Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Apolipoprotein E (apo-E) plays a crucial role in lipid metabolism.
• Understanding the structure of apo-E mRNA is essential for studying its expression and function.
• Previous studies lacked detailed sequence information for rat apo-E.

Purpose of the Study:

• To clone and characterize the rat liver apolipoprotein E (apo-E) mRNA.
• To determine the nucleotide and amino acid sequences of rat apo-E.
• To compare the rat apo-E sequence with human apo-E to identify conserved regions.

Main Methods:

• Construction of a rat liver cDNA library in pBR322.
• Hybrid selection and translation of mRNA to identify apo-E clones.
• Cloning of the 5'-terminal region using primed cDNA synthesis.
• Subcloning into M13mp7 for probe synthesis.
• RNA-blot hybridization to determine mRNA length and tissue distribution.
• Nucleotide sequencing of double-stranded cDNA.
• Inference of amino acid sequence and comparison with human apo-E.

Main Results:

• A 900-base pair clone of rat liver apo-E mRNA was isolated.
• Rat apo-E mRNA is approximately 1200 nucleotides long and present in both liver and intestine (lower level).
• The nucleotide sequence revealed a precursor protein of 311 amino acids, with the first 18 likely removed during processing.
• Mature rat apo-E (293 amino acids) shares 69% overall amino acid identity with human apo-E (299 amino acids), with conserved NH2-terminal and COOH-terminal domains.

Conclusions:

• The complete nucleotide sequence of rat liver apo-E mRNA has been determined.
• Rat apo-E exhibits significant sequence homology with human apo-E, particularly in functional domains.
• The findings provide a basis for further studies on apo-E structure-function relationships and its role in lipid transport.