First identification of caldesmon transcripts in bovine oviduct epithelial cells in vitro by means of an RNA differential display technique examining culture-induced expression changes.
Innovative molecular biology techniques enable the quick evaluation of distinct gene expression pattern of cells or tissues. Hitherto, a cell-type-specific behaviour has been difficult to evaluate. In addition to standard morphological and immunological criteria in this study the expression of in vitro-cultured bovine oviduct epithelial cells (BOECs) was compared with fresh cells by RNA arbitrarily primed polymerase chain reaction (RAP-PCR). The cultured cells showed mitotic activity during the whole culture period (6 days) until they had reached about 80% confluency. Remarkable results of a random transcript screening using fresh versus cultured BOECs were reported, in which a single PCR product appeared during culture, but was absent in fresh cells. Sequence analysis of the culture-induced 522 bp fragment revealed a high homology (87%) to caldesmon (CaD) of various species and a 92% homology to a short cDNA fragment of a bovine non-muscle CaD. Specific, cross-species PCR primers were used to elongate this partial sequence (1,036 bp). This resulting cDNA showed an open reading frame and was identified as a bovine non-muscle CaD isoform. When compared with human non-muscle CaDs (89% homology) a deletion of 2 codons was observed. According to sequential culture experiments, CaD expression was not found in fresh BOECs but specific transcripts appeared within 48-113 h under specific culture conditions. It is likely that augmented CaD expression in cultured BOECs may reflect the cell effort adapting to specific culture conditions. The hypothesis that increased CaD levels could be important to facilitate adherence and spreading by formation of new stress fibres can be favoured. This first identification of caldesmon expression being specifically induced during in vitro culture demonstrates the potential of RAP-PCR for the analysis and validation of cell culture techniques.
Einspanier
R
R
Institute of Physiology, FML-Weihenstephan, TU München, Freising, Germany. einspani@weihenstephan.de
Bieser
B
B
Reischl
J
J
Prelle
K
K
D000818:Animals; D001483:Base Sequence; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology; D005187:Fallopian Tubes / Q000166:cytology* / Q000502:physiology; D005260:Female; D005455:Fluorescent Antibody Technique / Q000662:veterinary; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012689:Sequence Homology, Nucleic Acid
D002148:Calmodulin-Binding Proteins; C034477:RNA primers; D012313:RNA