ISSN:
1365-2958
Quelle:
Blackwell Publishing Journal Backfiles 1879-2005
Thema:
Biologie
,
Medizin
Notizen:
The genome of the archaeal virus φCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100–800 nucleotides. A fraction of φCh1 genomes is modified within 5′-GATC-3′ and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene. The protein product was designated M.φCh1-I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the β-subgroup of the (N6-adenine) methyltransferase class. The mtase gene of φCh1 was classified as a ‘late’ gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of φCh1. After infection of cells, M.φCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the φCh1 progeny genomes after infection of N. magadii carry the M.φCh1-I methylation in contrast to 50% of virus genomes generated by induction of φCh1-lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5′-GATC-3′. Additionally, expression of M.φCh1-I in dam–E. coli cells led to a complete substitution of the function of M.Dam in DNA mismatch repair.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1046/j.1365-2958.2000.01786.x
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