Organization and expression of leghaemoglobin genes

Leghaemoglobin genes in soybean (Glycine max) are present as a moderately reiterated family of sequences. Since there are identical restriction site patterns of these sequences in DNA isolated from leaf, root, or nodule tissue, the data suggest that no major changes in the organization or methylation of leghaemoglobin genes occur during their induction. Cloned soybean leghaemoglobin-cDNA cross hybridized with RNA from root nodules of kidney bean (Phaseolus vulgaris), and to a lesser extent, of pea (Pisum sativum) indicating sequence homology in the leghaemoglobin genes of these species. Hybridization to the genomic DNA restriction fragments of two other species, Glycine soja and Vicia faba, also indicated interspecies sequence homologies. Several restriction fragments appear to be common to all species examined. The induction of these genes occurs following infection of the plant by Rhizobium and is independent of the appearance of nitrogenase activity in the nodules. The level of expression is, however, influenced by various mutations in Rhizobium that result in the development of ineffective (nonnitrogen fixing) nodules.     

Production of cloned cDNA from a Swedish barley yellow dwarf virus isolate

A cDNA library was produced from the RNA of a Swedish MAV-like isolate of barley yellow dwarf virus (BYDV). The procedure involved random priming and the ds cDNA was cloned into the EcoRl site of the plasmid pUC19. Among the clones obtained some hybridised specifically with MAV-like isolates whereas others also hybridised with PAV-like isolates. Only very weak hybridisation was observed with an RPV-like isolate. An Australian cDNA clone, reported to be PAV-specific (pBY82, Waterhouse, Gerlach & Miller, 1986), hybridised with Swedish MAV-like but not with PAV-like isolates. Probes prepared from the clones detected virus in plant extracts by dot-blot hybridisation with sensitivity greater than that of ELISA. Virus was also readily detected in extracts of viruliferous aphids.     

Characterization of nodule-specific cDNA clones from Sesbania rostrata and expression of the corresponding genes during the initial stages of stem nodules and root nodules formation

We have constructed a Sesbania rostrata stem nodule-specific cDNA library. By screening with heterologous probes from pea and soybean, we have isolated several nodulin cDNA clones. On the basis of nucleotide and amino acid sequence homology, two nearly full-length cDNA clones coding for two different leghemoglobin-like proteins have been identified. The inserts of two other clones reveal a high degree of amino acid sequence homology (81% and 72%) to the early nodulin Enod2 from soybean; the characteristic heptapeptide repeat units PPHEKPP and PPYEKPP of the soybean Enod2 are conserved in the proteins encoded by these Sesbania cDNA clones. The time course of Enod2 and leghemoglobin mRNA appearance during the formation of stem nodules and root nodules on S. rostrata was analyzed by northern blot hybridization. Significant differences were found for the initiation of mRNA accumulation of these nodulins between S. rostrata and soybean.     

Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans

Summary A cDNA clone (pcPvNGS-01) to glutamine synthetase (GS) mRNA from root nodules of Phaseolus vulgaris showed cross-hybridization to GS and mRNA from soybean root nodules, thus allowing its use as a probe to study the expression of GS genes during root nodule development in soybeans. Hybrid-select translation of root and nodule RNA of soybean with DNA from pcPvNGS-01, followed by 2D gel electrophoresis, showed six peptides in the root and an additional four peptides in the nodule which represent nodule-specific glutamine synthetase (GS_n) gene products. The GS_n gene products appeared for the first time between day 11 and 12 after infection, either concomitant with the onset of nitrogenase activity or immediately following it. The levels of expression of the GS_n and leghemoglobin genes were not affected in young Fix^- nodules formed by Bradyrhizobium japonicum strains that are defective in nitrogenase activity, suggesting that the induction of these two sets of host genes take place independent of nitrogenase activity. However, in Fix^- nodules that are incapable of maintaining the peribacteroid membrane, GS_n gene products were not detected while 1ba, 1bc₂ and 1bc₃ appeared. In both the timing of appearance during root nodule development and the effect of different bacterial mutations on the expression, GS_n genes differ from most other nodulin genes examined (30), suggesting different regulatory mechanisms.     

