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Example sentences for: pyub
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A cosmid corresponding to the one in the sur3 clone was isolated from the E. coli (pYUB178::H37Rv) library by probing colony blots with the 4.5 kb Bam HI/ Eco Rl fragment of the M. tuberculosis genomic DNA insert in the plasmid recovered from the sur3 clone.
The pYUB178 plasmid and the λ phage library of pYUB178::H37Rv cosmids were generously provided by Dr. William Jacobs, Albert Einstein University, New York, NY [ 20 ] . The cosmid library contains 30-50 kb fragments of M. tuberculosis genomic DNA generated by partial Sau 3A digestion cloned into Bcl I-digested pYUB178.
In this study, the enrichment process resulted in the isolation of two strains containing small, integrated plasmids rather than the expected 30-50 kb cosmids [ 20 ] . Small plasmids corresponding to the integrated ones were present in the original E. coli (pYUB178:H37Rv) cosmid library.
To enrich for clones with increased survival in human macrophages, the library was passed through THP-1 macrophages as shown schematically in Figure 1. THP-1 is a human monocyte-derived macrophage cell line [ 21 ] and wild-type M. smegmatis bacteria are rapidly killed by THP-1 macrophages [ 22 ] . After three rounds of enrichment, the resulting clones were analyzed individually by Southern blot to evaluate the number of different clones present as previously described [ 20 ] . Out of the 3000 colonies recovered, genomic DNAs from the bacteria of 90 randomly chosen colonies were digested with Pst I and hybridized with the pYUB178 vector.
Bacteria from ~4,000 colonies (representing ~20 genome equivalents) were recovered and pooled to generate the M. smegmatis (pYUB178::H37Rv) library.
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