Effects of mcr restriction of methylated CpG islands of the L1 transposons during packaging and plating stages of mammalian genomic library construction.
The use of optimally methylation-tolerant mcrA- mcrB- strains has been shown to produce an over tenfold increase in the plating efficiencies of mammalian genomic libraries, compared to a superior conventional phage host strain LE392 which is mcrB+. However, there is an even more significant effect of mcr restriction. Amongst the recombinants recovered with an mcrB+ host, we have found that there is an additional 30-fold reduction in the frequencies of clones containing the heavily methylated 5'-CpG island sequences of both the human and rat L1 repetitive elements. The mcrA product was also found to restrict clones of these methylated genomic segments, but not as strongly as mcrB. However, the use of packaging extracts made from mcrA+ lysogens did not result in convincing reductions in the recoveries of these dispersed methylated elements. The magnitude of mcr restriction during plating due to methylated dispersed elements is sufficient to make a significant proportion of mammalian genomes unclonable from genomic libraries constructed previously using conventional mcr+ hosts.
Doherty
J P
JP
Molecular Genetics, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Graham
M W
MW
Linsenmeyer
M E
ME
Crowther
P J
PJ
Williamson
M
M
Woodcock
D M
DM
D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D003851:Deoxyribonucleases / Q000235:genetics; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D015698:Genomic Library; D005838:Genotype; D006801:Humans; D008745:Methylation; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid
D004251:DNA Transposable Elements; D003851:Deoxyribonucleases; D015226:Dinucleoside Phosphates; C015772:CpG dinucleotide