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PubMed1988

Characterization of response elements for androgens, glucocorticoids and progestins in mouse mammary tumour virus.

We have characterized steroid response elements in mouse mammary tumour virus (MMTV) by transient transfection. Four partial inverted repeats of the sequence TGTTCT function as response elements for androgen, as well as for glucocorticoid and progestins, although the relative hormone inductions mediated by each oligonucleotide were different. Mutational analysis of the left half of the palindrome showed that a perfect dyad symmetry is not required for optimum activity as a steroid response element. To investigate potential interactions between steroid receptors and transcription factors we have analysed the minimum sequence requirements for a hormone response. Interestingly, a single 15 bp steroid response element and a TATA box are sufficient for steroid inductions. When the distance between the two elements was increased by up to two turns of the helix the hormone induction initially increased and then gradually declined with no obvious periodicity.

Animals↗
PubMed1989

Transcription regulation in vitro by an E. coli promoter containing a DNA cruciform in the '-35' region.

A promoter with the potential to adopt a 50 basepair (bp) cruciform spanning from -19 to -69 has been constructed in the plasmid pBR322 tetracycline resistance gene (tet) by forming an inverted repeat from '-35' sequences. Compared to a control promoter, the sequence of this cruciform promoter differs only by a 22 bp insertion between -48 and -69, upstream from the usual location of promoter sequences. The cruciform is extruded in a supercoil-dependent manner, and transcription from this promoter in vitro by RNA polymerase decreases as the negative supercoil density of the plasmid DNA increases. In contrast, transcription from the control promoter increases with negative supercoiling. Thus, DNA secondary structure in the '-35' region can affect promoter-polymerase interaction. The tet promoter cruciform also influences expression of the pBR322 beta-lactamase gene (bla). This apparently results when extrusion of the cruciform reduces the superhelicity of the plasmid molecule to a level that is below the optimum for expression from the bla promoter, illustrating one mechanism for how DNA secondary structure may effect action-at-a-distance. Transcription from both promoters in vivo does not differ from controls, suggesting that this cruciform is not generated to a significant extent intracellularly, most probably as a result of the slow kinetics of extrusion.

Base Composition↗
PubMed1995

Negative and positive regulation of Tn10/IS10-promoted recombination by IHF: two distinguishable processes inhibit transposition off of multicopy plasmid replicons and activate chromosomal events that favor evolution of new transposons.

Tn10 is a composite transposon; inverted repeats of insertion sequence IS10 flank a tetracycline-resistance determinant. Previous work has identified several regulatory processes that modulate the interaction between Tn10 and its host. Among these, host-specified DNA adenine methylation, an IS10-encoded antisense RNA and preferential cis action of transposase are particularly important. We now find that the accessory host protein IHF and the sequences that encode the IHF-binding site in IS10 are also important regulators of the Tn10 transposition reaction in vivo and that these determinants are involved in two distinguishable regulatory processes. First, IHF and the IHF-binding site of IS10, together with other host components (e.g., HU), negatively regulate the normal intermolecular transposition process. Such negative regulation is prominent only for elements present on multicopy plasmid replicons. This multicopy plasmid-specific regulation involves effects both on the transposition reaction per se and on transposase gene expression. Second, specific interaction of IHF with its binding site stimulates transposon-promoted chromosome rearrangements but not transposition of a short Tn10-length chromosomal element. However, additional considerations predict that IHF action should favor chromosomal transposition for very long composite elements. On the basis of these and other observations we propose that, for chromosomal events, the major role of IHF is to promote the evolution of new IS10-based composite transposons.

Bacterial Proteins↗
PubMed1990

Characterization of the Arabidopsis Adh G-box binding factor.

Protein-DNA interaction at an inverted repeat of the sequence 5'-GTGG-3' (G-box) has been associated with the transcription of several plant genes [Giuliano, G., et al. (1988). Proc. Natl. Acad. Sci. USA 85, 7089-7093; Ferl, R.J., and Laughner, B.H. (1989). Plant Mol. Biol. 12, 357-366; Schulze-Lefert, P., et al. (1989). EMBO J. 8, 651-656]. We characterized the binding of the Arabidopsis G-box binding factor (GBF) from whole-cell extracts and fractionated extracts to the G-box of alcohol dehydrogenase (Adh) using gel mobility shift assays. DNase I footprinting localized the region of GBF/G-box interaction to two sites, one apparent high-affinity binding site (-227 to -201) and a possible low-affinity binding site (-193 to -182). DNA-protein cross-linking demonstrated that the G-box is bound by proteins of two sizes, 31 kilodaltons and 18 kilodaltons. In addition, we found that in vitro the interaction of GBF from Arabidopsis suspension cultures or leaves with the Adh G-box is indistinguishable, and that there is evidence of multiple protein-protein interactions.

