Wound-released chemical signals may elicit multiple responses from an Agrobacterium tumefaciens strain containing an octopine-type Ti plasmid

The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The virH operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated virK. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ. Expression of these genes was also measured by nuclease S1 protection assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone. Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanch?e diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.     

Acidosis and glucocorticoids induce branched-chain amino acid catabolism

Chronic renal failure (CRF) is frequently complicated by malnutrition and wasting. The loss of lean body mass in CRF is the result of accelerated protein and amino acid degradation. Both appear to occur via acidosis-induced, glucocorticoid-dependent processes. In skeletal muscle, acidosis stimulates the activity of the rate-limiting enzyme in branched-chain amino acid metabolism, branched-brain ketoacid dehydrogenase (BCKAD). The activation of BCKAD in acidosis is likely to be glucocorticoid-dependent.     

The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F

The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.     

Identification of the replication region of the Lactobacillus acidophilus plasmid pLA106

Several naturally occurring antibiotic resistance plasmids were isolated from Pasteurella multocida type D strains. One plasmid, pPM1, was used to study transfer of DNA among P. multocida strains, and could be transferred into Escherichia coli and some P. multocida isolates. However, pPM1 could only be transferred into the toxigenic P. multocida LFB3 at very low frequency. Plasmid recovered from the electrotransformants could be transferred to LFB3 at high frequency. These plasmid DNAs were resistant to PstI, and sensitive to DpnI digestion. Sensitivity to DpnI was common to all the P. multocida DNAs, but resistance to PstI was confined to LFB3. Plasmid pPM1 treated with PstI methylase was able to transform LFB3 at an increased frequency compared to unmethylated DNA, suggesting that LFB3 has a restriction system which cleaves at or near PstI sites.     

Identification of the region required for monomerization of the rolling circle plasmid pKYM

A mild form of diabetes in young people was recognized in the pre-insulin era but was forgotten, probably because of Joslin's dictum that all young people with diabetes should have insulin as a safeguard against complications. After the introduction of sulphonylureas in the 1950s it was found, most notably by Fajans and Conn at the University of Michigan, that tolbutamide could improve or normalize carbohydrate tolerance in some young non-obese mildly diabetic patients. These experiments were not primarily of genetic interest because diabetes was regarded as homogeneous with young and old patients forming part of the same continuum. The question was whether treatment could prevent young subjects with mild diabetes progressing to a total loss of insulin reserve. By 1973, Fajans had shown that the carbohydrate intolerance of 45 patients diagnosed under age 25 had not progressed after up to 16 years on sulphonylureas. Nearly all (43 out of 45) these subjects had a first degree relative with diabetes. In 1974, under the title 'Mild familial diabetes with dominant inheritance' Tattersall described three families in which diabetes, although diagnosed in adolescence, could be treated with sulphonylureas over 40 years later and was dominantly inherited. Collaboration between Fajans and Tattersall established that 'chemical' diabetes in Michigan was also predominantly inherited and distinct from classical 'juvenile-onset' diabetes. In Paris in 1973 Lestradet also described a non-insulin-dependent form of childhood diabetes and later established that it was dominantly inherited. In 1974, Tattersall and Fajans coined the acronym MODY which was defined as 'fasting hyperglycaemia diagnosed under age 25 which could be treated without insulin for more than two years'.     

Electron cytochemical localization of gamma-aminobutyric acid catabolism in rat cerebellar cortex

An electron cytochemical technique is described for the localization of GABA-T, the enzyme which degrades the neurotransmitter GABA, in rat cerebellar cortex. The technique allows ultrastructural demonstration of GABA-T activity by the final deposition of an electron dense formazan precipitate at reaction sites, whilst maintaining adequate ultrastructural preservation for recognition of cellular and subcellular structures. Numerous electron dense precipitates are evident as discrete punctate deposits situated mainly in mitochondria of stellate cells, basket cells and astrocytic glial cells; they are also seen in axonal or dendritic profiles at some synaptic junctions. The technique enables the first cytochemical demonstration of the mitochondrial localization of GABA-T activity in nervous tissue to be presented. It establishes that GABA-T is present in supposed GABA neurones, in pre- or post-synaptic endings, or both, of presumed inhibitory synapses and in glial cells which may be associated with these synapses. From this seemingly ubiquitous distribution, functional aspects of GABA-T in these cells is considered.     

