Effective gene silencing in a microsporidian parasite associated with honeybee (Apis mellifera) colony declines.

Honeybee colonies are vulnerable to parasites and pathogens ranging from viruses to vertebrates. An increasingly prevalent disease of managed honeybees is caused by the microsporidian Nosema ceranae. Microsporidia are basal fungi and obligate parasites with much-reduced genomic and cellular components. A recent genome-sequencing effort for N. ceranae indicated the presence of machinery for RNA silencing in this species, suggesting that RNA interference (RNAi) might be exploited to regulate Nosema gene expression within bee hosts. Here we used controlled laboratory experiments to show that double-stranded RNA homologous to specific N. ceranae ADP/ATP transporter genes can specifically and differentially silence transcripts encoding these proteins. This inhibition also affects Nosema levels and host physiology. Gene silencing could be mediated solely by Nosema or in concert with known systemic RNAi mechanisms in their bee hosts. These results are novel for the microsporidia and provide a possible avenue for controlling a disease agent implicated in severe honeybee colony losses. Moreover, since microsporidia are pathogenic in several known veterinary and human diseases, this advance may have broader applications in the future for disease control.

Large-scale field application of RNAi technology reducing Israeli acute paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae).

The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.

Werner syndrome gene variants in human sarcomas.

Werner syndrome is an autosomal inherited disease that is characterized by premature aging. The gene mutated in Werner syndrome (WS), WRN, encodes both a 3' --> 5' DNA helicase and a 3' --> 5' DNA exonuclease. Among the WS phenotypes is an exceptionally high incidence of sarcomas. We asked whether spontaneous sarcomas, not known to be associated with WS, also harbor mutations or unreported single nucleotide polymorphisms (SNPs) in WRN. We analyzed RNA or DNA sequences within the helicase and exonuclease domains from 51 and 69 matched sarcoma and adjacent normal tissues, respectively. Among a total of 13 nucleotide variants detected, we identified three novel nonsynonymous substitutions: c.611C>T, c.809_810insT, and c.1882C>G. We further characterized one, c.611C>T, which results in substitution of an evolutionarily conserved proline at amino acid 204 in the exonuclease domain with leucine. We show that P204L WRN exhibits a reduction of WRN exonuclease activity; the specific activity is approximately 10-fold lower than that of wild-type WRN. In contrast, the helicase activity of P204L WRN is reduced less than twofold.

Mutations in DNA polymerase eta are not detected in squamous cell carcinoma of the skin.

The major etiological agent in skin cancer is exposure to UV-irradiation and the concomitant DNA damage. UV-induced DNA lesions, such as thymine dimers, block DNA synthesis by the major DNA polymerases and inhibit the progression of DNA replication. Bypass of thymine dimers and related lesions is dependent on the translesion polymerase DNA polymerase eta (Poleta). In the inherited disorder, xeroderma pigmentosum variant (XPV), inactivation of Poleta results in extreme sensitivity to UV light and a marked increase in the incidence of skin cancer. Here, we tested the hypothesis that somatic mutations and/or polymorphisms in the POLH gene that encodes Poleta are associated with the induction of UV-dependent skin cancers. We sequenced the exonic regions of the Poleta open reading frame in DNA from 17 paired samples of squamous cell skin carcinoma and adjacent histologically normal tissue. We analyzed approximately 120,000 nucleotides and detected no mutations in POLH in the tumors. However, we identified 6 different single-nucleotide polymorphisms, 3 of them previously undocumented, which were present in both the tumor and paired normal tissue. We conclude that neither mutations nor polymorphisms in the coding regions of POLH are required for the generation of human skin squamous cell carcinoma.

Is GAS1 a co-receptor for the GDNF family of ligands?

Glial-cell-line-derived neurotrophic factor (GDNF) is a survival and maintenance factor for dopamine-containing neurons and motoneurons. GDNF belongs to a family of structurally related factors that includes neurturin (NRTN), artemin (ARTN) and persephin (PSPN). An initial step in the activation of signaling via the GDNF family of ligands (GFLs) is their binding to their cognate co-receptor GFR alpha. GAS1, an apparently unrelated protein, exhibits homology to GFR alpha and thus we hypothesize that GAS1 can serve as an alternative receptor for GFLs. The functional similarity between GFR alpha and GAS1 extends to their role in embryogenesis, differentiation and glia maintenance, and is substantiated by overlap in their expression profile, subcellular localization and structural details. We propose that the relative expression and localization of the two remote receptors, GFR alpha and GAS1, on the membranes of neuronal and glial cells determines whether these cells survive or undergo apoptotic death.

Mitochondrial DNA integrity is not dependent on DNA polymerase-beta activity.

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-beta (Polbeta), which exhibits both 5'-deoxyribose-5-phosphate (5'-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-gamma (Polgamma) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polbeta or a "Polbeta-like" enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polbeta is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polgamma and the other to Polbeta, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polbeta from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polbeta activity. In a functional test for the presence of Polbeta function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polbeta null cells. In contrast, mtDNA from cells harboring mutations in the Polgamma exonuclease domain that abolish proofreading displayed a >or=17-fold increase in mutation frequency. We conclude that Polbeta is not an essential component of the machinery that maintains mtDNA integrity.

Amino acid substitutions at conserved tyrosine 52 alter fidelity and bypass efficiency of human DNA polymerase eta.

DNA polymerase eta (Pol eta) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase eta (hPol eta) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol eta Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to non-complementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol eta, one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.

In vitro production and screening of DNA polymerase eta mutants for catalytic diversity.

Mutant DNA polymerases have become an increasingly important tool in biotechnology. The ability to examine the activity and specific properties of enzymes has a crucial role in the characterization of the enzyme. We have developed several systems for characterizing DNA polymerases that combine random mutagenesis with in vivo selection systems. However in vivo screening systems for specific properties are sometimes unavailable. The ability to quickly screen for polymerase activity has many applications, including the identification of compounds that can inhibit polymerase activity, identifying the properties of newly discovered polymerases, and engineering new biological properties into existing polymerases. These applications can both expand the knowledge of the basic science of polymerases and can further industrial efforts to identify new drugs that specifically target polymerase activity. Here we present a high-throughput in vitro assay to select for active polymerases. We show the applicability of this assay by measuring the level of activity for a set of in vitro synthesized polymerase mutants and by screening for the incorporation of a fluorescent nucleotide analog by DNA polymerases.