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Isolation, sequence and molecular karyotype analysis of the actin gene of Cryptosporidium parvum
1Parasitology Laboratory, San Francisco General Hospital, CA.
Abstract:
Actin is an ubiquitous and highly conserved microfilament protein which is hypothesized to play a mechanical, force-generating role in the unusual gliding motility of sporozoan zoites and in their active penetration of host cells. We have identified and isolated an actin gene from a Cryptosporidium parvum genomic DNA library using a chicken beta-actin cDNA as an hybridization probe. The nucleotide sequences of two overlapping recombinant clones were identical and the amino acid sequence deduced from the single open reading frame was 85 % identical to the P. falciparum actin I and human gamma-actin proteins. The predicted 42 106-Da Cryptosporidium actin contains 376 amino acids and is encoded by a single-copy gene which contains no introns. The nucleic acid coding sequence is 72% biased to the use of A or T in the third position of codons. Chromosome-sized DNA released from intact C. parvum oocysts was resolved by OFAGE into 5 discrete ethidium bromide-staining DNAs ranging in size from 900 to 1400 kb; the cloned C. parvum actin gene hybridized to a single chromosomal DNA of approximately 1200 kb.
Insights
Researchers identified and isolated an actin gene from Cryptosporidium parvum, a parasite. This finding provides insights into parasite motility and host cell invasion mechanisms.
Area of Science:
- Molecular Biology
- Parasitology
- Genetics
Background:
- Actin is a conserved microfilament protein crucial for cell mechanics.
- In apicomplexan parasites like Cryptosporidium parvum, actin's role in motility and host cell invasion is hypothesized.
- Understanding parasite actin is key to developing novel control strategies.
Purpose of the Study:
- To identify and isolate the actin gene from Cryptosporidium parvum.
- To characterize the deduced amino acid sequence and gene structure.
- To determine the chromosomal location of the Cryptosporidium parvum actin gene.
Main Methods:
- Screening a Cryptosporidium parvum genomic DNA library using a chicken beta-actin cDNA probe.
- Nucleotide sequencing of recombinant clones.
- Deduction of amino acid sequence from the open reading frame.
- Analysis of gene structure (introns) and codon bias.
- Resolution of chromosomal DNA using OFAGE and Southern hybridization.
Main Results:
- An actin gene was successfully identified and isolated from Cryptosporidium parvum.
- The deduced amino acid sequence showed 85% identity to Plasmodium falciparum and human actin.
- The Cryptosporidium parvum actin gene is a single-copy, intronless gene encoding a 42,106-Da protein.
- The gene is located on a single chromosome of approximately 1200 kb.
Conclusions:
- The identification of the Cryptosporidium parvum actin gene provides a molecular basis for its hypothesized mechanical role.
- The high conservation suggests conserved functions across species.
- This work is foundational for further research into parasite motility and host-pathogen interactions.