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[Enzymatic synthesis and molecular cloning of the pigeon alpha-globin structural gene]
Abstract:
Double-stranded DNA synthesized from the pigeon globin mRNA by the subsequent actions of avian myeloblastosis virus reverse transcriptase and E. coli DNA polymerase I was split with nuclease S1 and inserted into PstI site in the plasmid pBR322 by poly(dG) times poly(dC) homopolymer extension technique using terminal deoxynucleotidyl transferase. E. coli transformants have been shown to contain pigeon globin sequences by colony hybridization with pigeon globin [32P]cDNA. The inserted DNA fragment deleted from recombinant DNA by PstI treatment hybridizes with globin cDNA. The maximal length of the inserted fragment measured in agarose gel was found to be 550--560 base pairs. Inserted sequences subjected to analysis by hybridization with alpha- and beta-[32P]cDNA have been ascribed to the pigeon alpha globin chain. EcoRI, HindIII, BglII, SalI, BamHI, PstI restriction enzymes did not cleave the inserted DNA fragment. Pigeon DNA coding alpha-globin chain contains recognition sites for AluI, HindII and HaeIII restriction enzymes. Part of the recombinant clones remains resistant to ampicillin and therefore in some of these clones the globin gene could be expressed.
Insights
Researchers synthesized pigeon alpha globin complementary DNA (cDNA) and cloned it into E. coli. The cloned pigeon alpha globin gene sequences were successfully identified and characterized.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Globin genes are crucial for oxygen transport.
- Understanding avian globin gene structure aids evolutionary and functional studies.
- Recombinant DNA technology allows for the isolation and analysis of specific genes.
Purpose of the Study:
- To synthesize and clone pigeon globin complementary DNA (cDNA).
- To characterize the cloned pigeon globin gene sequences.
- To investigate the potential expression of the pigeon globin gene in E. coli.
Main Methods:
- Double-stranded DNA synthesis using reverse transcriptase and DNA polymerase I.
- Nuclease S1 digestion and insertion into plasmid pBR322 via homopolymer extension.
- Transformation of E. coli and colony hybridization with pigeon globin cDNA.
- Restriction enzyme analysis and gel electrophoresis to determine fragment length and characteristics.
- Hybridization studies with alpha- and beta-globin cDNA probes.
Main Results:
- Successfully synthesized and cloned pigeon globin cDNA into E. coli.
- Identified recombinant clones containing pigeon globin sequences (550-560 base pairs).
- Confirmed the inserted sequences correspond to the pigeon alpha globin chain.
- Determined the inserted fragment lacks common restriction sites but contains sites for AluI, HindII, and HaeIII.
- Observed ampicillin resistance in some clones, indicating potential globin gene expression.
Conclusions:
- Pigeon alpha globin cDNA was successfully cloned and characterized.
- The cloned fragment represents the pigeon alpha globin gene.
- The study provides a foundation for further investigation into avian globin gene expression and regulation.