Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays.
Protein extracts obtained from male and female schistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3' end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generating relatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of protein occurred at the 5' end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide containing the F-10 HRE, evidenced proteins having MWS of 30, 45 and 65 kDa. These proteins are presumably involved in the regulation of transcription of the F-10 gene.
Engelender
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Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Cidade Universitária, Brasil.
Rumjanek
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FD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004527:Egg Proteins / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D011485:Protein Binding; D012550:Schistosoma mansoni / Q000235:genetics*; D012741:Sexual Maturation / Q000235:genetics; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D004527:Egg Proteins; D015801:Helminth Proteins; C084248:major eggshell protein F-10, Schistosoma mansoni; D004247:DNA; D003850:Deoxyribonuclease I