Products of a lagging DNA strand synthesis of nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA

There are three group I introns in the nuclear small subunit ribosomal RNA gene (SSU rDNA) of the ballistoconidiogenous anamorphic yeast-like fungus Tilletiopsis flava JCM 5186. The size of these sequences were 325 nt (position 516), 335 nt (position 1199) and 437 nt (position 1506), respectively. The introns at position 516 (T.flav516) and position 1199 (T.flav1199) belonged to subgroup IB3, and that of position 1506 (T.flav1506) belonged to subgroup IC1. The results of comparison with other group I introns found in SSU rDNA of eucaryotes showed that the positions 516 and 1199 were common positions to IB3 group I introns of fungi and green algae, and that positions 943, 1506 and 1512 were those to IC1 group I introns of fungi, and green and red algae. It is indicated that the insertion position of introns have close relationship with the nature of the subgroup to which they belonged. For phylogenetic analysis, we employed 9 IB3 introns, in which 7 were at position 516 and 2 were at position 1199, and 25 IC1 introns. The maximum likelihood tree based on the conserved region alignment showed that group I introns of subgroup IB3 were phylogenetically distant from those of subgroup IC1. T.flav516 (basidiomycete) constituted a subcluster with R.dacr516 (basidiomycete) and M.albo516 (ascomycete). T. flav1199 was located at the closer position of C.chlo1199 (green alga) than other IB3 introns at position 516. T.flav1506 was located at the subcluster, which was constituted by the 1506 introns found in SSU rDNA of fungi (B.yama1506, P.cari1506, and P.inou1506) and those of green algae (C.elli1506, C.mira1506, G.spir1506, and M.sacl1506) with IC1 introns at the position 1512 (D.parv1512 and C.sacc1512). The analysis of flanking regions showed that both 5' and 3' flanking sequences were well conserved in each insertion site, and indicated that the ancestors of the intron at different site had been inherited from the different origin. Therefore, the two IB3 introns found positions 516 and 1199, T.flav516 and T.flav1199, were supposed to have the independent ancestors. Our results supported the theory of the diversity of group I introns that group I introns had been transferred horizontally to the distinct insertion site, and were inherited and diverged vertically.     

Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recently we developed a positive selection vector for analysis of plasmid recombination events in B. subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Several lines of evidence suggest that single-strand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resemble potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.     

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilobases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.     

Characterization of polyamine synthesis pathway in Bacillus subtilis 168

Wnt-1, a secreted glycoprotein, participates in development of the nervous system and contributes to mammary oncogenesis when overexpressed. We show that GSK3 activity is decreased in mouse mammary cells transformed by Wnt-1. These cells also exhibit a substantial Wnt-1 dependent increase in the uncomplexed population of beta-catenin. Wnt-1 signaling does not change the steady state level of either GSK3 alpha or GSK3 beta but instead leads to an increased association between GSK3 beta and beta-catenin. HGF/SF treatment of mouse mammary cells also leads to a transient decrease in GSK3 activity and a parallel, selective increase in the uncomplexed pool of beta-catenin. Both Wnt-1 and HGF/SF lead to nuclear accumulation of beta-catenin and activation of a LEF/Tcf responsive reporter gene. This study defines a pivotal signal transduction pathway, activated by both Wnt-1 and HGF/SF, leading to decreased GSK3 beta activity and consequently an increase in the free pool and nuclear accumulation of beta-catenin and changes in gene expression.     

Gene vaccination with naked plasmid DNA: mechanism of CTL priming

The injection of naked plasmid DNA directly into the muscle cells of mice has been shown to induce potent humoral and cellular immune responses. The generation of a cytotoxic T lymphocyte (CTL) response after plasmid DNA injection may involve the presentation of the expressed antigen in the context of the injected myocytes' endogenous major histocompatibility (MHC)-encoded class I molecules or may use the MHC molecules of bone marrow-derived antigen presenting cells (APC) which are capable of providing co-stimulation as well. To resolve which cell type provides the specific restricting element for this method of vaccination we generated parent-->F1 bone marrow chimeras in which H-2bxd recipient mice received bone marrow that expressed only H-2b or H-2d MHC molecules. These mice were injected intramuscularly with naked plasmid DNA that encoded the nucleoprotein from the A/PR/8/34 influenza strain, which as a single antigen has epitopes for both H-2Db and H-2Kd. The resulting CTL responses were restricted to the MHC haplotype of the bone marrow alone and not to the second haplotype expressed by the recipient's myocytes. The role of somatic tissues that express protein from injected plasmids may be to serve as a reservoir for that antigen which is then transferred to the APC. Consequently, our data show that the mechanism of priming in this novel method for vaccination uses the MHC from bone marrow-derived APC, which are efficient at providing all of the necessary signals for priming the T cell.     

