Biomedical subjects

J L Key

Publications and source records attributed to J L Key.

At least 55 records · Page 3Linked to original sources

PubMed1985

DNA sequence and transcript mapping of a soybean gene encoding a small heat shock protein.

The DNA sequence of a gene (Gmhsp17.5-E) encoding a small heat shock protein of soybean, Glycine max, has been determined. Nuclease S1 mapping of the 5' terminus of the corresponding RNA indicates that the start site for transcription is located 82 bases upstream from the coding region and 24 bases downstream from a "TATA"-like region (-T-T-T-A-A-A-T-A-). The 5' flanking region of Gmhsp17.5-E contains two imperfect dyads that closely resemble regulatory elements present in the promoters of heat-inducible genes of Drosophila. One, positioned 18 bases upstream from the TATA-like region, shows 90% homology to the Drosophila heat shock consensus sequence. The other overlaps an upstream TATA sequence and is located at position -213. Analysis of the derived amino acid sequence indicates that the protein encoded by Gmhsp17.5-E is related structurally to the four small heat shock proteins of Drosophila. This relationship is most evident by comparison of hydropathy profiles; they show conservation of several major hydrophilic and hydrophobic regions, which suggests that these proteins have common structural features.

Journal Article↗

PubMed1985

An Analysis of Growth Regulator Interactions and Gene Expression during Auxin-Induced Cell Elongation Using Cloned Complementary DNAs to Auxin-Responsive Messenger RNAs.

We have examined the effects of cytokinin, fusicoccin, and ethylene on auxin-induced changes in gene expression during auxin-promoted cell elongation in soybean (Glycine max L. Merr. cv Wayne) using cloned cDNAs to two auxin-responsive mRNAs (Walker, Key 1982 Proc Natl Acad Sci USA 79: 7185-7989). RNA blot analyses demonstrate that under conditions of cytokinin inhibition of auxin-promoted cell elongation the levels of these two auxin-responsive mRNAs is unaltered. Fusicoccin-promoted elongation is not associated with an enhanced expression of these two mRNAs, suggesting that the increased levels of these mRNAs observed during auxin-promoted cell elongation are not simply due to enhanced rates of cell elongation. We have also determined that ethylene plays no apparent role in the regulation of expression of these mRNAs. However, the auxins indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and alpha-naphthalene acetic acid all enhance an accumulation of these mRNAs. We conclude that the regulation of these mRNAs is directly dependent on auxin. That auxin-promoted cell elongation is dependent upon the increased accumulation of these mRNAs remains to be determined.

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PubMed1985

Ribulose 1,5-Bisphosphate Carboxylase Synthesis during Heat Shock.

Ribulose 1,5-bisphosphate carboxylase (RuBPCase) was chosen as a model protein to study how heat shock (HS) affects both chloroplast protein synthesis and the nuclear-chloroplast interaction in production of chloroplast proteins. Experiments were performed using highly chlorophyllous, soybean (Glycine max L. Merr. var Corsoy) cell suspension cultures active in chloroplast protein synthesis. Synthesis of RuBPCase large (L) and small (S) subunits was followed by in vivo labeling, and corresponding mRNA levels were examined by Northern and dot hybridization analyses. Results demonstrate that L and S synthesis declines with increasing HS temperatures (33-40 degrees C) and reaches minimum levels (20-30% of control) at temperatures of maximum HS protein synthesis (39-40 degrees C). Recovery of L and S synthesis following a 2-hour HS at 38 or 40 degrees C was also studied. The changes in S synthesis during HS and recovery correlate with the steady state levels of S mRNA. In contrast, changes in L synthesis show little relationship to the corresponding mRNA levels; levels of L mRNA remain relatively unchanged by HS. We conclude that chloroplast protein synthesis shows no greater sensitivity to HS than is observed for cytoplasmic protein synthesis and that transport of proteins into the chloroplast (e.g.S subunit) continues during HS. Furthermore, there is no apparent coordination of L and S subunit mRNA levels under the conditions examined.

