Related Experiment Videos
Complexity of the early genetic response to growth factors in mouse fibroblasts
J M Almendral1, D Sommer, H Macdonald-Bravo
1European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Abstract:
Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.
Insights
Researchers identified genes involved in cell growth by analyzing RNA from stimulated NIH 3T3 cells. Cycloheximide enhanced gene expression, revealing a complex genetic program during cell proliferation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Growth factors regulate genes crucial for cell proliferation and differentiation.
- Identifying these genes is key to understanding cell cycle control mechanisms.
Purpose of the Study:
- To identify genes regulated by growth factors in NIH 3T3 cells.
- To characterize the transcriptional changes during serum-induced cell proliferation.
Main Methods:
- Constructed a lambda cDNA library from serum-stimulated NIH 3T3 cells in the presence of cycloheximide.
- Utilized differential screening to identify growth factor-regulated genes.
- Analyzed mRNA levels and transcription kinetics of induced genes.
Main Results:
- Identified 82 independent gene sequences, including known oncogenes c-fos and c-myc.
- Observed significant variations in induction kinetics and extent among different genes.
- Demonstrated that cycloheximide superinduces mRNA levels by inhibiting transcription and prolonging mRNA half-lives.
Conclusions:
- Serum stimulation initiates a complex genetic program during cell proliferation.
- Growth factor-regulated genes exhibit diverse transcriptional responses.
- Cycloheximide significantly impacts mRNA stability and transcription, aiding in the identification of regulated genes.