Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199.

The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from Sphingomonas aromaticivorans F199 has been determined. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene, m-xylene, and p-cresol are predicted to be distributed among 15 gene clusters. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. Conjugative transfer of pNL1 to another Sphingomonas sp. was demonstrated, and genes associated with this function were found in two large clusters. Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes.

Identification and sequence analysis of a 27-kilobase chromosomal fragment containing a Salmonella pathogenicity island located at 92 minutes on the chromosome map of Salmonella enterica serovar typhimurium LT2.

Using a genomic approach, we have identified a new Salmonella pathogenicity island, SPI-4, which is the fourth Salmonella pathogenicity island to be identified. SPI-4 was located at 92 min on the chromosome map and was flanked by the ssb and soxSR loci. The DNA sequence covering the entire SPI-4 and both boundaries was determined. The size of SPI-4 was about 25 kb and it contains 18 putative open reading frames (ORFs). Three of these ORFs encode proteins that have significant homology with proteins involved in toxin secretion. Another five ORFs encode proteins that have significant homology with hypothetical proteins from Synechocystis sp. strain PCC6803 or Acinetobacter calcoaceticus. The rest of the ORFs encode novel proteins, one of which has five membrane-spanning domains. SPI-4 is likely to carry a type I secretion system involved in toxin secretion. Furthermore, a previously identified locus (ims98), which is required for intramacrophage survival, was also mapped within the SPI-4 region. These findings suggested that SPI-4 is needed for intramacrophage survival.

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.

Power frequency magnetic fields do not contribute to transformation of JB6 cells.

The potential for power frequency magnetic fields to enhance neoplastic transformation has been investigated in vitro using promotion-sensitive mouse epidermal JB6 cells. In a soft agar assay, 60-Hz magnetic fields of 0.01, 0.1, 1.0, or 1.1 mT flux density did not induce anchorage-independent growth. In addition, these magnetic fields did not enhance tumor promoter-induced transformation showing no increase in the maximum number of transformed colonies and no shift in the dose-response curve. Thus, these data do not support the notion that environmental exposures to magnetic fields contribute to transformation.

Lung cancer response following inhaled radon in the A/J and C57BL/6J mouse.

A/J and C57BL/6J mice, strains which differ in susceptibility to chemical-induced lung tumours, were examined for differential sensitivity to radon-induced lung cancer. Sixty weeks after exposure to 952 WLM radon (3.34 J h1 m-3), no lung tumours were found in the control, sham- (one dust-) exposed, or radon-exposed C57BL/6J mice. In contrast, each of the A/J groups had a substantial number of tumours. Although there were no differences in overall lung tumour incidence or multiplicity associated with radon and ore dust exposure in A/J mice, distinct differences in tumour size and histopathological appearance were noted. The ratio of lung adenoma-to-carcinoma found in control A/J mice was 10:1, whereas the ratio in one dust-exposed animals was 10:8 and that in radon-exposed animals was 8:6. Based on these data, particulates appear to modify the promotion-progression of lung cancer in this strain. In addition, the appearance of two very large carcinomas histologically resembling Clara-cell-derived tumours only in the radon-exposed A/J mice and the possibility of alpha-particle-induced killing of target cells in the radon-exposed animals suggests that A/J but not the C57BL/6J, mice may have genes that modify susceptibility to radon-induced lung cancer.

Use of tagged random hexamer amplification (TRHA) to clone and sequence minute quantities of DNA--application to a 180 kb plasmid isolated from Sphingomonas F199.

We have developed a novel method to clone and sequence minute quantities of DNA. The method was applied to sequence a 180 kb plasmid pNL1. The first step was the production of a size distributed population of DNA molecules that were derived from the 180 kb plasmid pNL1. The first step was accomplished by a random synthesis reaction using Klenow fragment and random hexamers tagged with a T7 primer at the primer 5'-end (T7-dN6, 5'-GTAATACGACTCACTATAGGGCNNNNNN-3'. In the second step, Klenow-synthesized molecules were amplified by PCR using T7 primer (5'-GTAATACGACTCACTATAGGGC-3'). With a hundred nanograms starting plasmid DNA from pNL1, we were able to generate Klenow-synthesized molecules with sizes ranging from 28 bp to >23 kb which were detectable on an agarose gel. The Klenow-synthesized molecules were then used as templates for standard PCR with T7 primer. PCR products of sizes ranging from 0.3 to 1.3 kb were obtained for cloning and sequencing. From the same Klenow-synthesized molecules, we were also able to generate PCR products with sizes up to 23 kb by long range PCR. A total 232.5 kb sequences were obtained from 593 plasmid clones and over twenty putative genes were identified. Sequences from these 593 clones were assembled into 62 contigs and 99 individual sequence fragments with a total unique sequence of 86.3 kb.

Short exposures to 60 Hz magnetic fields do not alter MYC expression in HL60 or Daudi cells.

