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Background: All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA)., Results: Stenotrophomonas maltophilia is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. The availability of the whole DNA sequence of the S. maltophilia strain K279a allowed us to set up fast and accurate PCR-based diagnostic protocols based on the measurement of length variations of loci carrying a variable number of short palindromic repeats marking the S. maltophilia genome. On the basis of the amplimers size, it was possible to deduce the number of repeats present at 12 different loci in a collection of S. maltophilia isolates, and therefore label each of them with a digit. PCR-negative regions were labelled 0. Co-amplification of two pairs of loci provided a 4-digit code sufficient for immediate subtyping. By increasing the number of loci analyzed, it should be possible to assign a more specific digit profile to isolates. In general, MLVA data match genotyping data obtained by PFGE (pulsed-field gel electrophoresis). However, some isolates exhibiting the same PCR profiles at all loci display distinct PFGE patterns., Conclusion: The utilization of the present protocol allows to type several S. maltophilia isolates in hours. The results are immediately interpretable without the need for sophisticated softwares. The data can be easily reproducible, and compared among different laboratories.

YPALs (Yersinia palindromic sequences) are miniature DNA insertions scattered along the chromosomes of yersiniae. The spread of these intergenic repeats likely occurred via transposition, as suggested by the presence of target site duplications at their termini and the identification of syntenic chromosomal regions which differ in the presence/absence of YPAL DNA among Yersinia strains. YPALs tend to be inserted closely downstream from the stop codon of flanking genes, and many YPAL targets overlap rho-independent transcriptional terminator-like sequences. This peculiar pattern of insertion supports the hypothesis that most of these repeats are cotranscribed with upstream sequences into mRNAs. YPAL RNAs fold into stable hairpins which may modulate mRNA decay. Accordingly, we found that YPAL-positive transcripts accumulate in Yersinia enterocolitica cells at significantly higher levels than homologous transcripts lacking YPAL sequences in their 3' untranslated region.

Genome-wide analyses carried out in silico revealed that the DNA repeats called enterobacterial repetitive intergenic consensus sequences (ERICs), which are present in several Enterobacteriaceae, are overrepresented in yersiniae. From the alignment of DNA regions from the wholly sequenced Yersinia enterocolitica 8081 and Yersinia pestis CO92 strains, we could establish that ERICs are miniature mobile elements whose insertion leads to duplication of the dinucleotide TA. ERICs feature long terminal inverted repeats (TIRs) and can fold as RNA into hairpin structures. The proximity to coding regions suggests that most Y. enterocolitica ERICs are cotranscribed with flanking genes. Elements which either overlap or are located next to stop codons are preferentially inserted in the same (or B) orientation. In contrast, ERICs located far apart from open reading frames are inserted in the opposite (or A) orientation. The expression of genes cotranscribed with A- and B-oriented ERICs has been monitored in vivo. In mRNAs spanning B-oriented ERICs, upstream gene transcripts accumulated at lower levels than downstream gene transcripts. This difference was abolished by treating cells with chloramphenicol. We hypothesize that folding of B-oriented elements is impeded by translating ribosomes. Consequently, upstream RNA degradation is triggered by the unmasking of a site for the RNase E located in the right-hand TIR of ERIC. A-oriented ERICs may act in contrast as upstream RNA stabilizers or may have other functions. The hypothesis that ERICs act as regulatory RNA elements is supported by analyses carried out in Yersinia strains which either lack ERIC sequences or carry alternatively oriented ERICs at specific loci.

Pro-inflammatory cytokines, environmental stresses, as well as receptor tyrosine kinases regulate the activity of JNK. In turn, JNK phosphorylates Jun members of the AP-1 family of transcription factors, thereby controlling processes as different as cell growth, differentiation, and apoptosis. Still, very few targets of the JNK-Jun pathway have been identified. Here we show that JNK is required for the induction of c-myc expression by PDGF. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the promoter of the c-myc proto-oncogene that recruits in vivo the c-Jun and JunD AP-1 family members and controls the PDGF-dependent transactivation of the c-myc promoter. These findings suggest the existence of a novel biochemical route linking tyrosine kinase receptors, such as those for PDGF, and c-myc expression through JNK activation of AP-1 transcription factors. They also provide a novel potential mechanism by which both JNK and Jun proteins may exert either their proliferative or apoptotic potential by stimulating the expression of the c-myc proto-oncogene.

The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for RetM918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors RetC634Y and RetM918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.

