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PubMed1985

Isolation of a versatile Serratia marcescens mutant as a host and molecular cloning of the aspartase gene.

An extracellular nuclease-deficient, antibiotic-sensitive, and restrictionless mutant was isolated from the wild-type strain of Serratia marcescens Sr41 by four rounds of mutagenesis. The mutant was transformed efficiently with plasmid DNAs prepared from Escherichia coli and S. marcescens, and was used as a host for the cloning of the aspartase gene (aspA+) of S. marcescens. Cells carrying the cloned aspA+ gene on a multicopy plasmid produced ca. 39-fold more aspartase than did control cells, and the level of enzyme overproduction was in proportion to the copy number of the aspA+ recombinant plasmid. Aspartase was identified as a polypeptide of molecular weight 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Ammonia-Lyases↗
PubMed1992

Characterization of transformation-deficient mutants of Acinetobacter calcoaceticus.

Three Acinetobacter calcoaceticus transformation-deficient mutants, obtained by insertional mutagenesis with the nptll gene, have been characterized physiologically. One mutant (AAC211) was found to be completely transformation deficient, while two others, AAC213 and AAC214, were severely impaired in transformation efficiency (100-1000 times lower than the wild type). The latter applied to both chromosomal as well as plasmid DNA. Analysis of the chromosomal DNA fragments flanking the nptll gene in the mutants showed that mutants AAC213 and AAC214 had an insertion of the nptll gene in the same chromosomal region, but that they were the result of two independent mutational events, whereas the insertion in mutant AAC211 was at a different position. None of the three mutants showed phenotypic or genotypic characteristics typical of a RecA-deficient strain.

Acinetobacter calcoaceticus↗
PubMed1990

[Transformation and identification of recombinant plasmid pAT153 containing HCMV gene Hind III F fragment and its clinical application].

Transformation of the recombinant plasmid pAT153 containing human cytomegalovirus (HCMV) gene Hind III F fragment into E.coli strain HB 101 was made by the CaCl2 method with a 24% of transformation efficiency. The bacterial colonies containing the recombinant plasmid were selected in colony hybridization with 32P-labelled HCMV Hind III F fragment. The recombinant plasmid was isolated by the modified alkaline lysis method which is characterized by high quality, great quantity, and time-saving. HCMV Hind III F fragment has no homology to the other herpes virus. The 32P-labelled HCMV Hind III F fragment probe could detect 0.25 pg homological sequences of HCMV DNA. The use of this probe allows the detection of HCMV in clinical specimens with the advantages of quickness, good sensitivity and specificity.

Cytomegalovirus↗
PubMed2004

A novel plasmid (pEMCJH03) isolated from moraxella catarrhalis possibly useful as a cloning and expression vector within this species.

A preliminary screening study of six Moraxella catarrhalis isolates from primary school children in the Netherlands identified a small 3.5 kb plasmid (pEMCJH03), containing four open reading frames, which encoded three mobilizing and one replicase protein. Insertion of a kanamycin containing transposon (yielding pEMCJH04) allowed selection and isolation of the plasmid in Escherichia coli. Natural transformation of pEMCJH04 into M. catarrhalis was successful for 25% (3/12) of non-isogenic isolates, with no link between (lack of) transformability and genetic lineage or (lack of) transformability and complement phenotype, though the transformation efficiency was found to be rather low at approximately 615CFU/microg (range=60-1040CFU/microg ). This is only the second publication detailing a plasmid isolated from this important respiratory pathogen, and the ability to clone and express foreign proteins in M. catarrhalis using pEMCJH04 could help in the development of a vaccine expression vector, as well as providing a useful tool for studying promoter activity and in complementation studies of gene knockout mutants.

Bacterial Proteins↗
PubMed1999

Activation of leukotriene synthesis in human neutrophils by exogenous arachidonic acid: inhibition by adenosine A(2a) receptor agonists and crucial role of autocrine activation by leukotriene B(4).