Analysis of changes in gene expression induced by 60 Hz magnetic fields has been considered to support an association between exposure to magnetic fields and cancer risk. Several reports have indicated that these fields rapidly activate many genes in mammalian cells. However, previous studies in this area have not provided sufficient information to support the conclusions drawn. To clarify this controversial research, we have attempted to validate, under rigorously controlled conditions, key experiments on induction of gene expression by magnetic fields. An extensive series of experiments, incorporating critical improvements in experimental design, most notably blind exposures and internal standards, was performed with human HL60 and Daudi cells. Exposure conditions covered a range of flux densities (5.7 microT to 10 mT) and times (20-60 min). No alteration in the human MYC gene, commonly referred to as c-myc, or beta-actin steady-state mRNA levels was observed. The lack of an effect was not attributable to exposure geometry, timing of RNA preparation, or serum lot and concentration. To eliminate any remaining variables, exact replication was performed in one of the laboratories previously reporting gene expression effects; again, no evidence for altered MYC expression was found. Finally, differential display PCR indicated that extremely low-frequency magnetic field-induced changes in gene expression were not prevalent.

An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain.

Protein-protein interactions involving specific transactivation domains play a central role in gene transcription and its regulation. The promoter-specific transcription factor Sp1 contains two glutamine-rich transcriptional activation domains (A and B) that mediate direct interactions with the transcription factor TFIID complex associated with RNA polymerase II and synergistic effects involving multiple Sp1 molecules. In the present study, we report the complementary DNA sequence for an alternatively spliced form of mouse Sp1 (mSp1-S) that lacks one of the two glutamine-rich activation regions present in the full-length protein. Corresponding transcripts were identified in mouse tissues and cell lines, and an Sp1-related protein identical in size to that predicted for mSp1-S was detected in mouse nuclear extracts. Cotransfection analysis revealed that mSp1-S lacks appreciable activity at promoters containing a single Sp1 response element but is active when multiple Sp1 sites are present, suggesting synergistic interactions between multiple mSp1-S molecules. The absence of a single glutamine-rich domain does not fully explain the properties of the smaller protein and indicates that additional structural features account for its unique transcriptional activity. The functional implications of this alternatively spliced form of Sp1 are discussed.

Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199.

A supercoiled 180-kb plasmid, pNL1, has been isolated from the deep-subsurface, chemoheterotrophic Sphingomonas sp. strain F199, and a physical map was generated. Analysis of a pNL1-derived cosmid library indicated that catechol 2,3-dioxygenase activity was linked to two distinct regions of the plasmid. Thus, the genes for aromatic catabolism in this Sphingomonas strain are, at least in part, plasmid encoded.

Design and fabrication of well confined uniform magnetic field exposure systems.

Exposure systems that provide good magnetic field uniformity, minimum stray fields, and minimal heating, vibration, and hum, as well as capability for true sham exposure in which current flows in the coils, are needed to determine rigorously the biological effects of weak magnetic fields. Designs based on acrylic polymer coil support structures and twisted pair bifilary coil windings were employed to fabricate several different systems for the exposure of laboratory animals and cell cultures to magnetic fields. These systems exhibit excellent performance characteristics in terms of exposure field uniformity, stray field containment, and exposure field cancellation in the sham exposure mode. A custom-written computer program was used to determine the best arrangement for coils with regard to field uniformity in the exposure volume and stray field containment. For in vivo exposures, modules were made up of four Merritt four-coil sets, built into a single structure and positioned to form an octapole with fields directed in the horizontal plane. For in vitro applications, two different coil configurations were selected to produce the vertical fields required. A quadrupole system, comprising modules consisting of two Merritt four-coil sets arranged side by side to limit stray fields, was built as a prototype. In the second configuration, one Merritt four-coil set was positioned inside the other to form a concentric coil set. In both in vitro systems, exposure chambers were connected to remote commercial incubators in order to reduce ambient magnetic fields in the exposure volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Microwave-specific heating affects gene expression.

The effects of low-level microwave radiation on gene expression in Escherichia coli have been examined in a sensitive model. We confirm the previously reported existence of an increase in beta-galactosidase expression by microwave radiation--an increase not duplicated by bulk heating. However, the effect was not frequency dependent and appeared to be due to heating effects peculiar to microwaves. These results indicate that small thermal gradients may be a source of biological effects of non-ionizing radiation.

Developmental expression of Sp1 in the mouse.

The expression of the trans-acting transcription factor Sp1 in mice was defined by a combination of RNA analysis and immunohistochemical localization of the Sp1 protein. Although ubiquitously expressed, there was an unexpected difference of at least 100-fold in the amount of Sp1 message in different cell types. Sp1 protein levels showed corresponding marked differences. Substantial variations in Sp1 expression were also found in some cell types at different stages of development. Sp1 levels appeared to be highest in developing hematopoietic cells, fetal cells, and spermatids, suggesting that an elevated Sp1 level is associated with the differentiation process. These results indicate that Sp1 has a regulatory function in addition to its general role in the transcription of housekeeping genes.

Localization of the gene for the trans-acting transcription factor Sp1 to the distal end of mouse chromosome 15.