Simultaneous formation of functional leading and lagging strand holoenzyme complexes on a small, defined DNA substrate

The biochemical characterization of leading and lagging strand DNA synthesis by bacteriophage T4 replication proteins has been addressed utilizing a small, defined primer/template. The ATP hydrolysis activity of 44/62, the clamp loading complex responsible for holoenzyme assembly, was monitored during assembly of both the leading and lagging strand holoenzyme complex. The ATPase activity of 44/62 diminishes once a functional holoenzyme is assembled on both the leading and lagging strand. The assembly of the lagging strand holoenzyme is facilitated by several factors including biotinylated streptavidin blocks at the end of the fork strands, preassembly of the leading strand holoenzyme, and by the presence of the DNA primase with ribonucleoside triphosphates. The resultant minimal replicative complex consists of two holoenzymes and a primase nested on a model replication fork derived from a 62-mer template/34-mer primer/36-mer lagging strand in an apparent 2:2:1:1 ratio of 45 protein:polymerase:primase:forked DNA. The 44/62 protein complex does not remain associated with the complex. The primase alone slowly synthesizes pentaribonucleotides on the forked DNA when the lagging strand contains a nonannealed TTG initiation site with the rate of synthesis greatly stimulated by the addition of the 41 helicase. The addition of deoxy-NTPs to this complex results in leading strand synthesis, but extension of the synthesized RNA primer does not occur. DNA synthesis in both the leading and lagging strand directions is achieved, however, when a 6-mer DNA primer is annealed to the primase recognition site of the forked DNA substrate. A model is presented that describes how leading and lagging strand DNA synthesis might be coordinated as well as the associated molecular interactions of the replicative proteins.     

Comparison of strand breaks in plasmid DNA after positional changes of Auger electron-emitting iodine-125

To elucidate the kinetics of the induction of DNA strand breaks by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA after the decay of 125I (1) in proximity to DNA after minor-groove binding (125I-iodoHoechst 33342, 125IH) and (2) at a distance from DNA (125I-iodoantipyrine, 125IAP). Iodine-125 bound to the minor groove in DNA or free in solution is equally effective per decay in producing single-strand breaks (SSBs), while 125I bound to the minor groove is 6.7-fold more efficient than 125I free in solution in producing double-strand breaks (DSBs) (1.08 +/- 0.13 compared to 0.16 +/- 0.01 DSB/decay). Consequently, SSB to DSB ratios for 125IAP and gamma radiation (20.7 +/- 2.9 and 43.8 +/- 1.5, respectively) are greater than that for 125IH (2.9 +/- 0.4). Finally, the decay of 125IH leads to fragmentation of plasmid DNA beyond SSBs and DSBs.     

Genetic mapping of sporulation operons in Bacillus subtilis using a thermosensitive sporulation mutant

A thermosensitive sporulation mutant was used to determine the order of sporulation operonsin the urs region of the Bacillus subtilis chromosome. Data from three-factor transformation crosses and three- and four-factor transduction crosses established the order metC-SPO-96(SpoII)-spo-85(SpoV)-spo-279(SpoII)-furA-ura-cysC-spo-NG1.67(SpoIII). Previously, furA was thought to lie to the right of ura and cysC to the left (Dubnau, 1970; Young and Wilson, 1972).     

Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.     

ComEA is a DNA receptor for transformation of competent Bacillus subtilis

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.     

