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Biomedical subjects

H M Goodman

Publications and source records attributed to H M Goodman.

At least 19 recordsLinked to original sources
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↗
PubMed1992

The role of cytoreductive surgery in the management of stage IV epithelial ovarian carcinoma.

Patients with Stage IV epithelial ovarian carcinoma are generally treated in the same manner as are patients with disease confined to the abdomen--cytoreductive surgery followed by combination chemotherapy. Between 1980 and 1990, 35 women with histologically or cytologically documented Stage IV ovarian carcinoma were treated in this fashion. Sixteen women (45%) underwent optimal initial cytoreductive surgery, defined as less than 2 cm maximum residual disease. Eleven of the 19 women undergoing suboptimal initial procedures underwent interval cytoreduction after two to four cycles of chemotherapy, with 7 achieving an optimal status after the interval procedure. Overall, 23 of 35 patients (66%) were successfully cytoreduced to less than 2 cm either initially or at an interval procedure. Thirty-one of the 35 patients received combination regimens containing platinum as part of their initial therapy. Kaplan-Meier survival curves demonstrated no significant difference in survival between those groups of women cytoreduced intervally or initially, or between those groups of women optimally cytoreduced at some point during their initial therapy and those who were not. The 5-year survival for the entire group was less than 5%, with no significantly prolonged survival seen in those patients undergoing successful cytoreduction.

Chemotherapy, Adjuvant↗
PubMed1992

Isolation of the Arabidopsis ABI3 gene by positional cloning.

Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.

Amino Acid Sequence↗
PubMed1992

Expression of antisense or sense RNA of an ankyrin repeat-containing gene blocks chloroplast differentiation in arabidopsis.

The Arabidopsis AKR gene that encodes a protein with four ankyrin repeats (a 33-amino acid motif that appears in the 89K domain of the human protein ankyrin) was isolated and characterized. A short sequence outside the ankyrin repeats is similar to that of the protein of the Drosophila muscle segment homeobox (msh) gene. The expression of the AKR gene is light dependent, and transgenic Arabidopsis plants with two or more copies of an antisense or sense AKR construct became chlorotic in a developmentally regulated manner. The chlorotic phenotype was genetically transmitted to the next generation, although most chlorotic plants produced much less seed. Reduced presence of thylakoid membranes and loss of grana are found in the plastids of chlorotic leaves, indicating that antisense or sense AKR has blocked chloroplast differentiation. This study indicates the importance of ankyrin repeat-containing proteins, not only in yeast and animals, but in plants as well.

Amino Acid Sequence↗
PubMed1992

Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations.

Ionizing radiation is known to cause chromosomal alterations such as inversions and deletions and has been used extensively for inducing mutations. In Arabidopsis, two methods for the isolation of genes identified on the basis of mutant phenotypes--genomic subtraction and chromosome walking--either rely on or are greatly facilitated by the availability of these types of mutations. This article gives a detailed characterization of ionizing radiation-induced mutations in plants. The Arabidopsis genes encoding chalcone flavanone isomerase (CHI) and dihydroflavonol 4-reductase (DFR) were cloned and found to correspond to two transparent testa loci. A CHI allele, generated by fast-neutron irradiation, consisted of an inversion within the gene. A 272-bp fragment from 38 centimorgans away on the same chromosome was transferred to one end of this inversion. A DFR allele, induced by x-irradiation, contained two deletions and an inversion of the 2.8-centimorgan intervening region. Sequence analysis of the break points in both mutants indicate that repair of radiation-induced damage involves mechanisms similar or identical to those that mediate the integration of foreign sequences into the genome. The chromosome rearrangements found in these mutants have important implications for the use of ionizing radiation-induced alleles in classical and molecular genetic experiments in plants.

Alcohol Oxidoreductases↗
PubMed1992

A growth hormone (GH) analog can antagonize the ability of native GH to promote differentiation of 3T3-F442A preadipocytes and stimulate insulin-like and lipolytic activities in primary rat adipocytes.

