Abstract
We have constructed cDNA clones from size-selected mRNA of developing soybean seeds to identify genes encoding the Bowman-Birk protease inhibitor. Recombinant clones containing sequences coding for the Bowman-Birk inhibitor were identified by mRNA hybrid selected translation and immunoprecipitation. The nucleotide sequence of the insert of plasmid pB38 corresponds to the mRNA for the Bowman-Birk inhibitor, although it is missing a portion of the coding sequence at the 5' end. Northern hybridization analysis of total RNA isolated from developing soybean seeds showed that the mRNA for the inhibitor is approximately 450 nucleotides long and that it accumulates early in embryogeny. Southern hybridization analysis of restriction enzyme-digested soybean nuclear DNA indicated that the gene encoding the Bowman-Birk inhibitor is not highly reiterated in the genome. We have isolated a gene encoding the Bowman-Birk inhibitor from a soybean genomic library constructed in Charon 4A. DNA sequence analysis of the genomic clone reveals that it is similar, although not identical, to the cDNA clone and that it contains no intervening sequences.
Highlights
We have constructed cDNAclones from size-selected aka, 1977).The Kunitz inhibitor is a single polypeptide chain mRNA of developing soybean seeds to identify genes of M, = 21,500 that inhibitsboth trypsin and chymotrypsin
Construction and Identification of cDNA Clones-To increase theprobability of obtaining cDNA clones for soybean proteaseinhibitors, we isolated enrichedfractions of the mRNAs by dimethyl sulfoxide sucrosegradient centrifugation (Foard et al, 1982). These mRNAs were used to synthesize cDNAs ranging in size from 250 to 500 nucleotides
After transformation of E. coli HB101, several hundred clones were screened with a sscDNAprobeprepared from theenriched mRNA fraction
Summary
Nucleotides are numaa Ser Lys Pro Cys Cys Asp Gln Cys Ala Cys Thr Lys Ser Asn Pro Pro Gln Cy5 Arg Cys bered beginning with the first base in the initiation codon of the gene. TA the genomic clone.A colon indicates the A13.10 ACT GAT ATG AGA TTG AAT TCG TGC CAT TCA GCC TCT AAA TCG TGT CCG TCC CCA CTC TCC 180 start of the cDNA; only nucleotides that are different are indicated. The derived aa Ser Asp Met Arg Leu Asn S e r Cys His S e r Ala Cys Lys Ser Cys Ala Cys Ala Leu Ser 1 le amino acid sequenceof the gene is shown below the nucleotide sequence. PB 38 A13.10 ACT GAT GAC GACCAA GAA AAC TAA GAC AAA TGT AAA GAA CCC AAA ATA ACC TTC CCC TTT aa Ser Clu Asp Asp Lys G l u Asn STOP ( -COOH )
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