cDNA cloning and developmental expression of pea nodulin genes

A cDNA library prepared from pea nodule poly(A)⁺ RNA was screened by differential hybridization with cDNA probes synthesized from root and nodule RNA respectively. From the cDNA clones that hybridized exclusively with the nodule probe five clones, designated pPsNod 6, 10, 11, 13 and 14 and each containing unique sequences, were further characterized together with one leghemoglobin and one root-specific cDNA clone. In vitro translation of RNA selected by the pPsNod clones showed that the corresponding genes encode nodulins with molecular weights ranging from 5 800 to 19 000. During pea root nodule development expression of the five PsNod genes starts more or less concomitantly with the onset of nitrogen fixing activity in the nodules and the time course of appearance and accumulation of the nodulin mRNAs is similar to that of leghemoglobin mRNA. In ineffective pea root nodules expression of the PsNod genes is induced but the final accumulation levels of the mRNAs are markedly reduced to various degrees. The expression of another nodulin gene, designated ENOD2, was followed using a heterologous soybean cDNA clone as probe. In pea root nodules the ENOD2 gene is expressed at least five days before the PsNod and leghemoglobin genes, and in contrast to the PsNod mRNAs the concentration of the ENOD2 mRNA is the same in wild type and fix ^- nodules. The results described suggest that in root nodules several regulatory mechanisms exist which determine the final nodulin mRNA amounts accumulating in the root nodule.     

从吸收光谱的变化看H2O2对豆血红蛋白的定位损伤

豆血红蛋白（Ｌｂ）是一种含铁的蛋白质,在大豆（Ｇｌｙｃｉｎｅｍａｘ）的根瘤中含量很。从新鲜根瘤中提取制备的氧合豆血红蛋白（ＬｂＯ２,含二价铁的蛋白质）是Ｌｂ的活性形式。ＬｂＯ２在可见光区５７７ｎｍ和５４０ｎｍ有吸收峰,这两个峰与ＬｂＯ２中血红素（铁卟啉）的结构密切相关;另外,ＬｂＯ２在紫外区２８０ｎｍ处还有一个吸收峰,此峰与珠蛋白（ＬｂＯ２的蛋白质部分）的构象有关．ＬｂＯ２在一定浓度的Ｈ２Ｏ２作用下,可见光的特异吸收变化迅速,而紫外吸收相对稳定,因而推测Ｈ２Ｏ２对ＬｂＯ２的损伤是有着固定的位点,这个位点是在含二价铁的卟啉环上,而不在珠蛋白中．     

Analysis and in situ detection of cholecalcin messenger RNA (9000 Mr CaBP) in the uterus of the pregnant rat

Summary The molecular cloning of a cDNA fragment synthesised from rat duodenal mRNA coding for cholecalcin (calbindin), a 9000 Mr vitamin D-induced calcium-binding protein (CaBP), has been previously described. DNA/RNA hybridisation assays have been used to examine CaBP mRNA production in the uterine horns and duodena of pregnant (21 day) rats using the cloned CaBP cDNA. Northern hybridisation studies showed that the ³²P cDNA sequence hybridised to a single 500–600 nucleotide species in both the uterus and the duodenum, thus demonstrating identical CaBP mRNA processing in both tissues. Dot blot hybridisation studies showed that the CaBP mRNA concentration was greatest in the duodenum while that of the uterine horns was about 10% of the duodenal level. The observed differences in CaBP mRNA levels correlate well with the in vivo CaBP concentrations. In situ hybridisation histochemistry using ³H cDNA revealed that CaBP mRNA visualised by silver grains was found in all the parts of the endometrium and the myometrium. However, CaBP mRNA was more concentrated in the outer and inner muscular fibres and in the luminal cells of the endometrium than in the stroma cells. These results demonstrate that the CaBP gene is expressed in specific cells of the rat uterus.     

Distribution of root mRNA species in other vegetative organs of pea (Pisum sativum L.)

Summary A cDNA library was prepared from, poly(A)⁺ RNA from roots of pea (Pisum sativum L.). Twenty five clones were selected by use of random numbers and used as probes on Northern blots to analyse the distribution of their corresponding mRNA species in other vegetative pea organs: leaf, stem and developing cotyledon. Fifteen cDNA inserts hybridised to single mRNA species, five hybridised to two mRNA species and one hybridised to five homologous mRNAs. Four cDNA clones (16% of those selected) gave no hybridization signals, indicating that the steady state levels of mRNAs were below the detection limit (i.e.less than 2.5 x 10^-5% of poly(A)⁺ RNA). Most of the root mRNAs were represented in all four pea organs as sequences of low and medium abundance. All but two cDNAs encoded mRNA species enhanced in root. However, cDNA clones appeared not to encode mRNA species expressed in a strictly organ-specific manner, as no mRNA unique to root was found. Thus, if organ-unique mRNA species are present, they are only present at a very low level of abundance in the poly(A)⁺RNA population.     