Approx. 2% of the Neisseria meningitidis genome consists of small DNA insertion sequences known as Correia or nemis elements, which feature TIRs (terminal inverted repeats) of 26-27 bp in length. Elements interspersed with coding regions are co-transcribed with flanking genes into mRNAs, processed at double-stranded RNA structures formed by TIRs. N. meningitidis RNase III (endoribonuclease III) is sufficient to process nemis+ RNAs. RNA hairpins formed by nemis with the same termini (26/26 and 27/27 repeats) are cleaved. By contrast, bulged hairpins formed by 26/27 repeats inhibit cleavage, both in vitro and in vivo. In electrophoretic mobility shift assays, all hairpin types formed similar retarded complexes upon incubation with RNase III. The levels of corresponding nemis+ and nemis- mRNAs, and the relative stabilities of RNA segments processed from nemis+ transcripts in vitro, may both vary significantly.

Neisseria miniature insertion sequences (nemis) are miniature DNA insertion sequences found in Neisseria species. Out of 57 elements closely flanking cellular genes analyzed by PCR, most were conserved in Neisseria meningitidis but not in N. lactamica strains. Since mRNAs spanning nemis are processed by RNase III at hairpins formed by element termini, gene sets could selectively be regulated in meningococci at the posttranscriptional level.

Substitution of Cys-634 in the extracellular domain of the Ret tyrosine kinase receptor causes its dimerization and activation of its transforming potential. To gain further insight into the molecular basis leading to Ret activation we purified a mutant protein consisting of the entire ectodomain of the Ret carrying a Cys-634-->Tyr substitution (EC-Ret(C634Y)). The protein is glycosylated, like the native one, and is biologically active. By using an in vitro cell system we show that EC-Ret(C634Y) inhibits the membrane-bound receptor Ret(C634Y), interfering with its dimerization. Furthermore, we demonstrate that EC-Ret(C634Y) competes with the wild-type Ret receptor for ligand binding. The results presented support the notion of the possible involvment of glial cell line-derived neurotrophic factor (GDNF) with multiple endocrine neoplasia type 2A (MEN2A) tumours, and describe a useful tool for generating molecular mimetics directed towards specific mutations of the ret oncogene.

In bacteria, conditions that uncouple translation from transcription activate intragenic terminators located within cistrons. We analyzed the function of NusA in intragenic termination, making use of two tandem terminators located within the hisG cistron, GTTE1 and GTTE2. GTTE2 is a canonical Rho site, capable to terminate with Rho alone in vitro. By contrast, GTTE1 is a suboptimal terminator, featuring a boxA element and requiring a functional NusB to terminate efficiently in vivo. We found that a functional NusA is necessary for efficient termination events to occur at both GTTE1 and 2. To enhance termination at GTTE1 in conditions in which the transcript is free of ribosomes, NusA acts at the same step as NusB and NusE/S10. In the presence of concomitant translation, termination at GTTE1 is dependent on the relative position of the translation stop codon and boxA. If translation stops upstream of boxA, NusA acts at the same step as NusB enhance termination. Ribosomes terminating translation at boxA influence termination at GTTE1. Interactions of NusA and/or NusB with ribosomal components, including NusE/S10, might facilitate termination. Differently from what observed at GTTE1, the NusA-stimulated pausing seems to be sufficient for the occurrence of complete termination events at GTTE2. A functional NusA is also necessary to prevent premature termination of normally translated transcripts. Our data support the hypothesis that NusA may program a fraction of the RNA polymerase to terminate transcription upon interactions with specific sites on the nascent mRNA and either other Nuses or ribosomes.

The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized rne-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.

Previous studies have shown that the expression of the his operon of Salmonella typhimurium is regulated at the level of transcription initiation, transcription elongation and RNA processing. We have analyzed his RNA in both prototrophic strains or strains harboring regulatory and auxotrophic mutations grown under a variety of metabolic conditions that lead to differential expression of the operon. Under some of these conditions, there is an increase in the amount of prematurely released his-specific RNA, resulting in modulation of the relative amount of full-length transcripts. Under the same metabolic conditions, there is also a modulation of RNA processing events that generate a very stable RNA species comprising the five distal cistrons. These effects appear to be due to perturbation of the translation process caused by alterations in the intracellular pool of initiator transfer RNA.