Feedback loops involving Spo0A and AbrB in in vitro transcription of the genes involved in the initiation of sporulation in Bacillus subtilis

The keto acid 2-oxo-4[methylthio]butanoic acid (OMTB) is an intermediate in the conversion of synthetic feed grade methionine sources to L-methionine in vivo in poultry and other animals. Because methionine sources are utilized by the chick with considerably less than 100% efficiency as sources of L-methionine, it is important to determine what metabolic process may limit the utilization of these sources. Because OMTB is converted to L-methionine by transamination, a study was conducted to determine which amino acids might serve as nitrogen donors in the conversion of OMTB to L-methionine in the chicken. Dialyzed tissue homogenates, mitochondria, and cytosol from liver, kidney, intestine, and skeletal muscle were incubated with OMTB and individual L-amino acids (isoleucine, leucine, valine, glutamic acid, aspartic acid, alanine, glutamine, asparagine, and phenylalanine) and the methionine that accumulated was determined by ion exchange chromatography. Tissues differed in the conversion of OMTB to methionine: kidney was most active, liver and intestinal mucosa were intermediate, and skeletal muscle had lowest activity. All amino acids supported methionine synthesis. Branched-chain amino acids and glutamic acid were the most effective substrates in tissue cytosols except in intestinal mucosa, in which asparagine was also effective. The preferred substrates in mitochondria were glutamate in liver mitochondria, isoleucine and alanine in kidney mitochondria, and branched-chain amino acids and glutamic acid in skeletal muscle mitochondria. All amino acids except alanine supported methionine synthesis from OMTB in mitochondria of intestinal mucosa. We conclude that a wide variety of amino acids can serve as substrates for transamination of OMTB in the chicken, and that the availability of nitrogen donors is unlikely to be a limiting factor in the conversion of OMTB to methionine.     

Homology in natural cryptic plasmids of Bacillus subtilis

Peculiarities of DNA homology in a number of cryptic plasmids isolated from soil bacillary strains and related or identical to Bacillus subtilis were studied. Fragments generated after digestion of one of these plasmids, p1414, were employed as a probe for blot hybridization with identical fragments of other plasmids. The data obtained suggest that nearly all the studied plasmids possess a common homology site that occupies a significant plasmid portion. Regions of detectable and weak homology were located throughout this site. Moreover, plasmid p1414 was shown to carry a large site that lacks homology with plasmids belonging to two groups. Eventually, one of these plasmids demonstrates complete lack of homology with the remaining plasmids.     

Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes

The present study was conducted to examine the effect of activin A on growth of rat hepatocytes. EGF induced a 10-fold increase in DNA synthesis as assessed by [3H]thymidine incorporation in cultured hepatocytes. When activin A was added together with EGF, DNA synthesis induced by EGF was markedly inhibited. Inhibition was detected at a concentration of 10(-10) M, and 5 x 10(-9) M activin A almost completely blocked EGF-mediated DNA synthesis. Similarly, activin A completely blocked DNA synthesis induced by hepatocyte growth factor/scatter factor. Activin A was capable of inhibiting EGF-mediated DNA synthesis, even when added 36 h after the addition of EGF. With the same time interval, TGF-beta also blocked EGF-induced DNA synthesis. Although both activin A and TGF-beta inhibited growth of hepatocytes in a similar manner, either activin A or TGF-beta did not compete with each other in their binding when assessed by competitive binding using an iodinated ligand. When hepatocytes were incubated with EGF, release of bioactivity of activin A into culture medium was detected after 48 h or later. Activity of activin A was released from parenchymal cells but not from nonparenchymal cells. mRNA for beta A subunit of activin was detected only slightly in unstimulated hepatocytes, but markedly increased at 48 h after the addition of EGF. To determine whether endogenously produced activin A affects DNA synthesis, we examined the effect of follistatin, an activin-binding protein that blocks the action of activin A. An addition of follistatin significantly enhanced EGF-induced DNA synthesis. Finally, in partial hepatectomized rat, expression of mRNA for beta A subunit in liver was markedly increased 24 h after the partial hepatectomy. These results indicate that activin A inhibits initiation of DNA synthesis in hepatocytes by acting on its own receptor and that activin A acts as an autocrine inhibitor of DNA synthesis in rat hepatocytes.     

Plasmid DNA in a groundwater aquifer microcosm--adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis

Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation. This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats. A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat. In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I. In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals. The divalent cations required to form the association were those present in the GWA/GW microcosm. The association was stable to extended elution over one week at 23 degrees C. Upon adsorption, the DNA became highly resistant against enzymatic degradation. About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW. Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis. However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA. The studies also revealed that in the transformation of B. subtilis Mg2+ can be replaced by Na+, K+ or NH4+. The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.