Alcohol Dehydrogenase↗
PubMed2005

ISEcp1B-mediated transposition of blaCTX-M in Escherichia coli.

Several expanded-spectrum beta-lactamase bla(CTX-M) genes are associated with ISEcp1-like elements in Enterobacteriaceae. We found that ISEcp1B was able to mobilize the adjacent bla(CTX-M-19) gene by a transpositional mechanism in Escherichia coli by recognizing a variety of DNA sequences as right inverted repeats.

Anti-Bacterial Agents↗
PubMed1981

Reexamination of the genome size of myxobacteria, including the use of a new method for genome size analysis.

The genome sizes of two myxobacteria, Myxococcus xanthus and Stigmatella aurantiaca, were measured by renaturation analysis and also by a new method involving the quantitation of individual restriction fragments. In contrast to several previous reports, which indicate that M. xanthus has a genome size which is three to four times that of Escherichia coli, the present measurements indicated that the M. xanthus genome is only about 24 to 53% larger than that of E. coli. S. aurantiaca had a genome size nearly identical to that of M. xanthus. Of possible significance is the fact that the renaturation curves of M. xanthus and S. aurantiaca deoxyribonucleic acid both exhibited significant fractions which renatured with rapid, unimolecular kinetics. However, we were unable to establish that these fractions represented inverted repeats of repetitive sequences.

Chromosomes, Bacterial↗
PubMed1987

Identification of Tn4451 and Tn4452, chloramphenicol resistance transposons from Clostridium perfringens.

The recombinant plasmids pJIR45 and pJIR97 contain the chloramphenicol resistance determinants derived from the Clostridium perfringens R plasmids pIP401 and pJIR27, respectively. Escherichia coli cultures which harbored these recombinant plasmids rapidly became chloramphenicol sensitive when grown in the absence of chloramphenicol. The loss of resistance was associated with the loss of 6.2-kilobase (kb) segments from both plasmids. Detailed restriction analysis of E. coli- and C. perfringens-derived deletion plasmids indicated that deletion of these segments was essentially precise. Transposition of the 6.2-kb segments was demonstrated by cloning the determinants into a temperature-sensitive plasmid, curing the recombinant plasmids, and selecting chloramphenicol-resistant, plasmid-free clones. Southern hybridization analysis of chromosomal DNA isolated from these recA E. coli clones indicated that the 6.2-kb segments had transposed to different sites on the chromosome. Heteroduplex analysis and restriction mapping indicated that the transposons, Tn4451 (pIP401) and Tn4452 (pJIR27), were closely related and did not contain large inverted or directly repeated sequences. These transposons represent the first transposable elements from the clostridia to be identified and characterized.

Chloramphenicol↗
PubMed2000

Minimal and contributing sequence determinants of the cis-acting locus of transfer (clt) of streptomycete plasmid pIJ101 occur within an intrinsically curved plasmid region.

Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.

Bacterial Proteins↗
PubMed1999

Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes.

Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) in trans, and inverted terminal repeat (ITR) sequences in cis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.

Base Sequence↗
PubMed1994

Interactions between DNA-bound trimers of the yeast heat shock factor.

The heat shock transcription factor (HSF) is a trimer that binds to DNA containing inverted repeats of the sequence nGAAn. HSF can bind DNA with the sequence nGAAnnTTCn or with the sequence nTTCnnGAAn, with little preference for either sequence over the other. However, (nGAAnnTTCn)2 is considerably less active as a heat shock response element (HSE) than is (nTTCnnGAAn)2. The electrophoretic mobilities of DNA-protein complexes and chemical cross-linking between protein monomers indicate that the sequence (nGAAnnTTCn)2 is capable of binding a single HSF trimer. In contrast, the sequence with higher biological activity, (nTTCnnGAAn)2, is capable of binding two trimers. Thus, the ability of four-nGAAn-element HSEs to bind one or two trimers depends on the permutation with which the elements are presented. A survey of naturally occurring HSEs shows the sequence (nTTCnnGAAn)2 to be the more prevalent. We suggest that the greater ability of one permutation over the other to bind two HSF trimers accounts for the initial identification of the naturally occurring heat shock consensus sequence as a region of dyad symmetry.

