Rafael J. Yáñez

Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair

African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3′-5′-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase β (pol β), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol β-type DNA polymerase core, constituted by only the “palm” and “thumb” subdomains. It is worth noting that such an “unfingered” DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.

Two putative African swine fever virus helicases similar to yeast ‘DEAH’ pre-mRNA processing proteins and vaccinia virus ATPases D11L and D6R

Two open reading frames (ORFs) of African swine fever virus (ASFV) encoding putative helicases have been sequenced. The two genes, termed D1133L and B962L, are located in the central region of the viral genome, but are separated by about 40 kb of DNA. Both genes are expressed late during ASFV infection of Vero cells, after replication of viral DNA has begun. Contiguous to D1133L, three other ORFs (D129L, D79L and D339L), encoding putative proteins of unknown function, have been sequenced. Proteins D1133L and B962L contain the amino acid motifs that characterize helicases of superfamily II. D1133L is most similar to a group of putative helicases which includes two proteins of vaccinia virus (D11L and D6R) involved in transcription of the viral genome, their homologues in other poxviruses, the protein encoded by ORF 4 of the yeast plasmids, pGKL2 and pSKL, and the previously identified ASFV protein, Q706L. B962L resembles a group of RNA-helicase-like proteins which includes three proteins of Saccharomyces cerevisiae involved in pre-mRNA splicing (PRP2, PRP16 and PRP22), Drosophila melanogaster KURZ and MLE, and vaccinia virus 18R.

Induction of balbiani ring puffing changes by sugars and alcohols in Chironomus thummi

Abstract The capacity of several substances to induce in Chironomus thummi larvae the repression of Balbiani ring 2 (BR2) and the coordinated activation of Balbiani ring 1 (BR1) has been evaluated. Hexoses as well as some disaccharides, except lactose, were efficient inducers. The pentoses tested failed to provoke puffing changes. Alcohols, such as ethanol and glycerol, showed a moderate capacity as inducers, whereas methanol and isopropanol were ineffective. When a mixture of galactose and glycerol was used, a significant acceleration of the response, at the puffing as well as the translational level, was observed. To search for common effects of different inducers, an attempt was first made to test whether the lowering of the inorganic phosphate level in the haemolymph could play a causal role in the shift of BR1/BR2 puffing as it does in the response of the Camptochironomus group. In C. thummi , we have found effective inducers which increased, decreased or did not modify the inorganic phosphate concentration in the haemolymph thus suggesting the independence of the induced puffing changes and phosphate level modifications.