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PubMed1985

Presence of Heat Shock mRNAs in Field Crown Soybeans.

Our laboratory has extensively defined many parameters of the heat shock (HS) response in etiolated soybean (Glycine max [L.] Merr.) hypocotyls, including the identification of cDNA clones for mRNAs encoding several low molecular weight HS proteins. We have now investigated the response of mature plants to a HS in a growth chamber and to high temperature stress under field conditions. Soybean plants show induction of HS mRNAs when the temperature of the chamber is rapidly shifted from 28 degrees C to 45 degrees C. This temperature of induction is significantly higher than the optimal induction temperature for etiolated hypocotyls, probably reflecting the ability of mature plants to lower their leaf temperatures below the ambient air temperature through transpirational cooling. Samples of soybean leaves were taken from an irrigated and a nonirrigated field during a 24-h period when midday temperatures reached 40 degrees C. Several HS mRNAs were present in samples from both fields, although the levels of these mRNAs were much higher in nonirrigated leaves. This differential response of HS mRNA steady state levels was not a response to water stress, since water-stressed plants at 28 degrees C did not induce HS mRNAS. Rather, these quantitative differences are probably due to differences in actual leaf temperatures between irrigated and nonirrigated leaves. The presence of these HS mRNAS in field-grown plants suggests that HS proteins are produced as part of the normal plant response to high temperature.

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PubMed1985

Genes for low-molecular-weight heat shock proteins of soybeans: sequence analysis of a multigene family.

Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5- and 17.6-kilodalton HS proteins were determined. Nuclease S1 mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.

Amino Acid Sequence↗

PubMed1984

Acquisition of Thermotolerance in Soybean Seedlings : Synthesis and Accumulation of Heat Shock Proteins and their Cellular Localization.

When soybean Glycine max var Wayne seedlings are shifted from a normal growth temperature of 28 degrees C up to 40 degrees C (heat shock or HS), there is a dramatic change in protein synthesis. A new set of proteins known as heat shock proteins (HSPs) is produced and normal protein synthesis is greatly reduced. A brief 10-minute exposure to 45 degrees C followed by incubation at 28 degrees C also results in the synthesis of HSPs. Prolonged incubation (e.g. 1-2 hours) at 45 degrees C results in greatly impaired protein synthesis and seedling death. However, a pretreatment at 40 degrees C or a brief (10-minute) pulse treatment at 45 degrees C followed by a 28 degrees C incubation provide protection (thermal tolerance) to a subsequent exposure at 45 degrees C. Maximum thermoprotection is achieved by a 2-hour 40 degrees C pretreatment or after 2 hours at 28 degrees C with a prior 10-minute 45 degrees C exposure. Arsenite treatment (50 micromolar for 3 hours) also induces the synthesis of HSP-like proteins, and also provides thermoprotection to a 45 degrees C HS; thus, there is a strong positive correlation between the accumulation of HSPs and the acquisition of thermal tolerance under a range of conditions.During 40 degrees C HS, some HSPs become localized and stably associated with purified organelle fractions (e.g. nuclei, mitochondria, and ribosomes) while others do not. A chase at 28 degrees C results in the gradual loss over a 4-hour period of the HSPs from the organelle fractions, but the HSPs remain selectively localized during a 40 degrees C chase period. If the seedlings are subjected to a second HS after a 28 degrees C chase, the HSPs rapidly (complete within 15 minute) relocalize in the organelle fractions. The relative amount of the HSPs which relocalize during a second HS increases with higher temperatures from 40 degrees C to 45 degrees C. Proteins induced by arsenite treatment are not selectively localized with organelle fractions at 28 degrees C but become organelle-associated during a subsequent HS at 40 degrees C.

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PubMed1983

Fractionation of nucleoli from auxin-treated soybean hypocotyl into nucleolar chromatin and preribosomal particles.

Nucleoli from auxin-treated tissues (Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8-fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms.Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio of the chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones.The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles.

