Host-specific regulation of nodulation genes in Rhizobium is mediated by a plant-signal, interacting with the nodD gene product

We have identified a nodD gene from the wide host-range Rhizobium strain MPIK3030 (termed nodD1) which is essential for nodulation on Macroptilium atropurpureum (siratro). Experiments with nodA–lacZ gene fusions demonstrate that the MPIK3030 nodD1 regulates expression of the nodABC genes. Additionally, we used nodC–lacZ fusions of Rhizobium meliloti to show that the MPIK3030 nodD1 gene induces expression of these fusions by interacting with plant factors from siratro and from the non-host Medicago sativa (alfalfa). The R. meliloti nodD genes, however, only interact with alfalfa exudate. In line with these results, no complementation of MPIK3030 nodD1 mutants could be obtained on siratro with the R. meliloti nodD genes, while the MPIK3030 nodD1 can complement nodD mutants of R. meliloti on alfalfa. Furthermore, R. meliloti transconjugants harbouring the MPIK3030 nodD1 efficiently nodulate the illegitimate host siratro. When compared with other nodD sequences, the amino acid sequence of the MPIK3030 nodD1 shows a conserved aminoterminus, whereas the carboxy-terminus of the putative gene product diverges considerably. Studies on a chimeric MPIK3030/R. meliloti nodD gene indicates that the carboxy-terminal region is responsible for the interaction with plant factor(s) and may have evolved in different rhizobia specifically to interact with plant–host factors.     

Identification of host range determinants in the Rhizobium species MPIK3030

Summary R. meliloti primarily nodulates Medicago sativa but cannot nodulate Macroptilium atropurpureum. By introducing an 11.4 kb region into R. meliloti from the Symplasmid of Rhizobium strain MPIK3030, the host range of the R. meliloti transconjugants were shown to be extended to M. atropurpureum, one of the hosts of MPIK3030 but not normally nodulated by R. meliloti. The region responsible for host range extension was isolated by mass conjugating a clone bank from MPIK3030 into the R. meliloti wild type, and subsequent screening for nodulation on M. atropurpureum. Using deleted derivatives of a plasmid reisolated from endosymbiotic bacteria, the host range region was further narrowed down to three EcoRI fragments. Tn5 mutagenesis allowed the isolation of three discrete regions on an 11.4 kb section, which are involved in the extension of host range to M. atropurpureum. Finally, complementation experiments performed with R. meliloti common nod and hsn mutants indicated that none of the genes involved in the early steps of nodulation, including host-range functions, can be complemented by genes carried on the 11.4 kb fragment derived from MPIK3030.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis

Carbon catabolite repression of several catabolic operons in Bacillus subtilis is mediated by the repressor CcpA. An inactivation of the ccpA gene has two distinct phenotypes: (i) catabolite repression of catabolic operons is lost and (ii) the growth of bacteria on minimal medium is severely impaired. We have analyzed the physiological properties of a ccpA mutant strain and show that the ccpA mutation does not affect sugar transport. We have isolated extragenic suppressors of ccpA that suppress the growth defect (sgd mutants). Catabolite repression of beta-xylosidase synthesis was, however, not restored suggesting that the suppressor mutations allow differentiation between the phenotypes of the ccpA mutant. A close inspection of the growth requirements of the ccpA mutant revealed the inability of the mutant to utilize inorganic ammonium as a single source of nitrogen. An intact ccpA gene was found to be required for expression of the gltAB operon encoding glutamate synthase. This enzyme is necessary for the assimilation of ammonium. In a sgd mutant, gltAB operon expression was no longer dependent on ccpA, suggesting that the poor expression of the gltAB operon is involved in the growth defect of the ccpA mutant.     

Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells

The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-₁. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-₁and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-₁ (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-₁ stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-₁ andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-₁, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC.

     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Noisy-threshold control of cell death

Background  

Cellular responses to death-promoting stimuli typically proceed through a differentiated multistage process, involving a lag phase, extensive death, and potential adaptation. Deregulation of this chain of events is at the root of many diseases. Improper adaptation is particularly important because it allows cell sub-populations to survive even in the continuous presence of death conditions, which results, among others, in the eventual failure of many targeted anticancer therapies.     

Unravelling enzymatic discoloration in potato through a combined approach of candidate genes,QTL, and expression analysis

Enzymatic discoloration (ED) of potato tubers was investigated in an attempt to unravel the underlying genetic factors. Both enzyme and substrate concentration have been reported to influence the degree of discoloration and as such this trait can be regarded as polygenic. The diploid mapping population C × E, consisting of 249 individuals, was assayed for the degree of ED and levels of chlorogenic acid and tyrosine. Using this data, Quantitative Trait Locus (QTL) analysis was performed. Three QTLs for ED have been found on parental chromosomes C3, C8, E1, and E8. For chlorogenic acid a QTL has been identified on C2 and for tyrosine levels, a QTL has been detected on C8. None of the QTLs overlap, indicating the absence of genetic correlations between these components underlying ED, in contrast to earlier reports in literature. An obvious candidate gene for the QTL for ED on Chromosome 8 is polyphenol oxidase (PPO), which was previously mapped on chromosome 8. With gene-specific primers for PPO gene POT32 a CAPS marker was developed. Three different alleles (POT32-1, -2, and -3) could be discriminated. The segregating POT32 alleles were used to map the POT32 CAPS marker and QTL analysis was redone, showing that POT32 coincides with the QTL peak. A clear correlation between allele combinations and degree of discoloration was observed. In addition, analysis of POT32 gene expression in a subset of genotypes indicated a correlation between the level of gene expression and allele composition. On average, genotypes having two copies of allele 1 had both the highest degree of discoloration as well as the highest level of POT32 gene expression.