Molecular analysis of the F plasmid traVR region: traV encodes a lipoprotein

The nucleotide sequences of the conjugative F plasmid transfer region genes, traV and traR, have been determined. The deduced amino acid sequence of TraV indicated that it may be a lipoprotein; this was confirmed by examining the effect of globomycin on traV-encoded polypeptides synthesized in minicells. An open reading frame that may represent a previously undetected transfer gene, now designated trbG, was identified immediately upstream of traV. The deduced product of traR was found to share amino acid similarity with proteins from the bacteriophages 186 and P2 and with the dosage-dependent dnaK suppressor DksA.     

Impaired glycolysis and protein catabolism induced by acid in L6 rat muscle cells

BACKGROUND: In skeletal muscle, metabolic acidosis stimulates protein degradation and oxidation of branched-chain amino acids. This could occur to compensate for impairment of glucose utilization induced by acid. METHODS: To test this hypothesis, glycolysis and protein degradation (release of [14C]-phenylalanine) were measured in L6 skeletal muscle cells cultured in Eagle's minimum essential medium at pH 7.1 or 7.5 for up to 3 days. RESULTS: No marked changes in total DNA or in cell viability were detected, nor was there any significant effect on intracellular pH or the water content of the cells (which is thought to be a key regulator of protein turnover, especially in liver). In spite of this, acid stimulated protein degradation, induced net protein loss from the cultures, inhibited glucose uptake and glycolysis (lactate output) and was associated with increased [1-14C]-leucine oxidation. Effects on protein degradation and glycolysis were gradual, reaching a maximum after 20-30 h. To investigate whether glycolytic flux itself can influence protein degradation, increased glycolysis was simulated by adding glucose (20 mmol L-1) or pyruvate (1 mmol L-1) to the medium. At pH 7.1, neither addition had any effect on protein degradation. CONCLUSION: Although acid-induced protein wasting is associated with impaired glycolysis, no obligatory coupling exists between glycolytic flux and protein degradation.     

Analysis of the oxidative catabolism of retinoic acid in rat Dunning R3327G prostate tumors

Transforming growth factor-beta (TGF-beta) is a pleiotropic regulatory factor of tissue remodeling. Angiogenesis, a prerequisite of tissue repair and tissue expansion, is induced by TGF-beta in vivo, while proliferation and migration of cultured endothelial cells are inhibited by TGF-beta. Indirect mechanisms stimulating angiogenesis and modification of TGF-beta effects by cell-matrix interaction have been postulated to account for this paradigm. Because cellular behavior in tissue remodeling is decisively determined by cell-matrix interactions, which in turn is mediated via integrins, we investigated the effect of TGF-beta on matrix-dependent endothelial cell functions. Integrin expression of human dermal microvascular endothelial cells (HDMEC) was measured by Northern blot and fluorescence-activated cell sorter analysis after TGF-beta treatment and correlated to cell-matrix interactions, which were studied in a colorimetric cell attachment assay as well as the Boyden chamber chemotaxis assay. We found a cell-specific downregulation of integrin expression in HDMEC on the level of mRNA as well as on the cell surface. This effect correlated well with the reduction of integrin-dependent cell adhesion to several matrix proteins, in particular to fibronectin. Moreover, TGF-beta decreased fibronectin-induced chemotaxis of HDMEC. Thus, TGF-beta controls cell-matrix interaction of HDMEC by down-regulation of integrin expression. This effect of TGF-beta reflects direct and cell-specific control mechanisms on microvascular cells that may be critical for the coordinated process of angiogenesis requiring a balance of stimulatory and inhibitory factors.     