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PubMed1982

Isolation of cloned cDNAs to auxin-responsive poly(A)RNAs of elongating soybean hypocotyl.

Auxin-responsive cDNA clones have been isolated from a cDNA library prepared from elongating soybean hypocotyl poly(A)(+)RNA. The expression of two such sequences has been assessed by RNA blot hybridization analyses during normal developmental transitions in the soybean hypocotyl and during incubation of sections excised from the region of cell elongation. The concentrations of these poly(A)(+)RNAs are higher in the elongating zone than in the apical and mature zones of the hypocotyl. Both poly(A)(+)RNAs are depleted during incubation of the sections in the absence of auxin. The loss of one of these sequences (pJCW1) is prevented by the addition of auxin to the incubation medium while the other sequence (pJCW2) increases above the initial level in the presence of auxin. The addition of auxin to auxin-depleted tissue in which the sequences are depleted results in rapid accumulation of these poly(A)(+)RNAs; pJCW1 accumulates to the control level while pJCW2 increases well above the control level. These data along with others [Baulcombe, D. C. & Key, J. L. (1980) J. Biol. Chem. 255, 8907-8913] demonstrate directly a highly selective effect of auxin on the expression of a small number of mRNAs in tissues undergoing both cell elongation and cell division in response to auxin. Although the data are suggestive of a close association betwen auxin action and altered gene expression, a causal relationship is not established. It seems highly unlikely, however, that such specific effects of auxin on gene expression are unimportant in auxin physiology.

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PubMed1982

An analysis of mRNAs for a group of heat shock proteins of soybean using cloned cDNAs.

Hybridization studies carried out with poly(A)+ RNA and its corresponding cDNA showed the presence of a new highly abundant RNA class after heat shock (hs) at 40 degrees C in soybean hypocotyl compared to tissue incubated under normal conditions at 28 degrees C. cDNA clones complementary to RNAs of this class were isolated; eleven clones were characterized and used in the analysis of these abundant RNAs. The most abundant hs-sequences were found to be 800-900 nucleotides in length and present in about 19,000 copies per cell. Extensive sequence homology among hs-induced RNAs was indicated by cross-hybridization of cDNA clones and by common protein patterns generated in hybrid release translations. The existence of at least two different nucleotide sequences common to several different hs poly(A)+ mRNAs was documented by different, nonoverlapping protein patterns obtained by in vitro synthesis with hybrid selected RNAs. Four clones contained a sequence common to mRNAs for at least 13 proteins of 15,000-18,000 daltons; another sequence common to mRNA for three to four proteins of 21,000-23,000 daltons was selected by one clone. Two other clones selected a major hs-protein of about 18,000 daltons. The mRNAs of these low molecular weight hs-proteins accumulated rapidly after induction at either 40 degrees C or 42.5 degrees C and decreased rapidly during subsequent incubation at 28 degrees C.

Base Sequence↗

PubMed1981

Heat shock proteins of higher plants.

The pattern of protein synthesis changes rapidly and dramatically when the growth temperature of soybean seedling tissue is increased from 28 degrees C (normal) to about 40 degrees C (heat shock). The synthesis of normal proteins is greatly decreased and a new set of proteins, "heat shock proteins," is induced. The heat shock proteins of soybean consist of 10 new bands on one-dimensional NaDodSO(4) gels; a more complex pattern is observed on two-dimensional gels. When the tissue is returned to 28 degrees C after 4 hr at 40 degrees C, there is progressive decline in the synthesis of heat shock proteins and reappearance of a normal pattern of synthesis by 3 or 4 hr. In vitro translation of poly(A)(+)RNAs isolated from tissues grown at 28 and 40 degrees C shows that the heat shock proteins are translated from a new set of mRNAs induced at 40 degrees C; furthermore, the abundant class mRNAs for many of the normal proteins persist even though they are translated weakly (or not at all) in vivo at 40 or 42.5 degrees C. The heat shock response in soybean appears similar to the much-studied heat shock phenomenon in Drosophila.

