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Biomedical subjects

R N Beachy

Publications and source records attributed to R N Beachy.

At least 145 records · Page 8Linked to original sources
PubMed1985

Accumulation and assembly of soybean beta-conglycinin in seeds of transformed petunia plants.

A gene encoding the alpha'-subunit of beta-conglycinin, a seed storage protein of soybean (Glycine max), was transformed into petunia cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens, and plants were regenerated. Transcripts of the introduced gene accumulated in immature embryos but not in leaves of the transformed plants. Soybean protein was first detected immunologically in proteins extracted from embryos at 10 days post pollination (d.p.p.), concurrent with the accumulation of subunits of the major petunia seed proteins. Between 10 and 16 d.p.p. the primary soybean protein detected had an apparent mol. wt. of 55 kd. The 76-kd alpha'-subunit and several smaller polypeptides accumulated between 16 and 24 d.p.p., when seeds had matured. Polypeptides <76 kd probably resulted from specific proteolytic cleavage of the alpha'-subunit. The alpha'-subunit and the smaller polypeptides assembled into multimeric proteins with sedimentation coefficients of 7-9S, similar to the sedimentation coefficients of beta-conglycinins isolated from soybean seeds. This transformation and expression system should be ideally suited for testing gene mutations to alter the amino acid composition of these seed storage proteins.

Journal Article↗
PubMed1985

Degradation of beta-Conglycinin in Early Stages of Soybean Embryogenesis.

This study focuses on the role of protein turnover in the accumulation of the storage protein beta-conglycinin (7S protein) during soybean embryogenesis. The results of pulse:chase experiments using (3)H-leucine indicate that the turnover of the subunits of beta-conglycinin by proteolysis is more rapid in early stages of cotyledon maturation than in later stages.

Journal Article↗
PubMed1985

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons.

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the beta-subunit of beta-conglycinin, one of the major storage proteins of soybean seeds. The amount of the alpha'-and alpha-subunits of beta-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the beta-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and beta-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against beta-conglycinin, there was an increased amount of pre-beta-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-beta-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of beta-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the beta-subunit of beta-conglycinin in developing cultured soybean cotyledons.

Journal Article↗
PubMed1984

Detection of genomic-length soybean mosaic virus RNA on polyribosomes of infected soybean leaves.

Soybean mosaic virus (SMV)-related RNAs were examined in both polyribosomal and nonpolyribosomal fractions of systemically infected soybean leaves. Viral RNAs were detected by Northern blot hybridization analysis using two cloned SMV-cDNAs representing different regions of the viral genome as hybridization probes. Genomic length SMV-RNA (Mr of 3.3 X 10(6] was found in specific association with EDTA-sensitive polyribosomes of infected leaves, indicating that it functions as a messenger RNA in these cells. A smaller SMV-related RNA (Mr of 1.6 X 10(6] was sometimes detected in the polyribosomal fraction; however, reconstruction experiments indicate that this RNA is a breakdown product of the genomic-length RNA, generated during cell fractionation or RNA extraction. Two other SMV-related RNAs with Mr of 2.0 and 0.78 X 10(6) were sometimes detected in infected cells and were not generated from genomic SMV-RNA or intact virus particles in reconstruction experiments. However, these RNAs were exclusively associated with the EDTA-resistant, nonpolyribosomal fraction of infected cells. These data suggest that genomic-length SMV-RNA is the only viral RNA which is translated in these infected plants.

Cloning, Molecular↗
PubMed1984

Molecular characterization of a deletion mutation affecting the alpha'-subunit of beta-conglycinin of soybean.

A naturally occurring variety of soybean Glycine max cv. Keburi, which lacks the alpha'-subunits of the 7S seed storage protein (beta-conglycinin), was recently described by Kitamura and Kaizuma. Keburi beta-conglycinins contain a normal complement of alpha-, beta-, and gamma-subunits that accumulate in a temporally and spatially regulated manner comparable to that of the standard cultivar Provar. Poly(A)+ RNA isolated from mid-to-late maturation stage Keburi seeds was translated in vitro, yielding products equivalent to those of Provar poly(A)+ RNA except that Keburi mRNA did not produce the pre-alpha'-subunit polypeptide. The basis of the Keburi phenotype was determined by examining genomic DNA using Southern blot hybridization. Restriction fragments isolated from a cloned 11.5 kb EcoRI fragment of genomic DNA containing a normal alpha'-subunit gene (Gmg 17.1) were used as hybridization probes. Sequences far upstream of the alpha'-subunit gene were present in both Provar and Keburi cultivars. However, hybridization reactions with probes from within the gene demonstrated that a deletion had occurred in Keburi DNA beginning immediately 5' to the alpha'-subunit gene represented on this 11.5 kb fragment. The deletion extends through most of the coding sequences, producing the Keburi phenotype.

