The eukaryotic translation initiation factor eIF-4A is an ATP-dependent RNA helicase involved in ribosome attachment to the 5' end of mRNAs. Employing as a probe a Cryptosporidium parvum genomic amplicon encoding a partial polypeptide related to eIF-4A, we screened a C. parvum sporozoite cDNA library to clone the full length of the gene. Two complete cDNAs were characterized, Cp.F6 and Cp.F10, which consisted of 1,900 and 1,418 bp, respectively. The overlapping portions of the sequences shared 100% identity and encoded a polypeptide of 405 amino acids whose identity to known eIF-4A molecules ranged between 77 and 39%. The 2 cDNAs differed in the length of their respective 3' untranslated regions, of 577 bp in Cp.F6 and 72 bp in Cp.F10, in both of which a putative polyadenylation signal was identified. The structure of the cloned cDNAs, along with genomic Southern blot data indicating that eIF-4A is encoded by a single copy gene, strongly suggested that Cp.F6 and Cp.F10 reflect a differential 3' end processing of mRNA precursors, not observed so far in C. parvum. Northern blot analysis confirmed that the sporozoites express 2 eIF-4A mRNAs and showed that the lower molecular weight transcript is 10- to 20-fold more abundant. We also investigated the polymorphism of the eIF-4A gene and defined a novel polymerase chain reaction-restriction fragment length polymorphism marker discriminating between C. parvum isolates of genotypes 1 and 2.
The initiation translation factor eIF-4A of Cryptosporidium parvum is encoded by two distinct mRNA forms and shows DNA sequence polymorphism distinguishing genotype 1 and 2 isolates.
CRISANTI, Andrea
2000
Abstract
The eukaryotic translation initiation factor eIF-4A is an ATP-dependent RNA helicase involved in ribosome attachment to the 5' end of mRNAs. Employing as a probe a Cryptosporidium parvum genomic amplicon encoding a partial polypeptide related to eIF-4A, we screened a C. parvum sporozoite cDNA library to clone the full length of the gene. Two complete cDNAs were characterized, Cp.F6 and Cp.F10, which consisted of 1,900 and 1,418 bp, respectively. The overlapping portions of the sequences shared 100% identity and encoded a polypeptide of 405 amino acids whose identity to known eIF-4A molecules ranged between 77 and 39%. The 2 cDNAs differed in the length of their respective 3' untranslated regions, of 577 bp in Cp.F6 and 72 bp in Cp.F10, in both of which a putative polyadenylation signal was identified. The structure of the cloned cDNAs, along with genomic Southern blot data indicating that eIF-4A is encoded by a single copy gene, strongly suggested that Cp.F6 and Cp.F10 reflect a differential 3' end processing of mRNA precursors, not observed so far in C. parvum. Northern blot analysis confirmed that the sporozoites express 2 eIF-4A mRNAs and showed that the lower molecular weight transcript is 10- to 20-fold more abundant. We also investigated the polymorphism of the eIF-4A gene and defined a novel polymerase chain reaction-restriction fragment length polymorphism marker discriminating between C. parvum isolates of genotypes 1 and 2.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