The effect of amino acid substitutions introduced to the third alpha-helix in bovine GH (bGH) was investigated. A GH analog (bGH-M8), in which three amino acids were substituted to form an idealized amphiphilic alpha-helix, possessed the same specific binding affinity as wild-type bGH to cell membranes prepared from 3T3-F442A cells or rat adipocytes. However, bGH-M8 failed to stimulate preadipocyte differentiation, as measured by the level of glycerol-3-phosphate dehydrogenase activity. An equimolar concentration of bGH-M8 was inhibitory for this adipogenic effect caused by bGH at a concentration of 30 pM. bGH-M8 also failed to induce an insulin-like response and reduced lipolytic potency in rat primary adipocytes. A 10-fold excess of bGH-M8 abolished the effect of wild-type bGH in the insulin-like and lipolytic assays. Thus, bGH-M8 inhibited these actions of wild-type bGH and, therefore, appears to be a competitive antagonist. These results suggest that a major biologically active domain resides in the third alpha-helix of bGH, which is independent of amino acids important in the initial interaction of GH with its receptor.

3T3 Cells↗
PubMed1992

Growth hormone increases intracellular free calcium in rat adipocytes: correlation with actions on carbohydrate metabolism.

Adipocytes that have been preincubated for 3 h or more in hormone-free medium respond to GH with a transient insulin-like increase in glucose metabolism, followed by a period of refractoriness to further insulin-like stimulation. Adipocytes freshly isolated from normal rats and adipocytes that were exposed to GH in the first hour of a 4-h incubation period are refractory to this insulin-like effect. Because earlier studies revealed a relationship between refractoriness and cellular calcium, we examined the effects of GH on the intracellular calcium concentration ([Ca2+]i) under a variety of conditions in which sensitivity to insulin-like stimulation or refractoriness is known to be affected. A dual nitrogen laser imaging microscope with computer-assisted image processing to measure fluorescence changes in individual adipocytes loaded with fura-2 AM was used to measure [Ca2+]i. After prolonged incubation in vitro, resting [Ca+2]i was 120 +/- 6 nM and remained unchanged for more than 1 h after the addition of 100 ng/ml human GH (hGH), which doubled the rate of incorporation of [3-3H] glucose into triglycerides in this interval. Lipogenesis declined in the second hour, and [Ca+2]i slowly increased, reaching 324 +/- 49 nM by the end of the third hour (P less than 0.05). When added with GH, actinomycin-D, an inhibitor of RNA synthesis, caused the accelerated rate of lipogenesis to persist for at least 5 h and blocked the delayed increase in resting [Ca+2]i. Resting [Ca2+]i in freshly isolated, and hence refractory, adipocytes was 342 +/- 34 nM and declined to 112 +/- 11 nM concomitant with acquisition of insulin-like sensitivity to GH. The addition of 100 ng/ml hGH to these cells at the beginning of incubation, under conditions known to sustain refractoriness prevented the decline in resting [Ca2+]i and enabled them to exhibit a further acute increase in [Ca2+]i in response to a second exposure to hGH in the fourth hour. When added 60 min after GH, actinomycin-D blocked the ability to raise [Ca2+]i acutely in response to GH, but did not interfere with GH's action to sustain resting [Ca2+]i, which remained at about 300 nM. The concentration of GH needed to increase [Ca2+]i acutely in refractory cells is about 6-fold higher than that needed to maintain resting [Ca+2]i. Differences in the time of sensitivity to actinomycin-D and dose dependency suggest that sustaining resting [Ca2+]i and production of the acute increase in [Ca2+]i are separate phenomena.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1992

Characterization of the short isoform of the growth hormone receptor synthesized by rat adipocytes.

Two mRNA transcripts that are believed to be alternately spliced products of the GH receptor gene have been reported in a variety of rat tissues. The smaller (1.2 kilobases) transcript was cloned from an adipocyte library, sequenced, and found to encode a protein identical to the soluble GH-binding protein (GHBP) in plasma. An assay that is specific for the short isoform of the GH receptor, often referred to as the GHBP, has been developed using a rabbit antiserum that recognizes the unique amino acid sequence at its carboxyl end. The assay depends upon immunoprecipitation of a complex consisting of [125I]human GH, the binding protein, antiserum, and protein-A cross-linked to agarose beads. To validate the assay, samples of rat plasma were analyzed and found to contain sufficient binding protein to bind 1.46 pmol (32 ng) GH/ml, with an affinity of 2.7 x 10(9) M-1. In adipocyte extracts, binding protein activity was sufficient to bind 61 fmol GH/g tissue, with an affinity of 2.3 x 10(9) M-1. The binding protein was found primarily in the particulate fraction of adipocytes, and it is estimated that adipocytes contain approximately 7000 copies of the binding protein/cell. Only 10% of the binding activity was present in the high speed supernatant of adipocyte homogenates, and soluble binding protein did not appear to be released into the incubation medium when adipocytes were incubated in vitro. A 50-kilodalton (kDa) 35S-labeled protein that may be a glycosylated form of the binding protein was immunoprecipitated from both the soluble and particulate fractions of adipocyte extracts by the antiserum, and addition of the synthetic peptide antigen blocked immunoprecipitation of this protein. A 150-kDa protein in the high speed supernatant fraction was also specifically immunoprecipitated by the antiserum. Although it is unlikely to be a glycosylated form of the binding protein, it may cross-react with the antiserum or perhaps be coprecipitated, because it interacts with the binding protein. In addition, 38- and 42-kDa bands were specifically immunoprecipitated from the detergent-treated particulate fraction of adipocyte extracts that were enriched for the binding protein by adsorption to immobilized GH. We conclude that 1) adipocytes synthesize the short isoform of the GH receptor, and that this protein is primarily associated with a membrane fraction of the cells; and 2) the GHBP expressed in adipocytes is not released into the incubation medium and differs in size from the GHBPs in rat plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1991

Cloning and chromosomal mapping of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate-dehydrogenase from Arabidopsis thaliana.

Both cDNA and genomic clones for the nuclear genes encoding chloroplast (cp) (gapA and gapB) and cytosolic (gapC) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana have been isolated and characterized. Genomic Southern-blot analyses indicate that there is only one copy of each gapA, gapB and gapC gene in A. thaliana. Comparison of the deduced amino acid (aa) sequences shows that the A and B subunits are highly similar (80% positional aa identity), while there is less similarity between the cp and cytosolic subunits (45% aa identity). These relationships are consistent with the idea that the cp and cytosolic GAPDHs evolved from different lineages, as suggested in our previous study of tobacco GAPDHs [Shih et al., Cell 47 (1986) 73-80]. In addition, the chromosomal locations for the three gap genes were determined by restriction fragment length polymorphism mapping; the three gap genes are not closely linked, gapA (55.8 cM) and gapC (0.0 cM) are on chromosome 3, and gapB (51.3 cM) is on chromosome 1.

Amino Acid Sequence↗
PubMed1991

Refractoriness to growth hormone is associated with increased intracellular calcium in rat adipocytes.

In adipocytes that have been deprived of growth hormone (GH) for at least 3 hr, GH elicits a transient insulin-like response that is followed by a period of refractoriness to further insulin-like stimulation. Exposure of adipocytes to GH in the first hour of a 3-hr incubation prevents the appearance of insulin-like sensitivity. Intracellular Ca2+ concentration [( Ca2+]i) was measured in individual adipocytes that were loaded with fura-2 hexakis(acetoxymethyl) ester after preincubation in the presence (refractory) or absence (sensitive) of recombinant human GH at 100 ng/ml. Using a dual nitrogen laser imaging microscope with computer-assisted image processing to measure fluorescence changes, we observed that resting [Ca2+]i was 220 +/- 10 nM in refractory adipocytes and 110 +/- 6 nM in sensitive adipocytes (P less than 0.001). GH had no acute effect on [Ca2+]i in sensitive adipocytes but caused a sustained 3-fold increase in [Ca2+]i in refractory cells within 3 min (P less than 0.001). Insulin did not change [Ca2+]i in either sensitive or refractory adipocytes. In refractory cells treated with insulin and GH simultaneously, insulin completely blocked the rise in [Ca2+]i due to GH. Oxytocin elicited a prompt increase in [Ca2+]i followed by a quick return to resting levels in both sensitive and refractory cells. These findings indicate that basal [Ca2+]i is increased in refractory cells and that GH produces a sustained rise in [Ca2+]i only in refractory adipocytes. We suggest that the sustained increase in [Ca2+]i produced by GH in refractory cells prevents the expression of the insulin-like response.

