Isolation of a tomato alcohol dehydrogenase 2-encoding cDNA using phage-promoted antibody screening of a plasmid cDNA library.
We describe the cloning of a cDNA encoding tomato alcohol dehydrogenase 2 (Adh2) by screening plasmid cDNA clones in phage plaques. A cDNA library constructed in a plasmid vector containing a unique SstI site at the 5' end of the cDNA insert was transferred into the SstI site of the lacZ gene of phage lambda Charon16, and screened by anti-Adh2 antibody to identify reactive plaques. Plasmid cDNA clones were recovered by SstI digestion, ligation, and transformation from phage minipreps for subsequent characterization. This system preserves the original plasmid library for subsequent screening with nucleic acid probes to identify full-length, multiple independent, or related cDNA clones not subject to the selection pressure of phage growth or lysogeny, or negative antibody reactivity. Thirty-two cDNA clones were identified with polyclonal antiserum to Adh2. Three of these reacted with monoclonal anti-Adh2 and only those three hybridized to maize adh1 sequence. One of these cDNAs, Adh31, was further characterized as encoding Adh2 by hybrid-selected translation and high sequence homology with the maize adh1 gene.
Genez
A L
AL
Plant Cell Research Institute, Inc., Dublin, CA 94568.
Staraci
L C
LC
Alexander
D C
DC
Rejda
J M
JM
Williamson
V M
VM
Chase
T
T
Williams
B G
BG
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000906:Antibodies; D010582:Bacteriophage lambda; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D015723:Gene Library; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D010957:Plasmids; D011489:Protein Denaturation
D000906:Antibodies; D007527:Isoenzymes; D000426:Alcohol Dehydrogenase