Related Experiment Videos
Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein
Abstract:
Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.
Insights
Researchers cloned complementary DNAs (cDNAs) for rat prostatein subunits. Analysis revealed homologous sequences in the C3 subunit
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Prostatein is a major secretory protein in the rat ventral prostate.
- Understanding prostatein gene structure is crucial for studying its function and regulation.
Purpose of the Study:
- To clone and characterize complementary DNAs (cDNAs) encoding rat prostatein subunits.
- To determine the complete DNA coding sequence for the C3 subunit of prostatein.
- To identify and analyze the genomic organization of prostatein genes.
Main Methods:
- Cloning of rat ventral prostate poly(A) RNA using the "dG-dC tailing" procedure.
- Hybrid-arrested translation to identify clones for prostatein subunits.
- DNA sequencing and analysis of cDNA clones (E45 and E85).
- Screening of a rat genomic DNA library using a C3 cDNA probe.
- Restriction enzyme mapping and Southern blot analysis of genomic clones.
Main Results:
- Identified cDNAs for all three prostatein subunits (C1, C2, C3).
- Determined the composite DNA coding sequence for the Mr = 14,000 (C3) subunit.
- Discovered a homologous 12-nucleotide sequence in the C3 signal peptide, shared with other subunits.
- Isolated two unique genomic clones containing sequences for the C3 subunit.
- Found analogous restriction enzyme sites within coding regions but differences in intervening and flanking sequences, indicating two distinct genes.
Conclusions:
- The rat ventral prostate synthesizes prostatein from at least three subunits.
- The C3 subunit gene exhibits sequence homology in its signal peptide with other prostatein subunits.
- Two unique genes encode the C3 subunit of prostatein, suggesting potential differential transcription or regulation.
- Both identified prostatein genes are likely transcribed in vivo.