Adipose Tissue↗
PubMed1991

The centromere region of Arabidopsis thaliana chromosome 1 contains telomere-similar sequences.

We describe the structure of an Arabidopsis thaliana genomic clone containing two classes of repetitive DNA elements derived from the centromere region of chromosome 1. One class is comprised of tandem arrays of a highly reiterated repeat containing degenerate telomere sequence motifs. Adjacent to these telomere-similar repeats we found a dispersed repetitive element reiterated approximately five times in the A. thaliana genome. The nucleotide sequence of the dispersed repeat is unusual, being extremely AT-rich and composed of numerous, overlapping repeat motifs.

Base Sequence↗
PubMed1991

Functional analysis of the pathogenesis-related 1a protein gene minimal promoter region. Comparison of reporter gene expression in transient and in stable transfections.

Pathogenesis-related (PR) proteins are a heterogeneous group of host encoded, low-molecular-mass proteins that are induced in plants by various external stimuli, such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5'-flanking region of the tobacco PR-1a gene [Pfitzner U.M., Pfitzner, A.J.P. & Goodman, H.M. (1988) Mol. Gen. Genet. 211, 290-295] was joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene. Expression of the reporter gene was monitored in transient expression assays as well as in stable transformants. The PR-1a 5'-flanking sequences from -335, -149 or -71 to +28 are functional promoter elements in tobacco and carrot protoplasts, as determined by transient expression. These constructs direct correct initiation at the normal transcription-start site of the PR-1a gene. The level of gene expression was about twofold less than that obtained with the cauliflower mosaic virus 35S RNA promoter. Regulation of gene expression by acetylsalicylic acid, however, could not be detected in the transient assays. When the same constructs were stably integrated into the tobacco genome, neither constitutive nor induced beta-glucuronidase activity was observed. A comparison of the results from the transient and the stable transfection experiments suggests that expression of the reporter gene may be due to a constitutive transcriptional activity of the PR-1a 5'-flanking regions under the conditions of the transient assays and that the PR-1a promoter may contain at least two functional domains.

Aspirin↗
PubMed1991

Molecular analysis of two PR-1 pseudogenes from tobacco.

Two independent PR-1 lambda genomic clones (W38/1 and W38/3) were isolated and characterized from a tobacco (Nicotiana tabacum cv. Wisconsin 38) library. Neither clone is identical to the previously described PR-1 cDNA clones, and both clones carry mutations within the highly conserved PR-1 protein coding region. For example, clone W38/1 has a GAA Glu codon instead of the translation stop codon thus harbouring an open reading frame extended by 16 additional amino acids. Furthermore, both clones display considerable variations in the genomic flanking sequences when compared to the PR-1a gene. In order to test whether the encoded genes are active, their upstream sequences were fused to the E. coli beta-glucuronidase (GUS) reporter gene. While significant GUS activities as compared to the 35S RNA promoter from cauliflower mosaic virus (CaMV) were obtained with the W38/1 and W38/3 sequences in transient gene expression assays, no transcriptional activities could be observed upon stable transformation of the same constructs. In addition, the protein coding region of W38/1 was joined to the CaMV 35S RNA promoter and transgenic tobacco plants were generated. However, neither transcripts nor a protein could be detected deriving from the W38/1 structural gene with this chimaeric construct in the transformants. Taken together, these data indicate that the genes contained in lambda clones W38/1 and W38/3 are not active in planta.

Base Sequence↗