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PubMed1980

Polyadenylated RNA sequences which are reduced in concentration following auxin treatment of soybean hypocotyls.

Previous work has shown that any effect of exogenous auxin on gene expression in soybean hypocotyl tissue must be restricted to a relatively small fraction of the polyadenylated RNA. However, kinetic hybridization analysis with cDNA probes revealed that a minor abundant class of sequences is markedly reduced in concentrations in the auxin-treated polyadenylated RNA. Recombinant plasmids containing copies of polyadenylated RNA species were constructed using the G-C tailing procedure and clones of auxin-regulated sequences were detected by differential in situ hybridization with cDNA of polyadenylated RNA from auxin-treated or untreated hypocotyls. Although the 12 clones which were selected all contained different size inserts, and were therefore independent, 11 of these apparently hybridized to just two different RNA species. The rate constant of the auxin-sensitive abundant component of the untreated polyadenylated RNA/DNA hybridization was similar to that of the reaction between the two major groups of clones and untreated polyadenylated RNA. This indicates that these cloned sequences are homologous with that cDNA fraction. The twelfth clone is thought to be representative of a group of less abundnt auxin-regulated polyadenylated mRNA species which had been detected in an earlier analysis of the in vitro translation products of soybean hypocotyl RNA. Both the timing and the extent of the influence of auxin on the relative concentration of these cloned sequences are quite consistent with a close relationship between growth regulation by auxin and its effects on gene expression.

Base Sequence↗

PubMed1980

Histone Kinase from Soybean Hypocotyls: PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES.

A histone-type protein kinase (EC 2.7.1.37) has been partially purified (320-fold) from the crude extracts of soybean hypocotyls by means of a combination of gel filtration and anion exchange procedures. The purified enzyme fraction is devoid of the activities of phosphoprotein phosphatase (EC 3.1.3.16), histone protease, and casein (or phosvitin)-type kinase. The soybean histone kinase uses ATP to phosphorylate specifically lysine-rich histone H1 from either pea seedlings or calf thymus.The histone kinase requires free sulfhydryl group(s) for activity, but not stability. The pH optimum is around 9 to 10. The apparent K(m) values for histone H1 of pea seedlings and calf thymus are 0.4 and 0.9 micromolar, respectively. The K(m) values for ATP are 40 nanomolar with the optimal concentration of Mn(2+) (50 nanomolar) and 0.4 micromolar with that of Mg(2+) (5 millimolar). The estimated molecular weight of the kinase is 52,000 by gel filtration or 48,600 by sedimentation constant (3.2 S). cAMP does not alter the sedimentation velocity of the kinase. The enzyme activity is unaffected by cyclic nucleoside monophosphates and plant growth substances. Like arginine-rich histones, a variety of divalent cations and polycations (polyamines) are inhibitory.This cAMP-independent soybean histone kinase is not associated with the isolated ribosomes but shows highest specific activity in the nuclearchromatin fraction, suggesting that it may function in the regulation of histone H1 phosphorylation in the soybean hypocotyl.

Journal Article↗

PubMed1979

Sequence complexity of polyadenylated ribonucleic acid from soybean suspension culture cells.

The sequenc complexity of total poly(A) RNA from a higher plant system, soybean cultured cells, was determined. Labeled cDNA synthesized from the poly(A) RNA hybridized exclusively with the unique sequence component of total soybean DNA. Analysis of the hybridization reaction between cDNA and the poly(A) RNA template revealed three abundance classes in the poly(A) RNA. These classes represent 18, 44, and 38% of the poly(A) RNA and contain information for approximately 60, 1900 and 30,000 different 1400-nucleotide RNA molecules. From these results, the total sequence complexity of poly(A) RNA was estimated to be 4.5 X 10(7) nucleotides. Saturation hybridization of labeled unique DNA with RNA showed that the total cell RNA represents 12.4% of the unique DNA sequence complexity, or 6.4 X 10(7) nucleotides, while poly(A) RNA respresent 8.7% of the unique DNA sequence complexity, or 3.3 X 10(7) nucleotides. Thus, it is estimated that 50--70% of total RNA sequence complexity is contained in poly(A) RNA in these cells.