We report here that the apparent inability of isolated human polymorphonuclear leukocytes (PMNs) to efficiently transform arachidonic acid (AA) is the consequence of A(2a) receptor engagement by endogenous adenosine accumulating in incubation media. Indeed, when adenosine is eliminated from PMN suspensions by the addition of adenosine deaminase, or when cells are incubated with adenosine A(2a) receptor antagonists, important quantities (40-80 pmol/10(6) cells) of 5-lipoxygenase products are synthesized by PMN incubated with 1 to 5 microM exogenous AA. The selective A(2a) receptor agonist CGS21680 was a very potent inhibitor of the AA-induced leukotriene (LT) synthesis, showing an IC(50) of approximately 1 nM. The mechanism of AA-induced stimulation of LT synthesis observed in the absence of extracellular adenosine was investigated. In adenosine deaminase-treated PMN, exogenous AA induced Ca(2+) mobilization and the translocation of 5-lipoxygenase to nuclear structures. A time lag of 20 to 60 s (variable between PMN preparations) was observed consistently between the addition of AA and the elevation of intracellular Ca(2+) concentration (and LT synthesis), indicating that AA itself did not trigger the Ca(2+) mobilization in PMN. This AA-induced Ca(2+) mobilization, as well as the corresponding 5-lipoxygenase translocation and stimulation of LT synthesis, was blocked efficiently by the LT synthesis inhibitor MK0591, the LTB(4) receptor antagonists CP105696 and LY223982, and the LTA(4) hydrolase inhibitor SC57461A. These data demonstrate that AA is a highly potent and effective activator of LT synthesis and acts through a mechanism that requires an autocrine stimulatory loop by LTB(4).

Adenosine↗
PubMed1992

Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein.

To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication. Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay. Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites. Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids. Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent. A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity. A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population. A mutation in the middle RS2 site had no significant effect on pSC101 replication.

Bacterial Proteins↗
PubMed2026

Benzoic acid inhibits peach root growth and lateral root emergence by disrupting auxin homeostasis through salicylic acid accumulation.

We established a non-sterile root transformation system in peach seedlings. Using this system, we demonstrated that BA treatment inhibits plant growth and lateral root emergence by SA-mediated disruption of auxin distribution. Allelopathic autotoxins, particularly benzoic acid (BA), are recognized as primary contributors to peach (Prunus persica) replant disease; however, the molecular mechanisms by which BA disrupts root development remain poorly understood. BA treatment significantly reduced stem and root length and inhibited lateral root emergence without affecting lateral root initiation. To investigate the underlying mechanism at cellular resolution, we established a non-sterile Agrobacterium rhizogenes-based root transformation system achieving 27.11% transformation efficiency. Auxin biosynthesis (PpYUC10), influx transport (PpAUX1), and response (PpARF19) genes were markedly downregulated following BA treatment. Transgenic roots expressing the DR5::GUS auxin reporter exhibited reduced DR5 activity in root tips and suppressed expression in tissues surrounding lateral root primordia, indicating impaired auxin signaling at both developmental sites. Hormone profiling revealed a non-significant trend toward reduced auxin metabolites alongside significant accumulation of salicylic acid (SA), an auxin-antagonistic hormone, and its storage conjugate SA 2-O-β-glucoside. Supporting a causal role for SA, exogenous SA phenocopied BA-induced root growth inhibition, whereas co-treatment with IAA or the SA-biosynthesis inhibitor aminoindan-1-phosphonic acid (AIP) significantly rescued lateral root number and root fresh weight. Multi-treatment RNA-seq identified "response to auxin" and "response to salicylic acid" as the most enriched GO terms in BA-treated roots, and AIP treatment restored the expression of key auxin-related genes while reversing BA-induced SA-pathway changes. Together, these findings suggest that BA-induced SA accumulation suppresses auxin biosynthesis, transport, and signaling, thereby inhibiting peach root growth and lateral root emergence. This study elucidates the molecular basis of BA autotoxicity and establishes a transformation platform for functional genomic studies in Prunus.

Indoleacetic Acids↗
PubMed2004

Vitreoscilla hemoglobin overexpression increases submergence tolerance in cabbage.

Agrobacterium tumefaciens was used to deliver the vhb gene into cabbage (Brassica oleracea var. Cabitata) cv. Xiaguang's parent line, 103. Using hypocotyls and cotyledon petioles as explants for infection, we obtained a transformation efficiency of 3-5% based on the number of transgenic shoots produced from the number of explants used for infection. Molecular analysis indicated that the vhb gene was stably integrated into the cabbage genome and that the vhb gene was expressed at the RNA level. Characterization of the vhb over-expressing transgenic plants revealed that transgenic seeds germinated faster than the wildtype controls. More importantly, the transgenic plants showed tolerance to a prolonged submergence treatment, suggesting that the vhb gene may provide an excellent tool for creation of submergence/flooding-tolerant cultivars of agriculturally important crops.

Adaptation, Physiological↗
PubMed1998

Inactivation of p53 but not p73 by adenovirus type 5 E1B 55-kilodalton and E4 34-kilodalton oncoproteins.

The adenovirus E1B 55-kDa and E4 34-kDa oncoproteins bind and inactivate the p53 tumor suppressor gene product, resulting in cell transformation. A recently discovered cellular protein, p73, shows extensive similarities to p53 in structure and function. Here we show that the simultaneous transient expression of E1B 55-kDa and E4 34-kDa proteins is sufficient to drastically shorten the intracellular half-life of p53, leading to strongly reduced steady-state p53 levels. Concomitantly, the E1B 55-kDa and E4 34-kDa proteins act synergistically to inactivate the transcriptional activity of p53. Mutational analysis suggests that physical interactions between the E1B 55-kDa protein and p53 and between the E1B 55-kDa and E4 34-kDa proteins are both required for p53 degradation. In contrast, the ability of p53 to interact with the cellular mdm2 oncoprotein or with its cognate DNA element appears to be dispensable for its destabilization by adenovirus gene products. The adenovirus E1B 55-kDa protein did not detectably interact with p73 and failed to inhibit p73-mediated transcription; also, the E1B 55-kDa and E4 34-kDa proteins did not promote p73 degradation. When five amino acids near the amino termini were exchanged at corresponding positions between p53 and p73, this rendered p53 resistant and p73 susceptible to complex formation and inactivation by the E1B 55-kDa protein. Our results suggest that while p53 inactivation is a central step in virus-induced tumor development, efficient transformation can occur without targeting p73.

Adenovirus E1B Proteins↗
PubMed1989

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction.

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.

Cloning, Molecular↗
PubMed1993

Strategies in pathogenesis: mechanistic specificity in the detection of generic signals.

The virulence genes of the plant pathogen Agrobacterium tumefaciens are induced by more than 40 low-molecular-weight phenolic compounds. The prevailing opinion is that (i) wound-derived phenols produced on breach of the integrity of the cell wall act as the initiating signal in a series of events which results in host cell transformation, and (ii) a classical membrane receptor, putatively VirA, is responsible for the recognition of all such phenolic inducers. Here, we argue that the discovery of the subset of inducers that are relatives of the dehydrodiconiferyl alcohol glucoside (DCG) growth factors redirects our attention to work on the plant wound as a site of cell division, and suggests that we further explore the implications of early work on the relationship between transformation efficiency and the status of the cell cycle of the host. In addition, we argue that the significant structural diversity allowed in the para position of the phenol ring of inducers suggests that a receptor-ligand interaction based solely on structural recognition is insufficient, but that recognition followed by a specific proton transfer event may be sufficient to explain vir induction activity. Hence, the specificity of the response of A. tumefaciens may be a consequence of the features required for a chemical reaction to occur on the receptor surface. Finally, we review affinity labelling studies which exploit this phenol detection mechanism and which provide evidence that the phenol receptor may be other than VirA, the sensory kinase of the two component regulatory system implicated in Agrobacterium virulence.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetophenones↗
PubMed1993

Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica.

Two DNA segments exhibiting ARS (autonomously replicating sequence) activity in the dimorphic yeast Yarrowia lipolytica were cloned from its chromosome on an integrative LEU2 plasmid. These ARS segments, designated Y1ARS1 and Y1ARS2, conferred on the hybrid plasmids high transformation efficiency and enabled extrachromosomal transmission of the plasmids in 1 or 2 copies per yeast cell under selective conditions. Deletion analysis showed that at least 728-1003 bp for Y1ARS1 and 1377-1629 bp for Y1ARS2 were required for full function. Both of these regions contained two 10/11 matches to an ARS core consensus in Saccharomyces cerevisiae, whereas neither was similar to the S. cerevisiae centromere sequence. Significantly, both Y1ARS elements contained at, or close to, their boundaries a 13 bp sequence, 5'-TATATTCAAGCAA-3', which resembles the cleavage site for topoisomerase II in Drosophila. A central 524 bp ClaI fragment of Y1ARS2 contained four stretches of a 17 bp direct repeat sequence, 5'-GAAAAACAAAAACAGGC-3', and exhibited the electrophoretic behavior typical of bent DNA.

Base Sequence↗
PubMed2005

A VicRK signal transduction system in Streptococcus mutans affects gtfBCD, gbpB, and ftf expression, biofilm formation, and genetic competence development.

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the vicRK signal transduction system in Streptococcus mutans. A vicK (putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a vicR (putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the vicRKX operon (Smuvic+). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the vicK mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic+. Based on real-time PCR, Smuvic+ showed increased gtfBCD, gbpB, and ftf expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the gtfB, gtfC, and ftf genes. Also, transformation efficiency in the presence or absence of the S. mutans competence-stimulating peptide was altered for the vic mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of vicK did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the vicRK signal transduction system in regulating several important physiological processes in S. mutans.

Animals↗
PubMed1991

Transformation and genomic restriction mapping of Rochalimaea spp.

Transformation procedures using electroporation were established for Rochalimaea quintana. Several cosmid/plasmids possessing the RK2 or RSF1010 origin of replication were successfully inserted. Plasmid retention and replication were verified by antibiotic resistance and Southern blot analysis. The highest level of transformation was obtained at a voltage field strength of 12.5 kV/cm with a pulse time of 10 miliseconds. Transformation efficiency was low (0.3%) with approximately 10(5) transformants/microgram of DNA. One construct, designated pAG10, reached sufficient levels in R. quintana to be isolated by density gradient centrifugation. Analysis of this plasmid after several cycles of growth in R. quintana revealed no obvious modifications. Physical maps of Rochalimaea spp. chromosomal DNA using pulse-field electrophoresis are being developed. Digestion of R. vinsonii chromosomal DNA with NotI or SfiI resulted in three and one fragments, respectively. When R. quintana was digested in a similar manner, both NotI and SfiI produced four fragments. Double digestion of R. quintana DNA with NotI and SfiI yield seven fragments ranging in size from 11 to 925 kb. Summing the fragments indicate an approximate genome size of 2.1 x 10(6) bp for R. vinsonii and 1.7 x 10(6) bp for R. quintana chromosomal DNA.

Electrophoresis, Gel, Pulsed-Field↗
PubMed1998

Direct interaction of Jak1 and v-Abl is required for v-Abl-induced activation of STATs and proliferation.

In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl-Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abl oncogene.

Animals↗
PubMed1994

The EBNA-2 arginine-glycine domain is critical but not essential for B-lymphocyte growth transformation; the rest of region 3 lacks essential interactive domains.

