ABSTRACT
Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages - PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with â¼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.
Subject(s)
Bacteriolysis/physiology , Mycobacteriophages/isolation & purification , Mycobacteriophages/metabolism , Mycobacterium tuberculosis/virology , Base Composition , DNA, Viral/genetics , Genes, Viral/genetics , Genome, Viral , India , Mycobacteriophages/classification , Mycobacteriophages/genetics , Mycobacterium smegmatis/virology , Open Reading Frames , Siphoviridae/classification , Siphoviridae/genetics , Siphoviridae/isolation & purificationABSTRACT
The expansion of CGG repeats in the 5'-untranslated region (5'UTR) of FMR1 gene is the molecular basis of fragile X syndrome in most of the patients. The nature of the flanking sequences in addition to the length and interruption pattern of repeats is predicted to influence CGG repeat instability in the FMR1 gene. We investigated nucleosome occupancy as a contributor to CGG repeat instability in a transgenic mouse model containing unstable (CGG)(26,) from human FMR1 cloned downstream of nucleosome-excluding sequence. We observe that the transgene has an open chromatin structure compared to the stable endogenous mouse Fmr1 within the same nucleus. CGG repeats in mouse Fmr1 are flanked by nucleosomes unlike the repeats in the transgene in all the tissues examined. Further in vitro chromatin reconstitution experiments show that DNA fragment without the SV40ori/EPR (nucleosome-excluding sequence) forms more stable chromatin than the one containing it, despite having the same number of CGG repeats. The correlation between nucleosomal organisation of the FMR1 gene and CGG repeat instability was supported by significantly lower frequency of repeat expansion in mice containing an identical transgene without the SV40ori/EPR. Our studies demonstrate that flanking DNA sequences can influence repeat instability through modulation of nucleosome occupancy in the region.
Subject(s)
Fragile X Mental Retardation Protein/genetics , Fragile X Syndrome/genetics , Nucleosomes/genetics , Nucleosomes/metabolism , Trinucleotide Repeats/genetics , Animals , Base Sequence , Chromatin/genetics , Chromatin/metabolism , Disease Models, Animal , Female , Gene Deletion , Gene Expression Regulation/genetics , Gene Order , Gene Targeting , Genetic Vectors/genetics , Genomic Instability , Humans , Male , Mice , Mice, Transgenic , Molecular Sequence Data , Pedigree , Sequence Alignment , Transgenes/geneticsABSTRACT
The expression of genes in transgenic mice is known to be influenced by the site of integration even when they carry their own promoter elements and transcription factor binding sites. The cytomegalovirus (CMV) promoter, a strong promoter often used for transgene expression in mammalian cells in culture, is known to be silenced by DNA methylation and histone deacetylation but there is no report on the role of histone methylations in its regulation. We generated two transgenic lines carrying green fluorescence protein coding gene as reporter driven by cytomegalovirus major immediate-early promoter/enhancer. We observe that silencing of CMV promoter is dependent on the site of transgene integration, except in testis, and the nature of DNA and histone methylations strongly correlate with the expression status of the reporter. We find that silenced CMV promoter interacts in vivo, with Methyl CpG binding protein 2 (MeCP2), a recruiter of histone deacetylases (HDACs) and histone (H3K9) methyl transferase. Histone H3K4methylation, the active chromatin mark, is also associated with silenced promoter, suggesting bivalent marking of the promoter and its susceptibility to reactivation on induction.
Subject(s)
Antigens, Viral/genetics , Cytomegalovirus/genetics , Epigenesis, Genetic , Gene Expression Regulation, Viral , Immediate-Early Proteins/genetics , Promoter Regions, Genetic/genetics , Animals , Antigens, Viral/metabolism , Cells, Cultured , Chromatin/metabolism , Chromatin Immunoprecipitation , CpG Islands , DNA Methylation , Gene Silencing , Green Fluorescent Proteins/metabolism , Histone Deacetylases , Histones/metabolism , Humans , Immediate-Early Proteins/metabolism , Kidney/cytology , Kidney/metabolism , Methyl-CpG-Binding Protein 2/genetics , Methyl-CpG-Binding Protein 2/metabolism , Mice , Mice, Transgenic , Regulatory Sequences, Nucleic Acid , Virus ReplicationABSTRACT
The proteins belonging to SWI2/SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression. We have identified a human gene with a putative DNA binding domain, which belongs to the INO80 subfamily of SWI2/SNF2 proteins. Here we report the cloning, expression, and functional activity of the domains from hINO80 gene both in terms of the DNA dependent ATPase as well as DNA binding activity. A differential expression of the various domains within this gene is detected in human tissues while a ubiquitous expression is detected in mice. The intranuclear localization is demonstrated using antibodies directed against the DBINO domain of hINO80.
