Gene disruption of a G4-DNA-dependent nuclease in yeast leads to cellular senescence and telomere shortening.

The yeast gene KEM1 (also named SEP1/DST2/XRN1/RAR5) produces a G4-DNA-dependent nuclease that binds to G4 tetraplex DNA structure and cuts in a single-stranded region 5' to the G4 structure. G4-DNA generated from yeast telomeric oligonucleotides competitively inhibits the cleavage reaction, suggesting that this enzyme may interact with yeast telomeres in vivo. Homozygous deletions of the KEM1 gene in yeast block meiosis at the pachytene stage, which is consistent with the hypothesis that G4 tetraplex DNA may be involved in homologous chromosome pairing during meiosis. We conjectured that the mitotic defects of kem1/sep1 mutant cells, such as a higher chromosome loss rate, are also due to failure in processing G4-DNA, especially at telomeres. Here we report two phenotypes associated with a kem1-null allele, cellular senescence and telomere shortening, that provide genetic evidence that G4 tetraplex DNA may play a role in telomere functioning. In addition, our results reveal that chromosome ends in the same cells behave differently in a fashion dependent on the KEM1 gene product.

Disruption of the gene for hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in thermotolerance.

The alpha-crystallin-related heat shock proteins are produced by all eukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disrupted expression of the single-copy hsp30 gene of Neurospora crassa, using repeat-induced point mutagenesis, and we generated and characterized mutant strains that were deficient in hsp30 synthesis. These strains could grow at high temperature and they acquired thermotolerance from a heat shock. However, the hsp30-defective strains proved to be extremely sensitive to the combined stresses of high temperature and carbohydrate limitation, enforced by the addition of a nonmetabolizable glucose analogue. Under these conditions, their survival was reduced by 90% compared with wild-type cells. This sensitive phenotype was reversed by reintroduction of a functional hsp30 gene into the mutant strains. The mutant cells contained mitochondria from which a 22-kDa protein was readily extracted with detergents, in contrast to its retention by the mitochondria of wild-type cells. Antibodies against hsp30 coimmunoprecipitated a protein also of approximately 22 kDa from wild-type cells. Results of this study suggest that hsp30 may be important for efficient carbohydrate utilization during high temperature stress and that it may interact with other mitochondrial membrane proteins and function as a protein chaperone.

Disruption of the adenosine deaminase gene causes hepatocellular impairment and perinatal lethality in mice.

We have generated mice with a null mutation at the Ada locus, which encodes the purine catabolic enzyme adenosine deaminase (ADA, EC 3.5.4.4). ADA-deficient fetuses exhibited hepatocellular impairment and died perinatally. Their lymphoid tissues were not largely affected. Accumulation of ADA substrates was detectable in ADA-deficient conceptuses as early as 12.5 days postcoitum, dramatically increasing during late in utero development, and is the likely cause of liver damage and fetal death. The results presented here demonstrate that ADA is important for the homeostatic maintenance of purines in mice.