Identification and cloning of a developmentally regulated Cryptosporidium parvum gene by differential mRNA display PCR.
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.
Schroeder
A A
AA
Veterinary Patho, Biology, University of Minnesota, St. Paul, MN 55108, USA.
Brown
A M
AM
Abrahamsen
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D016785:Cryptosporidium parvum / Q000235:genetics* / Q000254:growth & development; D016054:DNA, Protozoan / Q000032:analysis; D004847:Epithelial Cells / Q000469:parasitology; D018507:Gene Expression Regulation, Developmental; D017125:Genes, Protozoan / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011499:Protein Processing, Post-Translational / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D016053:RNA, Protozoan / Q000032:analysis; D017422:Sequence Analysis, DNA
D016054:DNA, Protozoan; C114757:HC23 protein, Cryptosporidium parvum; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan