Schistosoma mansoni tropomyosin: cDNA characterization, sequence, expression, and gene product localization.
We have defined the polypeptide pattern of 3-hr Schistosoma mansoni schistosomula on nonequilibrium two-dimensional gels (NEPHGE). An acidic group of polypeptides with a molecular weight of about 40 kDa and a pI value of around 5.0 (numbered 48/59/53) were identified as antigens on Western blots probed with chronic human infection sera or vaccinated mouse sera. Polypeptides 48/49/53 from silver-stained NEPHGE gels produced antisera that were specific as demonstrated by Western blot analysis and immunoprecipitations of in vitro translation products. A cDNA clone (clone 1) from a S. mansoni adult worm pBR322 library was isolated by using cDNA probes made from size-fractionated mRNA and defined as encoding polypeptide 49 by hybridization selection of the mRNA which was in vitro translated and immunoprecipitated with specific mouse antiserum. A lambda gt 11 expression clone which contained an insert close to the full length mRNA was isolated from a S. mansoni cercariae library. The complete sequence of the mRNA was determined by sequencing the insert of this clone as well as primer extension of total RNA. The only open reading frame coding for 284 amino acids in the 1316 nucleotide sequence showed a 44.76 to 55.44% homology with the amino acid sequences of 18 different tropomyosins from various species. Computer-predicted secondary structure of schistosome tropomyosin was mainly alpha-helix which was very similar to other tropomyosins. Northern analysis showed the mRNA to be about 1.5 kb in size and detectable at much higher levels in the adult worm stage as compared to the cercariae and the egg stages. Western blot analysis likewise showed that greater amounts of tropomyosin were detected in extracts from adult worm stage as compared to extracts from cercariae and egg stages. Immunocytochemical analysis shows that tropomyosin is strongly associated with the tegument of adult worms. The restriction digestion pattern given by genomic Southern analysis suggests the existence of introns and/or multiple gene copies. Thus polypeptide 49, an immunodominant antigen, represents schistosome tropomyosin.
Xu
H
H
Department of Microbiology, School of Medicine, State University of New York, Buffalo 14214.
Miller
S
S
van Keulen
H
H
Wawrzynski
M R
MR
Rekosh
D M
DM
LoVerde
P T
PT
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000276:immunology; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D012550:Schistosoma mansoni / Q000032:analysis / Q000235:genetics* / Q000254:growth & development / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid; D014335:Tropomyosin / Q000032:analysis / Q000235:genetics* / Q000276:immunology
D000947:Antigens, Helminth; D010455:Peptides; D012333:RNA, Messenger; D014335:Tropomyosin; D004247:DNA