Identification and DNA sequence of the mobilization region of the 5-nitroimidazole resistance plasmid pIP421 from Bacteroides fragilis

The nucleotide sequence of the DNA mobilization region of the 5-nitroimidazole resistance plasmid pIP421, from strain BF-F239 of Bacteroides fragilis, was determined. It contains a putative origin of transfer (oriT) including three sets of inverted repeats and two sequences reminiscent of specific integration host factor binding sites. The product of the mobilization gene mob421 (42.2 kDa) is a member of the Bacteroides mobilization protein family, which includes the MobA of pBI143, NBUs, and Tn4555. Sequence similarity suggests that it has both oriT binding and nicking activities. The transfer frequency of pIP421 in a B. fragilis donor strain possessing a Tc(r) or Tc(r) Em(r)-like conjugative transposon was significantly enhanced by tetracycline. Moreover, the mobilization region of pIP421 confers the ability to be mobilized from Escherichia coli by an IncP plasmid.     

Deep drawable ultra low carbon Ti IF steels hot rolled in the ferrite region

The control of the amount of solute carbon in ultra low carbon Ti IF steels during ferrite rolling and subsequent recrystallization is of prime importance for the development of an appropriate recrystallization texture and for the production of thin deep drawable hot strips. In the present work, the effect of the solute carbon content and the rolling conditions on the recrystallization texture after ferrite rolling and on the corresponding Lankford value was quantified. Therefore, ultra low carbon Ti IF steels with different sulphur and titanium contents were rolled in the ferrite region, in order to obtain a variation in solute carbon content (from 0 to about 10 ppm) at the ferritic rolling temperatures. It was shown that a deep drawing grade (r_mean> 1.4) can be obtained if the chemical composition of the steel guarantees a complete stabilisation of the solute carbon in the austenitic temperature region and if sufficient strain (85%) is given in the finishing train at temperatures lower than about 800°C. It can be concluded that the sulphur and titanium contents have to be chosen slightly higher in comparison to the conventional Ti IF steel grades used for cold rolling and annealing.     

A study of branched-chain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched-chain amino acids in Saccharomyces cerevisiae

The specific activity of branched-chain amino acid aminotransferase was highest when S. cerevisiae was grown in minimal medium containing a branched-chain amino acid as nitrogen source. Growth in complex media with glycerol or ethanol gave moderately high levels, whereas with glucose and fructose the specific activity was very low. Mutagenesis defined three genes (BAA1 to BAA3) required for branched-chain amino acid catabolism. The baa1 mutation reduced the specific activity of the aminotransferase, the stationary phase density in YEPD and caused gross morphological disturbance. Branched-chain amino acid aminotransferase is essential for sporulation.     

Characterization of the acc operon from the nopaline-type Ti plasmid pTiC58, which encodes utilization of agrocinopines A and B and susceptibility to agrocin 84

The acc locus from the Ti plasmid pTiC58 confers utilization of and chemotaxis toward agrocinopines A and B (A+B), as well as susceptibility to a highly specific antiagrobacterial antibiotic, agrocin 84. DNA sequence analyses revealed that acc is composed of eight open reading frames, accR and accA through accG. Previous work showed that accR encodes the repressor which regulates this locus, and accA codes for the periplasmic binding protein of the agrocinopine transport system (S. Beck Von Bodman, G. T. Hayman, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 89:643-647, 1992; G. T. Hayman, S. Beck Von Bodman, H. Kim, P. Jiang, and S. K. Farrand, J. Bacteriol. 175:5575-5584, 1993). The predicted proteins from accA through accE, as a group, have homology to proteins that belong to the ABC-type transport system superfamily. The predicted product of accF is related to UgpQ of Escherichia coli, which is a glycerophosphoryl diester phosphodiesterase, and also to agrocinopine synthase coded for by acs located on the T-DNA. The translated product of accG is related to myoinositol 1 (or 4) monophosphatases from various eucaryotes. Analyses of insertion mutations showed that accA through accE are required for transport of both agrocin 84 and agrocinopines A+B, while accF and accG are required for utilization of the opines as the sole source of carbon. Mutations in accF or accG did not abolish transport of agrocin 84, although we observed slower removal of the antibiotic from the medium by the accF mutant compared to the wild type. However, the insertion mutation in accF abolished detectable uptake of agrocinopines A+B. A mutation in accG had no effect on transport of the opines. The accF mutant was not susceptible to agrocin 84 although it took up the antibiotic. This finding suggests that agrocin 84 is activated by AccF after being transported into the bacterial cell.     

