Purification of the mRNA for chicken very low density lipoproteinII and molecular cloning of its full-length double-stranded cDNA.
The mRNA coding for the small apo-Very Low Density Lipoprotein (apo-VLDLII) from chicken serum was highly enriched by oligo(dT) chromatography and preparative gel electrophoresis of estrogenised liver RNA. Double-stranded cDNA was synthesised by the subsequent actions of reverse transcriptase and DNA polymerase, and used for a preliminary characterisation of the structural gene. Molecular cloning of dC-tailed ds-cDNA into the Pst I site of plasmid pBR 322 yielded several recombinant clones. Five chimeric DNAs were selected and characterised by restriction enzyme mapping and electron microscopy of R-loops. At least two of them (pVLDLII 3.33 and pVLDLII 4.82) contain an almost full-length ds-transcript of VLDLII mRNA in which no more than 10-20 bases at the 5'- end are missing.
D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism*; D007700:Kinetics; D008079:Lipoproteins, VLDL / Q000096:biosynthesis*; D008099:Liver / Q000378:metabolism; D008297:Male; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D008079:Lipoproteins, VLDL; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes