Strain selection and improvement of gene transfer for genetic manipulation of Pseudomonas savastanoi isolated from olive knots

Research on diseases of herbaceous plants caused by Pseudomonads has been rapidly progressing; however, for most pathovars which infect woody plants, strains accessible to genetic manipulation have not yet been reported. At present, few studies have reported the transfer of genes to Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease. A collection of P. savastanoi pv. savastanoi isolates was tested for its ability to receive, by conjugation, the broad-host range plasmid pBBR1MCS-2; four of them, showing conjugation frequencies higher than 10(-3) transconjugants/recipient, were selected. Differences in motility, colony size and morphology, and knot formation in olive explants were observed among the selected strains; nonetheless, amplification and sequencing of the 16S rRNA gene confirmed that they belonged to P. savastanoi species. Transformation frequency by electroporation of pBBR1MCS-2 into these strains was improved up to four orders of magnitude using plasmids isolated from a P. savastanoi strain and from an Escherichia coli modification/restriction-deficient strain. Three of the selected strains maintained pBBR1MCS-2 stably and compatibly with their native plasmids during at least 90 generations, allowing the use of this vector for gene expression studies. Transposition via conjugation of different mini-Tn5, with or without the reporter genes gfp or luxAB, yielded frequencies varying from 1 x 10(-5) to 2.4 x 10(-9) transconjugants/recipient. Southern analysis of mutants obtained in strain NCPPB 3335 using a collection of DNA sequence tag transposons indicated that transposition occurs randomly, and in most cases at single sites in the genome of this strain, allowing the utilization of transposon tools for cell tagging and for the construction of insertional mutations. Knots developed on one-year-old plants inoculated with a Gfp-tagged strain clearly showed green fluorescence.     

Inactivation of the rhlA gene in Pseudomonas aeruginosa prevents rhamnolipid production,disabling the protection against polymorphonuclear leukocytes

Many of the virulence factors produced by the opportunistic human pathogen Pseudomonas aeruginosa are quorum‐sensing (QS) regulated. Among these are rhamnolipids, which have been shown to cause lysis of several cellular components of the human immune system, e.g. monocyte‐derived macrophages and polymorphonuclear leukocytes (PMNs). We have previously shown that rhamnolipids produced by P. aeruginosa cause necrotic death of PMNs in vitro. This raises the possibility that rhamnolipids may function as a ‘biofilm shield’in vivo, which contributes significantly to the increased tolerance of P. aeruginosa biofilms to PMNs. In the present study, we demonstrate the importance of the production of rhamnolipids in the establishment and persistence of P. aeruginosa infections, using an in vitro biofilm system, an intraperitoneal foreign‐body model and a pulmonary model of P. aeruginosa infections in mice. Our experimental data showed that a P. aeruginosa strain, unable to produce any detectable rhamnolipids due to an inactivating mutation in the single QS‐controlled rhlA gene, did not induce necrosis of PMNs in vitro and exhibited increased clearance compared with its wild‐type counterpart in vivo. Conclusively, the results support our model that rhamnolipids are key protective agents of P. aeruginosa against PMNs.     

Mutations in the holocarboxylase synthetase gene HLCS

Holocarboxylase synthetase (HLCS) deficiency is an autosomal recessive disorder. HLCS is an enzyme that catalyzes biotin incorporation into carboxylases and histones. Since the first report of the cDNA sequence, 30 mutations in the HLCS gene have been reported. Mutations occur throughout the entire coding region except exons 6 and 10. The types of mutations are one single amino acid deletion, five single nucleotide insertions/deletions, 22 missense mutations, and two nonsense mutations. The only intronic mutation identified thus far is c.1519+5G>A (also designated IVS10+5G>A), which causes a splice error. Several lines of evidence suggest that c.1519+5G>A is a founder mutation in Scandinavian patients. Prevalence of this mutation is about 10 times higher in the Faroe Islands than in the rest of the world. The mutations p.L237P and c.780delG are predominant only in Japanese patients. These are probably founder mutations in this population. Mutations p.R508W and p.V550M are identified in several ethic groups and accompanied with various haplotypes, suggesting that these are recurrent mutations. There is a good relationship between clinical biotin responsiveness and the residual activity of HLCS. A combination of a null mutation and a point mutation that shows less than a few percent of the normal activity results in neonatal onset. Patients who have mutant HLCS with higher residual activity develop symptom after the neonatal period and show a good clinical response to biotin therapy.     

