Open AccessPurification of the mRNA for chicken very low density lipoproteirtIIand molecular cloning of its full-length double-stranded cDNA
Author(s) -
Bé Wieringa,
Willem Roskam,
Annika C. Arnberg,
Janneke van der Zwaag-Gerritsen,
Geert Ab,
M. Gruber
Publication year - 1979
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/7.8.2147
Subject(s) - biology , complementary dna , microbiology and biotechnology , recombinant dna , reverse transcriptase , molecular cloning , plasmid , restriction enzyme , messenger rna , gel electrophoresis , nucleic acid thermodynamics , restriction map , dna , oligonucleotide , rna , gene , biochemistry
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.