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Molecular cloning of G1 phase mRNAs from a subtractive G1 phase cDNA library
Abstract:
Many G1-phase-specific mRNAs have been identified from various normal or transformed cells based on serum induction and re-entry into the cell cycle from quiescence. However, these mRNAs may not represent some important genes expressed during G1 phase in continuously cycling cells. The eukaryotic cell cycle possesses two cdk (cyclin-dependent kinase) dependent regulatory gates through which cells pass during late G1 phase and G2 phase of each cycle. Subtractive hybridization was employed to synthesize a high R0t fraction cDNA library enriched in sequences expressed during G1 phase prior to passage through the G1-phase gate. To prepare G1-phase cells from continuously cycling cell populations, G1-phase HeLa cells were collected by centrifugal elutriation and highly synchronous S phase cells were obtained by double thymidine block followed by centrifugal elutriation. A G1-phase subtractive cDNA library was prepared by subtracting G1-phase cDNA with a 10-fold excess of S-phase mRNA. Single-stranded, G1-phase cDNAs were isolated by oligo(dA) chromatography. The library was screened with a high R0t fraction subtractive probe population. Following two rounds of screening, 20 positive clones were obtained. Northern blot analysis indicated that six of these clones were enhanced in expression level during G1 phase when compared with S phase. Nucleotide sequence comparison of each clone with the GenBank data base revealed that hG1.11 was highly homologous (99%) to the apoferritin light chain gene and clones hG1.6, hG1.10, hG1.17, and hG1.18 represented new G1-phase-enriched members of four human ribosomal protein gene families (71-95% homology). The last clone, hG1.1, encoded a highly charged polypeptide not previously identified. Additional study of these G1-phase-enriched mRNAs will be required to determine their role in cell cycle progression and the G1-phase gateway through which cells transit as they proceed through the cell cycle.
Insights
Researchers identified novel G1-phase-specific mRNAs in continuously cycling cells using subtractive hybridization. These findings offer new insights into cell cycle regulation and G1-phase progression.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Many G1-phase specific mRNAs are identified from quiescent cells, but may not represent continuously cycling cells.
- The eukaryotic cell cycle has two cyclin-dependent kinase (CDK) regulated checkpoints in late G1 and G2 phases.
- Understanding G1-phase gene expression is crucial for cell cycle progression.
Purpose of the Study:
- To identify novel G1-phase specific genes in continuously cycling cells.
- To characterize G1-phase enriched mRNAs using subtractive hybridization.
- To investigate the role of these genes in cell cycle regulation.
Main Methods:
- Synchronized G1 and S phase HeLa cells were obtained using centrifugal elutriation and double thymidine block.
- A G1-phase subtractive cDNA library was created by subtracting G1 cDNA with excess S-phase mRNA.
- Northern blot analysis and nucleotide sequence comparison were used to screen and identify positive clones.
Main Results:
- A G1-phase enriched cDNA library was successfully synthesized.
- Twenty positive clones were obtained after screening, with six showing enhanced G1-phase expression.
- Identified clones included apoferritin light chain and novel G1-phase enriched ribosomal protein genes, along with a new polypeptide.
Conclusions:
- This study identified novel G1-phase specific mRNAs in continuously cycling cells.
- The identified genes, including ribosomal proteins and apoferritin, likely play roles in G1-phase progression.
- Further research is needed to elucidate the precise functions of these G1-phase enriched mRNAs in cell cycle regulation.