A putative second adenylate kinase-encoding gene from the yeast Saccharomyces cerevisiae.

Sequencing of the region upstream from the yeast RAD3 gene has revealed an open reading frame (ORF) of 225 amino acids (aa) that could encode a 25.3-kDa polypeptide. The predicted aa sequence of this ORF is homologous with that of several eukaryotic adenylate kinase (Adk)-encoding genes, including the yeast gene, ADK1. These findings suggest that the yeast Saccharomyces cerevisiae has a second Adk-encoding gene, tentatively designated as ADK2.     

Effect of the Mi Gene in Tomato on Reproductive Factors of Meloidogyne chitwoodi and M. hapla

The effect of the Mi gene on the reproductive factor of Meloidogyne chitwoodi and M. hapla, major nematode pests of potato, was measured on nearly isogenic tomato lines differing in presence or absence of the Mi gene. The Mi allele controlled resistance to reproduction of race 1 of M. chitwoodi and to one of two isolates of race 2. No resistance to race 3 of M. chitwoodi or to M. hapla was found. Variability in response to isolates of race 2 may reflect diversity of virulence genotypes heretofore undetected. Resistance to race 1 of M. chitwoodi could be useful in potato if the Mi gene were functional following transferral by gene insertion technology into potato. Since the Mi gene is not superior to RMc₁ derived from Solarium bulbocastanum, the transferral by protoplast fusion appears to offer no advantage.     

Differentiation of Meloidogyne incognita and M. arenaria novel resistance phenotypes in Lycopersicon peruvianum and derived bridge-lines

Lycopersicon peruvianum PI 270435 clone 2R2 and PI 126443 clone 1MH were crossed reciprocally with three L. esculentum-L. peruvianum bridge-lines. The incongruity barrier between the two plant species was overcome; F₁ progeny were obtained from crosses between four parental combinations without embryo-rescue culture. Hybridity was confirmed by leaf and flower morphology and by the production of nematode-resistant F₁ progeny on homozygous susceptible parents. Clones of the five F₁ bridgeline hybrids were highly resistant to Mi-avirulent root-knot nematode (Meloidogyne incognita) at both 25°C and 30°C soil temperatures. However, only clones from PI 270435-3MH and PI 126443-1MH, and hybrids from PI 126443-1MH, were resistant to Mi-virulent M. incognita isolates at high soil temperature. Clones and hybrids from PI 270435-2R2 were not resistant to two Mi-virulent M. incognita isolates at high soil temperature. A source of heat-stable resistance was identified in bridge-line EPP-2, and was found to be derived from L. peruvianum LA 1708. Accessions of the L. peruvianum Maranon races, LA 1708 and LA 2172, and bridge-line EPP-2, segregated for heat-stable resistance to Mi-avirulent M. incognita, but were susceptible to Mi-virulent M. incognita isolates. Clone LA 1708-I conferred heat-stable resistance to M. arenaria isolate W, which is virulent to heat-stable resistance genes in L. peruvianum PI 270435-2R2, PI 270435-3MH, and PI 126443-1MH. Clone LA 1708-I has a distinct heat-stable factor for resistance to Mi-avirulent M. arenaria isolate W, for which the gene symbol Mi-4 is proposed. A Mi-virulent M. arenaria isolate Le Grau du Roi was virulent on all Lycopersicon spp. accessions tested, including those with novel resistance genes.     

Expression of Saccharomyces adenylate kinase gene in Candida boidinii under the regulation of its alcohol oxidase promoter

The methylotrophic yeast, Candida boidinii, was investigated as a new efficient host for heterologous gene expression. The Saccharomyces cerevisiae adenylate kinase gene (ADK1) was used as the first example for heterologous enzyme production in C. boidinii. C. boidinii cells were transformed with plasmids harboring the S. cerevisiae ADK1 gene under the alcohol oxidase (C. boidinii AOD1) promoter. The chromosome-integrant strains produced adenylate kinase protein corresponding to 22%–28% of the total soluble proteins in an enzymatically active form. When the three-copy integrative transformant was grown for 60 h on methanol-glycerol medium in a 1.5-l jar fermentor, adenylate kinase was produced intracellularly with a yield of up to 2 g/l culture medium. As the expression of the S. cerevisiae ADK1 in C. boidinii was under similar regulation to that of the C. boidinii AOD1, the previously cloned 1.7-kb AOD1 promoter fragment was proved to harbor sufficient cis elements for AOD1 regulation and found to be an efficient promoter for heterologous gene expression.     

