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Identification and isolation of Cryptosporidium parvum genes encoding microtubule and microfilament proteins
1Parasitology Laboratory, Department of Medicine, San Francisco General Hospital, University of California 94143.
Abstract:
Microtubules and microfilaments are highly conserved cytoskeletal polymers hypothesized to play essential biomechanical roles in the unusual gliding motility of Apicomplexan zoites and in their invasion of, and development within, host epithelial cells. We have identified and isolated Cryptosporidium parvum genes encoding the microtubule proteins alpha- and beta-tubulin and the microfilament protein actin by screening a lambda gt11 C. parvum genomic DNA library with degenerate oligonucleotide and heterologous cDNA hybridization probes respectively. The alpha- and beta-tubulin genes have been partially sequenced and the deduced peptide sequences show greatest homology with the tubulins of the related parasites, T. gondii and P. falciparum. The complete nucleic acid sequence of the actin gene predicts a 376 amino acid, 42 kDa protein having 85% sequence identity with the P. falciparum actin I and the human gamma-actin proteins. Each of these cytoskeletal protein genes was demonstrated to be of cryptosporidial origin by Southern analyses of C. parvum chromosomes fractionated by pulsed field gel electrophoresis; the cloned alpha- and beta-tubulin genes hybridized with chromosomes of ca. 1,200 and 1,500 kb respectively and the cloned actin gene also hybridized with a 1,200 kb chromosome.
Insights
Researchers identified key cytoskeletal genes, including alpha- and beta-tubulin and actin, in Cryptosporidium parvum. These findings shed light on the parasite's motility and host cell invasion mechanisms.
Area of Science:
- Molecular Biology
- Parasitology
- Cell Biology
Background:
- Microtubules and microfilaments are crucial cytoskeletal polymers.
- These structures are essential for Apicomplexan zoite motility and host cell invasion.
Purpose of the Study:
- To identify and isolate genes encoding microtubule and microfilament proteins in Cryptosporidium parvum.
- To analyze the homology and genetic origin of these cytoskeletal genes.
Main Methods:
- Screening a Cryptosporidium parvum genomic DNA library using degenerate oligonucleotide and heterologous cDNA probes.
- Partial sequencing of alpha- and beta-tubulin genes and complete sequencing of the actin gene.
- Southern blot analysis of fractionated Cryptosporidium parvum chromosomes using pulsed field gel electrophoresis.
Main Results:
- Identified and isolated genes for alpha-tubulin, beta-tubulin, and actin in Cryptosporidium parvum.
- Sequenced tubulin genes showed homology to related parasites like T. gondii and P. falciparum.
- Sequenced actin gene predicted a protein with high identity to P. falciparum actin I and human gamma-actin.
- Southern analyses confirmed the cryptosporidial origin of these genes, mapping them to specific chromosomes.
Conclusions:
- The identified genes are of cryptosporidial origin and encode proteins homologous to those in other apicomplexans and eukaryotes.
- These cytoskeletal proteins likely play significant roles in Cryptosporidium parvum's biological functions, including motility and host cell interactions.