Germin. Molecular cloning of cDNA that selects germin mRNA from bulk wheat mRNA.
(1) Bulk mRNA from germinated wheat embryos was denatured with methylmercury and subjected to electrophoresis in agarose gel to obtain a fraction of mRNA that was modestly enriched with respect to its complement of translatable germin mRNA. This fraction of mRNA was used as a source of primary templates for preparing a cDNA library. (2) Escherichia coli JM101 was transfected with recombinant pUC8 plasmids containing cDNA inserts. Colonies of transformed bacteria (ca. 4 x 10(3)) were differentially screened by hybridizing them with cDNA probes that were prepared from RNA populations containing different proportions of translatable germin mRNA. (3) A 160 base pair (bp) cDNA, which hybridized more strongly to the probe made from the RNA population containing the greater proportion of translatable germin mRNA in colony hybridizations, also hybridized more strongly to the RNA population containing the greater proportion of translatable germin mRNA when it was used as a probe for Northern analysis. (4) As judged by peptide mapping of a protein made by cell-free translation, the 160-bp cDNA selected virtually pure germin mRNA from the bulk mRNA of germinated wheat embryos when it was used in "hybrid release" experiments. The same 160-bp cDNA was used to select a "full length" germin cDNA from a library prepared by the Gubler-Hoffman method.
Rahman
S
S
Biochemistry Department, University of Toronto, Ontario, Canada.
Grzelczak
Z
Z
Kennedy
T
T
Lane
B
B
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D014908:Triticum / Q000196:embryology / Q000235:genetics*
D018076:DNA, Complementary; D006023:Glycoproteins; D010940:Plant Proteins; D012333:RNA, Messenger; C058814:germin