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.

Nature, incidence, and cause of work-related amputations in Minnesota.

BACKGROUND: The Minnesota Sentinel Event Notification System for Occupational Risks (SENSOR) has collected data on the nature, incidence, and cause of work-related amputation injuries that have taken place since 1992. METHODS: SENSOR defined an amputation as any finger amputation or the loss of any other body part; 832 workers were identified as having amputation injuries between 1994 and 1995 and 72% of these workers completed telephone interviews. RESULTS: The amputation injury rate for Minnesota workers was 39 per 100,000 workers, with agriculture and manufacturing having the highest rates. Sixty-six percent of the injuries involved one finger; 14% involved two or more fingers. Persons working with machinery reported 73% of the injuries. CONCLUSIONS: A closer examination of the incidence and causes for amputation injuries shows that these were not random events. Reliance on human reactions to prevent injury is inadequate; therefore, additional research needs to be conducted.

Medical, personal, and occupational outcomes for work-related amputations in Minnesota.

BACKGROUND: The Minnesota Sentinel Event Notification System for Occupational Risks (SENSOR) surveillance system has collected data on the medical, personal, and occupational outcomes associated with work-related amputations since 1992. METHODS: SENSOR defined amputations as any finger amputation or the loss of any other body part; 832 workers were identified as having amputation injuries between 1994 and 1995 and 72% of these workers completed a telephone interview. RESULTS: Twenty percent of those injured required overnight hospitalization. Ninety-one percent of the cases reported having missed work, with 56% reporting missing ten or more days. Individuals working on their usual jobs at the time of injury were more likely to report less serious medical and occupational outcomes. CONCLUSIONS: Severe injuries were significantly associated with worse medical, personal, and occupational outcomes. Two groups of machines, material handling, and powered handtools were associated with a higher proportion of severe injuries.

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.

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.

Peer assessment of social reputation in community samples of disruptive and nondisruptive children: utility of the revised class play method.

Examined the psychometric properties of the Revised Class Play (RCP), a peer assessment measure of social reputation, in epidemiological samples of disruptive (n = 220) and nondisruptive (n = 104) children in Grades 2 through 5. Principal components analyses yielded a four-factor solution that was similar for disruptive and nondisruptive children and to previous research with this instrument. Discriminative function analyses demonstrated that the four RCP dimensions were each successful in predicting group membership, with the leadership and social etiquette dimensions best able to differentiate disruptive and nondisruptive groups. Regression modeling showed that the variance accounted for by the four RCP dimensions was large and varied for specific dimensions based on the criterion variable chosen. The advantages of the RCP as a devise for tracking social competence and peer reputation in high-risk disruptive children are discussed.

Production and reutilization of an extracellular phosphatidylinositol catabolite, glycerophosphoinositol, by Saccharomyces cerevisiae.

Phosphatidylinositol catabolism in Saccharomyces cerevisiae is known to result in the formation of extracellular glycerophosphoinositol (GroPIns). We now report that S. cerevisiae not only produces but also reutilizes extracellular GroPIns and that these processes are regulated in response to inositol availability. A wild-type strain uniformly prelabeled with [3H] inositol displayed dramatically higher extracellular GroPIns levels when cultured in medium containing inositol than when cultured in medium lacking inositol. This difference in extracellular accumulation of GroPIns in response to inositol availability was shown to be a result of both regulated production and regulated reutilization. In a strain in which a negative regulator of phospholipid and inositol biosynthesis had been deleted (an opi1 mutant), this pattern of extracellular GroPIns accumulation in response to inositol availability was altered. An inositol permease mutant (itr1 itr2), which is unable to transport free inositol, was able to incorporate label from exogenous glycerophospho [3H]inositol, indicating that the inositol label did not enter the cell solely via the transporters encoded by itr1 and itr2. Kinetic studies of a wild-type strain and an itr1 itr2 mutant strain revealed that at least two mechanisms exist for the utilization of exogenous GroPIns: an inositol transporter-dependent mechanism and an inositol transporter-independent mechanism. The inositol transporter-independent pathway of exogenous GroPIns utilization displayed saturation kinetics and was energy dependent. Labeling studies employing [14C]glycerophospho[3H] inositol indicated that, while GroPIns enters the cell intact, the inositol moiety but not the glycerol moiety is incorporated into lipids.