Base Sequence↗

PubMed1979

Stability of polysome-associated polyadenylated RNA from soybean suspension culture cells.

The half-life of polysome-associated, poly(A)-RNA in exponentially growing soybean (Glycine max) suspension culture cells was determined with pulse-chase experiments. Based on a best fit from a computer analysis of the data, two decay components for poly(A)-RNA were found. One component had a half-life of approximately 0.6 h, while the other had a half-life of about 30 h, similar to the doubling time of the cultures. At the beginning of the chase period, the short-lived component represented approximately 90% of the total poly(A)-RNA in the polysomes. This percentage decreased with time so that, under steady-state conditions, the long-lived component probably represented the majority of poly(A)-RNA.

Cells, Cultured↗

PubMed1979

Sequence organization of the soybean genome.

The total complexity of one constituent soybean (Glycine max) genome is estimated to be 1.29 . 10(9) nucleotide pairs, as determined by analysis of the reassociation kinetics of sheared (0.47 kilobase) DNA. Single copy sequences are estimated to represent from 53 to 64% of the genome by analysis of hydroxyapatite binding of repetitive DNA as a function of fragment length. From 65 to 70% of these single copy sequences have a short period interspersion with 1.11--1.36 kilobase lengths alternating with 0.3--0.4 kilobase repetitive sequence elements. The repetitive sequences of soybean DNA are interspersed both among themselves and among single copy regions of the genome.

Base Sequence↗

PubMed1978

2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins.

In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4-dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [gamma-(32)P]adenosine 5'-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F(1) is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F(1) but its identity is unknown. Urea-acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins.

Journal Article↗

PubMed1978

Isolation and Characterization of a Chromatin-associated Protein Kinase from Soybean.

A chromatin-associated casein-type protein kinase has been purified 500-fold from soybean (Glycine max, var. Wayne) tissue. The enzyme can be completely dissociated from isolated chromatin in 250 millimolar (NH(4))(2)SO(4). After purification, the kinase preparation is stable for at least 6 months at 0 C. The enzyme will phosphorylate casein, phosvitin, and denatured chromatin proteins, but not histones. Only ATP will serve as a phosphate donor with an apparent K(m) of 8 micromolar. Five millimolar Mg(2+) is required for maximal activity, but Mn(2+) will support phosphorylation at a lower level. The average molecular weight as determined by sucrose gradient sedimentation and gel filtration is approximately 55,000. Under conditions of low ionic strength [less than 250 millimolar (NH(4))(2)SO(4)] soybean casein kinase forms higher molecular weight aggregates with other chromosomal proteins in the preparation. The enzyme activity is not affected by cyclic AMP. Casein kinase shows a broad optimum between 7 and 8 and the isoelectric point is approximately 9. Preliminary data indicate that soybean casein kinase will not phosphorylate soybean RNA polymerases I or II, nor does it have any obvious effect on in vitro chromatin transcription by endogenous RNA polymerases.

Journal Article↗

PubMed1978

Isolation and preliminary characterization of a casein kinase from cauliflower nuclei.

A casein-type protein kinase has been isolated from cauliflower (Brassica cauliflora Gars.) nuclei and purified to a specific activity of 23,000 units/milligram of protein (1 unit is defined as the transfer of 1 picomole of (32)Pi from gamma-[(32)P]ATP to substrate per minute at 28 C). The enzyme has a molecular weight of approximately 39,000 as judged by sucrose density gradient sedimentation. The casein kinase requires ATP as the phosphate donor and will phosphorylate casein and phosvitin, but not histones. The enzyme activity is not affected by cAMP or cGMP. The casein kinase appears to be analogous to casein kinases described in other plant and animal systems.

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