Structural characterization of the symbiotically important low-molecular-weight succinoglycan of Sinorhizobium meliloti.
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The production of succinoglycan by Sinorhizobium meliloti Rm1021 is required for successful nodule invasion by the bacterium of its host plant, alfalfa. Rm1021 produces succinoglycan, an acidic exopolysaccharide composed of an octasaccharide repeating unit modified with acetyl, succinyl, and pyruvyl moieties, in both low- and high-molecular-weight forms. Low-molecular-weight (LMW) succinoglycan, previously thought to consist of monomers, trimers, and tetramers of the repeating unit, has been reported as being capable of promoting the formation of nitrogen-fixing nodules by succinoglycan-deficient derivatives of strain Rm1021. We have determined that the three size classes of LMW succinoglycan species are in fact monomers, dimers, and trimers of the repeating unit and that the trimer is the species active in promoting nodule invasion. A detailed structural analysis of the components of LMW succinoglycan by using various chromatographic techniques, along with nuclear magnetic resonance analyses, has revealed that there is considerable heterogeneity within the LMW succinoglycan oligomers in terms of noncarbohydrate substitutions, and we have determined the structural basis of this heterogeneity. (+info)
Epitope identification for a panel of anti-Sinorhizobium meliloti monoclonal antibodies and application to the analysis of K antigens and lipopolysaccharides from bacteroids.
(34/896)
In two published reports using monoclonal antibodies (MAbs) generated against whole cells, Olsen et al. showed that strain-specific antigens on the surface of cultured cells of Sinorhizobium meliloti were diminished or absent in the endophytic cells (bacteroids) recovered from alfalfa nodules, whereas two common antigens were not affected by bacterial differentiation (P. Olsen, M. Collins, and W. Rice, Can. J. Microbiol. 38:506-509, 1992; P. Olsen, S. Wright, M. Collins, and W. Rice, Appl. Environ. Microbiol. 60:654-661, 1994). The nature of the antigens (i.e., the MAb epitopes), however, were not determined in those studies. For this report, the epitopes for five of the anti-S. meliloti MAbs were identified by polyacrylamide gel electrophoresis-immunoblot analyses of the polysaccharides extracted from S. meliloti and Sinorhizobium fredii. This showed that the strain-specific MAbs recognized K antigens, whereas the strain-cross-reactive MAbs recognized the lipopolysaccharide (LPS) core. The MAbs were then used in the analysis of the LPS and K antigens extracted from S. meliloti bacteroids, which had been recovered from the root nodules of alfalfa, and the results supported the findings of Olsen et al. The size range of the K antigens from bacteroids of S. meliloti NRG247 on polyacrylamide gels was altered, and the epitope was greatly diminished in abundance compared to those from the cultured cells, and no K antigens were detected in the S. meliloti NRG185 bacteroid extract. In contrast to the K antigens, the LPS core appeared to be similar in both cultured cells and bacteroids, although a higher proportion of the LPS fractionated into the organic phase during the phenol-water extraction of the bacteroid polysaccharides. Importantly, immunoblot analysis with an anti-LPS MAb showed that smooth LPS production was modified in the bacteroids. (+info)
Human 76p: A new member of the gamma-tubulin-associated protein family.
(35/896)
The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level. (+info)
Evaluation of berseem clover in diets of ruminants consuming corn crop residues.
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Berseem clover hay was compared to alfalfa hay provided at 0, 25, and 50% of the diet DM in mixtures with corn crop residues to wether lambs. Berseem clover hay had lower (P < .05) concentrations of NDF, ADF, and CP than alfalfa hay. The digestibility of DM, DMI, and N balance did not differ (P > .05) between diets containing alfalfa hay or berseem clover hay. To evaluate stockpiled berseem clover as a supplement for grazed corn crop residues, berseem clover and oats were incorporated into a corn-corn-oat/berseem clover crop rotation for 3 yr in replicated 6.1-ha fields. Two cuttings of oat-berseem clover hay were harvested each summer before forage was stockpiled for winter grazing. After corn grain harvest, multiparous and primiparous crossbred cows in midgestation were allotted to each field at 1.01 ha/cow to strip-graze corn crop residues with or without stockpiled berseem clover or allocated to replicated drylots for 98 to 140 d. Each group was offered alfalfa-grass hay as large bales to maintain a mean body condition score of 5 on a 9-point scale. Mean rates of total and digestible OM disappearance from grazed and ungrazed field areas of berseem clover and corn crop residues did not differ over the 3 yr. In vitro organic matter disappearance (IVOMD) tended to decrease more rapidly (P = .13) and NDF and ADF concentrations increased more rapidly (P < .05) in berseem clover than in corn crop residues. Seasonal BW change did not differ (P > .05) between winter management systems in any year, and seasonal body condition score changes did not differ (P > .05) between cows grazing corn crop residues and berseem clover and those maintained in a drylot in yr 2 and 3. Cows grazing corn crop residues with or without berseem clover required less (P < .05) hay than those maintained in drylot. Although the effects of berseem clover hay supplementation on the intake and digestibility of corn crop residues do not differ from alfalfa hay, the nutritional value of stockpiled berseem clover decreases rapidly during winter, limiting its value as a standing supplement for corn crop residues in late winter. (+info)
Meristem-localized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development in pea and alfalfa.