Since deletion of region 3 (amino acids [aa] 333 to 425) of Epstein-Barr virus nuclear protein 2 (EBNA-2) results in EBV recombinants which cannot transform primary B lymphocytes (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991), the role of domains of region 3 was investigated. Deletion of the Arg-Gly repeat domain, R-337GQSRGRGRGRGRGRGKG354, results in EBV recombinants that transform primary B lymphocytes with modestly decreased activity. The transformed cells grow slowly and are difficult to expand. EBNA-2 deleted for the Arg-Gly domain does not associate with the nuclear chromatin fraction. The Arg-Gly repeat has an intrinsic ability to bind to histone H1, to other proteins, including EBNA-1, and to nucleic acids, especially poly(G). Two independent deletions of each part of the rest of region 3 (aa 359 to 383 and 385 to 430) have little effect on transformation, while deletion of the rest of region 3 (aa 361 to 425) as a single segment substantially reduces transformation efficiency. EBNA-2 deleted for all of region 3 can still transactivate the LMP1 promoter in transient expression assays but is less active than EBNA-2 in transactivating the BamHI-C promoter. EBNA-2 deleted for the Arg-Gly domain is better than EBNA-2 at transactivating the LMP1 promoter and is as active as EBNA-2 in transactivating the BamHI-C promoter. These data are most compatible with a model in which the Arg-Gly domain of region 3 is a modulator of EBNA-2 interactions and activities, while the rest of region 3 is important in positioning the region 2 J kappa binding domain relative to the region 4 acidic transactivating domain. Despite the null phenotype of the region 3 deletion, region 3 is unlikely to mediate essential interactions with other proteins.

Amino Acid Sequence↗
PubMed2006

The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib.

Mutations in the Bcr-Abl kinase domain are a frequent cause of imatinib resistance in patients with advanced CML or Ph+ ALL. The impact of these mutations on the overall oncogenic potential of Bcr-Abl and on the clinical course of the disease in the absence of imatinib is presently unclear. In this study, we analyzed the effects of the Bcr-Abl P-loop mutation Y253H and the highly imatinib resistant T315I mutation on kinase activity in vitro and transforming efficiency of Bcr-Abl in vitro and in vivo. Immunoprecipitated Bcr-AblY253H and Bcr-AblT315I proteins displayed similar kinase activities and substrate phosphorylation patterns as Bcr-Abl wildtype. We directly compared the proliferative capacity of mutant to wildtype Bcr-Abl in primary BM cells in vitro and in a murine transplantation model of CML by using a competitive repopulation assay. The results implicate that in the absence of imatinib, there is no growth advantage for cells carrying Bcr-AblT315I or Bcr-AblY253H compared to Bcr-Ablwt, whereas imatinib treatment clearly selects for leukemic cells expressing mutant Bcr-Abl both in vitro and in vivo. Thus, the analysed Bcr-Abl mutants confer imatinib resistance, but do not induce a growth advantage in the absence of imatinib.

Amino Acid Substitution↗
PubMed1998

Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa.

Downregulation of the major autolysin in Streptococcus pneumoniae leads to penicillin tolerance, a feature that is characterized by the ability to survive but not grow in the presence of antibiotic. Screening a library of mutants in pneumococcal surface proteins for the ability to survive 10x minimum inhibitory concentration (MIC) of penicillin revealed over 10 candidate tolerance genes. One such mutant contained an insertion in the known gene psaA, which is part of the psa locus. This locus encodes an ABC-type Mn permease complex. Sequence analysis of adjacent DNA extended the known genetic organization of the locus to include two new open reading frames (ORFs), psaB, which encodes an ATP-binding protein, and psaC, which encodes a hydrophobic transmembrane protein. Mutagenesis of psaB, psaC, psaA and downstream psaD resulted in penicillin tolerance. Defective adhesion and reduced transformation efficiency, as reported previously for a psaA- mutant, were phenotypes shared by psaB-, psaC- and psaD- knockout mutants. Western blot analysis demonstrated that the set of mutants expressed RecA, but none of them showed translation of the autolysin gene, which is located downstream of recA. The addition of manganese (Mn) failed to correct the abnormal physiology. These results suggest that this ABC-type Mn permease complex has a pleiotropic effect on pneumococcal physiology including adherence and autolysis. These are the first genes suggested as being involved in triggering autolysin. The results raise the possibility that loss of function of PsaA, by vaccine-induced antibody for instance, may promote penicillin tolerance.

ATP-Binding Cassette Transporters↗