Transcription of the halophage phi H repressor gene is abolished by transcription from an inversely oriented lytic promoter.

The temperate phage phi H of the extremely halophilic archaebacterium Halobacterium salinarium encodes a repressor, Rep, which in the immune state represses the production of an early lytic transcript, denoted T4. Rep acts at the transcriptional level by blocking the promoter for T4. The promoter for the rep gene itself is positioned back to back to the promoter for T4, in a manner analogous to that of the cI/cro genes in bacteriophage lambda. Transcription of the rep gene does not occur when the phase is growing lytically. We show that this repressor of rep transcription during lytic growth is due to the transcription per se from the stronger, oppositely oriented promoter for T4, without the need of a phage gene product.

Silica exposure is associated with increased risk of developing rheumatoid arthritis: results from the Swedish EIRA study.

OBJECTIVE: To study the association between silica exposure and rheumatoid arthritis and how it is modified by cigarette smoking. METHODS: Data were analysed from 276 male cases and 276 male controls aged 18 to 70 years, included in a Swedish population based study between May 1996 and June 2001. A case was defined as a person recently diagnosed with rheumatoid arthritis according to the ACR criteria. Controls were selected from the study base as a stratified random sample accounting for age, sex, and residency. Men with a self reported history of work with rock drilling, stone crushing, or exposure to stone dust in general were defined as silica exposed. Rheumatoid factor (RF) status among cases was recorded. RESULTS: Silica exposed men had increased risk of rheumatoid arthritis, with an odds ratio (OR), adjusted for age, residential area, and smoking, of 2.2 (95% confidence interval, 1.2 to 3.9) among men aged 18 to 70 years, and 2.7 (1.2 to 5.8) among those aged 50 to 70 years. Men who had worked with rock drilling or stone crushing (regarded as highly exposed) had a slightly greater increase in risk of rheumatoid arthritis than silica exposed men in general, with an OR of 3.0 (1.2 to 7.6). The joint effects of silica exposure and smoking were compatible with synergy between these two exposures in the development of rheumatoid arthritis but this was not conclusive. CONCLUSIONS: Silica exposure is associated with increased risk of developing rheumatoid arthritis. This association is not explained by smoking habits.

Structure specific ds/ss-RNase activity in the extreme halophile Halobacterium salinarium.

A ds/ss-RNA processing activity involved in antisense-RNA mediated gene regulation in the extremely halophilic archaebacterium Halobacterium salinarium was investigated in vivo. H.salinarium cells were transformed with DNA encoding an RNA species complementary to a part of the major lytic transcript, termed T4, of the H.salinarium phage phi H. The transformants transcribing this construct, when infected by phage were able to process T4 in a similar way to the processing of the lytic transcript denoted T1, in the natural sense-antisense system. Processing of T4 was not observed under normal phage growth on wild-type cells. Thus the antisense-RNA mediated processing activity earlier reported is dependent on the presence of an RNA duplex and is not sequence specific.

Antisense RNA mediates transcriptional processing in an archaebacterium, indicating a novel kind of RNase activity.

Strains of the extremely halophilic archaebacterium Halobacterium salinarium that are lysogenic for the phage phi H produce an antisense RNA transcript complementary to the first 151 nucleotides (nt) of the early lytic phage transcript T1. This is the first case of antisense control of gene expression in an archaebacterium. We show through transformation of H. salinarium that the antisense RNA functions in trans, rendering the early lytic phage transcript T1 susceptible to specific cleavage by an unidentified RNase of unique endonucleolytic activity. The single-stranded ends of RNA are cut off at the ends of the 151 nt RNA duplex, removing the ribosomal binding sites from the first open reading frame of transcript T1 but without concomitant digestion of the products.

In vivo studies on the effects of immunity genes on early lytic transcription in the Halobacterium salinarium phage phi H.