Base Sequence↗
PubMed1989

Analysis of the 5'-flanking regions of rat inhibin alpha- and beta-B-subunit genes suggests two different regulatory mechanisms.

The genes encoding rat inhibin alpha- and beta-B-subunits were isolated and characterized. Both genes contain one intron that interrupts the region coding for the precursor portion of the alpha- and beta-B-subunits. The transcription start sites of alpha- and beta-B-subunit genes were determined by primer extension and nuclease mapping assay using mRNA from rat ovary and testis. Transcription of the alpha-subunit gene initiates predominantly at three adjacent sites with similar intensity. Several potential transcription start sites of beta-B-subunit gene are spread over 150 nucleotides upstream from translation initiation site. Neither of these two genes contains obvious TATA or CCAAT boxes. The alpha-subunit gene contains many GA clusters in the promoter region, while beta-B-subunit gene is highly GC rich. Several GGGCGG repeats and their inverted sequences, which are the potential binding sites for transcription factor Spl, were observed at the 5'-end as well as at the coding region of the beta-B-subunit gene. The potential cAMP-responsive element CTGCGTCAG was identified in alpha-but not beta-B-subunit gene. This sequence is identical to the cAMP- and phorbol ester-inducible DNA fragment found in human preproenkephalin gene. The different structure of the promoter region of rat alpha- and beta-B-subunit genes and the presence of a potential cAMP-inducible DNA sequence in alpha- but not beta-B-subunit gene is consistent with the hypothesis that transcription of alpha- and beta-B-subunit genes in rat is regulated by different mechanisms.

Amino Acid Sequence↗
PubMed2000

Utility of 17 chloroplast genes for inferring the phylogeny of the basal angiosperms.

Sequences from 14 slowly evolving chloroplast genes (including three highly conserved introns) were obtained for representative basal angiosperm and seed-plant taxa, using novel primers described here. These data were combined with published sequences from atpB, rbcL, and newly obtained sequences from ndhF. Combined data from these 17 genes permit sturdy, well-resolved inference of major aspects of basal angiosperm relationships, demonstrating that the new primers are valuable tools for sorting out the deepest events in flowering plant phylogeny. Sequences from the inverted repeat (IR) proved to be particularly reliable (low homoplasy, high retention index). Representatives of Cabomba and Illicium were the first two successive branches of the angiosperms in an initial sampling of 19 exemplar taxa. This result was strongly supported by bootstrap analysis and by two small insertion/deletion events in the slowly evolving introns. Several paleoherb groups (representatives of Piperales) formed a strongly supported clade with taxa representing core woody magnoliids (Laurales, Magnoliales, and Winteraceae). The monophyly of the sampled eudicots and monocots was also well supported. Analyses of three major partitions of the data showed many of the same clades and supported the rooting seen with all the data combined. While Amborella trichopoda was supported as the sister group of the remaining angiosperms when we added Amborella and Nymphaea odorata to the analysis, a strongly conflicting rooting was observed when Amborella alone was added.

Journal Article↗
PubMed1997

Structural and functional analysis of the chick chondroitin sulfate proteoglycan (aggrecan) promoter and enhancer region.

Aggrecan is a large chondroitin sulfate proteoglycan, the expression of which is both tissue-specific and developmentally regulated. Here we report the cloning and sequencing of the 1.8-kilobase genomic 5' flanking sequence of the chick aggrecan gene and provide a functional and structural characterization of its promoter and enhancer region. Sequence analysis reveals potential Sp1, AP2, and NF-I related sites, as well as several putative transcription factor binding sites, including the cartilage-associated silencers CIIS1 and CIIS2. A number of these transcription factor binding motifs are embedded in a sequence flanked by prominent inverted repeats. Although lacking a classic TATA box, there are two instances in the 1.8-kb genomic fragment of TATA-like TCTAA sequences, as have been defined previously in other promoter regions. Primer extension and S1 protection analyses reveal three major transcription start sites, also located between the inverted repeats. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively reduced portions of the aggrecan promoter region allowed mapping of chondrocyte-specific transcription enhancer and silencer elements that are consistent with the sequence analysis. These findings suggest the importance of this regulatory region in the tissue-specific expression of the chick aggrecan gene.