Inactivation of Crithidia fasciculata carbamoyl phosphate synthetase II by the antitumor drug acivicin

In Crithidia fasciculata, carbamoyl phosphate synthetase II, which catalyses the first step of de novo pyrimidine biosynthesis, was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. The antitumor drug acivicin competitively inhibited the synthetase II activity with respect to L-glutamine, yielding an apparent Ki of 2 microM. In the absence of L-glutamine, acivicin resulted in a selective, time-dependent inactivation of L-glutamine-dependent activity of the enzyme, with an inactivation constant (Kinact) of 100 microM and a minimum inactivation half-time (T) of 0.2 min. L-Glutamine protected the enzyme from inactivation. These results are consistent with a postulate that acivicin is an active site-directed affinity analogue of L-glutamine, achieving irreversible inactivation. The inactivated enzyme retained ammonia-dependent activity. Acivicin stimulated the ammonia-dependent activity by increasing the Vmax value of the enzyme; apparent Km values for ammonia and MgATP were not affected. Differential action of acivicin on the Crithidia and mammalian synthetase II is discussed.     

Mutational analysis of the coat protein gene of tobacco mosaic virus in relation to hypersensitive response in tobacco plants with the N' gene

Tomato strain L of tobacco mosaic virus (TMV-L) induces a hypersensitive response (necrotic local lesions) on tobacco plants with the N' gene. A factor responsible for induction of the hypersensitive response has been mapped to the coat protein gene. We have constructed several mutants which have insertions or deletions in the coat protein gene. Frame-shift mutants which cause premature termination of translation of the coat protein caused no necrotic local lesions on N' plants. Mutants which result in the expression of coat protein derivatives with one amino acid inserted after residue 56, 101, or 152 caused necrotic local lesions on N' plants. Deletion mutants lacking the coding region for fewer than the C-terminal 13 amino acid residues caused necrotic local lesions, whereas mutants lacking the coding region for the C-terminal 38 residues caused no necrotic local lesions. These results show that modifications of the coat protein gene affect its ability to induce the hypersensitive response in N' plants.     

Inactivation of the methicillin resistance gene mecA in vancomycin-resistant Staphylococcus aureus

Acquisition of high-level resistance to vancomycin in the laboratory mutant VM50 (vancomycin MIC increased from 1.5 to 100 microg/ml) was accompanied by the appearance of a heterogeneous phenotype and a virtual loss in methicillin resistance: in most cells of cultures of VM50 the methicillin MIC of the parental strain was reduced from 800 to 1.5 microg/ml with only a subpopulation (10(-5)) retaining methicillin resistance at near the parental level (MIC of 400 microg/ml). Interestingly, the vancomycin MIC of this subpopulation was less (25 microg/ml) than that of VM50 (100 microg/ml). A similar antagonism between methicillin and vancomycin resistance levels was observed upon introduction of an intact mecA into VM50 on a plasmid vector: methicillin resistance of the majority of cells increased from 1.5 to 100 microg/ml while the vancomycin MIC declined from 100 to 12/25 microg/ml. Membrane preparations from mutant VM50 showed no detectable penicillin-binding protein (PBP) 2A by the fluorographic assay. Sequencing of the mecA gene resident in mutant VM50 indicated the presence of a 19-bp duplication between nucleotide residues 280-298, leading to the generation of a stop codon TAA starting at nucleotide position 286.     