Antigens, Plant↗
PubMed1982

Closely related families of genes code for the alpha and alpha' subunits of the soybean 7S storage protein complex.

Nineteen cloned cDNAs encoding the alpha and alpha'-subunits of the 7S seed storage protein in the soybean, Glycine max, have been isolated from a recombinant cDNA library constructed with mRNA from maturing seeds. In addition, a gene encoding an alpha'-subunit has been isolated from a recombinant Charon 4A phage library containing genomic Glycine max DNA. The cloned DNAs have been divided, on the basis of their endonuclease sites, into two main classes of sequences which differ in approximately 6% of their nucleotides. Whereas the proteins encoded within each DNA class are nearly identical, the proteins encoded by the two different classes of soybean DNAs are distinct and correspond to alpha and alpha'-subunits. Thus, the alpha and alpha'-subunits are coded for by two closely related multigene families. The amino acid differences in the portions of the alpha and alpha'-subunits presented in this paper occur primarily near the carboxyl-terminus. The 3' noncoding nucleotides of the cloned alpha and alpha'-subunit DNAs are more highly conserved than are the coding nucleotides. This conservation suggests that the 3' untranslated sequences of the alpha and alpha'-subunit mRNAs are functional in the expression of the alpha and alpha'-subunit proteins or in the stabilization of the 7S subunit mRNAs.

Amino Acid Sequence↗
PubMed1982

Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences.

Amino Acid Sequence↗
PubMed1982

The biosynthesis and processing of high molecular weight precursors of soybean glycinin subunits.

The predominant storage protein of soybean seed, glycinin, is composed of two heterogeneous classes of related subunits, the acidics (Mr approximately 38,000) and the basics (Mr approximately 22,000). Immunoreaction of polypeptides translated in vitro from isolated seed mRNA using antibodies prepared against either purified acidic or basic subunit groups precipitated precursor polypeptides of Mr = 60,000 to Mr = 63,000. High pressure liquid chromatography fingerprinting of trypsin-generated fragments from in vitro synthesized precursors showed fragments specific to both acidic and basic subunits. No mature acidic or basic subunits were detected in vitro translation reactions by either immunoprecipitation or high pressure liquid chromatography fingerprinting. Pulse-labeling of cotyledons growing in culture with [3H]glycine showed rapid accumulation of label in glycinin precursors of Mr = 59,000 to Mr = 62,000. Although in vivo synthesized precursors had slightly greater electrophoretic mobility than in vitro synthesized precursors, little label initially appeared in mature glycinin subunits. After several hours of continued cotyledon growth in absence of label, precursors were processed and label accumulated in both acidic and basic subunit groups. Recombinant plasmids were prepared by reverse transcription of soybean seed mRNA, and clones which encode glycinin precursors were identified by heteroduplex-hybridization of translatable messages. Northern blot analysis of seed mRNA shows the mRNA-encoding glycinin precursors to migrate at Mr = 0.71 X 10(6) on agarose gels, corresponding to approximately 2050 nucleotides. This is sufficiently large to encode a polypeptide consisting of both a glycinin acidic and basic subunit.

Chromatography, High Pressure Liquid↗
PubMed1981

Biosynthesis of subunits of the soybean 7S storage protein.

