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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↗
PubMed1988

Isolation, characterization and sequencing of a novel repetitive DNA from the mung bean Vigna radiata.

A family of highly reiterated, small (approx. 300 bp) sequences has been identified in DNA of the mung bean Vigna radiata. The members are extensively interspersed throughout the chromosomes with some clustering. They also occur extrachromosomally. There is no tissue-specificity to the repeat family but it is highly species-specific. The repetitive DNA hybridizes to total RNA as well as to polyadenylated RNA isolated from germinated mung beans. It has analogy with the human AluI family in the mode of isolation, size, genomic distribution, copy number and transcribability though they do not share any sequence homology. A repetitive DNA clone was selected from a shotgun genomic library of mung bean DNA in pBR322. The average copy number of the cloned repeat is estimated to be 8 x 10(4) per haploid genome, and thus constitutes approx. 5% of the total mung bean genome. The genomic organization and transcription of the cloned repeat is reported. Sequencing of the cloned repetitive DNA reveals the presence of the number of direct and inverted repeats and some short palindromic sequences.

Base Sequence↗
PubMed2000

The novel insertion sequences IS1417, IS1418, and IS1419 from Burkholderia glumae and their strain distribution.

Three insertion sequences, IS1417, IS1418, and IS1419, were isolated from Burkholderia glumae (formerly Pseudomonas glumae), a gram-negative rice pathogenic bacterium, on the basis of their abilities to activate the expression of the neo gene of the entrap vector pSHI1063. The 1335-bp IS1417 element with 17-bp imperfect terminal inverted repeats was found to be flanked by 5-bp direct repeats of the vector sequence. IS1418 is 865 bp in length and carries 15-bp inverted repeats with a target duplication of 3 bp. The 1215-bp IS1419 sequence is bounded by the 36-bp terminal inverted repeats of the element and 7-bp direct repeats of the vector sequence. IS1417 and IS1418 belong to the IS2 subgroup of the IS3 family and the IS427 subgroup of the IS5 family, respectively, whereas IS1419 does not appear to be a member of any known IS family. Southern blot analysis of DNAs from B. glumae field isolates indicated that those IS elements are widely distributed, but the host range of the three IS elements appears to be limited to B. glumae and some other related species such as B. plantarii. The polymorphisms exhibited in B. glumae isolates suggest that those elements are useful for molecular epidemiological studies of B. glumae infections.

Amino Acid Sequence↗
PubMed1985

The kinetic properties of cruciform extrusion are determined by DNA base-sequence.

The extrusion kinetics of two cruciforms derived from unrelated DNA sequences differ markedly. Kinetic barriers exist for both reactions, necessitating elevated temperatures before extrusion proceeds at measureable speeds, but the dependence upon temperature and ionic strength is quite different for the two sequences. One, the ColE1 inverted repeat, exhibits a remarkably great temperature dependence of reaction rate and is suppressed by moderate amounts of NaCl or MgCl2. In contrast, the other, a synthetic inverted repeat present in pIRbke8, shows more modest temperature dependence and has a requirement for the presence of salt, with optimal concentrations being 50 mM NaCl or 100 microM MgCl2. Under optimal conditions, cruciform extrusion rates are fast (t1/2 less than 60m) at 37 degrees C for both sequences at native superhelix densities. In 50 mM NaCl the pIRbke8 inverted repeat is characterised by an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole -1. The differences in kinetic properties between the two sequences indicate that DNA base sequence is itself an important factor in determining cruciform kinetics, and possibly even in the selection of the mechanistic pathway.

Base Sequence↗
PubMed1989

Nucleotide sequence of IS492, a novel insertion sequence causing variation in extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica.

The complete nucleotide sequence of insertion element IS492, which causes reversible inactivation of extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica, is presented. Insertion of IS492 results in the EPS- phenotype, and excision results in restoration of EPS+. DNA sequencing of the site of insertion in the eps locus showed that insertion of IS492 generates a 5-base-pair repeat and that its excision is precise. IS492 is 1,202 nucleotides in length and contains one large open reading frame encoding a protein of 318 amino acids, a candidate for transposition function. No similarity between IS492 and other transposable elements has been found. Unlike the situation with other insertion sequences, no direct or inverted repeats exist at the termini of IS492.

Amino Acid Sequence↗
PubMed1989

A host-encoded DNA-binding protein promotes termination of plasmid replication at a sequence-specific replication terminus.

We have purified approximately 6600-fold an approximately 40-kDa protein (Ter protein) encoded by Escherichia coli that specifically binds to two sites at the 216-base-pair replication terminus (tau) of the plasmid R6K. Chemical footprinting experiments have shown that the Ter protein binds to two 14- to 16-base-pair sequences that exist as inverted repeats in the tau fragment. Site-directed mutagenesis of one of the terminus sequences (tau R) resulted in a mutant tau R that failed to bind to the Ter protein. The same mutant terminus also failed to terminate DNA replication in vivo. These experiments strongly suggest that the interaction of the Ter protein with tau sequences plays an essential role in the termination of DNA replication, specifically at tau.

Base Sequence↗
PubMed1986

Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.

When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.

Chromosome Deletion↗
PubMed1996

Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions.

The adeno-associated virus type 2 (AAV) genome can be successfully rescued from recombinant plasmids following transfection in adenovirus-infected human cells. However, following rescue, the AAV genome undergoes preferential replication and encapsidation, whereas little replication and packaging of the vector DNA sequences occur. In view of the crucial role in the rescue, replication, and packaging of the proviral genome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the HP-formation, we evaluated the involvement of the individual ITRs as well as their components in the selective viral DNA replication and encapsidation. A number of recombinant AAV plasmids that contained deletions-substitutions in different regions of the individual ITRs were constructed and examined for their potential to allow rescue, replication, and/or packaging in adenovirus-infected human cells in vivo. The results reported here document that (ii) two HP structures and one D-sequence are sufficient for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA sequences, (iv) one HP structure alone or two D-sequences, but not one D-sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs in the absence of the HP structures and the D-sequences, (vi) in the absence of the D-sequences, the HP structures are insufficient for successful encapsidation of the AAV genomes, and (vii) the AAV genomes containing only one ITR structure can be packaged into biologically active virions. Thus, the D-sequence plays a crucial role in the efficient rescue and selective replication and encapsidation of the AAV genome. Furthermore, the D-sequence specifically interacts with a hitherto unknown host-cell protein that we have designated the D-sequence-binding protein (D-BP). These studies illustrate that the D-sequence-D-BP interaction constitutes an important step in the AAV life cycle.

Base Sequence↗
PubMed1977

Sequence arrangement of the rDNA of Drosophila melanogaster.

The sequence arrangement of genes coding for stable rRNA species and of the interspersed spacers on long single strands of rDNA purified from total chromosomal DNA of Drosophila melanogaster has been determined by a study of the structure of rRNA:DNA hybrids which were mounted for electron microscope observation by the gene 32-ethidium bromide technique. One repeat unit contains the following sequences in the order given. First, an 18 S gene of length 2.13 +/- 0.17 kb. Second, an internal transcribed spacer (Spl) of length 1.58 +/- 0.15 kb. A short sequence coding for the 5.8S and perhaps the 2S rRNA species is located within this spacer. Third, the 28S gene with a length of 4.36 +/- 0.23 kb. About 55% of the 28S genes are unbroken or continuous (C genes). However, about 45% of the 28S genes contain an insertion of an additional segment of DNA that is not complementary to rRNA (l genes). The insertion occurs at a reproducible point 2.99 +/- 0.26 kb from the junction with Spl. The insertions are heterogeneous in length and occur in three broad size classes: 1.42 +/- 0.47, 3.97 +/- 0.55, and 6.59 +/- 0.62 kb. Fourth, an external spacer between the 28S gene and the next 18S gene which is presumably mainly nontranscribed and which has a heterogeneous length distribution with a mean length and standard deviation of 5.67 +/- 1.92 kb. Short inverted repeat stems (100-400 nucleotide pairs) occur at the base of the insertion. It is known from other studies that I genes occur only on the X chromosome. The present study shows that the I and C genes on the X chromosomes are approximately randomly assorted. The sequence arrangement on the plasmid pDm103 containing one repeat of rDNA (Glover et al., 1975) has been determined by similar methods. The I gene on this plasmid contains an inverted repeat stem. The occurrence of inverted repeat sequences flanking the insertion supports the speculation that these sequences are translocatable elements similar to procaryotic translocons.

