Abstract
A novel cDNA fragment, named cca1 (confluent 3Y1 cell-associated 1), was previously isolated on the basis of preferential accumulation of the corresponding mRNA in growth-arrested confluent but not in growing subconfluent rat 3Y1 cells. The cca1 cDNA was found to consist of 5022 nucleotides with an open reading frame of 1905 nucleotides, encoding a protein of 635 amino acids. Unlike the 3Y1 cell case, cca1 mRNA was not detected in confluent 3Y1 BU, 3Y1 BU/pTK, 3Y1-16E6, or F2408 cells, whose growth patterns monitored by phalloidin staining and bromodeoxyuridine incorporation were different from those of the confluent 3Y1 cells. A restoration of the confluent 3Y1-type growth pattern was observed in the cca1 cDNA-introduced 3Y1 BU and 3Y1 BU/pTK cells after reaching confluence but not in the cDNA-introduced 3Y1-16E6 or F2408 cells. The results allow us to conclude that cca1 is required but not sufficient for formation of growth-arrested confluent monolayer of 3Y1 cells.
Highlights
Introduction of a firstATG-deleted cDNA into the 3Y1 BU/ pTK cells failed to restore the confluent 3Y1-type growth pattern after reaching confluence
Obtained cca1 cDNA consisted of 5022 nt (Fig. 1), which was compatible with the size of the corresponding mRNA estimated by Northern blot analysis (5 kb) [6]
Homology searches indicated that cca1 cDNA and the predicted amino acid sequences have no similarities to genes and proteins accessible in the data bases of GenBanky and EMBL nucleotide sequence and SWISS-PROT, PIR, and PRF protein sequence
Summary
Cell Lines and Culture Conditions—The cells were grown in Eagle’s minimal essential medium supplemented with 10% fetal calf serum except where specified. To construct pLRNL-cca1(-atg), the six nucleotides (nt 360 –365) containing the first ATG site of the cDNA were replaced with a BamHI site, and the BamHI fragment containing 3550 nt (nt 366 –3915) of the cDNA was cloned into the BamHI site of the pLRNL vector By using this first ATG-minus construct, the N-terminal region-deleted protein (222 amino acids (aa); aa 414 – 635) would be expected to be produced from the ATG (nt 1602) for the second methionine. The rat gapdh sequence was amplified with specific primers (CLONTECH) by PCR, using oligo(dT) primed 3Y1 cDNAs as template DNAs. The PCR product was cloned into pMOSBlue-T vector (Amersham Corp.), and the resulting plasmid, pMOS-gapdh, was used for the preparation of the control probe for the RNase protection assay. The organization of the cytoskeletal actin filaments of the cells was examined under a fluorescent microscope
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