Biosynthesis of the alpha', alpha, and beta subunits of the soybean 7S storage protein (conglycinin) was studied by in vivo and in vitro experiments. One hour in vivo labeling produced polypeptides of 72 kilodaltons (kD) and 78 kD which, in a subsequent 1-h chase period, gave rise to a heterodisperse band of polypeptides of 76-83 kD, the apparent molecular weights of mature alpha and alpha', respectively. After a 3-h chase period only mature alpha and alpha' were labeled. The pre-alpha' (80 kD) and pre-alpha (78 kD) polypeptides produced by in vitro translation of total seed poly(A)+ RNA did not coelectrophorese with polypeptides labeled in vivo. The mature beta-subunit (53 kD), produced in vivo during the 1-h chase period, apparently was translated as a 50 kD polypeptide in vitro. The data suggest that, in vivo, primary translation products undergo both cotranslational and posttranslational modifications during the formation of mature subunits. cDNA clones complementary to soybean seed poly(A)+ RNA were prepared and subsequently identified by hybrid-release and immunoprecipitation experiments. Clone pGmc 236 (550 base pairs) (bp) was shown to contain sequences complementary to mRNAs for the alpha', alpha, and beta subunits on the basis of hybrid-release experiments, corroborating tryptic fingerprints that demonstrate that these subunits are closely related to each other. Although selected by a single cDNA clone, the mRNAs are not identical because the alpha' mRNA was eluted from the filter-bound cDNAs at a lower temperature than were the alpha beta mRNAs, pGmc 236 hybridized on Northern blots to mRNAs of approximately 2500 nucleotides, sufficient size to code for the alpha' or alpha subunit of the 7S storage protein.

Antigens, Plant↗
PubMed1980

In Vitro Synthesis of the alpha and alpha' Subunits of the 7S Storage Proteins (Conglycinin) of Soybean Seeds.

Messenger RNAs (mRNAs), isolated from immature soybean (Glycine max L., Merr.) seeds, that bound to oligo(dT)-cellulose were fractionated by centrifugation in sucrose density gradients containing dimethyl sulfoxide. mRNAs with sedimentation values between 21S and 25S coded for the in vitro translation of polypeptides with electrophoretic mobilities similar to those of the alpha' and alpha subunits of the 7S seed storage protein. High pressure liquid chromatographic analyses of the trypsin-induced fragments ("column fingerprinting") verified that the polypeptides produced in vitro were closely related to authentic alpha' and alpha subunits.The fractions of RNA that coded for the in vitro synthesis of these subunits contained three major species of nonribosomal RNA (molecular weights of 1.1, 0.84, and 0.75 x 10(6)) sufficiently large to code for the synthesis of the alpha' and alpha subunits.

Journal Article↗
PubMed1978

Isolation of polyribosomes and messenger RNA active in in vitro synthesis of soybean seed proteins.

Polyribosome preparations containing low proportions of monosomes to polyribosomes have been isolated from developing seeds of Glycine max L. Merrill using a high pH-high KCl buffer. The polyribosomes were functional in in vitro protein synthesis reactions using wheat germ 23,000g supernatant preparations. Results of experiments using aurintricarboxylic acid indicated that most or all of the amino acid incorporation in vitro resulted from the completion of nascent polypeptides associated with the isolated polyribosmes. RNA purified from polyribosome preparations by affinity chromatography on oligo(dT)-cellulose was also active in vitro, and had different Mg and K requirements for translation than did the polyribosomes. Translation of oligo(dT)-cellulose-purified mRNA was inhibited by the addition of 7-methylguanosine 5'-phosphate, suggesting that soybean mRNAs are "capped" at their 5' ends. Some, but not all, of the products of these reactions were identical in electrophoretic mobility to radioactive polypeptides of storage proteins produced in soybean cotyledons grown in culture.

Journal Article↗
PubMed1976

Changes in soluble proteins in callus cells of hypersensitive tobacco inoculated with tobacco mosaic virus.

Protein changes occurred in callus cells of hypersensitive tobacco (Nicotiana tabacum var. Xanthi-nc) 72 hr after inoculation with tobacco mosaic virus and incubation on a minimal growth medium. Two protein bands, serologically related to viral coat protein, were obtained from extracts of infected cells following electrophoresis on 7% and 10% polyacrylamide gels. An additional, slower migrating protein, perhaps due to virus-induced stimulation of a host protein, also was detected. Although local lesions appeared on callus after 40 hr of incubation, four proteins previously reported in lesion-bearing hypersensitive tobacco leaves were not found. The possible significance of this and the usefulness of a callus-TMV system as a tool to study virus-induced protein changes are discussed.

Cells, Cultured↗