JBC, Vol. 252, Issue 7, 2209-2217, Apr, 1977
Cloning and amplification of rabbit alpha- and beta-globin gene sequences into Escherichia coli plasmids
F. Rougeon and B. Mach
cDNA, synthesized from rabbit globin mRNA, was used in a self-priming
reaction, with avian myeloblastosis virus DNA polymerase, for the synthesis
of double-stranded DNA. Globin DNA ranging from about 400 to 650 base pairs
was elongated with dG tails using deoxypolynucleotide transferase and was
annealed to linear Escherichia coli plasmic pCR1, elongated with dC tails.
Preparation of the plasmid DNA involved an enzymatic reconstruction of one
EcoRI-specific site on each side of the molecule. After transformation of
E. coli cells to kanamycin resistance with the hybrid molecules, bacterial
clones harboring recombinant plasmids were studied for the presence of
globin-specific DNA. Plasmids containing either alpha or beta rabbit globin
gene sequences were obtained. There was a 4-fold excess of recombinant
plasmids containing beta-globin sequences over those with alpha-globin DNA.
The longest beta-globin sequences found in plasmids were about 550 to 600
pairs long, and correspond therefore to the entire beta-globin structural
gene and to some of the untranslated regions. The alpha-globin sequences
were 400 to 450 base pairs long. Treatment of clone pCR1betarG 19 with
EcoRI endonuclease released two DNA fragments (410 and 210 base pairs)
resulting from cleavage at two reconstructed external EcoRI sites and at
one internal EcoRI site within the rabbit globin gene. The same treatment
of pCR1alpharG 11 released one fragment. In most other recombinant plasmids
studied however, no fragment was released by EcoRI digestion.
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