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N₂ fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.     

Control of leghaemoglobin synthesis in snake beans

1. The finding that the plant is the genetic determinant of leghaemoglobin production in legume nodules was further tested by inoculating snake beans with two strains of Rhizobium selected to give large genetic differences. Carbohydrate requirement patterns, immunological techniques and DNA base ratio determinations were used to demonstrate genetic differences between the two rhizobial strains. 2. Partially purified preparations of the haemoglobins from the nodules produced by the two strains showed no differences when examined by electrophoresis, isoelectric focusing or ion-exchange chromatography. 3. Two different leghaemoglobins from each type of nodule were separated by chromatography on DEAE-cellulose. One of these was isolated in the Fe(3+) form and accounted for two-thirds of the total leghaemoglobin. When it was examined in the analytical ultracentrifuge and by amino acid analysis, this major component did not vary with the inoculant rhizobial strain. The molecule had an s(20,w) of 1.88S, a diffusion coefficient of 10.7x10(-7)cm(2).s(-1) and a mol. wt. of 16700. 4. These results strongly support the hypothesis that the mRNA for leghaemoglobin is transcribed from plant DNA.     

Ferric Leghemoglobin in Plant-Attached Leguminous Nodules

Leghemoglobin (Lb) is essential for nitrogen fixation by intact leguminous nodules. To determine whether ferric Lb (Lb3+) was detectable in nodules under normal or stressed conditions, we monitored the status of Lb in intact nodules attached to sweet clover (Melilotus officinalis) and soybean (Glycine max [L.] Merr.) roots exposed to various conditions. The effects of N2 and O2 streams and elevated nicotinate levels on root-attached nodules were tested to determine whether the spectrophotometric technique was showing the predicted responses of Lb. The soybean and sweet clover nodules' Lb spectra indicated predominantly ferrous Lb and LbO2 in young (34 d) plants. As the nodule aged beyond 45 d, it was possible to induce Lb3+ with a 100% O2 stream (15 min). At 65 d without inducement, the nodule Lb status indicated the presence of some Lb3+ along with ferrous Lb and oxyferrous Lb. Nicotinate and fluoride were used as ligands to identify Lb3+. Computer-calculated difference spectra were used to demonstrate the changes in Lb spectra under different conditions. Some conditions that increased absorbance in the 626 nm region (indicating Lb3+ accumulation) were root-fed ascorbate and dehydroascorbate, plant exposure to darkness, and nodule water immersion.     

Chromosomal arrangement of leghemoglobin genes in soybean.

A cluster of four different leghemoglobin (Lb) genes was isolated from AluI-HaeIII and EcoRI genomic libraries of soybean in a set of overlapping clones which together include 45 kilobases (kb) of contiguous DNA. These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-Lb psi-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus. A sequence probably common to the 3' region of Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main Lb locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is primarily expressed in root and leaf is found 5' to two Lb loci. Overall, the main leghemoglobin locus is similar in structure to the mammalian globin gene loci.     

Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant

A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the CaMV—35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes . Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing β-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.     

Determination of 16S ribosomal RNA gene copy number in Streptococcus uberis, S. agalactiae, S. dysgalactiae and S. parauberis

Abstract Species-specific oligonucleotide probes and a universal oligonucleotide probe derived from sequences of 16S rRNA were hybridised to chromosomal DNA from Streptococcus agalactiae, S. dysgalactiae, S. parauberis and S. uberis following digestion with Eco RI. Due to the presence of a unique Eco RI site in each 16S rRNA gene, the number of hybridised fragments was indicative of the number of 16S rRNA genes. Southern hybridisation indicated six 16S rRNA genes in ten isolates of S. agalactiae , five genes in ten isolates of S. uberis , five genes in six isolates and six in another isolate of S. dysgalactiae , and six genes in four isolates of S. parauberis . For a fifth isolate of S. parauberis , six 16S rRNA genes were indicated by the universal probe but only five when hybridised to the species-specific probe, indicating sequence variation (microheterogeneity) within the probe target region.