A cDNA clone encoding a glycinin A1a subunit precursor of soybean.

A cDNA clone covering the whole coding region for a glycinin subunit precursor containing the A1a acidic subunit, one of the A2 family, has been identified from a library of soybean cotyledonary cDNA clones using a mixed oligonucleotide probe. Analysis of the cDNA insert revealed that it contained 1746 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 54 nucleotides, a signal peptide region corresponding to 19 amino acids, an acidic subunit region (A1a) corresponding to 291 amino acids followed by a basic subunit region corresponding to 185 amino acids, and a 3'-terminal nontranslated region of 207 nucleotides. By comparing the predicted protein sequence of this precursor with that of the legumin A precursor of pea, it was found that glycinin A2 subunit family appeared to be more closely related to the legumin than to the A3 subunit family, and that the evolutional rearrangement of glycinin genes has occurred.     

The amino acid sequence of the A2B1a subunit of glycinin

The amino acid sequences of the acidic and basic components of the A2B1a subunit of glycinin, the major seed reserve protein of the soybean (Glycine max L. Merr.), were determined. They contain 278 and 180 amino acids, respectively, and have molecular weights of 31,600 +/- 100 and 19,900 +/- 100. The molecular weight of the acidic component is considerably less than that estimated by sodium dodecyl sulfate-gel electrophoresis (37,000). Sequence heterogeneity was detected at several positions scattered throughout the primary structures of both components, indicating that the preparation sequenced was composed of several nearly identical polypeptides. These data, in conjunction with a recently determined nucleotide sequence of the 3'-terminal two-thirds of the analogous glycinin subunit gene, illustrate the complexity of the gene family responsible for synthesis of glycinin subunits.     

A complete cDNA coding for the sequence of glycinin A2B1a subunit precursor

Analysis of the A₂B_1a subunit precursor, one of the A₂-subunit family of glycinin, the main storage protein of soybean, revealed that it is composed of a signal peptide segment (18 amino acids), the A₂ acidic polypeptide (282 amino acids), followed by the B_1a basic polypeptide (185 amino acids). There was overall 63% homology between this subunit complex and pea legumin, which is an analogous protein to glycinin. As this degree of homology is rather higher than that in the A₃B₄ subunit, one of the A₃ subunit family, it seems that the genes encoding the A₂ subunit family are phylogenetically more strongly related to the legumin genes.     

Effect of structural modifications on the assembly of a glycinin subunit.

A Gy4 glycinin cDNA was modified and used to produce structurally altered 11S storage protein subunits. We evaluated these modified subunits for their ability to assemble into oligomers. Alterations made in the acidic polypeptide changed the subunit solubility characteristics but did not eliminate assembly. Modifications in the basic polypeptide usually eliminated assembly of subunits into trimers. A region exhibiting high natural variability located at the COOH terminus of the acidic polypeptide that we have designated the hypervariable region was also studied. Extensive deletions and insertions were tolerated in the hypervariable region without perturbing subunit assembly. Some of the insertions significantly increased the methionine content in the Gy4 glycinin subunit. Together, our results indicated that the structure of the basic polypeptide was more critical for assembly of trimers than that of the acidic polypeptide, an observation that implies that the basic polypeptides direct trimer formation. The assembly assays described here will be useful in efforts to improve seed quality. Using them, the effects of modifications to the storage protein subunits can be rapidly evaluated before introducing the mutated genes into plants.     

