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Identification and cloning of a developmentally regulated Cryptosporidium parvum gene by differential mRNA display
A A Schroeder1, A M Brown, M S Abrahamsen
1Veterinary Patho, Biology, University of Minnesota, St. Paul, MN 55108, USA.
Abstract:
To identify Cryptosporidium parvum genes expressed during intracellular development, differential mRNA display was used to detect differences in gene expression between mock-infected and C. parvum-infected human epithelial cells. A reproducible band present only in C. parvum-infected cells, ddHC-23, was isolated and cloned. Southern blot analysis demonstrated that ddHC-23 represented a C. parvum gene. RT-PCR revealed that HC-23 mRNA levels decreased from 6 to 12h post-infection (pi), were maximally expressed at 24h pi, and returned to low levels at 48 and 72h pi. Northern blot analysis determined that the approx. 3.6kb transcript is expressed by sporozoites prior to invasion of epithelial cells. Screening of a C. parvum genomic library with ddHC-23 isolated a genomic subclone which contained a 2790bp ORF, uninterrupted by introns. Sequence analysis indicated that the encoded protein, which displayed no similarity to any sequences in the public databases, contained a high proportion of polar amino acids, with the most abundant being Asp (17.3%), Ser (15.8%) and Gly (8.1%). Numerous potential sites for posttranslational modification were present including: casein kinase II and protein kinase C phosphorylation sites, N-myristolation sites and N-glycosylation sites. These findings demonstrate the usefulness of differential mRNA display for identifying developmentally regulated C. parvum genes within the background of genes expressed by the host cell. 1998 Elsevier Science B.V.
Insights
Researchers identified a novel Cryptosporidium parvum gene, HC-23, crucial for intracellular development. This gene
Area of Science:
- Molecular Biology
- Parasitology
- Genomics
Background:
- Cryptosporidium parvum is a significant human pathogen causing diarrheal disease.
- Understanding gene expression during C. parvum's intracellular lifecycle is key to developing interventions.
- Host cell gene expression can obscure pathogen-specific gene activity.
Purpose of the Study:
- To identify novel Cryptosporidium parvum genes expressed during intracellular development.
- To characterize the expression profile and sequence of a newly identified C. parvum gene.
- To demonstrate the utility of differential mRNA display for parasite gene discovery.
Main Methods:
- Differential mRNA display to compare gene expression in infected versus mock-infected human epithelial cells.
- Isolation and cloning of differentially expressed bands (ddHC-23).
- Southern blot, RT-PCR, Northern blot, and genomic library screening for gene characterization.
Main Results:
- A novel C. parvum gene, ddHC-23 (later referred to as HC-23), was identified and isolated.
- HC-23 mRNA expression peaked at 24 hours post-infection and was also detected in sporozoites prior to invasion.
- The gene encodes a protein with no known database similarity, rich in polar amino acids and potential post-translational modification sites.
Conclusions:
- Differential mRNA display is effective for identifying pathogen-specific genes during host-parasite interactions.
- The identified gene HC-23 is developmentally regulated and expressed early in the C. parvum lifecycle.
- The novel protein may play a role in intracellular development and warrants further functional investigation.