Inactivation of the E-cadherin gene in sporadic diffuse-type gastric cancer.

Loss of the cell adhesion molecule E-cadherin has been observed in a variety of human carcinomas, and germline E-cadherin mutations have been found in several familial cases of diffuse gastric cancer. We sought to determine the prevalence and nature of E-cadherin alterations in "sporadic" gastric carcinomas. We performed comprehensive sequencing of the coding region, loss of heterozygosity (LOH) analysis, and immunohistochemical protein expression determination on 40 sporadic gastric adenocarcinomas. In total, 7 of 25 diffuse-type cancers harbored genetic alterations in the E-cadherin gene. Novel mutations predicted to significantly compromise protein function were found within 4 of these cancers, 2 of which harbored alterations resulting in biallelic inactivation of the gene product. Three diffuse cancers failed to amplify Exon 8 of E-cadherin, suggesting the presence of a homozygous abnormality. Notably, one germline E-cadherin mutation was also identified within these "sporadic" diffuse cancers. Significant gene mutations were not found in the 14 intestinal-type or histologically mixed cancer. Immunohistochemistry revealed aberrant or negative protein expression in seven diffuse-type tumors, four of which correlated with the genetic alterations. Both diffuse and intestinal-type tumors exhibited low rates of LOH, suggesting that allelic loss at the locus is not a common mechanism for E-cadherin inactivation during gastric tumorigenesis. Our observations suggest that inactivation of the E-cadherin gene occurs only in a subset of diffuse-type gastric cancers, as the majority of cases did not contain genetic alterations or identifiable protein abnormalities. Germline E-cadherin alterations, although rare, may underlie some diffuse gastric cancer cases that have important biologic and practical implications     

Disruption of the cyclosporin synthetase gene of Tolypocladium niveum

Cyclosporin A is a potent and clinically-important immunosuppressive drug (Sandimmun^R). It is produced by the fungus Tolypocladium niveum. A transformation system for T. niveum ATCC34921 based on hygromycin selection was established. In order to obtain a T. niveum promoter, the cyclophilin gene was isolated using the Neurospora crassa gene as probe. A plasmid vector was constructed in which the promoter region of the T. niveum cyclophilin gene was fused to a bacterial hygromycin phosphotransferase gene. Protoplasts were transformed with this plasmid and hygromycin-resistant transformants were isolated. Using this transformation system, mutants of T. niveum with disrupted versions of the cyclosporin synthetase gene (simA) were engineered by DNA-mediated transformation. Disruption of the gene resulted in loss of the ability to produce cyclosporins.     

Diagnostics in patients with glutathione synthetase deficiency but without mutations in the exons of the GSS gene

The synthesis of the ubiquitous tripeptide glutathione is impaired in patients with glutathione synthetase deficiency. The defect is inherited in an autosomal recessive manner, and the diagnosis is based on clinical, biochemical, and genetic criteria. In seven of our 30 index cases, however, no disease causing mutations could be identified in the coding exons or exon-intron boundaries of the glutathione synthetase gene GSS. These patients had severely decreased glutathione synthetase activities in lysates of cultured fibroblasts, and the levels of the enzyme were undetectable using a polyclonal antibody raised against human glutathione synthetase. RT-PCR mediated sequence analysis revealed previously not reported splice mutations in all patients. Thus, we conclude that in the investigation of patients with glutathione synthetase deficiency, and probably other genetic diseases as well, it might be time saving to initiate mutation analysis with sequencing of mRNA.     

glnA mutations define the structural gene for glutamine synthetase in Aspergillus

Summary Five allelic glutamine auxotrophs of Aspergillus have been assayed for glutamine synthetase activity. The amounts of glutamine synthetase activity present in these strains were proportional to their ability to grow in the absence of L-glutamine, whereas the amount of -glutamyl transferase did not correlate with the amount of synthetase or with the growth properties. Two out of this sample of mutants have been shown to have a glutamine synthetase with a K _m for L-glutamate which differs from the wild type.     