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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. (+info)
Proteins isolated from lucerne roots by affinity chromatography with sugars analogous to Nod factor moieties.
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Nod factors are important elicitors in legume-bacterium symbiosis. Any candidate plant receptor(s) for these lipo-oligosaccharides can be expected to show some lectin-like properties. A novel protein (P60), a native tetramer with 60 kDa monomers, has been isolated from a membrane fraction of Medicago sativa (lucerne, alfalfa) roots by using affinity chromatography with either GlcNAc or N,N', N"-triacetyl-(1-->4)-beta-d-chitotriose [(GlcNAc)(3)] grafted to agarose beads as the matrix and, in a second step, Sephadex G-200 gel filtration. With (GlcNAc)(3)-agarose an additional protein of 78 kDa was isolated. P60 showed haemagglutination activity with specificity for GalNAc, GalN, GlcNAc and GlcN. Binding experiments with radioactive GlcNAc gave a K(d) of 95 nM and one binding site per monomer of P60; Nod factor competed strongly for this binding. In native PAGE, protein incubated with O-sulphated Nod factors had a higher electrophoretic mobility as a consequence of binding. However, the largest modification was observed with a natural mixture of Nod factors, containing the O-acetylated and O-sulphated tetrasaccharidic NodRm-IV(Ac,S) (in which Ac stands for an O-acetylated group at the non-reducing end and S for O-sulphation at the reducing end) in addition to the non-O-acetylated NodRm-IV(S) (which alone had little effect) and NodRm-V(S). The native PAGE study was also performed with known lectins from other sources, but only the 34 kDa lectin of Phytolacca americana (pokeweed) showed any such interaction, although without discrimination between Nod factors. Finally, one peptide of each isolated protein was sequenced; the peptide from P60 showed some similarity with dihydrolipoamide dehydrogenase and ferric leghaemoglobin reductase, whereas the peptide from P78 was identical with an analogous region of 70 kDa heat shock protein. (+info)
The complete nucleotide sequence of apple mosaic virus (ApMV) RNA 1 and RNA 2: ApMV is more closely related to alfalfa mosaic virus than to other ilarviruses.
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The complete nucleotide sequences of apple mosaic virus RNA 1 and 2 have been characterized. Apple mosaic virus RNA 1 is 3476 nucleotides in length and encodes a single large open reading frame (ORF), whereas apple mosaic virus RNA 2 is 2979 nucleotides in length and also encodes a single ORF. The amino acid sequences encoded by RNA 1 and 2 show similarity to all of the other ilarviruses for which sequence data are available, but both are more closely related to alfalfa mosaic virus (AMV) than to other ilarviruses. Points of similarity include the absence of ORF 2b, present on the RNA 2 of all previously characterized ilarviruses. The close relationship to AMV also occurs in the movement protein, encoded by RNA 3, but not with the coat protein. These data suggest that the present taxonomy should be revised, and that AMV should be considered an aphid-transmissible ilarvirus. (+info)
Effects of a return chewing gum/packaging material mixture on in situ disappearance and on feed intake, nutrient digestibility, and ruminal characteristics of growing steers.
(40/896)
In situ and in vivo digestibility experiments were conducted to determine the acceptability, digestibility, and safety of a return chewing gum/packaging (G/P) material mixture when fed to steers. In the in situ experiment, both ruminal and intestinal disappearances were measured. Two ruminally and duodenally cannulated steers, which were given free access to alfalfa hay (AH), were used in this study. Duplicate Dacron bags containing the G/P were incubated in the rumen for 0, 3, 6, 12, 24, and 48 h. After ruminal incubation, the 12-, 24-, and 48-h bags were placed in the duodenum and collected in the feces to determine intestinal disappearance. In situ ruminal DM disappearance was greater than 70% for all substrates tested at 0 h, indicating high solubility of the substrates in water, and began to reach a plateau after 12 h of incubation. Intestinal in situ disappearance was not different (P>.25) from zero. In the digestion trial, four ruminally cannulated steers (337+/-21.3 kg BW; mean +/- SD) were used in a 4x4 Latin square design with the following treatments: 0) 50% corn (C), 50% AH; 10) 45% C, 45% AH, 10% G/P; 20) 40% C, 40% AH, 20% G/P; 30) 35% C, 35% AH, 30% G/P. Steers fed G/P-containing diets had greater (P<.01) DMI than the control steers. Increasing the G/P resulted in a linear (P<.05) increase in DMI. Apparent DM digestibility tended to be higher (P<.10) for the G/P-containing diets than for the control. A quadratic effect (P<.05) on digestible DMI was observed, with greater (P<.01) digestible DMI values for G/P-containing diets (4.8 vs. 5.8 kg/d). Digestible organic matter and total nonstructural carbohydrate intakes followed trends similar to those of DM. Apparent aluminum digestibility of G/P-containing diets was not different (P>.13) from zero. The level of G/P in the diet had no effect (P>.2) on total VFA concentration or ruminal pH. There was a linear decrease (P<.01) in the molar percentage of isobutyrate and isovalerate in addition to a linear increase (P<.01) in butyrate and valerate with increasing levels of G/P. There was a quadratic effect (P<.01) on molar proportions of acetate and propionate and on the acetate:propionate ratio. Results of both experiments suggest that G/P may be fed to safely replace up to 30% of corn-alfalfa hay diets for growing steers with advantages in improving DMI and digestibility. (+info)