RESUMO
To analyze the methylation status of wild-type adeno-associated virus type 2 (AAV2), bisulfite PCR sequencing (BPS) of the packaged viral genome and its integrated form was performed and 262 of the total 266 CG dinucleotides (CpG) were mapped. In virion-packaged DNA, the ratio of the methylated cytosines ranged between 0â»1.7%. In contrast, the chromosomally integrated AAV2 genome was hypermethylated with an average of 76% methylation per CpG site. The methylation level showed local minimums around the four known AAV2 promoters. To study the effect of methylation on viral rescue and replication, the replication initiation capability of CpG methylated and non-CpG methylated AAV DNA was compared. The in vitro hypermethylation of the viral genome does not inhibit its rescue and replication from a plasmid transfected into cells. This insensitivity of the viral replicative machinery to methylation may permit the rescue of the integrated heavily methylated AAV genome from the host's chromosomes.
Assuntos
Ilhas de CpG , Metilação de DNA , Genoma Viral , Parvovirinae/genética , Dependovirus , Sequenciamento de Nucleotídeos em Larga Escala , Parvovirinae/fisiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Vírion/genética , Montagem de Vírus , Replicação ViralRESUMO
A novel Gram-stain-positive bacterial strain, designated as K13T, was isolated from compost and characterized using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, the strain showed highest similarity (93.8â%) to Paenibacillus nanensis MX2-3T. Cells of strain K13T were aerobic, motile rods. The major fatty acids were anteiso C15â:â0 (34.4â%), iso C16â:â0 (17.3â%) and C16â:â0 (10.0â%). The major menaquinone was MK-7, the polar lipid profile included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and an aminophospholipid. The DNA G+C content was 52.3â%. Based on phenotypic, including chemotaxonomic characteristics and analysis of the 16S rRNA gene sequences, it was concluded that strain K13T represents a novel genus, for which the name Xylanibacillus gen. nov., sp. nov. is proposed. The type species of the genus is Xylanibacillus composti, the type strain of which is strain K13T (=DSM 29793T=NCAIM B.02605T).
Assuntos
Bacillales/classificação , Compostagem , Filogenia , Microbiologia do Solo , Bacillales/genética , Bacillales/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
We provide evidence that a prokaryotic insertion sequence (IS) element is active in a vertebrate system. The transposase of Escherichia coli element IS30 catalyzes both excision and integration in extrachromosomal DNA in zebrafish embryos. The transposase has a pronounced target preference, which is shown to be modified by fusing the enzyme to unrelated DNA binding proteins. Joining the transposase to the cI repressor of phage lambda causes transposition primarily into the vicinity of the lambda operator in E. coli, and linking to the DNA binding domain of Gli1 also directs the recombination activity of transposase near to the Gli1 binding site in zebrafish. Our results demonstrate the possibility of fusion transposases to acquire novel target specificity in both prokaryotes and eukaryotes.