Global transposon mutagenesis and a minimal mycoplasma genome

Science

Volumn 286, Issue 5447, 1999, Pages 2165-2169

  Hutchison III, Clyde A ^a,b   Peterson, Scott N ^a   Gill, Steven R ^a   Cline, Robin T ^a   White, Owen ^a   Fraser, Claire M ^a   Smith, Hamilton O ^a,c   Venter, J Craig ^a,c  

^a J CRAIG VENTER INSTITUTE   (United States)

^b UNIVERSITY OF NORTH CAROLINA SCHOOL OF MEDICINE   (United States)

^c CELERA GENOMICS   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

DNA FRAGMENT;

ARTICLE; BACTERIAL GENE; DNA REPLICATION; GENETIC COMPLEMENTATION; GENOME; MUTAGENESIS; MYCOPLASMA; MYCOPLASMA GENITALIUM; MYCOPLASMA PNEUMONIAE; PRIORITY JOURNAL; SEQUENCE ANALYSIS; TRANSPOSON;

MYCOPLASMA GENITALIUM; MYCOPLASMA PNEUMONIAE;

EID: 0032764518     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5447.2165     Document Type: Article

References (34)

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21. 9. This procedure was designed to make the subsequent cloning of transposition events from nonviable cells highly improbable. Genomic DNA was isolated from mid-log cultures; 2 μg of DNA was digested with Dra I. The genomic DNA restriction digests were diluted to 5 ng/μl and fragments were circularized using DNA ligase. Transposon junctions were amplified using inverse PCR with two primers specific for the end of the transposon Tn4001. Reaction products containing oligonucleotide-encoded Eco RI and Hind III sites were digested with these enzymes and cloned into the corresponding sites in pUC18. DNA sequencing templates were prepared from selected colonies and sequences generated as described (2). Transposon junction sequences were aligned with the appropriate genomic sequence to establish the site in the genome of transposon insertion.
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33. The figure shows 600 M. genitalium sites that were unambiguously mapped onto the M. pneumoniae genome. The whole-genome alignment method of Delcher et al. (79) mapped 317 sites, but does not map sites close to insertion/deletion and rearrangement differences between the chromosomes. An additional 201 sites were mapped by searching the M. pneumoniae genome for matches to short sequences (200 to 400 base pairs) containing each M. genitalium insertion site. An additional 83 sites that were not mappable by the above methods (because of matches to several related M. pneumoniae sequences) were mapped to the corresponding position within the orthologous M. pneumoniae gene. The M. pneumoniae-specific regions are from (3), with minor modifications. Each pink highlighted region contains a block of M. pneumoniae-specific genes and arbitrarily includes the intergenic regions flanking it.
34. In the M. genitalium experiments, the growth medium is SP-4 supplemented with either glucose or fructose as indicated. In the M. pneumoniae experiment, the cells were grown in Hayflick's medium supplemented with glucose or fructose. Cells were grown five passages (split 1 to 10) under the indicated condition before preparation of DNA for PCR templates. This is approximately equivalent to nine doublings.