Differential mRNA display cloning and characterization of a Cryptosporidium parvum gene expressed during intracellular development.
Differential mRNA display was used to detect differences in gene expression between mock-infected and Cryptosporidium parvum-infected human adenocarcinoma cells. A reproducible band present only in C. parvum-infected cells, ddHC-10 was isolated and cloned. Northern blot analysis was used to confirm the differential expression of the HC-10 mRNA. As differential mRNA display does not differentiate between parasite and host mRNAs, Southern blot analysis was used to demonstrate that ddHC-10 represented a C. parvum gene. Northern blot analysis demonstrated that HC-10 mRNA is expressed by sporozoites prior to invasion of host cells. Screening of a C. parvum genomic library identified 2 different genomic clones, HC-10-13C and HC-10-6C. The combined genomic sequence contained a predicted open reading frame of 2,952 base pairs (bp), coding for a protein of 984 amino acids with a predicted molecular weight of approximately 106 kDa. Reverse transcription polymerase chain reaction mapping of the HC-10 transcript demonstrated that the HC-10 gene lacks introns, and the approximately 4,789-bp mRNA contains relatively large 5' (approximately 1,390-bp) and 3' (approximately 440-bp) untranslated regions. The predicted polypeptide contained a high proportion of polar amino acids, with the most abundant amino acids being serine (10.5%), threonine (9.8%), and cysteine (7.6%). The C-terminal region of the predicted polypeptide is characterized by a threonine-rich region containing multiple repeats of the sequence TTTTRP. This repeat motif is similar to that found in the mucin-like genes of vertebrates and lower eukaryotes that have been shown to play important roles in cell-cell interactions in multicellular organisms and invasion of host cells by unicellular parasites.
Schroeder
A A
AA
University of Minnesota, St. Paul 55108, USA.
Lawrence
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CE
Abrahamsen
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002417:Cattle; D016785:Cryptosporidium parvum / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D018076:DNA, Complementary; D018507:Gene Expression Regulation, Developmental; D017125:Genes, Protozoan; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D006790:Host-Parasite Interactions; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D016053:RNA, Protozoan / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D006023:Glycoproteins; C118853:HC-10 protein, Cryptosporidium parvum; D010750:Phosphoproteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan