Search PubMed⌕ Search

Biomedical subjects

H M Goodman

Publications and source records attributed to H M Goodman.

At least 55 records · Page 3Linked to original sources
PubMed1995

An Arabidopsis thaliana root-specific kinase homolog is induced by dehydration, ABA, and NaCl.

Genomic and cDNA clones have been isolated for an Arabidopsis thaliana gene, ARSK1, that encodes a protein with structural similarities to serine/threonine kinases. Expression of ARSK1 is root specific and is induced by exposing roots to air during growth or by treatment of roots with ABA or NaCl. ARSK1 gene expression in transgenic plants is confined to cells in the tissues of the root as measured by beta-glucuronidase (GUS) expression from an ARSK1 gene promoter-GUS gene construct. Transverse sections of the stained roots further defined the tissue-specificity; high levels of expression in the epidermal, endoepidermal and cortex regions, but no or very little expression in the vascular system. Another feature of the expression pattern of the ARSK1 gene was a gradual increase in the expression expression level along the root with the highest level of expression in the region closest to the root meristem. These studies suggest that ARSK1 may have a role in the signal transduction pathway of osmotic stress.

Abscisic Acid↗
PubMed1995

Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis.

Eleven loci that play a role in the synthesis of flavonoids in Arabidopsis are described. Mutations at these loci, collectively named transparent testa (tt), disrupt the synthesis of brown pigments in the seed coat (testa). Several of these loci (tt3, tt4, tt5 and ttg) are also required for the accumulation of purple anthocyanins in leaves and stems and one locus (ttg) plays additional roles in trichome and root hair development. Specific functions were previously assigned to tt1-7 and ttg. Here, the results of additional genetic, biochemical and molecular analyses of these mutants are described. Genetic map positions were determined for tt8, tt9 and tt10. Thin-layer chromatography identified tissue- and locus-specific differences in the flavonols and anthocyanidins synthesized by mutant and wild-type plants. It was found that UV light reveals distinct differences in the floral tissues of tt3, tt4, tt5, tt6 and ttg, even though these tissues are indistinguishable under visible light. Evidence was also uncovered that tt8 and ttg specifically affect dihydroflavonol reductase gene expression. A summary of these and previously published results are incorporated into an overview of the genetics of flavonoid biosynthesis in Arabidopsis.

Acyltransferases↗
PubMed1995

Isolation of the Arabidopsis GA4 locus.

Progeny from a transgenic Arabidopsis plant generated by the Agrobacterium root transformation procedure were found to segregate for a gibberellin (GA)-responsive semidwarf phenotype. Complementation analysis with genetically characterized GA-responsive mutants revealed that the transgenic plant has an insertional mutation (ga4-2) that is an allele of the ga4 locus. The semidwarf phenotype of ga4-2 is inherited as a recessive mutation that cosegregates with both the T-DNA insert and the kanamycin resistance trait. DNA gel blot analysis indicated that the insertion site contains a complex T-DNA unit. A genomic library was constructed with DNA from the tagged ga4 mutant; a DNA clone was isolated from the library that flanks the T-DNA insert. The plant sequence isolated from this clone was used to isolate the corresponding full-length genomic and cDNA clones from wild-type libraries. DNA sequence comparison of the clones to the existing data bases suggests that they encode a hydroxylase. This conclusion is in agreement with a biochemical study that indicated that the ga4 mutant is deficient in 3 beta-hydroxylase in the GA biosynthetic pathway of Arabidopsis. RNA gel blot analysis showed that the message is ubiquitously expressed in different tissues of Arabidopsis but most abundantly in the silique. Unexpectedly, a higher level of transcription was detected in the ethyl methanesulfonate-induced ga4 mutant, and this overexpression was repressed by treatment with exogenous GA.

Alleles↗
PubMed1994

Growth hormone stimulates tyrosine phosphorylation of insulin receptor substrate-1.

Growth hormone (GH) produces insulin-like effects in rat adipocytes that have been deprived of GH for at least 3 h. The effect of a saturating concentration of GH is qualitatively and quantitatively similar to that produced by 2-4 ng/ml insulin but differs from that of insulin in the respect that adipocytes become refractory to prolonged or repeated stimulation with GH. Since activation of tyrosine kinase is an early event in the action of both hormones, we investigated the possibility that GH stimulation of tyrosine phosphorylation of some protein in the insulin transduction cascade might result in the similar effect of the two hormones. Adipocytes were preincubated for 3 h in the absence of hormones and then reincubated without or with 500 ng/ml GH or 4-400 ng/ml insulin for 10 min. The cells were lysed with an equal volume of buffer containing 1% SDS and preheated to 100 degrees C. Proteins were separated by electrophoresis on 7.5% polyacrylamide gels and transferred to nitrocellulose membranes, and tyrosine-phosphorylated proteins were detected using anti-phosphotyrosine antiserum coupled to horseradish peroxidase and reagents to produce chemiluminescence. The faint band seen at 185 kDa in control lanes was increased by GH treatment in five independent experiments. Insulin produced a similar effect at a concentration of 4 ng/ml, and phosphorylation increased in a dose-related manner in cells treated with higher concentrations of insulin. A prominent approximately 95-kDa band that is probably not the beta subunit of the insulin receptor was also seen in GH-treated cells. The beta subunit of the insulin receptor has similar electrophoretic mobility to the 95-kDa protein, but was not phosphorylated to an extent that allowed detection when insulin was added at concentrations below 400 ng/ml. Phosphorylation of the 185- and 95-kDa bands was evident within 1 min after addition of GH, persisted for at least 30 min, and was equally prominent in sensitive and refractory cells. Antiserum to IRS-1 immunoprecipitated the tyrosine-phosphorylated 185-kDa protein. The data suggest that IRS-1 is a substrate for a GH-activated tyrosine kinase, possibly JAK-2, which may account for the insulin-like effects of GH. The data further suggest that refractoriness to insulin-like stimulation by GH may result from an additional GH-dependent action that is distinct from phosphorylation of IRS-1.

Adipocytes↗
PubMed1994

Differential expression in Arabidopsis of Lhca2, a PSI cab gene.

A cDNA clone of the gene Lhca2 encoding a photosystem I (PSI) type II chlorophyll a/b-binding protein was isolated from Arabidopsis thaliana. The isolation of this, the fourth PSI cab gene from Arabidopsis, confirms a previous report [1] that indicated Arabidopsis may contain all four PSI cab genes identified in other plant species. Lhca2 is a single-copy gene as are the other known Arabidopsis PSI cab genes. The patterns of developmental expression and tissue-specific regulation of Lhca2 are similar to those of other PSI and PSII cab genes, but the light induction pattern and the steady-state mRNA level of Lhca2 are distinct. This suggests that a different mechanism may be employed to regulate the expression of Lhca2.

Amino Acid Sequence↗
PubMed1994

Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana.

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.

Arabidopsis↗
PubMed1994

Subcellular distribution of the long and short isoforms of the growth hormone (GH) receptor in rat adipocytes: both isoforms participate in specific binding of GH.

In rodents the GH receptor gene encodes long and short isoforms of the receptor that arise from alternate splicing of the mRNA transcript. To determine whether the long and short isoforms reside on the plasma membrane, we compared their sensitivity to mild digestion of adipocytes with trypsin. Immunofunctional assays were used to measure each isoform in adipocyte extracts. The GH-binding capacity of the short isoform in cell extracts was more than 5 times that of the long. Digestion of freshly isolated adipocytes with trypsin decreased the specific binding of [125I]human GH by 77%. The GH-binding capacities of the long and short isoforms in cell extracts were diminished by 68% and 18%, respectively. The relative insensitivity of the short isoform to digestion with trypsin suggests that much of it may be inaccessible to trypsin because it is located within the cell. When cells were allowed to recover for 2 h after digestion with trypsin, GH binding was restored. Analysis of cell extracts indicated net synthesis of the long isoform and further loss of the short. Adipocytes lost more than half of the short isoform during a 2-h incubation in vitro regardless of whether the cells were first digested with trypsin. Loss of the short isoform during this period appeared to be limited to the intracellular component, as the amount of the trypsin-sensitive component remained constant. The abundance of RNA transcripts that encode the long and short isoforms of the GH receptor remained unchanged when adipocytes were incubated in vitro for 3 h in the presence or absence of actinomycin-D. Therefore, the acute changes seen in these experiments do not reflect regulation of GH receptor gene transcription. Adipocytes appear to maintain a fixed ratio of the long and short isoforms of the GH receptor on their surface. Each isoform accounts for approximately half of the cell's GH-binding capacity.

Adipocytes↗
PubMed1994

Overexpression of the short form of the growth hormone receptor in 3T3-L1 mouse preadipocytes.

