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Molecular cloning and characterization of a cDNA showing negative regulation in v-src-transformed 3Y1 rat fibroblasts
1Division of Biochemistry, Chiba Cancer Center Research Institute, Japan.
Abstract:
A differential screening procedure was used to isolate genes that are specifically down-regulated in transformed cells. After screening a cDNA library derived from normal rat fibroblast (3Y1) cells, we obtained several independent cDNAs whose mRNA level was substantially lower in 3Y1 cells transformed with Rous sarcoma virus than in untransformed 3Y1 cells. Among them, N03 cDNA has been characterized extensively. N03 mRNA was also absent from v-mos- or simian virus 40-transformed 3Y1 cells but was present in v-Ha-ras-transformed 3Y1 cells. Nucleotide sequence analysis showed that the N03 protein is composed of 178 amino acid residues and does not have any sequence similarities with proteins in the data base. A putative zinc-finger domain is located in the central part of the sequence and a proline-rich domain is in the C-terminal region. N03 mRNA was detected by Northern blot analysis in several tissues, including lung, kidney, intestine, and brain, but not in liver. Genomic Southern blot hybridization revealed that the N03 gene exists as a single copy in the rat genome and that closely related, single-copy genes are also present in chicken and human.
Insights
Researchers identified N03, a novel gene significantly down-regulated in Rous sarcoma virus-transformed cells. This gene
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Cancer research often involves identifying genes altered during cellular transformation.
- Understanding gene expression changes is crucial for distinguishing normal from cancerous cells.
Purpose of the Study:
- To isolate and characterize genes specifically down-regulated in Rous sarcoma virus-transformed rat fibroblast (3Y1) cells.
- To investigate the expression patterns and genomic presence of a novel down-regulated gene, N03.
Main Methods:
- Differential screening of a cDNA library from normal rat 3Y1 cells.
- Analysis of N03 mRNA levels in various transformed and untransformed 3Y1 cell lines.
- Nucleotide sequence analysis of the N03 protein.
- Northern blot analysis for tissue-specific N03 mRNA expression.
- Genomic Southern blot hybridization to determine N03 gene copy number.
Main Results:
- Identified N03 cDNA, with significantly lower mRNA levels in Rous sarcoma virus-transformed 3Y1 cells compared to normal cells.
- N03 mRNA was absent in v-mos- and simian virus 40-transformed cells but present in v-Ha-ras-transformed cells.
- The N03 protein (178 amino acids) showed no database similarity, featuring a putative zinc-finger and proline-rich domain.
- N03 mRNA was detected in rat lung, kidney, intestine, and brain, but not liver.
- The N03 gene is a single copy in the rat genome, with related single-copy genes in chicken and human.
Conclusions:
- N03 represents a novel gene product potentially involved in cellular transformation processes.
- Its differential expression suggests a role in distinguishing between certain types of viral oncogene-induced transformation.
- The conserved nature of N03 across species indicates a fundamental biological function.