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Cloning and sequence of several alpha 2u-globulin cDNAs
Summary
Researchers developed a straightforward cloning method for alpha 2u-globulin, identifying male-specific clones. This technique efficiently yields alpha 2u-globulin cDNA, enabling complete amino acid sequence determination and analysis of its synthesis and processing.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Alpha 2u-globulin is a male-specific rat liver protein.
- Previous methods for cloning alpha 2u-globulin were complex, requiring mRNA enrichment or specific probes.
Purpose of the Study:
- To develop a simple and efficient cloning procedure for alpha 2u-globulin.
- To determine the complete nucleotide and amino acid sequence of alpha 2u-globulin.
- To investigate the synthesis and processing of alpha 2u-globulin.
Main Methods:
- Cloning of alpha 2u-globulin cDNA using total adult male liver poly(A)+RNA.
- Screening of recombinant colonies by comparing hybridization to male and female hepatic mRNA probes.
- In vitro translation of cloned alpha 2u-globulin cDNA and processing with pancreatic microsomes.
- Nucleotide sequencing of the alpha 2u-globulin cDNA.
Main Results:
- A simple cloning procedure was established, yielding predominantly alpha 2u-globulin clones.
- The complete amino acid sequence of alpha 2u-globulin and the leader sequence of its precursor (pro-alpha 2u-globulin) were elucidated.
- In vitro synthesized pro-alpha 2u-globulin was processed to the mature form, and its sequence matched previously determined data.
- Analysis revealed two potential polyadenylylation sites and specific codon usage in the leader sequence.
Conclusions:
- The developed cloning method is efficient for obtaining alpha 2u-globulin cDNA.
- The complete sequence provides insights into the protein's structure, synthesis, and post-translational modifications.
- The findings contribute to understanding the regulation and function of alpha 2u-globulin.