We have studied in vivo the effects of putative immunity genes on the expression of an early lytic gene of the Halobacterium salinarium phage. phi H. We transformed an H. salinarium host with DNA coding for a putative repressor gene, the transcript of which has been designated T6. We show that, in vivo, this gene specifically shuts off production of the early lytic transcript T4. A construct carrying the DNA transcribed as T4, but without its putative repressor binding sequences, shows T4 transcription enhanced to a level comparable to that observed in lytic growth of mutant phages capable of growing on immune H. salinarium strains. This transcript is insensitive to the action of the T6 product. The product of this 'unrepressed' T4 transcript is able to complement in trans the repressed T4 on superinfecting phi H-sensitive phages, allowing these to grow on a strain containing the repressor gene. It has, however, no effect on the production of repressor. We also mapped the start and end points of two other transcripts, T9 and T10, which are expressed only in the lysogenic state by cells immune to superinfection by phage, cloned the coding DNA and used it to transform H. salinarium. This DNA, though transcribed by the transformants, has no detectable effect on the cells, which remain susceptible to phage infection.

In vivo and in vitro analysis of transcription of the L region from the Halobacterium salinarium phage phi H: definition of a repressor-enhancing gene.

The 12-kb L region of the Halobacterium salinarium phage phi H is able to replicate as a plasmid, conferring a certain immunity to the host cell. We show here that the whole region is utilised for transcription at one stage or another in the phage life cycle. The DNA segment between the lytic transcripts T4 and T1 is shown to be constitutively transcribed. The effects of transcription on immunity were investigated using transformation experiments with H. salinarium. In this way, we show in vivo that the immune transcript T9, which on its own has no influence on phage growth or immunity, has a co-operative effect on the phi H repressor. The immunity effects analysed are sufficient to account for the degree of immunity conferred by the L region.

Mutational analysis of the regulatory region of the Mycobacterium plasmid pAL5000.

The regulatory region of the Mycobacterium fortuitum plasmid pAL5000 was studied in vivo and in vitro by mutational analysis. This region comprises the origin of replication for the plasmid and the start point of transcription for the repA/B genes, which encode the two replication proteins RepA and RepB. In this region there are two binding sites for RepB: a low-affinity site which is probably the origin of replication and a high-affinity-site which overlaps the promoter and implies an autoregulated expression of RepB. The high-affinity site contains two 8 bp palindromes, as well as an inverted repeat structure. By introducing point mutations into each of these motifs and monitoring changes to RepB binding in a gel-retardation assay, it was shown that the central, GC-rich palindrome (the GC-box) is the most important motif for protein binding. Mutations in the second, AT-rich palindrome (the AT-box) had no effect on protein binding and the inverted repeat structure per se was not needed, though some single-base changes affected binding to one or other of the DNA strands. These mutations were subsequently tested in vivo for their effects on plasmid replication in Mycobacterium smegmatis. Any change to the GC-box abolished replication, but changes to the other motifs were dependent on the position of the changed base, again indicating that the inverted repeats are not essential and that the AT-box is part of the promoter and not primarily recognised by RepB. The mutated plasmids did not show any changes in copy number to that of the wild-type. The expression of RepB was boosted by introducing a stronger promoter upstream of the repA/B genes. The resulting plasmid was capable of increasing to a degree in trans the copy number of other plasmids carrying the ori region, but was unstable when present on its own in M.smegmatis.

Functional definition of regions necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid pAL5000.

Different parts of the Mycobacterium fortuitum plasmid pAL5000 necessary for plasmid replication and incompatibility were defined and studied. Two ORFs, named repA and repB, were defined which are necessary for replication. A pAL5000 derivative deleted in these genes can be made to replicate by providing the gene products in trans. A 435 bp fragment was defined which was necessary in cis for replication and which had an influence on copy number. This region (inc), which contains several repeated motifs, was also able to confer a degree of incompatibility when cloned into an otherwise unrelated mycobacterial replicon. pAL5000-derived plasmids carrying two copies of the inc region had a lower copy number and were less stable than the wild-type. These effects were only observed when the two regions were in the same orientation. Plasmids carrying only the inc region and no other parts of pAL5000 could be made to replicate if repA and repB were supplied in trans from another plasmid. Based on these findings, systems for selectively curing cells of one plasmid of a pair were designed and shown to be functional in Mycobacterium smegmatis. These have potential as a simple delivery system for achieving transposon mutagenesis or gene replacement in mycobacteria.

Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000.

Plasmid pAL5000 from Mycobacterium fortuitum encodes two proteins necessary for replication: RepA (307 amino acid residues) and RepB (119 residues). A single RNA species encoding these proteins was characterized, and its 5' end was defined. The proteins were expressed as maltose-binding protein fusions in Escherichia coli. The RepB protein was shown in vitro to bind specifically to a previously defined 435-bp region of pAL5000 containing the origin of replication (ori). The precise RepB binding sites were defined by DNase I footprinting experiments. RepB binds to two motifs in the ori region: a high-affinity site within its own promoter region, implying autoregulation of its expression, and a low-affinity site further upstream, presumably the origin of replication itself. The binding to the latter motif seems to occur on one DNA strand only. The high-affinity binding site contains several palindromic sequences. Gel retardation assays were performed with the different binding sites as templates, and the binding constant to each site was estimated from protein titrations. This is the first molecular dissection of mycobacterial DNA-binding proteins and their interactions with their targets.

Genes for DNA cytosine methyltransferases and structural proteins, expressed during lytic growth by the phage phi H of the archaebacterium Halobacterium salinarium.

Lytic genes and transcription from the Halobacterium salinarium phage phi H were studied. Genes for three structural proteins were located to the left arm of the linear phage genome. The right arm was shown to encode three DNA cytosine methyltransferases, the first such sequences reported from an archaebacterium. One cytosine methyltransferase is of the N(4)-methyltransferase type. The other two open reading frames (ORFs) seem to be parts of the same gene, which has been split by a recombination event. This gene product is of the C5-methyltransferase type. The methyltransferase genes are the first phi H genes detected showing high homology to eubacterial proteins. Five of the six described gene products have a higher proportion acidic over basic amino acid residues, a common characteristic of halobacterial proteins. Lytic phi H transcription was shown to produce three RNA species, two shorter species encoding the methyltransferase genes and one large species transcribed from both the right and the left phage arm and subsequently being processed upstream of the region encoding the structural proteins.

Identification of promoter elements in mycobacteria: mutational analysis of a highly symmetric dual promoter directing the expression of replication genes of the Mycobacterium plasmid pAL5000.

The 120 bp origin of replication (ori) for the Mycobacterium plasmid pAL5000 has been shown to comprise the binding sites for the replication protein RepB as well as the start site of transcription for the repA and repB genes, encoding the replication proteins RepA and RepB. In this work it is demonstrated that a third gene product, Rap, is involved in replication in addition to the previously described proteins. Mycobacterium smegmatis cells transformed with replicons carrying the rap gene recover markedly faster upon electroporation than those transformed with the minimal replicon, which lacks rap. The rap gene, oppositely orientated to repA/B, was shown to be transcribed from a promoter orientated back-to-back to and overlapping the repA/B promoter. As a consequence of the extensive dyad symmetry in this region the two promoters share several elements, most of which are situated inside the high-affinity RepB-binding motif in the ori. Transcription of rap runs through the low-affinity RepB-binding site, which is part of the ori and necessary for replication. Both promoters were shown to be repressed by RepB. These divergent promoters were studied through site-specific mutagenesis in a xylE reporter gene assay. The analysis furnished evidence supporting the existence of a distal as well as a proximal element in mycobacterial promoters.

The immunity-conferring plasmid p phi HL from the Halobacterium salinarium phage phi H: nucleotide sequence and transcription.

The complete nucleotide sequence of the plasmid p phi HL, composing the central 12,041-bp L-region from the temperate phage phi H of Halobacterium salinarium is presented. Transcripts mapped to the p phi HL and the L-region produced under immune conditions, under lytic growth or constitutively, are described. The sequences upstream of the transcription start points show homology to the consensus sequence for archaeal (formerly archaebacterial) promoters. Lytic transcription is shown to be strictly time-dependent, with an early gene product required for the expression of late genes.