DNA↗
PubMed2006

The DcMaster Transposon Display maps polymorphic insertion sites in the carrot (Daucus carota L.) genome.

DcMaster is a family of PIF/Harbinger-like class II transposable elements identified in carrot. We present a modified Transposon Display molecular marker system allowing amplification of genomic regions containing DcMaster elements. We scored 77 DcMaster Transposon Display (DcMTD) amplicons, of which 54 (70%) were segregating in the F(2) progeny from the cross between wild and cultivated carrot. Segregating amplicons were incorporated into a previously developed molecular linkage map of carrot. Twenty-eight markers were attributed to the wild parent, 23 originated from the cultivated parent, and three markers remained unlinked. The markers were evenly distributed among the nine linkage groups. However, differences in the distribution pattern of DcMaster insertion sites in the genomes of the wild and cultivated parent were observed. Specificity of the obtained amplicons was confirmed by sequencing and three putative DcMaster subfamilies, differing in the sequence of their terminal inverted repeats, were revealed.

Base Sequence↗
PubMed2003

Insertion sequence ISEcp1B is involved in expression and mobilization of a bla(CTX-M) beta-lactamase gene.

The genetic structures (ca. 10-kb DNA fragment) surrounding the plasmid-borne extended-spectrum beta-lactamase bla(CTX-M-19) gene in a Klebsiella pneumoniae clinical isolate were determined. This beta-lactamase gene was part of a 4,797-bp transposon inserted inside orf1 of Tn1721. Inside this transposon, bla(CTX-M-19) was bracketed upstream and downstream by insertion sequences ISE cp1B and IS903D, respectively, and further downstream by a truncated gene encoding an outer membrane protein for iron transport. The single-copy ISEcp1B element was probably involved alone in the mobilization process that led to a 5-bp duplication at the target site of the transposed fragment. This mobilization event probably involved one inverted repeat of ISE cp1B and a second sequence farther away, resembling its second inverted repeat. Additionally, ISEcp1B provided -35 and -10 promoter sequences, contributing to the high-level expression of the bla(CTX-M-19) gene. Southern blot analysis failed to identify a reservoir of ISEcp1-like sequences among a series of gram-negative and gram-positive bacterial species usually found in the skin and intestinal human floras. The ability of ISEcp1-like elements to mobilize and to promote the expression of beta-lactamase genes may explain, in part, the current spread of CTX-M-type enzymes worldwide.

Anti-Bacterial Agents↗
PubMed2001

Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1.

The hig (host inhibition of growth) gene system of plasmid Rts1 belongs to the plasmid-encoded proteic killer gene family. Among the proteic killer genes described so far, hig is unique in that the toxin gene (higB) exists upstream of the antidote gene (higA). There are two promoters in the hig locus, Phig and PhigA, and only the former, which expresses both higB and higA genes, is negatively controlled by HigA and HigB proteins. In this study, we purified HigA protein by means of GST fusion. The electrophoretic mobility shift assay using the purified protein revealed that HigA specifically bound to the Phig region, but not to PhigA. The HigA-binding sequence was determined by DNase I footprinting assay to be a 56-bp sequence that completely covered the -35 and -10 boxes of Phig. The presence of two inverted repeats in the binding sequence and the identification of a dimer form of HigA by cross-linking experiment suggested that the protein bound to the Phig region as a dimer. HigB was purified as a GST fusion protein as well, though it was achieved only in the presence of HigA. HigA and GST-HigB formed a highly stable complex where the two proteins were present in an equimolar ratio.

Amino Acid Sequence↗
PubMed1995

DNA-binding and transactivation properties of Pax-6: three amino acids in the paired domain are responsible for the different sequence recognition of Pax-6 and BSAP (Pax-5).