In vivo analysis of expression of the chloroplast rDNA cluster during somatic embryogenesis of Daucus carota (D.carota) was performed by Northern-blot analysis with different DNA probes, spanning both the 16S rRNA gene, the 16S-23S rRNA spacer, which contains the two mosaic tRNA genes tRNA(Ile) and tRNA(Ala), and the region upstream of the 16S rRNA gene, where a tRNA(Val) maps. We show that expression both of the spacer tRNAs tRNA(Ile) and tRNA(Ala) is not significantly regulated during development whereas the amount of the transcript corresponding to tRNA(Val) is not detectable during early embryonic stages and progressively accumulates during late phases. Multiple transcription start sites have been identified upstream of the tRNA(Val) gene by S1 mapping analysis, which are activated late during the embryogenesis. These data indicate that developmental control mechanisms act on plastid gene expression during embryogenesis in carrot.

Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message. Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons. Given the absence of 5' to 3' exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3' to 5' exoribonucleases are involved in chemical decay of mRNA. As the 3' to 5' exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3' ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3' to 5' exoribonucleases. Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities. A considerable number of bacterial messages decay with a net 5' to 3' directionality. Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5' to 3' processive '5' binding nuclease'. The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts.

A 3457-base pair fragment of Azospirillum brasilense DNA which complemented mutations in the hisA and hisF genes of Escherichia coli was sequenced. The sequence analysis revealed the presence of six major contiguous open reading frames (ORF). The comparison of the predicted amino acid sequence of these ORF with those encoded by the eubacterial, archaebacterial and eukaryotic his genes sequenced thus far revealed that four of them have a significant degree of homology with the E. coli hisH, hisA, hisF and the C-terminal domain of the hisI gene products. S1 mapping experiments indicated that the putative transcription start site coincided with the AUG translational initiation codon of the hisBd gene, the first gene of the A. brasilense his operon. Downstream from the last ORF, a sequence was identified which functions as a Rho-independent transcription terminator. Comparison of amino acid sequences, gene order and organization and evolutionary aspects of the A. brasilense his cluster are discussed.

We have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild-type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis-acting element and translation of the cistron immediately downstream of the 5' end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter.

We have constructed an expression vector carrying the Escherichia coli his operon control region to study the ability of defined segments of DNA to cause rho factor-mediated transcription termination both in vivo and in vitro. We have previously identified a consensus motif consisting of a region of high cytosine over guanosine content common to several cryptic intracistronic transcription termination elements unmasked by polar mutations. We show that a DNA fragment possessing features similar to the ones previously identified is capable of causing rho-mediated mediated release of transcripts in vivo and in vitro. The efficiency of termination depends on the length and efficiency of termination depends on the length and relative cytosine over guanosine ratio of the element.

We used an expression vector plasmid containing the Escherichia coli K-12 histidine operon regulatory region to subclone the E. coli hisC gene. Analysis of plasmid-coded proteins showed that hisC was expressed in minicells. A protein with an apparent molecular weight of 38,500 was identified as the primary product of the hisC gene. Expression was under control of the hisGp promoter and resulted in very efficient synthesis (over 100-fold above the wild-type levels) of imidazolylacetolphosphate:L-glutamate aminotransferase, the hisC gene product. The complete nucleotide sequence of the hisC gene has been determined. The gene is 1,071 nucleotides long and codes for a protein of 356 amino acids with only one histidine residue.

A typical Rho-independent terminator of transcription was found at the end of the histidine operon of Salmonella typhimurium. This site is used to terminate, in addition to the his operon mRNA, a 1,200-nucleotide RNA of unknown function transcribed on the opposite strand. The efficiency of termination of transcription at this site was investigated in vivo and in vitro by cloning of the terminator structure in either orientation in vector systems used to study regulatory signals. Termination of transcription at this site was very efficient, both in vivo and in vitro, and in both orientations.

The operator-distal genes hisBHAFI(E) of the Escherichia coli K-12 histidine operon were mapped on a DNA fragment 4,500 base pairs long. This fragment, originally present in a lambda transducing phage, was cloned in the vector plasmid pBR313. A restriction map was determined, allowing identification of the orientation of the genes in the fragment. The cloned genes were expressed in appropriate hosts, independent of the orientation of the DNA fragment, as shown by transformation tests and by enzyme assays of one of the gene products, hisB, histidinol phosphatase. An internal transcription initiation site was identified by isolation of the cellular RNA, hybridization to specific DNA probes, and mapping by S1 nuclease.