A novel cold-inducible gene from Arabidopsis,RCI3, encodes a peroxidase that constitutes a component for stress tolerance

Myrothamnus flabellifolia, a short woody shrub from southern Africa, can survive severe desiccation of its vegetative organs. We studied mechanisms protecting this plant from oxidative damage during desiccation for 2 weeks, 4 and 8 months, and also during subsequent rehydration. This plant retains high concentrations of chlorophyll during desiccation, and these chlorophyll molecules are probably a source for potentially harmful singlet oxygen production. Desiccation triggered substantial increases in zeaxanthin and redox shifts of the antioxidants glutathione and ascorbate towards their oxidised forms. Simultaneously, the concentrations of violaxanthin, beta-carotene, ascorbate, alpha-tocopherol, and glutathione reductase activity progressively decreased. Antheraxanthin, gamma-tocopherol, lutein, neoxanthin and glucose-6-phosphate dehydrogenase displayed less pronounced changes in response to desiccation. Even after 4 months of desiccation, Myrothamnus flabellifolia recovered rapidly upon rehydration. Re-watering induced formation of ascorbate and glutathione, simultaneous reduction of their oxidised forms, and rapid production of alpha-tocopherol and of various carotenoids. Only after 8 months of desiccation did the antioxidant system of M. flabellifolia break down; 3 weeks after the onset of rehydration, these plants abscised their leaves, but even then they were still able to recover and develop new ones. Ascorbate, beta-carotene and alpha-tocopherol were totally depleted after 8 months of desiccation and did not recover upon rehydration; glutathione was partly maintained, but only in the oxidised form. We present a model demonstrating which parts of antioxidant pathways break down as oxidative stress becomes detrimental and we discuss some potential implications of our results for the genetic modification of crop plants to improve their drought tolerance.     

Developmental and pathogen-induced expression of three barley genes encoding lipid transfer proteins

Clones for three barley non-specific lipid transfer proteins (LTP2, LTP3, and LTP4; formerly Cw18, Cw20 and Cw21, respectively) which had been previously shown to inhibit growth of plant pathogens, were selected and characterized from a cDNA library derived from young etiolated leaves. Genes Ltp2 and Ltp4 were located in chromosome 3H and gene Ltp3 was assigned to chromosome 7H by Southern blot analysis of wheat—barley disomic addition lines, using gene-specific probes (3'-ends of cDNAs). These assignments were confirmed by the polymerase chain reaction, using specific primers. The three genes were expressed in stem, shoot apex, leaves and roots (at low levels) throughout development. Genes Ltp3 and Ltp4 were expressed at high levels, and Lpt2 at low levels, in the spike (rachis, lemma plus palea and grain coats). Neither of the mRNAs was detected in endosperm. The proteins were localized by tissue-printing with polyclonal antibodies in the outer cell layer of the exposed surfaces of the plant, throughout the embryo, and in vascular tissues. Expression levels in leaves were moderately increased by 0.34 M NaCl and by 0.1 mM abscisic acid and were not affected by cold, drought, salicylate, 2,6-dichloro-isonicotinic acid, ethylene or ethephon. Methyl Jasmonate (10 µM) switched off all three genes. Inoculation with Av6 or vir6 isolates of the fungal pathogen Erysiphe graminis increased the three mRNAs, especially that of LTP4, which reached a maximum nine-fold increase 12–16 h after infection.     

The highly conserved serine threonine kinase StkP of Streptococcus pneumoniae contributes to penicillin susceptibility independently from genes encoding penicillin-binding proteins

Background  

The serine/threonine kinase StkP of Streptococcus pneumoniae is a major virulence factor in the mouse model of infection. StkP is a modular protein with a N-terminal kinase domain a C-terminal PASTA domain carrying the signature of penicillin-binding protein (PBP) and prokaryotic serine threonine kinase. In laboratory cultures, one target of StkP is the phosphoglucosamine mutase GlmM involved in the first steps of peptidoglycan biosynthesis. In order to further elucidate the importance of StkP in S. pneumoniae, its role in resistance to β-lactams has been assessed by mutational analysis in laboratory cultures and its genetic conservation has been investigated in isolates from infected sites (virulent), asymptomatic carriers, susceptible and non-susceptible to β-lactams.     

