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Biomedical subjects

A M Hirsch

Publications and source records attributed to A M Hirsch.

At least 19 recordsLinked to original sources
PubMed2001

Expression of MsLEC1- and MsLEC2-antisense genes in alfalfa plant lines causes severe embryogenic, developmental and reproductive abnormalities.

Although it has been proposed that plant lectins play a number of roles, the function of these proteins in normal plant growth and development has been unclear. To analyze the functions of putative alfalfa lectin genes, lines of transgenic alfalfa plants expressing approximately half of the open reading frame of MsLEC1 or MsLEC2, in the antisense or sense orientation, were established and analyzed. The antisense plants displayed severe abnormalities in embryogenesis, and both vegetative and reproductive development were perturbed. Some differences were observed between MsLEC1- and MsLEC2-antisense plants, and abnormalities were especially severe during the early stages of development in both the primary and secondary transgenic generations. In contrast, vector-control and sense-transgene plants exhibited normal growth and development. MsLEC1 and MsLEC2 mRNA accumulation levels were reduced in cognate antisense plants, especially during the later stages of embryogenesis, but also tended to be low in MsLEC1 sense-transgene plants. However, correlated with the phenotypic abnormalities observed in the MsLEC1-antisense plants was the specific reduction in the accumulation of a candidate MsLEC1 protein. Our results suggest that the MsLEC1 and MsLEC2 gene products, in addition to being important for embryogenesis, are required throughout alfalfa development.

Antisense Elements (Genetics)↗
PubMed2001

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae.

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.

Carbohydrate Metabolism↗
PubMed2000

Dg93, a nodule-abundant mRNA of Datisca glomerata with homology to a soybean early nodulin gene.

We have isolated a 590-bp full-length cDNA clone designated Dg93, an mRNA that is highly expressed in symbiotic root nodules of the actinorhizal host Datisca glomerata. Dg93 mRNA encodes a deduced polypeptide of 105 amino acids with significant identity (74%) to the soybean (Glycine max) early nodulin (ENOD) gene GmENOD93 (Kouchi and Hata, 1993). Dg93 mRNA is abundant in nodules at 4 weeks post inoculation, the earliest time assayed, and steady-state mRNA levels remain elevated 11 weeks after inoculation. Spatial patterns of Dg93 mRNA expression are complex, with transcript accumulation in the nodule lobe meristem, early infection zone, periderm, and cells of the vascular cylinder, but not in the surrounding uninfected cortical cells. Dg93 is encoded by a small gene family in D. glomerata. To our knowledge, this is the first report of a gene from an actinorhizal host that is expressed in the nodule meristem and that shares sequence homology with an early nodulin gene from a legume.

Amino Acid Sequence↗
PubMed2000

Oxygen regulation of a nodule-located carbonic anhydrase in alfalfa.

Control of the permeability to oxygen is critical for the function of symbiotic nitrogen fixation in legume nodules. The inner cortex (IC) seems to be a primary site for this regulation. In alfalfa (Medicago sativa) nodules, expression of the Msca1 gene encoding a carbonic anhydrase (CA) was previously found to be restricted to the IC. We have now raised antibodies against recombinant Msca1 protein and used them, together with antibodies raised against potato leaf CA, to demonstrate the presence of two forms of CA in mature nodules. Each antibody recognizes a different CA isoform in nodule tissues. Immunolocalization revealed that leaf-related CAs were localized primarily in the nitrogen-fixing zone, whereas the Msca1 protein was restricted exclusively to the IC region, in indeterminate and determinate nodules. In alfalfa nodules grown at various O(2) concentrations, an inverse correlation was observed between the external oxygen pressure and Msca1 protein content in the IC, the site of the putative diffusion barrier. Thus Msca1 is a molecular target of physiological processes occurring in the IC cells involved in gas exchange in the nodule.

Blotting, Western↗
PubMed1999

Role of lectins (and rhizobial exopolysaccharides) in legume nodulation.