Inactivation of the PTEN tumor suppressor gene is associated with increased angiogenesis in clinically localized prostate carcinoma

The PTEN tumor suppressor gene encodes a dual-specificity protein phosphatase that may play a key role in modulating integrin-mediated signals. Inactivation of the PTEN gene has been detected in a small percentage of clinically localized prostate cancers but is common in metastatic disease. It has been shown in glioblastoma cell lines that loss of chromosome 10q, where the PTEN gene is located, is associated with increased angiogenic activity in the conditioned medium attributable to downregulation of thrombospondin-1, a negative regulator of angiogenesis. Therefore, we wished to determine whether inactivation of PTEN might be associated with increased angiogenesis in prostate cancers, because increased angiogenesis in localized cancers is associated with development of metastatic disease. Angiogenesis was assessed by counting microvessels in areas of maximal neovascularization after immunostaining with anti-factor VIII-related antigen antibodies in eight cases with proven homozygous deletion of the PTEN gene and 24 control cases. There was a statistically significant correlation between PTEN inactivation and increased microvessel counts. The microvessel density was higher at all Gleason scores in the cases with PTEN inactivation compared with control cases with the same score. To determine whether the increased angiogenesis in cases with PTEN inactivation was caused by downregulation of expression of the angiogenesis inhibitor thrombospondin-1, we analyzed a subset of the cases by immunostaining with anti-thrombospondin-1 antibody. Approximately 25% of cases showed decreased staining of prostate cancer cells, but there was no correlation with PTEN inactivation. Thus, PTEN inactivation is associated with increased angiogenesis, but the increased angiogenesis is not attributable to downregulation of thrombospondin-1 expression.     

Inactivation of the cystatin E/M tumor suppressor gene in cervical cancer

We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5'aza 2-deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.     

Comparison of RAG gene expression in normal and transformed precursor lymphocytes

Analyses of mechanisms that regulate V(D)J recombination haverelied heavily on the use of transformed precursor lymphocytecell lines. We now show that such lines have highly variableand frequently low levels of recombination activating genes(RAG)–1 and –2 gene expression. We also show thatexpression levels of the RAG genes can vary > 100-fold betweendifferent subcloned cells of an individual pre-B line. We discussthese findings in the context of normal regulation of RAG geneexpression and the implication for the use of transformed pre-Bcell lines as models for studying control of V(D)J recombinationactivity.     

Effects of single-nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis

Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96 L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.     

二氢叶酸还原酶和谷氨酰胺合成酶双基因筛选扩增系统对外源基因表达的影响

目的 研究二氢叶酸还原酶（DHFR，D）与谷氨酰胺合成酶（GC，G）单基因和二氢叶酸还原酶与谷氨酰胺合成酶（DHFR GS，DG）双基因筛选扩增系统对外源基因表达的影响。方法 选择N端部分TPO基因（T184）作为靶基因，以DHFR和GS基因作为筛选及扩增基因构建重组质粒pDCT184与pGCT184，将两种质粒分别转染CHOdhfr^-细胞形成单基因筛选扩增系统，两种质粒共转染CHOdhfr^-细胞形成双基因筛选扩增系统，在双基因筛选扩增系统中设计三种药物加压方式，第一种为DG方式；先用DHFR系统压力（MTX）筛选，再用GS系统压力（MSX）筛选；第二种为GD方式；先用GS系统压力筛选（MSX），再用DHFR系统压力筛选（MTX）；第三种为共加压方式；同时利用DHFR与GS系统压力筛选（MTX MSX）。通过比较T184基因拷贝数及表达水平来研究DHFR和GS双基因筛选扩增系统对外源基因表达的影响。结果 T184基因拷贝数及表达水平在DG双基因选择方式没有明显差异。结论 采用DHFR GS双基因筛选扩增系统共加压方式是提高CHOdhfr^-细胞表达外源基因的有效途径。