Related Experiment Videos
Expression cDNA cloning of a novel oncogene with sequence similarity to regulators of small GTP-binding proteins
A M Chan1, E S McGovern, G Catalano
1Laboratory of Cellular & Molecular Biology, National Cancer Institute, Bethesda, Maryland, MD 20892.
Abstract:
We generated a cDNA expression library from a human mammary epithelial cell line for detection of novel oncogenes by focus formation assay in NIH3T3 cells. A morphologically unique focus was identified and the transforming plasmid was isolated. The transforming gene, designated TIM, encoded a predicted protein species of 60 kDa containing a Dbl-Homology (DH) motif. This motif is also present in other growth regulatory molecules including Bcr, Cdc24, Vav, Ras-grf, and Ect2 which have been implicated as regulators of small GTP-binding proteins. NIH3T3 cells transfected with TIM expression plasmid showed altered growth properties in vitro and were tumorigenic when injected into nude mice. The 6.5 kilobasepair (kb) transcript of the TIM gene was mainly expressed in kidney, liver, pancreas, lung, and placenta. By analysing a panel of human-hamster somatic cell hybrids, we localized the TIM gene to human chromosome 7.
Insights
Researchers identified a novel oncogene, TIM, from human mammary cells. This gene promotes cell transformation and tumor formation, and is located on human chromosome 7.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- Oncogenes drive cancer development.
- Identifying novel oncogenes is crucial for understanding tumorigenesis.
- Human mammary epithelial cells are a source for discovering cancer-related genes.
Purpose of the Study:
- To identify and characterize novel oncogenes involved in cell transformation.
- To investigate the functional properties of a newly discovered transforming gene.
- To determine the genetic location of the identified oncogene.
Main Methods:
- cDNA expression library generation from human mammary epithelial cells.
- Focus formation assay in NIH3T3 cells to detect transforming activity.
- Plasmid isolation, gene sequencing, and protein analysis (Dbl-Homology motif identification).
- In vitro cell growth property assessment and in vivo tumorigenicity testing in nude mice.
- Gene transcript expression analysis and somatic cell hybrid mapping for chromosomal localization.
Main Results:
- A novel transforming gene, designated TIM, was isolated.
- TIM encodes a 60 kDa protein with a Dbl-Homology (DH) motif, known to regulate small GTP-binding proteins.
- TIM expression in NIH3T3 cells induced altered growth and tumorigenicity.
- The TIM gene transcript is primarily expressed in kidney, liver, pancreas, lung, and placenta.
- The TIM gene was mapped to human chromosome 7.
Conclusions:
- The novel TIM gene acts as an oncogene, promoting cell transformation and tumor formation.
- The Dbl-Homology motif in TIM suggests a role in small GTPase signaling pathways.
- TIM's expression pattern and chromosomal localization provide insights into its biological function and potential role in cancer.