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Rat aldolase isozyme gene
Abstract:
Rat aldolase B mRNA was partially purified from liver polysomes by an immunochemical technique followed by oligo(dT)-cellulose column chromatography. Double-stranded cDNA, synthesized from this mRNA, was inserted into the PstI site of plasmid pBR322 employing the oligo(dC)-oligo(dG) tailing method. Clones containing aldolase B cDNA inserts were selected by colony hybridization using 32P-labeled purified mRNA as a specific probe. Several recombinant plasmids containing 600 to 1000 base pair inserts were isolated. Hybrid selection-translation experiments showed that they hybridize specifically with aldolase B mRNA. By overlapping restriction maps of several individual cDNA inserts, it was found that they spanned 1200 base pairs, which represented about 70% of the aldolase B mRNA sequence. The nucleotide sequence of the cDNA was then determined and the sequence of 180 amino acids from the COOH terminus and the entire 3' untranslatable nucleotide sequence were clarified. Although the complete amino acid sequence of rat aldolase B has not yet been reported, it was found that several amino acids neighboring the COOH-terminal tyrosine obtained by carboxypeptidase digestion completely coincided with those determined from the cDNA sequence; i.e. -Ser-Leu-Phe-Thr-Ala-Ser-Tyr-Thr-Tyr. Furthermore, a putative active site peptide appeared and is extensively homologous to those of rabbit aldolases A and B.
Insights
Researchers synthesized complementary DNA (cDNA) from rat aldolase B messenger RNA (mRNA), creating clones that represent 70% of the aldolase B gene sequence for further study.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Aldolase B is a key enzyme in glycolysis and gluconeogenesis.
- Understanding the genetic sequence of aldolase B is crucial for studying its function and related metabolic disorders.
Purpose of the Study:
- To construct and characterize complementary DNA (cDNA) clones representing the rat aldolase B gene.
- To elucidate the nucleotide sequence and identify key functional regions of the aldolase B mRNA.
Main Methods:
- Partial purification of rat aldolase B messenger RNA (mRNA) from liver polysomes.
- Synthesis of double-stranded cDNA and insertion into the pBR322 plasmid vector.
- Colony hybridization and hybrid selection-translation assays for clone screening.
- Nucleotide sequencing and restriction mapping of cDNA inserts.
Main Results:
- Isolation of recombinant plasmids with aldolase B cDNA inserts ranging from 600 to 1000 base pairs.
- Construction of a contiguous cDNA sequence spanning 1200 base pairs, representing approximately 70% of the aldolase B mRNA.
- Determination of the 3' untranslatable region and the sequence of 180 amino acids from the COOH terminus.
- Identification of a putative active site peptide with homology to other aldolase enzymes.
Conclusions:
- The generated cDNA clones provide a significant portion of the rat aldolase B gene sequence.
- The sequence data offers insights into the protein's C-terminal structure and active site.
- This work facilitates further investigations into aldolase B gene expression and function.