Aggrecans↗
PubMed1996

Identification of putative nonautonomous transposable elements associated with several transposon families in Caenorhabditis elegans.

Putative nonautonomous transposable elements related to the autonomous transposons Tc1, Tc2, Tc5, and mariner were identified in the C. elegans database by computational analysis. These elements are found throughout the C. elegans genome and are defined by terminal inverted repeats with regions of sequence similarity, or identity, to the autonomous transposons. Similarity between loci containing related nonautonomous elements ends at, or near, the boundaries of the terminal inverted repeats. In most cases the terminal inverted repeats of the putative nonautonomous transposable elements are flanked by potential target-site duplications consistent with the associated autonomous elements. The nonautonomous elements identified vary considerably in size (from 100 bp to 1.5 kb in length) and copy number in the available database and are localized to introns and flanking regions of a wide variety of C. elegans genes.

Animals↗
PubMed1985

The short unique region of the B95-8 Epstein-Barr virus genome.

The 12-kbp short unique region of the B95-8 Epstein-Barr virus (EBV) genome has been sequenced and analysed for latent and lytic cycle transcripts. Two latent and three late mRNAs have been detected, the largest of the late transcripts potentially encoding a 143-kDa protein. The region containing oriP, the putative origin of replication of the genome as a plasmid in latently infected B lymphocytes, is shown to contain 21 direct repeats of a 30-bp A+T-rich sequence and a related large inverted repeat.

Base Sequence↗
PubMed1992

A HSV-1 variant (1720) generates four equimolar isomers despite a 9200-bp deletion from TRL and sequences between 9200 np and 97,000 np in inverted orientation being covalently bound to sequences 94,000-126,372 np.

The genome structure of a spontaneously generated HSV-1 strain 17 variant, 1720, has been determined by restriction endonuclease and Southern blot analysis. The short segment of 1720 is unaltered compared to the parental strain 17 genome, whereas the long segment is extensively rearranged. Almost all of TRL (approximately 9.2 kb) has been deleted and consequently IRL is converted into unique sequence. Sequences from approximately 9200 nucleotide position (np) to 97,000 np are present in inverted orientation, covalently bound to sequences in the prototype orientation from approximately 94,000 np to the L/S junction at 126,372 np. Thus, sequences from 94,000 np to 97,000 np are now diploid, with one copy in the normal orientation and location, and the other at the long terminus as an inverted repeat; no inversion of the intervening unique sequences occurs about this novel inverted repeat. In contrast, normal inversions of the long and short segments occur to give four equimolar genomic isomers, indicating that the novel long terminus has gained an "a" sequence. The duplication of sequences between 94,000 np and 97,000 np results in a genome containing two copies of UL43 and one complete and one partial copy each of genes UL42 and UL44 encoding the 65 kD DNA-binding protein and glycoprotein C, respectively. The variant has been shown to grow normally in vitro following high multiplicity infection.

Base Sequence↗
PubMed1999

Function and regulation of glnA in the methanogenic archaeon Methanococcus maripaludis.

The glnA gene in the domains Bacteria and Archaea encodes glutamine synthetase, a universally distributed enzyme that functions in ammonia assimilation and glutamine synthesis. We investigated the regulation and function of glnA in the methanogenic archaeon Methanococcus maripaludis. The deduced amino acid sequence of the gene demonstrated its membership in class GSI-alpha of glutamine synthetases. The gene appeared to be expressed as a monocistronic operon. glnA mRNA levels and specific activities of glutamine synthetase were regulated similarly by nitrogen. Three transcription start sites were identified, corresponding to two overlapping nitrogen-regulated promoters and one weaker constitutive promoter. An inverted repeat immediately upstream of the regulated transcription start sites mediated repression under noninducing conditions. Thus, mutations that altered the sequence of the inverted repeat resulted in derepression. The inverted repeat had sequence similarity with a repeat that we previously identified as the nif operator of M. maripaludis, suggesting a common mechanism of nitrogen regulation. Efforts to produce a glnA null mutant failed, suggesting that glnA is an essential gene in M. maripaludis.

Base Sequence↗