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Published on: April 4, 2016
Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae
Abstract:
Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.
Insights
Researchers enriched yeast ribosomal protein messenger RNA (mRNA) and identified five clones containing ribosomal protein genes. These findings suggest yeast ribosomal protein genes are not tightly linked.
Area of Science:
- Molecular Biology
- Yeast Genetics
Background:
- Ribosomal proteins are essential for protein synthesis.
- Understanding the organization of ribosomal protein genes is crucial for comprehending gene regulation and genome structure.
Purpose of the Study:
- To enrich for yeast ribosomal protein messenger RNA (mRNA).
- To identify and clone yeast ribosomal protein genes.
- To investigate the linkage and copy number of these genes.
Main Methods:
- Isolation of poly(A)+ small mRNA from small polysomes for enrichment.
- Cell-free translation and two-dimensional gel electrophoresis to assess enrichment.
- Differential colony hybridization using a yeast plasmid bank to screen for gene candidates.
- Analysis of cloned genes for copy number and linkage.
Main Results:
- A 5-10 fold enrichment of ribosomal protein mRNA was achieved.
- Five distinct clones containing yeast ribosomal protein gene sequences were identified.
- The mRNAs from these clones code for polypeptides that co-migrate with yeast ribosomal proteins.
- Three of the cloned genes' mRNA levels are regulated by the RNA2 locus.
- No evidence for tight linkage of yeast ribosomal protein genes was found.
- Gene copy number analysis revealed single-copy genes, genes with two copies, and one clone with potential repetitive sequences.
Conclusions:
- The study successfully enriched for and identified yeast ribosomal protein genes using molecular cloning techniques.
- The findings indicate that yeast ribosomal protein genes are not tightly clustered in the genome.
- Further investigation is needed to clarify the repetitive nature of sequences in one of the identified clones.
