Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein gene transpositions in trypanosome DNA.
Telomere-linked genes coding for the variant surface glycoproteins (VSGs) of African trypanosomes have been difficult to clone because their flanking regions frequently lack restriction sites. Therefore, we constructed a genomic DNA library of fragments generated by digestion of purified trypanosome DNA with mung bean nuclease, an enzyme that cleaves before and after genes in Plasmodium falciparum DNA (McCutchan, T. F., Hansen, J. L., Dame, J. B., and Mullins, J. A. (1984) Science 225, 625-628). Southern hybridizations with several gene probes showed that under the appropriate conditions mung bean nuclease produces discrete trypanosome DNA fragments that are as clearly resolved on an agarose gel as restriction fragments. The majority of VSG genes are on fragments of about 1.7 kilobase pairs. To examine the sites of mung bean nuclease cleavage, the insert boundary sequences of eight recombinant clones in the library containing VSG genes were determined. In general, mung bean nuclease cleaved 300-800 base pairs in front of the VSG start codon and within 50 base pairs on either side of the termination codon. These regions also form the boundaries of VSG gene conversion events indicating that the enzyme recognizes, in part, a conformational structure rather than a specific sequence. The analyzed clones included both telomere-linked and interior basic copy VSG genes indicating that the library potentially contains all of the telomere-linked VSG genes in the genome.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004274:DNA, Recombinant; D003851:Deoxyribonucleases / Q000378:metabolism*; D007887:Fabaceae; D006023:Glycoproteins / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D014643:Variant Surface Glycoproteins, Trypanosoma
D004274:DNA, Recombinant; D006023:Glycoproteins; D008565:Membrane Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; D004247:DNA; D003851:Deoxyribonucleases