroduplexes from the complex. Similar results with intrinsically bent duplexes make it unlikely that recognition is conferred by a bend alone. Heteroduplexes bound by IMR do not contain any obvious damage. These findings are consistent with the idea that yeast contains a distinct recognition factor, IMR that is specific for insertion/deletion mismatches.

Base Composition↗
PubMed1994

The Golgi guanosine diphosphatase is required for transport of GDP-mannose into the lumen of Saccharomyces cerevisiae Golgi vesicles.

The Saccharomyces cerevisiae Golgi lumenal guanosine diphosphatase is hypothesized to generate GMP which in turn allows entry of GDP-mannose into the lumen to serve as substrate for mannosylation of proteins and lipids. We have recently shown in studies in vivo that this GDPase is required for protein and sphingolipid mannosylation in the Golgi lumen of S. cerevisiae. We have now isolated Golgi-vesicles from wild type and gda1 null mutants (GDPase defective) and have found that the initial rate of GDP-mannose entry into mutant vesicles was 5-fold lower than into those of wild type. Because the concentration of GDP within vesicles is insufficient to inhibit Golgi lumenal mannosyltransferases and the null mutant vesicles are impaired in synthesis of Golgi mannoproteins, the above results demonstrate that the reduced availability of GDP-mannose in the null mutants is the cause for altered Golgi mannosylation of macromolecules.

Biological Transport↗
PubMed1993

Characterization of a DNA mismatch-binding activity in yeast extracts.

An activity present in nuclear extracts of the yeast Saccharomyces cerevisiae binds specifically to oligonucleotides containing DNA mismatches, as judged by a band shift assay. The specificity of this activity for mismatched DNA was confirmed by competition experiments; binding to radiolabeled heteroduplexes was abolished in the presence of excess unlabeled heteroduplex but not when excess unlabeled homoduplex was added. Both T/G and T/- (single base deletion) mispairs were recognized in each of two sequence contexts. Binding was also observed with G/G, G/A, A/C, and T/C mismatches, but recognition of a C/C mispair was very weak. Competition studies with the various mismatches were consistent with the idea that a single activity recognizes all mispairs tested. Extracts from strains mutant in either or both of two putative mismatch recognition functions, MSH2 and MSH3, were also tested. Mismatch-binding activity was present in extracts of msh3- strains but completely absent in msh2- strains. The molecular weight of the major binding protein was estimated by UV cross-linking experiments to be approximately 110 kDa, in good agreement with the size predicted for Msh2 protein (Reenan, R. A. and Kolodner, R. D. (1992) Genetics 132, 963-973).

Base Composition↗
PubMed1992

Polyamines and cell wall organization in Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae 179-5, an ornithine decarboxylase mutant (spe-1), showed several ultrastructural abnormalities when cultivated in the absence of polyamines. Besides the appearance of microvacuole-like spaces in the cytoplasm and of deformed nuclei, the most important alterations seemed to be located in the cell wall, which was thicker and of heterogeneous texture, and in the cell membrane, of irregular contour. These modifications could not be evoked by general stress conditions elicited by lack of nutrients. The relative levels of cell wall polysaccharides were altered in polyamine-deprived organisms, giving an envelope with increased mannan and decreased glucan content; this cell wall was incompletely attacked by the lytic enzyme zymolyase. Polyamine depletion led also to some abnormalities in the budding pattern. The above observations suggest the involvement of polyamines in the correct structure and organization of the yeast cell.

Cell Wall↗
PubMed1986

Altered heat-shock response in polyamine-depleted bacteria.

A polyamine-auxotrophic mutant of E. coli was cultivated in the presence or absence of putrescine and submitted to heat shock over 3 different ranges of temperature. In all cases, protein synthetic capacity measured in comparison to that of cultures at the preshift temperature was much higher in polyamine-depleted bacteria under thermic stress. Addition of putrescine only before the shift-up was able to restore gradually normal control of the relative protein synthetic capacity.

Bacterial Proteins↗
PubMed1985

Effect of alpha-difluoromethylornithine in lung metastases before and after surgery of primary adenocarcinoma tumors in mice.

The depletion of polyamines by alpha-difluoromethylornithine (DFMO) treatment of mice after subcutaneous inoculation of adenocarcinoma M3 cells caused a remarkable inhibition in the growth rate of primary tumors as well as in the occurrence and number of lung metastases with a concomitant increase in survival time. Tumor-bearing mice submitted to the surgical removal of primary tumors and then treated with alpha-difluoromethylornithine also showed a significant reduction of lung metastases. In addition, a lower number of lung metastatic nodules correlated with decreased levels of polyamines in the same tissue. The described approach provides a useful experimental model for studies in human cancer therapy.

Adenocarcinoma↗