The mouse chromosomal location for the gene (Sp1-1) encoding the trans-acting transcription factor Sp1 has been determined. Analysis of restriction fragment length polymorphisms in recombinant inbred, congenic, and interspecific backcross mice using human and mouse cDNA probes demonstrated that Sp1-1 is a single gene closely linked to the mammary tumor virus integration site-1 (Int-1) on the distal end of chromosome 15. Sp1 is a zinc finger protein, but Sp1-1 is not closely linked to any of the other zinc finger protein genes that have been mapped in mouse. Int-1 and other markers flanking the Sp1-1 locus are part of a conserved linkage group represented on human chromosome 12q.

SV40 stimulates expression of the transacting factor Sp1 at the mRNA level.

Expression of the trans-acting transcription factor Sp1 increased almost 10-fold after infection of cells by simian virus 40. This alteration, attributable to an early viral protein, occurred at the mRNA level beginning at 12 hr postinfection, shortly after the appearance of viral T antigen, and reached a plateau at 20 hr postinfection. The enhanced level of Sp1 message was accompanied by a marked increase in Sp1 protein in the cell nuclei. Furthermore, we have demonstrated that stimulation of Sp1 levels elevates expression from viral and cellular promoters. Enhancing the amount of this trans-acting factor may play a role in aiding the viral life cycle and in neoplastic transformation of infected cells.

Ultraviolet shadowing nucleic acids on nylon membranes.

We describe a method for the direct visualization of nucleic acids on nylon membranes. Nylon is weakly fluorescent under short wave ultraviolet light allowing membrane-bound nucleic acids to be detected with a sensitivity of 10 ng. This procedure involves no staining or destaining of the gels prior to transfer, does not require duplicate sample lanes or blots, and does not interfere with transfer of the nucleic acid to the membrane or subsequent hybridization.

A negative regulatory element with properties similar to those of enhancers is contained within an Alu sequence.

A negative regulatory element has been found within a member of the African green monkey Alu family of interspersed repeated sequences. This "reducer" element decreased transcription in both directions from a cellular simian virus 40-like bidirectional promoter independently of both orientation and position. The reducer was not promoter specific since it also decreased expression from the simian virus 40 early and human beta-globin promoters. In addition, the reducer decreased transcription from a polymerase III promoter. The reducer was contained in 38 base pairs of an Alu family member and interacted specifically with a monkey cell nuclear protein.

Sensitive model with which to detect athermal effects of non-ionizing electromagnetic radiation.

To clarify the potential of non-ionizing electromagnetic radiation to cause biological effects by athermal mechanisms, and to initiate elucidation of those mechanisms, a model system amenable to scrutiny at the molecular level has been designed and characterized. Assessment of beta-galactosidase activity in E. coli JM101 containing the plasmid pUC8 provides a sensitive assay with many important advantages. The ability to examine at the molecular level each of the processes involved in producing beta-galactosidase should permit elucidation of the molecular mechanism(s) that give rises to an observed effect.

Expression of class II-MHC antigens by tumor-associated and peritoneal macrophages: systemic induction during tumor growth and tumor rejection.

It was shown previously that combination therapy of tumor-bearing mice resulted in amplification of antitumor responses at the site of tumor rejection and involved some of the classical components of the immunological network. As a continuation of this work, we now show that amplification of immune responses involves an increase in class II-MHC antigen (Ia) expression both at the tumor site and peripherally in the peritoneal cavity. Of further interest, however, was the observation that there was also an increase in Ia expression by tumor-associated macrophages (TAM) and peritoneal macrophages (PMs) in mice bearing progressing tumors. In these experiments, Ia expression by TAM and PMs was assessed in C57BL/6J mice bearing the progressing syngeneic MCA/76-9 sarcoma as well as in mice that had received combination therapy consisting of an intraperitoneal injection of cyclophosphamide (CY) and an intravenous injection of tumor-sensitized T lymphocytes (immune cells). When progressing tumors were analyzed, it was seen that by the fourth day after tumor cell implantation, 45-60% TAM expressed Ia, a level that was sustained throughout the course of tumor growth. The treatment of tumor-bearers with CY had no effect on Ia expression by TAM. However, the number of Ia-expressing TAM increased significantly after combination therapy and, as the tumors regressed, reached a peak of up to 100% in the second week after therapy. TAM were shown by Northern blot hybridization to contain mRNA encoding for the Ia beta chain. When Ia expression by PMs was assessed, it was seen that during tumor progression there was an increased expression of Ia over background (from less than 10 to about 40%), beginning 9-12 days after tumor cell implantation and continuing for the duration of the experiments. This was not influenced by CY injection. Combination therapy significantly increased the number of PMs expressing Ia (up to 80%). Ia expression by PMs could be induced rapidly in vivo by injecting normal B6 mice intraperitoneally (ip) with T cells isolated from tumors induced to regress by combination therapy. PMs isolated up to 15 days after injection were shown to express Ia. Moreover, the ip injection of a mixture of specific MCA/76-9 tumor cells and these tumor-associated lymphocytes resulted in a more rapid rate and a higher level of Ia expression by PMs compared with the level induced by either treatment alone.(ABSTRACT TRUNCATED AT 400 WORDS)