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A A Schroeder

Publications and source records attributed to A A Schroeder.

9 recordsLinked to original sources
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

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.

Amino Acid Sequence↗