What precedes development of rheumatoid arthritis?

Studies on aetiology of inflammatory diseases such as rheumatoid arthritis (RA) need to investigate the potential environmental triggers that are active before onset of disease, the genetic context in which these triggers act, and whether the presence of such triggers in an arthritis prone genetic context will give rise to the immune reactions associated with/preceding RA. Such knowledge would help not only to address much better the issue of causality of these potential triggers and the immune reactions, but also to carry out various interventions aimed at influencing the disease provoking immune events before development of clinical signs of disease. This short report summarises recent data demonstrating (a) the presence of anticitrullin antibodies or rheumatoid factors in between a third and half of patients with RA before development of clinical signs; (b) long term smoking is associated with a high risk of future development of seropositive but not seronegative RA; and (c) a strong gene-environment interaction between smoking and SE genes in the development of seropositive RA. We conclude that, in a certain genetic context, smoking is a potential trigger of RA, and a combination of the two factors is associated with the occurrence of immune reactions long before the onset of RA.

Quantification of the influence of cigarette smoking on rheumatoid arthritis: results from a population based case-control study, using incident cases.

OBJECTIVE: To quantify the influence of cigarette smoking on the risk of developing rheumatoid arthritis (RA). METHODS: 679 cases and 847 controls included during May 1996-June 2000 in a case-control study, using incident cases, comprising the population aged 18-70 years of a defined area of Sweden, were investigated. A case was defined as a person from the study base who received for the first time a diagnosis of RA using the 1987 American College of Rheumatology criteria, and controls were randomly selected from the study base. Self reported smoking habits among cases and controls, and rheumatoid factor status among cases were registered. The incidence of RA in current smokers, ex-smokers, and ever-smokers, respectively, was compared with that of never-smokers. RESULTS: Current smokers, ex-smokers, and ever-smokers of both sexes had an increased risk for seropositive RA (for ever-smokers the odds ratio was 1.7 (95% confidence interval (95% CI) 1.2 to 2.3) for women, and 1.9 (95% CI 1.0 to 3.5) for men), but not for seronegative RA. The increased risk was only apparent among subjects who had smoked > or =20 years, was evident at an intensity of smoking of 6-9 cigarettes/day, and remained for up to 10-19 years after smoking cessation. The risk increased with increasing cumulative dose of smoking. CONCLUSION: Smokers of both sexes have an increased risk of developing seropositive, but not seronegative, RA. The increased risk occurs after a long duration, but merely a moderate intensity, of smoking and may remain for several years after smoking cessation.

Genomic organization of the human ELF3 (ESE-1/ESX) gene, a member of the Ets transcription factor family, and identification of a functional promoter.

We recently isolated a novel member of the Ets transcription factor/oncogene family, ESE-1/ESX/ELF3, with features distinct from any other Ets-related factor. ELF3 is the prototype of a new subclass of Ets factors, contains two DNA-binding domains, and, in contrast to any known Ets factor, is expressed exclusively in epithelial cells. ELF3 expression is induced during differentiation of the epidermis, indicating a role in the regulation of terminal differentiation genes in the epidermis. Due to the important role that other Ets factors play in cellular differentiation, ELF3 is expected to be a critical regulator of epithelial gene expression. We report here the cloning and the structural organization of the human ELF3 gene. The human ELF3 gene contains nine exons, which span approximately 5.8 kb of genomic DNA. Intron/exon borders and number of exons are almost identical to those in the mouse ELF3 gene. Comparison of the immediate promoter regions of the human and mouse ELF3 genes demonstrates the presence of TATA and CCAAT boxes as well as potential binding sites for Ets factors and NF-kappaB. Transfection experiments demonstrate that a 1.5-kb fragment of the 5' upstream region acts as a strong promoter in two epithelial cell lines.