Cloning and expression of two autolysin genes, cwlU and cwlV, which are tandemly arranged on the chromosome of Bacillus polymyxa var. colistinus

The cwlV gene, which encodes Bacillus polymyxa var. colistinus autolysin was cloned and sequenced. cwlV comprises a 1497-bp ORF and encodes a polypeptide of 499 amino acid (aa) residues (M_r of 53,707 Da). The N-terminal sequence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermined aa); this processed form corresponds to the C-terminal portion (183?aa, M_r of 20,050?Da) of the cwlV ORF. Sequencing of the flanking region revealed that another putative autolysin gene, cwlU, is located upstream of cwlV. cwlU encodes a polypeptide of 524 aa and its deduced sequence is 34.9% identical to the full-length sequence of CwlV. Downstream of cwlV, the genes for a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-homologous OrfX, a glutathione peroxidase homolog (Gpx), and the N-terminal region of OrfZ containing a ATP/GTP-binding site motif were found. Northern blotting and primer-extension analyses revealed that cwlU is transcribed as a single cistron, but cwlV is transcribed with orfW. The unprocessed forms of CwlV and CwlU (VΔS and UΔS, respectively) and their predicted mature forms (Vcat and Ucat, respectively) were expressed in, and purified from, Escherichia coli. Enzyme analysis indicated that VΔS and Vcat exhibit low and high cell wall hydrolase activities toward B. polymyxa cell wall, respectively, but UΔS and Ucat exhibit almost no and low cell wall hydrolase activities, respectively.     

Reproduction of Mi-Virulent Meloidogyne incognita Isolates on Lycopersicon spp.

Selection of detectable numbers of Mi-virulent root-knot nematodes has necessitated a greater understanding of nematode responses to new sources of resistance. During the course of this research, we compared the reproduction of four geographically distinct Mi-virulent root-knot nematode isolates on three resistant accessions of Lycopersicon peruvianum. Each accession carried a different resistant gene, Mi-3, Mi-7, or Mi-8. All nematode isolates were verified as Meloidogyne incognita using diagnostic markers in the mitochondrial genome of the nematode. Reproduction of Mi-virulent isolates W1, 133 and HM, measured as eggs per g of root, was greatest on the Mi-7 carrying accession and least on the Mi-8 carrying accession. In general, Mi-3 behaved similar to the Mi-8 carrying accession. Reproduction of the four nematode isolates was also compared on both Mi and non-Mi-carrying L. esculentum cultivars and a susceptible L. peruvianum accession. Resistance mediated by Mi in L. esculentum still impacted the Mi-virulent nematodes with fewer eggs per g of root on the resistant cultivar (P ≤ 0.05). Preliminary histological studies suggests that Mi-8 resistance is mediated by a hypersensitive response, similar to Mi.     

IS1549 from Mycobacterium smegmatis Forms Long Direct Repeats upon Insertion

A new insertion element, IS1549, was identified serendipitously from Mycobacterium smegmatis LR222 during experiments using a vector designed to detect the excision of IS6110 from between the promoter region and open reading frame (ORF) of an aminoglycoside phosphotransferase gene. Six of the kanamycin-resistant isolates had a previously unidentified insertion element upstream of the ORF of the aph gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C content in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS231, which is a member of the IS4 family of insertion elements. IS1549 contains 11-bp terminal inverted repeats and is characterized by the formation of unusually long and variable-length (71- to 246-bp) direct repeats of the target DNA during transposition. Southern blot analysis revealed that five copies of IS1549 are present in LR222, but not all M. smegmatis strains carry this element. Only strains with a 65-kDa antigen gene with a PCR-restriction fragment length polymorphism type identical to that of M. smegmatis 607 contain IS1549. None of 13 other species of Mycobacterium tested by PCR with two sets of primers specific for IS1549 were positive for the expected amplified product.     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined ?16 to ?25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.     

