ABSTRACT
A DNA sequence composed of 1281 nucleotides (nt) consisting of a single open reading frame (ORF) encoding a putative 20S proteasome beta1-type subunit was isolated from clones derived from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Southern blot analysis suggested that the sequenced DNA exists in the C. parvum genome as a single copy; transcription was verified through reverse transcription-polymerase chain reaction (RT-PCR) performed on total RNA isolated from C. parvum sporozoites. The predicted protein consists of 210 amino acids (aa), contains characteristic amino acids common to all proteasomal subunits, and shares stronger similarity to the beta1-type subunit of yeast than to other types of beta-subunits.
Subject(s)
Cryptosporidium parvum/genetics , Cysteine Endopeptidases/genetics , Multienzyme Complexes/genetics , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cysteine Endopeptidases/chemistry , Molecular Sequence Data , Multienzyme Complexes/chemistry , Open Reading Frames , Proteasome Endopeptidase Complex , Protein Subunits , Restriction Mapping , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parium, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.