The lectin recognition hypothesis proposes that plant lectins mediate specificity in the Rhizobium-legume symbiosis. Although the hypothesis was developed eight years before nod genes were identified in rhizobia and sixteen years before Nod factor was shown to be a major determinant of host specificity, experiments performed recently using transgenic lectin plants support its main tenets.

Fabaceae↗
PubMed1999

Meristem-localized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development in pea and alfalfa.

PsUGT1, which encodes a microsomal UDP-glucuronosyltransferase, was cloned from root tips of Pisum sativum. PsUGT1 expression is correlated with mitosis and strongly induced in dividing cells. A region at the C terminus of the encoded protein is closely related to the UDP-glucuronic acid binding site consensus sequence, and the protein encoded by PsUGT1 catalyzes conjugation of UDP-glucuronic acid to an unknown compound. Overexpression of PsUGT1 sense mRNA has no detectable effect on transgenic pea hairy root cultures or regenerated alfalfa. However, inhibiting PsUGT1 expression by the constitutive expression of antisense mRNA (under the control of the cauliflower mosaic virus 35S promoter) markedly retards growth and development of transgenic alfalfa. Cell structure and organization in the antisense plants are similar to those of controls, but plant growth is reduced and development is delayed. This inhibition in growth is correlated with a twofold delay in the time required for completion of a cell cycle and with a >99% inhibition of border cell production. Inhibition of PsUGT1 expression by meristem-localized inducible expression of PsUGT1 antisense mRNA (under the control of its own promoter) is lethal both in pea hairy roots and in transgenic alfalfa plants. These results indicate that PsUGT1 expression is required for normal plant growth and development, and they are consistent with the hypothesis that this UDP-glycosyltransferase regulates activity of a ligand(s) needed for cell division.

Amino Acid Sequence↗
PubMed1998

Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in transgenic alfalfa.

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40-1 and MsENOD40-2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40-1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40-2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40-1 promoter construct, whereas pseudonodules from plants containing the MsENOD40-2 promoter construct stained blue. A 616-bp region at the distal 5' end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.

Bacterial Proteins↗
PubMed1997

Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved.

Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses-nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

Journal Article↗
PubMed1997

The ribosomal protein P0 of soybean (Glycine max L. Merr.) has antigenic cross-reactivity to soybean seed lectin.

Soybean (Glycine max L. Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le-. A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le- soybean cultivars. Earlier work suggested that this protein was a novel lectin. Clones isolated from a Le- soybean root cDNA library produced a cross-reacting protein of the same size in Escherichia coli. Sequence analysis of these clones revealed a high degree of similarity to the ribosomal protein P0. The cross-reacting protein co-purified with ribosomes, and a monoclonal antibody raised to purified brine shrimp P0 cross-reacted to the same protein. The protein showed no lectin activity in a hemagglutination assay, nor did it bind to an N-acetyl-D-galactosamine affinity column. On the basis of this evidence, we conclude that the SBL-cross-reacting protein is not a lectin but a homologue of the ribosomal protein P0. Consequently, Le- soybeans must produce a lectin that is dissimilar to SBL at both the DNA and amino acid levels and we suggest that it is this lectin which is involved in nodulation.

Amino Acid Sequence↗
PubMed1996

Analysis of partial sequences of genes coding for 16S rRNA of actinomycetes isolated from Casuarina equisetifolia nodules in Mexico.

Filamentous bacteria isolated from surface-sterilized nodules of Casuarina equisetifolia trees in México were capable of reducing acetylene, a diagnostic test for nitrogenase, but were unable to nodulate their host. Analysis of partial 16S rRNA gene sequences suggests that the Mexican isolates are not Frankia strains but members of a novel clade.

Actinomycetales↗
PubMed1996

Unilateral pulmonary artery thrombotic occlusion: is distal arteriopathy a consequence?