Interaction Studies Between Meloidogyne Javanica and Fusarium Oxysporum f. Sp. lycopersici (Fol) Race 3 on Different Isolines of Tomato Cv. Tasti Lee

The Mi gene in tomato confers resistance to Meloidogyne javanica, M. incognita, and M. arenaria, the most common tropical root-knot nematode (RKN) species found in Florida. Fusarium wilt (Fol) is another major problem in Florida tomatoes which may interact with RKN and cause more plant damage. To study the interactions between RKN, Fusarium, and Mi in tomato, two greenhouse experiments were conducted. Both experiments used different isolines (with and without I-3 and Mi genes) of the tomato cultivar Tasti Lee^®. In the first experiment, all four isolines were subjected to two levels of RKN (~10,000 eggs/pot and no eggs) and two levels of Fol (1000 cc soil with 1,000 cfu/g at planting and no Fol), both applied at planting. In the second experiment, the two isolines without I-3 were exposed to the same two levels of RKN as described above and three levels of Fol (50 ml Fol with 1×10⁶ cfu/m at planting, at 10 DAT, and no Fol). Fol reduced root-knot infection and reproduction when both Fol and RKN were inoculated at planting but not when Fol was inoculated 10 days later. Plant damage from Fol was exacerbated in the presence of RKN, especially when both pathogens were present at planting. Isolines with I-3 grew better in Fol-inoculated soil but had no effect when Fol and RKN were both present. Isolines with Mi gene reduced RKN infection and reproduction but did not affect plant damage caused by Fol. In summary, while RKN reproduction was reduced in the presence of Fol, the overall plant damage was more severe when both pathogens were present.     

Resistance in Lycopersicon peruvianum to Isolates of Mi Gene-Compatible Meloidogyne Populations

Root-knot nematode resistance of F₁ progeny of an intraspecific hybrid (Lycopersicon peruvianum var. glandulosum Acc. No. 126443 x L. peruvianum Acc. No. 270435), L. esculentum cv. Piersol (possessing resistance gene Mi), and L. esculentum cv. St. Pierre (susceptible) was compared. Resistance to 1) isolates of two Meloidogyne incognita populations artificially selected for parasitism on tomato plants possessing the Mi gene, 2) the wild type parent populations, 3) four naturally occurring resistance (Mi gene)-breaking populations of M. incognita, M. arenaria, and two undesignated Meloidogyne spp., and 4) a population of M. hapla was indexed by numbers of egg masses produced on root systems in a greenhouse experiment. Artificially selected M. incognita isolates reproduced abundantly on Piersol, but not (P = 0.01) on resistant F₁ hybrids. Thus, the gene(s) for resistance in the F₁ hybrid differs from the Mi gene in Piersol. Four naturally occurring resistance-breaking populations reproduced extensively on Piersol and on the F₁ hybrid, demonstrating ability to circumvent both types of resistance. Meloidogyne hapla reproduced on F₁ hybrid plants, but at significantly (P = 0.01) lower levels than on Piersol.     

An additional copy of the adenylate cyclase-encoding gene relieves developmental defects produced by a mutation in a vegetative incompatibility-controlling gene in Podospora anserina

To identify cellular functions involved in vegetative incompatibility in filamentous fungi, we have initiated the cloning of Podospora anserina (Pa) mod genes. These genes interfere with the lethal reaction triggered by interaction between incompatible het genes. A gene (Pa AC) has been cloned by complementation of developmental defects caused by a mutation in the mod-D gene. This gene encodes a protein of 2145 amino acids (aa) that exhibits strong similarities with many adenylate cyclases (AC). About 65% aa identity has been found between the sequence of the polypeptide encoded by this Pa AC gene and the AC of Neurospora crassa. The organization of peptidic domains in the polypeptide encoded by Pa AC is closely related to that of Saccharomyces cerevisiae CYRI. Restriction-fragment-length polymorphism (RFLP) and genetic analysis have shown that Pa AC and mod-D are distinct genes     

Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase

We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169° on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment. This fragment contained an incomplete and two complete open reading frames (ORFs). The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25 038 Da), was found to be responsible for the observed reduced mannitol fermentation. The 3′ part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identity. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72 889 Da. PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases. Several of the essential aa in the loops known to form the active site of cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA. After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue.     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

Partial purification and properties of the cytoplasmic factors modulating adenylate cyclase activity in rat lung plasma membranes

The adult rat lung supernatant contains some factors which markedly enhance adenylate cyclase activity in membranes (Nijjar, M.S. (1979) Biochim. Biophys. Acta 584, 43–50). These factors were separated into two less active components (peaks 1 and 2) by DEAE-cellulose chromatography. However, their recombination restored the full activation of adenylate cyclase. Further purification and characterization of these factors revealed that the activation in peak 1 contained two proteins of low (14 500) and high (65 000) molecular weight whereas the activator in peak 2 contained only one protein of 65 000. The kinetics of adenylate cyclase activation revealed that both the K_m and V values were affected. The data also demonstrate that calmodulin was not involved in the cytoplasmic activation of adenylate cyclase in rat lungs.     