Phylogenetic and functional analysis of Arabidopsis RCI2 genes

Six new Arabidopsis thaliana genes (AtRCI2C-H) have been identified that show high homology to AtRCI2A and AtRCI2B. Sequence comparisons revealed that AtRCI2-related genes are widely spread among very different organisms, including other plant species, prokaryotes, fungi, and simply organized animals, and are also organized in gene families. Most RCI2 genes show a similar exon-intron organization, which indicates that they have been structurally conserved during evolution, and encode small, highly hydrophobic proteins containing two putative transmembrane domains. Consistently, the majority of AtRCI2 proteins localize in the plasma membrane. RCI2 proteins exhibit an elevated level of sequence similarity and seem to have evolved from a common ancestor. In spite of their high similarity, conserved subcellular localization, and common origin, experimental evidence is presented suggesting that different RCI2 proteins may have distinct functional roles. Thus, as previously demonstrated for AtRCI2A and AtRCI2B, the newly identified AtRCI2 genes (AtRCI2C-H) are differentially regulated in Arabidopsis organs and in response to abiotic stresses and ABA treatment. Furthermore, only the AtRCI2 proteins that do not contain the C-terminal hydrophilic tail (i.e. AtRCI2A-C and AtRCI2H) are able to complement for the loss of the yeast AtRCI2-related gene PMP3. On the basis of these results, different aspects on the evolution and roles of RCI2 genes are discussed.     

Cloning and expression of two genes encoding auxin-binding proteins from tobacco

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.     

Comparison of the tissue-specific expression and developmental regulation of two closely linked rodent genes encoding cytosolic retinol-binding proteins

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are two highly homologous cytoplasmic proteins that bind all-trans-retinol. We have recently demonstrated that the mouse genes encoding CRBP and CRBP II are closely linked on chromosome 9 and that both human genes are located on chromosome 3 (Demmer, L.A., Birkenmeier, E.H., Sweetser, D.A., Levin, M.S., Zollman, S., Sparkes, R.S., Mohandas, T., Lusis, A.J., and Gordon, J.I. (1987) J. Biol. Chem. 262, 2458-2467). We have now used RNA blot hybridization analysis to assess the degree to which these genes are coordinately expressed in fetal, suckling, weaning, and adult rat tissues. Both genes exhibit different developmental patterns of expression in liver, intestine, lung, kidney, testes, and placenta. In the intestine, CRBP mRNA was detected during the 16th day of gestation--prior to the development of a well-differentiated absorptive epithelium--and remained essentially unchanged throughout the peri- and postpartum periods. By contrast, the pattern of intestinal CRBP II mRNA accumulation closely parallels the times of first appearance, and subsequent proliferation, of the intestinal absorptive columnar epithelium, supporting the hypothesis that CRBP II is involved in the intestinal uptake or intracellular trafficking of this hydrophobic vitamin. In the fetal liver, both genes were expressed by gestational day 16. Whereas the concentration of hepatic CRBP mRNA increased markedly during the suckling and early weaning periods, CRBP II mRNA levels fell abruptly immediately after birth. These peripartum changes were not paralleled by remarkable alterations in the steady state levels of hepatic retinol. Marked changes in the expression of CRBP in the liver and of CRBP II in the intestine were also documented in pregnant and lactating female rats. These differences in CRBP/CRBP II gene expression strongly suggest that their proteins serve different physiological functions. The peripartum liver may provide a useful model for dissecting the relative roles played by these homologous proteins in retinoid metabolism as well as the factors which modulate activation and repression their genes.     

Protein sequences of linked genes are highly conserved in two bacterial species

It has been shown that proteins encoded by linked genes have similar rates of evolution and that clusters of essential genes are found in regions with low recombination rates. We show here that proteins encoded by linked genes in two closely related bacterial species, namely Escherichia coli K12 and Salmonella typhimurium LT2, evolve more slowly when compared with proteins encoded by genes that are not linked as assessed by protein sequence similarity. The proteins encoded by the identified linked genes share an average sequence identity of 82.5% compared with a 46.5% identity of proteins encoded by genes that are not linked.     

Molecular characterization and concerted evolution of two genes encoding RING-C2 type proteins in rice

RING (Really Interesting New Gene) finger proteins are believed to play a critical role in mediating the transfer of ubiquitin to heterogeneous substrate(s). While the two canonical types, RING-H2 and RING-HC, have been well-characterized, the molecular functions of the modified types, particularly the RING-C2 types, remain elusive. We isolated two rice genes harboring the RING-C2 domain on the distal parts of rice chromosomes 11 and 12, termed OsRINGC2-1 and OsRINGC2-2, respectively. A comparison of sequence divergences between 10 duplicate pairs on the distal parts of rice chromosomes 11 and 12 and randomly selected duplicate pairs suggested that OsRINGC2-1 and OsRINGC2-2 have evolved in concert via gene conversion. An in vitro ubiquitination assay revealed that both proteins possess E3 ligase activity, suggesting that the innate functions of these RING domains have not been affected by their modifications during evolution. Subcellular localizations were strikingly different; OsRINGC2-1 was found only in the cytoplasm with many punctate complexes, whereas OsRINGC2-2 was observed in both the nucleus and cytoplasm. The expression patterns of both genes showed striking differences in response to salt stress, whereas plants heterogeneous for both genes mediated salt tolerance in Arabidopsis, supporting the notion of concerted evolution. These results shed light on the molecular functions of OsRINGC2-1 and OsRINGC2-2 and provide insight into their molecular evolution.     

Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi

Borrelia burgdorferi causes Lyme disease, a multisystem illness that can persist in humans for many years. We describe recombination between homologous genes encoding the major outer surface proteins (Osps) A and B of B. burgdorferi which both deletes osp gene sequences and creates chimaeric gene fusions. Recombinant osp genes occur in multiple strains and encode unique proteins that lack some characteristic Osp epitopes. Antigenic variation in Osp through recombination may be relevant to the persistence of B. burgdorferi in an infected host, and has important implications for the utility of OspA and OspB as diagnostic or vaccine candidates for Lyme disease. We also describe Osp variation arising from nonsense mutations and sequence divergence, which may also represent significant sources of Osp polymorphism.     

A highly conserved hydrophobic motif in the exofacial vestibule of fructose transporting SLC2A proteins acts as a critical determinant of their substrate selectivity

The substrate specificity of the facilitated hexose transporter, GLUT, family, (gene SLC2A) is highly varied. Some appear to be able to translocate both glucose and fructose, while the ability to handle 2-deoxyglucose and galactose does not necessarily correlate with the other two hexoses. It has become generally accepted that a central substrate binding/translocation site determines which hexoses can be transported. However, a recent study showed that a single point mutation of a hydrophobic residue in GLUTs 2, 5 & 7 removed their ability to transport fructose without affecting the kinetics of glucose permeation. This residue is in the 7th transmembrane helix, facing the aqueous pore and lies close to the opening of the exofacial vestibule. This study expands these observations to include the other class II GLUTs (9 & 11) and shows that a three amino acid motif (NXI/NXV) appears to be critical in determining if fructose can access the translocation mechanism. GLUT11 can also transport fructose, but it has the motif DSV at the same position, which appears to function in the same manner as NXI and when all three residues are replaced with NAV fructose transport lost. These results are discussed in relation to possible roles for hydrophobic residues lining the aqueous pore at the opening of the exofacial vestibule. Finally, the possibility that the translocation binding site may not be the sole determinant of substrate specificity for these proteins is examined.     

A highly conserved hydrophobic motif in the exofacial vestibule of fructose transporting SLC2A proteins acts as a critical determinant of their substrate selectivity

The substrate specificity of the facilitated hexose transporter, GLUT, family, (gene SLC2A) is highly varied. Some appear to be able to translocate both glucose and fructose, while the ability to handle 2-deoxyglucose and galactose does not necessarily correlate with the other two hexoses. It has become generally accepted that a central substrate binding/translocation site determines which hexoses can be transported. However, a recent study showed that a single point mutation of a hydrophobic residue in GLUTs 2, 5 & 7 removed their ability to transport fructose without affecting the kinetics of glucose permeation. This residue is in the 7th transmembrane helix, facing the aqueous pore and lies close to the opening of the exofacial vestibule. This study expands these observations to include the other class II GLUTs (9 & 11) and shows that a three amino acid motif (NXI/NXV) appears to be critical in determining if fructose can access the translocation mechanism. GLUT11 can also transport fructose, but it has the motif DSV at the same position, which appears to function in the same manner as NXI and when all three residues are replaced with NAV fructose transport lost. These results are discussed in relation to possible roles for hydrophobic residues lining the aqueous pore at the opening of the exofacial vestibule. Finally, the possibility that the translocation binding site may not be the sole determinant of substrate specificity for these proteins is examined.     

Polymorphism in two genes for B2 high sulfur proteins of wool

Variation in the nucleotide sequence of the B2 high-sulfur protein genes has not been reported previously. This paper reports 15 nucleotide substitutions in each of the genes for the B2A and B2C proteins and a length of polymorphism in the B2A gene which translates to the insertion/deletion of one 30-nucleotide repeat sequence. Evidence is presented for gene conversion occurring within the B2 high-sulfur multigene family. These DNA polymorphisms may account for some of the microheterogeneity observed in the B2 high-sulfur proteins and may also be useful genetic markers of the B2 high-sulfur protein gene loci for future use in analysing wool fibre characteristics.