Preferential localization of DNA damage induced by depurination and bleomycin in a plasmid containing a scaffold-associated region

Recent evidence suggests that DNA damage of various origins is not randomly distributed in the genome but appears to be clustered in unidentified hypersensitive regions of the chromatin. A model was proposed that stipulates that unpaired DNA stretches, such as those found in scaffold- (or matrix)-associated regions (SARs) under torsional strain, are candidate regions of hypersensitivity to DNA damage in vivo. In this study, we assessed in vitro the relative susceptibility of supercoiled plasmids containing a SAR or chromatin loop DNA segment to DNA damage induced by acid-catalyzed depurination or FeIII-bleomycin. Single-strand specific S1 nuclease was used in combination with 3'-end-labeling to detect single-strand breaks or gaps, after cleavage of abasic sites or removal of 3'-phosphoglycolates by Escherichia coli endonuclease IV. The optimal conditions of DNA cleavage specificity by S1 nuclease were determined. Using these conditions, the DNA cleavage patterns obtained showed (i) a preferential localization of S1 hypersensitive sites in the SAR DNA as compared with plasmid or chromatin loop DNA and (ii) a strikingly similar localization of DNA damage with the two clastogenic treatments.     

Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315

The complete nucleotide sequence of the 8.7-kb theta-replicating plasmid pUCL287 from Tetragenococcus halophilus (formerly Pediococcus halophilus) ATCC33315 has been determined. The replication region was identified and analyzed. Its nucleotide sequence contains an untranslated region, the replication origin, followed by two open reading frames (ORFs) encoding two proteins of 311 (RepA287) and 168 (RepB287) amino acids, respectively. Evidence is presented to show that RepA287 represents the plasmid replication protein. RepB287, which is non-essential for replication, is involved in the plasmid copy-number control and segregational stability. The roles of lactococcal proteins homologous to RepB287 have not been defined so far. Nevertheless, the structural organization of the pUCL287 replication region is remarkably similar to those of well known theta-replicating lactococcal plasmids despite the absence of homology of the replication origin and of the replication protein, and this suggests that pUCL287 uses the same mechanism of replication. Nucleotide sequence comparisons show that pSMB74, a pediococcal plasmid encoding bacteriocin production, is a member of the pUCL287 replicon family.     

Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance

Electron paramagnetic resonance spectroscopy was employed to monitor the dynamics associated with a B-Z transition in both a linear (dG-dC)n and a modified pUC8 plasmid. A spin label consisting of cytidine substituted in position C5 with an 11-atom-tethered 5-membered ring nitroxide (DCAVAP) was incorporated into linear (dG-dC)n with Micrococcus luteus DNA polymerase or into a specific 34-bp Z-DNA-forming region of the 2.7-kb plasmid pRDZ8 with Thermus aquaticus DNA polymerase (Stoffel fragment). Although DCA-VAP is a modified nucleotide, it was an excellent substrate for both enzymes. The Z conformation was induced by changing the salt concentration from 0.01 to 4.5 M. The EPR line shape changed in response to the switch in DNA conformation. The degree of change was quantitatively similar for both the linear polymer and the plasmid with its Z-DNA-forming region. A motional analysis which focuses on local dynamics indicates that the order parameter S for the spin-labeled systems increases upon conversion from B-DNA to Z-DNA. This decrease in motional freedom is consistent with the observation that Z-DNA is more rigid than B-DNA.     

Arginine catabolism and the arginine succinyltransferase pathway in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.