In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHRL) and a smaller isoform, GHRs that consists of the extracellular domain and a unique hydrophilic carboxyl terminus. We examined the hypothesis that GHRs may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHRs was ligated into the mammalian expression vector pcDNA-I/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects. Sixteen of 24 neomycin resistant clones secreted at least twice as much GHRs in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold. The amount of GHRL in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHRL. The transfected cDNA for GHRs directs the synthesis of a 50 kDa protein. Cross-linking of [125I]hGH to transfected 3T3-L1 cells indicated a 3.5-fold increase in a 72 kDa GHRs[125I]hGH complex. In the presence of 10% newborn calf serum, incorporation of [35S]methionine into cellular proteins was similar in transfected clones and control cells. Deprivation of serum for 2 hr decreased protein synthesis by approximately 70% in control cells, but in the transfected cells, protein synthesis was reduced only by approximately 50% or 30% in cells exhibiting 2x or 3x increases in GH binding. In all cells, 1 nM IGF-1 restored protein synthesis to the serum replete level. Similarly, although 3H-2-deoxy-D-glucose (2DG) uptake was comparable in all cells after 2 hr of serum deprivation, 18 hr of serum deprivation decreased uptake by approximately 70% in control cells, but only by approximately 30% in cells with increased GH binding. One nanomolar IGF-1 restored 2DG uptake to levels seen after 2 hr or serum deprivation. IGF-1 had no effect after only 2 hr of serum deprivation. Measurement of IGF-1 secreted into the medium, revealed that clones which overexpress GHRs produce greater amounts of IGF-1 than control cells or transfected clones that failed to overexpress GHRs. We conclude that GHRs contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHRs in 3T3-L1 cells altered cell function in the absence of GH.

3T3 Cells↗
PubMed1994

The significance of the short form of the growth hormone receptor in rat adipocytes.

An isoform of the growth hormone (GH) receptor that lacks the transmembrane and intracellular domains is formed in adipocytes by alternate splicing of mRNA. This isoform is identical to the circulating GH-binding protein. The short isoform is about six times as abundant as the long isoform in rat adipocytes. It is located largely in the cytosolic compartment in association with intracellular membranes, but about 20% is present on the adipocyte surface as judged by the susceptibility to digestion by trypsin. In contrast, about 80% of the long isoform of the receptor is present on the cell surface. The two isoforms turn over at different rates and appear to be independently regulated. Both appear to contribute equally to GH binding. In preliminary studies, immunoneutralization of the short isoform decreased the magnitude of the effect of GH on glucose metabolism, suggesting that the short isoform may mediate some of the responses to GH.

Adipocytes↗
PubMed1993

Molecular genetic evidence of a unifocal origin for human serous ovarian carcinomas.

The hypothesis that ovarian cancer is multifocal in origin was examined using molecular genetic techniques. Patterns of allelic deletion on chromosome 17 were studied in 16 informative cases of Stage III serous epithelial ovarian carcinoma. DNA was extracted from specimens collected from the omentum and both ovaries, and the specific alleles and chromosomal loci involved in the deletion were identified and compared. In all cases, the patterns of allelic deletion were identical for the tumors that had been collected from different sites in the same patients. In addition, 4 of the 16 cases were heterozygous for the hypoxanthine phosphoribosyl transferase (HPRT) gene on the X-chromosome and were examined for methylation status. In all 4, the same parental allele of the HPRT gene was methylated in tumor cells collected from both ovarian and omental sites, suggesting that the patterns of inactivation of the X-chromosome are identical. This pattern of allelic deletion and HPRT-gene methylation in tumor samples collected from different sites implies that ovarian carcinomas have a unifocal origin.

Alleles↗
PubMed1993

Characterization of an Arabidopsis thaliana gene (TMKL1) encoding a putative transmembrane protein with an unusual kinase-like domain.

We have characterized a novel Arabidopsis gene, designated TMKL1 (for Transmembrane Kinase-Like). The encoded protein is predicted to contain an N-terminal signal peptide, an extracellular domain with seven imperfect leucine-rich repeats, a single hydrophobic transmembrane segment, and a kinase-like intracellular domain which lacks, however, several of the key conserved residues essential for catalytic activity. The TMKL1 protein thus represents a unique and functionally intriguing member of the family of plant proteins related to animal receptor kinases. Northern blot analysis indicates that the TMKL1 gene is transcriptionally active in a variety of organs, and is developmentally regulated during silique maturation. Its possible functions are discussed.

Amino Acid Sequence↗
PubMed1993

An Arabidopsis gene homologous to mammalian and insect genes encoding the largest proteasome subunit.