Cloning and sequence analysis of human genomic DNA encoding gamma subunit precursor of muscle acetylcholine receptor

The limited proteolysis of human low-molecular-mass kininogen by kallikrein from tissue sources has been studied. Porcine pancreatic kallikrein applied in catalytic amounts split the kininogen molecule (apparent mass 68 kDa) with the release of lysyl-bradykinin (1 kDa). This generated a nicked kininogen molecule with a heavy chain and light chain interconnected via disulfide bridging. Following reductive cleavage of the disulfide bonds, the heavy chain of apparent mass 62 kDa was isolated by preparative sodium dodecyl sulfate electrophoresis, and the light chain of 5 kDa by reversed-phase high-performance liquid chromatography. The light chain was found to be composed of 38 amino acids with a single half-cystine residue. Amino-terminal sequence analysis revealed that the light chain is derived from the carboxy terminus of the kininogen molecule [Lottspeich et al. (1984) Eur. J. Biochem. 142, 227-232]. Immunological characterization of the isolated L chain indicated that it harbours antigenic site(s) unique for low-Mr kininogen as well as sites common to high-Mr and low-Mr kininogen.     

Identification and characterization of DNA clones encoding group-II glycinin subunits

Summary DNA clones that encode the group-II subunits of soybean glycinin were identified and compared with clones for group-I subunits. The group-I clones hybridize weakly to those from group-II at low stringency, but fail to hybridize with them at moderate or high stringency. The genes for the group-II subunits are contained in 13 and 9 kb EcoRI fragments of genomic DNA in cultivar CX635-1-1-1. These fragments contain genes for subunits A₅A₄B₃ and A₃B₄, respectively. The larger size of mature group-II subunits compared with group-I subunits is correlated with a larger sized mRNA. However, the gross arrangement of introns and exons within the group-II coding regions appears to be the same as for the genes which encode group-I subunits. Messenger RNA for both groups of glycinin subunits appear in the seed at the same developmental interval, and their appearance lags slightly behind that of mRNAs for the a/a subunits of -conglycinin. These data indicate that the glycinin gene family is more complex than previously thought.Abbreviations bp base pairs - kb kilobase pairs - SDS sodium dodecyl sulfate Cooperative research between USDA/ARS and the Indiana Agric. Expt. Station. This work was supported in part by grants from the USDA Competitive Grants Program and the American Soybean Association Research Foundation. This is Journal Paper No. 10,078 from the Purdue Agricultural Experiment Station     

Cloning and sequence analysis of a zebrafish cDNA encoding DNA (cytosine-5)-methyltransferase-1

The zebrafish has become a well-established animal model for the analysis of development and of several disease phenotypes. Several of the favorable traits that make it a popular model organism would also be beneficial for the study of normal and abnormal vertebrate development in which DNA methylation may play a role. We report the determination of the full-length cDNA sequence corresponding to the zebrafish DNA (cytosine-5-) methyltransferase gene, Dnmt1. It is 4,907 bases long and has an open reading frame predicted to encode a 1,499 amino acid protein that is similar in size and sequence to a number of other methyltransferases identified in other organisms.     

Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide

Chicken -natriuretic peptide (-chNP) has been identified in chicken heart, which showed higher homology to brain natriuretic peptide (BNP) than to atrial natriuretic peptide (ANP) [1]. Complementary DNA (cDNA) clone encoding a chNP precursor (pre-chNP) precursor (pre-chNP) was isolated from cardiac cDNA library and sequenced. Pre-chNP was 140-residue signal peptide at the N-terminus and -chNP at the C-terminus, and did not exhibit high homology to poreine BNP except for the C-terminal region. However, a characteristic AT-rich nucleotide sequence commonly found in mammalian BNPs was also present in the 3′-untranslated region. Thus, chNP is concluded to be classified into the BNP-type     

Molecular analysis of a Leptospira borgpetersenii gene encoding an endoflagellar subunit protein

A flagellin gene, flaB, from Leptospira borgpetersenii (formerly L. interrogans) serovar hardjo was cloned and expressed in Escherichia coli. Expression of the 32 kDa FlaB protein was dependent upon the lacZ promoter from pUC18. Nucleotide sequence data showed an open reading frame encoding 283 amino acid residues, corresponding to a protein of molecular mass 31.3 kDa. The G + C content of the flaB gene was 54.7 mol%. Comparison of the deduced FlaB amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum FlaB proteins.     