A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

Cytosine bases can be deaminated spontaneously to uracil, causing DNA damage. Uracil-DNA glycosylase (UDG), a ubiquitous uracil-excising enzyme found in bacteria and eukaryotes, is one of the enzymes that repair this kind of DNA damage. To date, no UDG-coding gene has been identified in Methanococcus jannaschii, although its entire genome was deciphered. Here, we have identified and characterized a novel UDG from M.jannaschii designated as MjUDG. It efficiently removed uracil from both single- and double-stranded DNA. MjUDG also catalyzes the excision of 8-oxoguanine from DNA. MjUDG has a helix–hairpin–helix motif and a [4Fe–4S]-binding cluster that is considered to be important for the DNA binding and catalytic activity. Although MjUDG shares these features with other structural families such as endonuclease III and mismatch-specific DNA glycosylase (MIG), unique conserved amino acids and substrate specificity distinguish MjUDG from other families. Also, a homologous member of MjUDG was identified in Aquifex aeolicus. We report that MjUDG belongs to a novel UDG family that has not been described to date.     

Molecular cloning,characterization of one key molecule of teleost innate immunity from orange-spotted grouper (Epinephelus coioides): Serum amyloid A

The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.     

Stimulation of diamondback moth (Plutella xylostella) adenylate cyclase activity by a carbodiimide

1. DFCD or 3-(2,6-diisopropyl-4-phenoxyphenyl)-1-tert-butylcarbodiimide, a toxic metabolite of the thiourea acaricide/insecticide diafenthiuron, stimulated adenylate cyclase activity in preparations from heads of adult diamondback moths, Ptutella xylostella (L.).2. Depending upon assay conditions, DFCD gave a biphasic response with K_a, values of 0.025 and 1.25 μM for high and low affinity components, respectively, or a monophasic response with a K_a of 0.4 μM.3. Studies with potential agonists and antagonists suggested that octopamine-sensitive (K_a 7.5 μM) and dopamine-sensitive (K_a, 1.0μM) adenylate cyclases were present.4. At maximally effective concentrations, the activity of DFCD was nonadditive to that of octopamine or dopamine.5. It appeared that DFCD was binding to octopamine- and dopamine-sensitive adenylate cyclases and that affinity of the carbodiimide was higher for the former than for the latter.6. These actions on biogenic amine-sensitive adenylate cyclases likely are involved in the toxicity of diafenthiuron to diamondback moths.     

A second type-I PKS gene cluster isolated from Streptomyces hygroscopicus ATCC 29253, a rapamycin-producing strain

Analysis of a 32.8-kb segment of DNA from the rapamycin (Rp) producer, Streptomyces hygroscopicus ATCC 29253, revealed a new type-I polyketide synthase (PKS) cluster consisting of four open reading frames (ORF 1–4), each encoding a single PKS module. The four ORFs are transcribed in the same direction and are flanked by several smaller ORFs (ORF 5–9), which may be related to the PKS cluster. The first PKS-containing ORF has a ligase domain at the N-terminus of the polypeptide. This domain has 55% aa identity to the CoA ligase domain of the Rp PKS (Schwecke et al., 1995. Proc. Natl. Acad. Sci. 92, 7839–7843) which is also encoded in this strain (Lowden et al., 1996. Angew. Chem. Int. Ed. Engl. 35, 2249–2251). ORF5 (340 aa) and ORF6 (924 aa) were found to be homologous to RapK (41% aa identity) and RapH (35% aa identity), which are hypothesized to be a pteridine-dependent dioxygenase and a regulatory protein, respectively (Molnar et al., 1996. Gene 169, 1–7). In addition, ORF7 (391 aa) was found to have up to 42% aa identity to a number of plant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases (DAHPS) and 47% aa identity to PhzF, a bacterial DAHPS involved in phenazine antibiotic synthesis. The proximity of the DAHPS-encoding gene to the PKS cluster containing a Rp-like ligase domain suggests that a derivative of shikimate may be used as the PKS starter. ORF8 (283 aa) was found to have homology (32% aa identity) to a Synechocystis sp. gene of unknown function. The N-terminal portion of ORF9 was found to be similar to a tetracycline 6-hydroxylase (34% aa identity) from Streptomyces aureofaciens.