A gene encoding a protein with extensive homology to the largest subunit of the multicatalytic proteinase complex (proteasome) has been identified in Arabidopsis thaliana. This gene, referred to as AtPSM30, is entirely encompassed within a previously characterized radiation-induced deletion, which may thus provide the first example of a proteasome null mutation in a higher eukaryote. However, the growth rate and fertility of Arabidopsis plants do not appear to be significantly affected by this mutation, even though disruption experiments in yeast have shown that most proteasome subunits are essential. Analysis of mRNA levels in developing seedlings and mature plants indicates that expression of AtPSM30 is differentially regulated during development and is slightly induced in response to stress, as has been observed for proteasome genes in yeast, Drosophila, and mammals. Southern blot analysis indicates that the Arabidopsis genome contains numerous sequences closely related to AtPSM30, consistent with recent reports of at least two other proteasome genes in Arabidopsis. A comparison of the deduced amino acid sequences for all proteasome genes reported to date suggests that multiple proteasome subunits evolved in eukaryotes prior to the divergence of plants and animals.

Amino Acid Sequence↗
PubMed1993

The origin of the bifunctional dihydrofolate reductase-thymidylate synthase isogenes of Arabidopsis thaliana.

In most prokaryotic and eukaryotic organisms dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are encoded by independent genes. Evidence is presented here that the higher plant Arabidopsis thaliana has two bifunctional DHFR-TS genes. The structure of the genes, DHFR at the amino terminus and TS at the carboxy terminus, is identical to their organization in protozoa, the only other known organisms with bifunctional genes. Sequence alignments suggest that the bifunctional genes from protozoa and higher plants may have different evolutionary origins. The positions of the introns support the complementary hypothesis that the DHFR domain of the bifunctional plant genes and the monofunctional DHFR gene of vertebrates derive from a common, intron-containing progenitor, although the structure (bifunctional or monofunctional) of the ancestral gene remains indeterminate. Comparison of the two bifunctional genes of Arabidopsis indicates that the DHFR and TS domains evolved at different rates; each following the evolutionary history of their monofunctional counterparts. In contrast to the DHFR domain, the evolution of the TS domain shows a higher level of nucleotide and amino acid sequence conservation, but a remarkable variability in the intron positions.

Amino Acid Sequence↗
PubMed1993

Effects of blue and red light on expression of nuclear genes encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana.

We have characterized the effects of different light spectra on expression of the nuclear genes (GapA and GapB) encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase in Arabidopsis thaliana. Steady-state mRNA levels for both genes in etiolated seedlings increased after a short exposure to red or blue light. However, these increases could not be reversed by immediate far-red light following the initial light treatment. In mature plants, a short light pulse, regardless of its spectrum, had no apparent effect on GapA or GapB mRNA levels in dark-adapted plants. In contrast, continuous exposure to red, blue, or white light resulted in increases of GapA and GapB mRNA levels, with blue and white light being far more efficient than red light. Similarly, continuous exposure of etiolated seedlings to red, blue, or white light also resulted in increased GapA and GapB mRNA levels. In addition, we show that illumination of red light-saturated Arabidopsis plants with continuous blue light results in further increases of GapA and GapB mRNA levels. Based on these results, we conclude that both blue light photoreceptor- and phytochrome-mediated pathways are involved in light regulation of GapA and GapB genes in Arabidopsis, with blue light acting as the dominant regulator.

Arabidopsis↗
PubMed1992

Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis.

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase.

Amino Acid Sequence↗
PubMed1992

Cloning, genetic mapping, and expression analysis of an Arabidopsis thaliana gene that encodes 1-aminocyclopropane-1-carboxylate synthase.

A genomic clone of one member of the Arabidopsis thaliana (L.) Heynh. 1-aminocyclopropane-1-carboxylate (ACC) synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) gene family (AT-ACC1) was isolated and sequenced. A region of homology was found in the 5'-untranslated region with the promoter of a zucchini and a tomato ACC synthase gene. Comparison of its primary structure with other ACC synthases revealed conservation of seven peptide regions as well as similarity with 11 amino acids of the catalytic site of aminotransferases. Genomic DNA gel blotting suggested the existence of an ACC synthase multigene family in Arabidopsis, possibly with three other members, none of which is very closely related to AT-ACC1. The existence of at least one other gene was confirmed by the isolation of a cDNA (AT-ACC2) from a flower-specific cDNA library. The AT-ACC1 gene was mapped on the Arabidopsis restriction fragment length polymorphism map and is located on the top of chromosome 1. This position does not correspond to any known mutation on the genetic map. Expression of the AT-ACC1 gene was studied by reverse transcription-PCR on total RNA. Messenger accumulation was strong in young leaves and flowers. The gene was not induced by wounding of young leaves or in seedlings in the presence of auxin. Ethylene exposure of mature plants led to an induction of AT-ACC1 gene expression. It is suggested that AT-ACC1 protein has a role in developmental control of ethylene synthesis.

Amino Acid Sequence↗