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Molecular cloning of a DNA sequence complementary to creatine kinase M mRNA from chickens
Abstract:
We have cloned and identified a DNA sequence complementary to the mRNA of creatine kinase (CK) isozyme M, although the mRNA is a minor species of the total mRNA in developing myoblasts. Poly(A)+RNA from breast and thigh muscle of 5-week-old chicks was enriched for CK mRNA by a novel procedure of sucrose gradient centrifugation in the presence of methylmercuric hydroxide. DNA complementary to this mRNA was inserted into pBR322, and colonies containing the recombinant plasmids were screened for the ability of the plasmid DNA to hybridize with and rescue CK mRNA from total muscle mRNA. Three plasmids, pCS195, pCS192, and pM35-4, could specifically rescue CK-M mRNA. CK-M mRNA was detected by in vitro translation and specific immunoprecipitation. The identity of the in vitro translation product was further confirmed by its migration in two-dimensional gels at the isoelectric point and molecular weight of CK-M. The heterogeneity of CK-M observed in vivo also was found upon translation of the CK-M mRNA which hybridizes to the plasmid.
Insights
Researchers cloned DNA complementary to creatine kinase (CK) M mRNA, a minor species in developing myoblasts. This advancement aids in studying CK-M gene expression and its role in muscle development.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Creatine kinase (CK) isozyme M is crucial for cellular energy metabolism, particularly in muscle tissue.
- CK-M mRNA is a minor component of total mRNA in developing myoblasts, posing challenges for isolation and study.
Purpose of the Study:
- To clone and identify a DNA sequence complementary to creatine kinase (CK) isozyme M mRNA.
- To develop a method for enriching and isolating CK-M mRNA for further molecular analysis.
Main Methods:
- Enrichment of poly(A)+RNA from chick muscle using sucrose gradient centrifugation with methylmercuric hydroxide.
- Insertion of complementary DNA into the pBR322 plasmid vector.
- Screening of recombinant plasmids by hybridization and rescue of CK-M mRNA from total muscle mRNA.
- Detection and confirmation of CK-M mRNA using in vitro translation and immunoprecipitation, followed by two-dimensional gel electrophoresis.
Main Results:
- Three plasmids (pCS195, pCS192, pM35-4) were identified that specifically rescue CK-M mRNA.
- In vitro translation of rescued mRNA produced a protein product identical in molecular weight and isoelectric point to CK-M.
- The heterogeneity of CK-M observed in vivo was also detected in the in vitro translation product.
Conclusions:
- A successful method for cloning CK-M mRNA was established, providing a tool for further research.
- The cloned DNA sequence serves as a probe for studying CK-M gene expression.
- This work facilitates a deeper understanding of CK-M's role in muscle physiology and development.