The characteristics and postoperative outcomes of a unique group of eleven patients with total unilateral pulmonary artery (PA) thromboembolic occlusion were compared with those of some 400 patients who underwent bilateral thromboendarterectomies during the same time period. Preoperative historical, physical, and laboratory features and postoperative outcomes of these two groups were analyzed. The unilateral group had no distinct historical features. However, they were younger (32 +/- 10 yr) than the bilateral group (51 +/- 15 yr), dominantly female (10 of 11) versus a male predominance (62%) in the bilateral group; had significantly lower preoperative PA mean pressures (30 +/- 12 versus 46 +/- 12 mm Hg) and calculated pulmonary vascular resistance (360 +/- 293 versus 901 +/- 467 dynes/s/cm-5); and small lung by chest radiograph was common (8 of 11). Postoperatively, four unilateral patients developed unilateral rethrombosis (two immediate; two late, at several years postsurgery); this occurred in only one bilateral patient. Furthermore, of six patients with unilateral occlusion present more than 1 yr, reperfusion was poor in four despite an adequate thromboendarterectomy in all. Postoperative pulmonary angiograms in two of these disclosed apparent atrophy of central and distal pulmonary arteries. Prior animal investigation models indicate that unilateral PA occlusion is followed by development of a postobstructive arteriopathy in the resistance arteries of the occluded lung. The unusual outcomes in these 11 patients suggest that they may develop a similar arteriopathy which requires special management considerations at surgery and postoperatively.

Adult↗
PubMed1995

Assessing horizontal transfer of nifHDK genes in eubacteria: nucleotide sequence of nifK from Frankia strain HFPCcI3.

The structural genes for nitrogenase, nifK, nifD, and nifH, are crucial for nitrogen fixation. Previous phylogenetic analysis of the amino acid sequence of nifH suggested that this gene had been horizontally transferred from a proteobacterium to the gram-positive/cyanobacterial clade, although the confounding effects of paralogous comparisons made interpretation of the data difficult. An additional test of nif gene horizontal transfer using nifD was made, but the NifD phylogeny lacked resolution. Here nif gene phylogeny is addressed with a phylogenetic analysis of a third and longer nif gene, nifK. As part of the study, the nifK gene of the key taxon Frankia was sequenced. Parsimony and some distance analyses of the nifK amino acid sequences provide support for vertical descent of nifK, but other distance trees provide support for the lateral transfer of the gene. Bootstrap support was found for both hypotheses in all trees; the nifK data do not definitively favor one or the other hypothesis. A parsimony analysis of NifH provides support for horizontal transfer in accord with previous reports, although bootstrap analysis also shows some support for vertical descent of the orthologous nifH genes. A wider sampling of taxa and more sophisticated methods of phylogenetic inference are needed to understand the evolution of nif genes. The nif genes may also be powerful phylogenetic tools. If nifK evolved by vertical descent, it provides strong evidence that the cyanobacteria and proteobacteria are sister groups to the exclusion of the firmicutes, whereas 16S rRNA sequences are unable to resolve the relationships of these three major eubacterial lineages.

Amino Acid Sequence↗
PubMed1994

Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcript levels occur in young roots and root tips.

Flavonoids are involved in several different interactions between plants and microorganisms. In the Rhizobium-legume symbiosis, they play an important role as inducers of rhizobial nodulation (nod) genes. We have identified from an alfalfa cDNA library four clones for chalcone synthase (CHS) and two clones for chalcone isomerase (CHI); CHS and CHI are key enzymes in flavonoid biosynthesis. In Medicago sp., CHS is encoded by 8-12 genes, and CHI is encoded by 1-2 genes. Here we report the DNA sequence of these clones as well as their relatedness to other legume CHS and CHI clones. In addition, we report on the expression patterns of two CHS gene family members as well as the CHI gene in M. sativa cv. Iroquois. While CHS and CHI transcript levels are high in root tips and entire young roots, they are low in effective nodules elicited by wild-type strains of Rhizobium meliloti and very low in aerial portions of the plant (stems, leaves, flowers). However, wounding the cotyledons results in a rapid increase in transcript levels of both chalcone synthase and chalcone isomerase genes in these organs.

Acyltransferases↗