Variable and conserved structural elements of trypanosome variant surface glycoproteins

Biological Variation Among African Trypanosomes: I. Clonal Expression of Virulence Is Not Linked to the Variant Surface Glycoprotein or the Variant Surface Glycoprotein Gene Telomeric Expression Site

“…Immunological and biochemical examinations of these organisms showed that T. brucei rhodesiense LouTat 1 and 1A both express a similar surface coat phenotype (Inverso et al, 1988;Reinitz et al, 1992). These findings suggested, but did not prove, that virulence is not associated with the VSG molecule.…”

“…Trypanosome clones LouTat 1 and 1A express the same VSG gene Full-length cDNAs representing the VSG genes expressed in clones LouTat 1 and 1A were derived from poly(A þ ) RNA of the respective organisms by selective PCR amplification, as previously described (Reinitz et al, 1992). Briefly, a 26-bp oligonucleotide primer corresponding to the 5 0 spliced leader sequence (mini-exon-derived sequence) of trypanosome mRNAs and a 28-bp oligonucleotide primer corresponding to a conserved 3 0 sequence common to trypanosome VSG transcripts were used to amplify VSG cDNA.…”

“…The 484-bp fragment generated by PCR for use as a probe in Southern analyses is denoted by solid underline within the DNA sequence. The accession number for the LouTat 1 VSG sequence is X56643 (Reinitz et al, 1992).…”

“…Preparation of LouTat 1=1A VSG cDNA probes A 395-bp VSG cDNA fragment, contained in the recombinant pGEM-3Z plasmid pTbrVSG A -4 (Reinitz et al, 1992), was isolated by double digestion with BamHI and EcoRI, purified from 1.2% low-melting-temperature agarose gels, and digested with HpaII to generate a 187-bp sequence representing an internal, variant specific sequence within the cDNA (Fig. 1).…”

“…Cloning of full-length VSG cDNA was accomplished by methods previously described (Reinitz et al, 1992). Polyadenylated RNA was isolated from frozen trypanosome pellets by conventional procedures of polytron homogenization in 8 M guanidinium hydrochloric acid (HCl), centrifugation through 5.7 M CsCl cushions, and poly(Aþ) RNA selection on oligo dT columns.…”