The genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA polymerases slipping during replication, and that some types of colorectal cancer may reflect mutations in genes involved in DNA mismatch repair.
In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD ؉ , with a twofold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8 m reversion assay, with no increase in nuclear mutations. Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases.
Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations (2,3,6), whereas the msh3 mutation has only a 2-fold effect on the mutation rate (5); reversion rates of a frameshift mutation are elevated about 1000-fold by MSH2, MLH1, or PMS1 and about 10-fold by MSH3. Homologues to the yeast MSH3 gene exist in mice (7) and humans (8).Alterations in the length of simple repetitive DNA tracts in yeast are likely to reflect DNA polymerase slippage events (9, 10). In wild-type strains, the displaced repeats resulting from DNA polymerase slippage (Fig. 1) are often corrected by excising the mismatched region from the newly synthesized strand and repairing the gap by using information derived from the template strand. Although repeats are displaced on only one strand in each slippage event, it should be noted that both strands reflect a distortion of the helix. As expected from this model, yeast strains with pmsl, msh2, or mlhl mutations have greatly increased levels of instability in microsatellite sequences (9).Mutations in human homologs of MSH2, MLH1, and PMS1 lead to a variety of hereditary tumors (11)(12)(13)(14)(15) (9) and contained the assay plasmids pSH31 (MS85) or pSH91 (MS96). MS128 was derived from AMY125 by insertion of an msh2 deletion allele [constructed by using the plasmid pll-2-Tn1OLUK7-7 (6) obtained from E. Alani, Cornell University, Ithaca, NY], transformation with the assay plasmid pSH91, and insertion of a LEU2 gene by using CV9. MS111 was constructed by transforming AMY125 with CV9 (MS71), transforming MS71 with pEN33 (strain GCY140; contains deletion mutation of MSH3), and transforming GCY140 with pSH91. MS125 was constructed by transforming GCY178 with pSH91; GCY178 was made by doing a two-step transplacement of GCY141 (an msh3 mutant strain isogenic with GCY140) with the plasmid p3O6M2RIA to introduce the msh2 mutation. MS110 and MS111 are isogenic, as are MS124 and MS125, except for the assay plasmid. MS94 was constructed by transforming AMY125 with the plasmid pII-2-Tn1OLUK7-7 to introduce the msh2 mutation, followed by transformation with the assay plasmid pSH31.Assays of Microsatellite Instability. Two plasmids, pSH31 (22) and pSH91 (9), were used to measure microsatellite instability. Since the methods used to monitor tract instability have been published (22), the description of these methods is abbreviated. The plasmid pSH31 contained an out-of-frame 29-bp poly(GT) tract within the coding sequence of 13-galactosidase (Fig. 2a). Strains containing this plasmid formed white colonies on plates containing 5-bromo-4-chloro-3-indolyl f-D-galactoside (X-Gal); strains that had an alteration in the poly(GT) tract length that restored the correct reading frame of the 13-galactosidase gene, however, formed blue colonies (as confirmed by DNA sequence analysis). To determine a rate of instability, we determined the frequency of blue colonies in 15-20 cultures. If any of the cultures had no blue Abbreviations: X-Gal, ...
Most pollination in large-scale agriculture is dependent on managed colonies of a single species, the honey bee Apis mellifera. More than 1 million hives are transported to California each year just to pollinate the almonds, and bees are trucked across the country for various cropping systems. Concerns have been raised about whether such “migratory management” causes bees undue stress; however to date there have been no longer-term studies rigorously addressing whether migratory management is detrimental to bee health. To address this issue, we conducted field experiments comparing bees from commercial and experimental migratory beekeeping operations to those from stationary colonies to quantify effects on lifespan, colony health and productivity, and levels of oxidative damage for individual bees. We detected a significant decrease in lifespan of migratory adult bees relative to stationary bees. We also found that migration affected oxidative stress levels in honey bees, but that food scarcity had an even larger impact; some detrimental effects of migration may be alleviated by a greater abundance of forage. In addition, rearing conditions affect levels of oxidative damage incurred as adults. This is the first comprehensive study on impacts of migratory management on the health and oxidative stress of honey bees.
Western honey bees, Apis mellifera, live in highly eusocial colonies that are each typically headed by a single queen. The queen is the sole reproductive female in a healthy colony, and because long-term colony survival depends on her ability to produce a large number of offspring, queen health is essential for colony success. Honey bees have recently been experiencing considerable declines in colony health. Among a number of biotic and abiotic factors known to impact colony health, disease and queen failure are repeatedly reported as important factors underlying colony losses. Surprisingly, there are relatively few studies on the relationship and interaction between honey bee diseases and queen quality. It is critical to understand the negative impacts of pests and pathogens on queen health, how queen problems might enable disease, and how both factors influence colony health. Here, we review the current literature on queen reproductive potential and the impacts of honey bee parasites and pathogens on queens. We conclude by highlighting gaps in our knowledge on the combination of disease and queen failure to provide a perspective and prioritize further research to mitigate disease, improve queen quality, and ensure colony health.
Several investigators have reported that transcription stimulates some types of mitotic recombination in the yeast Saccharomyces cerevisiae. We find that mutations that reduce the rate of transcription of the yeast HIS4 gene in vegetative cells reduce the frequency of mitotic, but not meiotic, recombination events.
A number of nuclear mutations have been identified in a variety of mitochondrial diseases including progressive external ophthalmoplegia (PEO), Alpers syndrome and other neuromuscular and oxidative phosphorylation defects. More than 50 mutations have been identified in POLG, which encodes the human mitochondrial DNA (mtDNA) polymerase gamma, PEO and Alpers patients. To rapidly characterize the effects of these mutations, we have developed a versatile system that enables the consequences of homologous mutations, introduced in situ into the yeast mtDNA polymerase gene MIP1, to be evaluated in vivo in haploid and diploid cells. Overall, distinct phenotypes for expression of each of the mip1-PEO mutations were observed, including respiration-defective cells with decreased viability, dominant-negative mutant polymerases, elevated levels of mitochondrial and nuclear DNA damage and chromosomal mutations. Mutations in the polymerase domain caused the most severe phenotype accompanied by loss of mtDNA and cell viability, whereas the mutation in the exonuclease domain showed mild dominance with loss of mtDNA. Interestingly, the linker region mutation caused elevated mitochondrial and nuclear DNA damage. The cellular processes contributing to these observations in the mutant yeast cells are potentially relevant to understanding the pathologies observed in human mitochondrial disease patients.
The ongoing decline of honey bee health worldwide is a serious economic and ecological concern. One major contributor to the decline are pathogens, including several honey bee viruses. However, information is limited on the biology of bee viruses and molecular interactions with their hosts. An experimental protocol to test these systems was developed, using injections of Israeli Acute Paralysis Virus (IAPV) into honey bee pupae reared ex-situ under laboratory conditions. The infected pupae developed pronounced but variable patterns of disease. Symptoms varied from complete cessation of development with no visual evidence of disease to rapid darkening of a part or the entire body. Considerable differences in IAPV titer dynamics were observed, suggesting significant variation in resistance to IAPV among and possibly within honey bee colonies. Thus, selective breeding for virus resistance should be possible. Gene expression analyses of three separate experiments suggest IAPV disruption of transcriptional homeostasis of several fundamental cellular functions, including an up-regulation of the ribosomal biogenesis pathway. These results provide first insights into the mechanisms of IAPV pathogenicity. They mirror a transcriptional survey of honey bees afflicted with Colony Collapse Disorder and thus support the hypothesis that viruses play a critical role in declining honey bee health.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.