Cloning and sequence analysis of human pituitary cDNA encoding the novel polypeptide 7B2

Application of a differential hybridization technique led to the isolation of a human pituitary cDNA clone encoding the complete structure of the polypeptide 7B2. This protein of unknown function, which is sorted to secretory granules, appears to be present selectively in neurons and endocrine cells. The polypeptide chain of human 7B2, preceded by a cleaved signal peptide, comprises 185 amino acids (a calculated Mr of 20,793). Interesting features of the highly-conserved 7B2 structure include (i) a serine phosphorylation consensus sequence, (ii) the occurrence of three pairs of dibasic amino acids representing potential proteolytic cleavage sites and, in particular, (iii) the presence of three regions homologous to GTP-binding domains giving 7B2 structural characteristics of a GTP-binding protein.     

Cloning and characterization of B delta, a novel regulatory subunit of protein phosphatase 2A.

Variable regulatory subunits of protein phosphatase 2A (PP2A) modulate activity, substrate selectivity and subcellular targeting of the enzyme. We have cloned a novel member of the B type regulatory subunit family, B delta, which is most highly related to B alpha. B delta shares with B alpha epitopes previously used to generate subunit-specific antibodies. Like B alpha, but unlike B beta and B gamma which are highly brain-enriched, B delta mRNA and protein expression in tissues is widespread. B delta is a cytosolic subunit of PP2A with a subcellular localization different from B alpha and may therefore target a pool of PP2A holoenzymes to specific substrates.     

Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A

Type 2A serine/threonine protein phosphatases (PP2A) are key components in the regulation of signal transduction and control of cell metabolism. The activity of these protein phosphatases is modulated by regulatory subunits. While PP2A activity has been characterized in plants, little is known about its regulation. We used the polymerase chain reaction to amplify a segment of a cDNA encoding the B regulatory subunit of PP2A from Arabidopsis. The amplified DNA fragment of 372 nucleotides was used as a probe to screen an Arabidopsis cDNA library and a full-length clone (AtB) of 2.1 kbp was isolated. The predicted protein encoded by AtB is 43 to 46% identical and 53 to 56% similar to its yeast and mammalian counterparts, and contains three unique regions of amino acid insertions not present in the animal B regulatory subunit. Genomic Southern blots indicate the Arabidopsis genome contains at least two genes encoding the B regulatory subunit. In addition, other plant species also contain DNA sequences homologous to the B regulatory subunit, indicating that regulation of PP2A activity by the 55 kDa B regulatory subunit is probably ubiquitous in plants. Northern blots indicate the AtB mRNA accumulates in all Arabidopsis tissues examined, suggesting the protein product of the AtB gene performs a basic housekeeping function in plant cells.     

Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis

A gene library constructed from large (20 kb) fragments of total DNA from the geldananmycin-producing strain Streptomyces hygroscopicus 3602 cloned in the plasmid vector pIJ61 were used to transform S. lividans TK24. Three transformants of about 800 tested were found to have acquired the ability to produce an antibiotic lethal to a geldanamycin-sensitive strain of Bacillus subtilis. The plasmids isolated from these transformants, pIA101, pIA102 and pIA103, each contained an insert of 15 kb. A 4.5 kb DNA fragment from the insert in pIA102 hybridised to DNA from S. hygroscopicus 3602 and to DNA encoding part of the erythromycin polyketide synthase but not to S. lividans TK24 DNA. The integration-defective phage vector C31 KC515 containing this 4.5 kb fragment was able to lysogenise S. hygroscopicus 3602 to produce lysogens defective in geldanamycin production. Loss of the prophage restored the ability to produce geldanamycin. Extracts of fermentation broth cultures of S. lividans containing pIA101, pIA102 and pIA102 and pIA103 analysed by thin-layer chromatography (TLC) contained compounds identical or very similar to purified geldanamycin, which were not present in S. lividans. These compounds showed a mass spectrum indistinguishable from geldanamycin. The evidence suggests that the clones contain DNA sequences encoding functions required for geldanamycin biosynthesis including components of the polyketide synthase.