Pax-6 is known to be a key regulator of vertebrate eye development. We have now isolated cDNA for an invertebrate Pax-6 protein from sea urchin embryos. Transcripts of this gene first appear during development at the gastrula stage and are later expressed at high levels in the tube foot of the adult sea urchin. The sea urchin Pax-6 protein is highly homologous throughout the whole protein to its vertebrate counterpart with the paired domain and homeodomain being virtually identical. Consequently, we found that the DNA-binding and transactivation properties of the sea urchin and mouse Pax-6 proteins are very similar, if not identical. A potent activation domain capable of stimulating transcription from proximal promoter and distal enhancer positions was localized within the C-terminal sequences of both the sea urchin and mouse Pax-6 proteins. The homeodomain of Pax-6 was shown to cooperatively dimerize on DNA sequences consisting of an inverted repeat of the TAAT motif with a preferred spacing of 3 nucleotides. The consensus recognition sequence of the Pax-6 paired domain deviates primarily only at one position from that of BSAP (Pax-5), and yet the two proteins exhibit largely different binding specificities for individual, naturally occurring sites. By creating Pax-6-BSAP fusion proteins, we were able to identify a short amino acid stretch in the N-terminal part of the paired domain which is responsible for these differences in DNA-binding specificity. Mutation of three Pax-6-specific residues in this region (at positions 42, 44, and 47 of the paired domain) to the corresponding amino acids of BSAP resulted in a complete switch of the DNA-binding specificity from Pax-6 to BSAP. These three amino acids were furthermore shown to discriminate between the Pax-6- and BSAP-specific nucleotide at the divergent position of the two consensus recognition sequences.

Amino Acid Sequence↗
PubMed1982

Micronuclear DNA sequences of Oxytricha fallax homologous to the macronuclear inverted terminal repeat.

The macronucleus of the protozoan Oxytricha fallax is generated from a micronucleus following conjugation. While the micronucleus contains high molecular weight DNA, the macronucleus contains only short linear DNA molecules which all end in the same 20 bp inverted terminal repeat (Ma-ITR). The Ma-ITR was radioactively labeled and purified for use as a probe in hybridizations to micronuclear and macronuclear DNA. Sequences homologous to the Ma-ITR were detected in micronuclear DNA. The copy number of the repeat in the micronuclear genome is approximately that required to encode the macronuclear DNA termini. The micronuclear copies are found embedded in repeated long sequence blocks.

Animals↗
PubMed1991

Bidirectional promoter and terminator regions bracket mmr, a resistance gene embedded in the Streptomyces coelicolor A3(2) gene cluster encoding methylenomycin production.

Low- and high-resolution nuclease mapping of in vivo transcripts, and in vitro transcription reactions using purified RNA polymerase, were used to analyse transcription of and around the mmr gene, which specifies resistance of Streptomyces coelicolor A3(2) to methylenomycin (Mm) and is located in the middle of a cluster of Mm-production-encoding genes. Transcription of mmr is from a single major start point (tsp) which is separated by only 81 bp from a divergent tsp. A pattern of direct and inverted repeats in the nucleotide sequence in this region may play a part in regulation of these promoters. The 3' end of the mmr transcript overlaps by 20-30 bp the 3' end of an RNA molecule involved in Mm production. The converging transcripts both terminate at the same large inverted repeat in the DNA. Purified RNA polymerase terminated transcription at this sequence in vitro (albeit only in one orientation).

Anti-Bacterial Agents↗
PubMed1994

Two inverted repeats in the nodD promoter region are involved in nodD regulation in Rhizobium leguminosarum.

In Rhizobium leguminosarum (R.l.) biovar viciae, the nodulation gene nodD encodes a transcriptional activator (NodD) which binds to highly conserved DNA sequences (nod-boxes) in the promoters of other nod operons. In addition, NodD represses nodD transcription and this occurs at the divergent and overlapping nodA-nodD promoters. We mutagenised this region with hydroxylamine, and by cloning the mutagenised DNA into a vector carrying the lacZ reporter gene downstream from the cloning site identified mutations affecting nodD expression and repression. The resulting plasmids were transferred to R. l. viciae strains containing or lacking nodD. Two classes of promoter mutants were identified: those in which nodD transcription was altered and those in which NodD-dependent repression was altered. The nucleotide (nt) changes in the promoter region were found to be located within two inverted repeat sequences (A2 and A3) which are about 70 bp apart. A2 is important for nodD transcription and A3 (which is upstream from A2) is involved in NodD-dependent repression. The nt sequence at A3 shows some homology to the nod-box region of the nodA promoter. It is proposed that the NodD-dependent repression occurs as a result of NodD binding to both A3 and the nodA nod-box, forming a loop which prevents transcription of nodD from its promoter, A2, which lies between A3 and the nod-box. This model is supported by the observation that there are at least three sites for NodD binding in the nodA-nodD promoter region.

Bacterial Proteins↗
PubMed1985

TU elements: a heterogeneous family of modularly structured eucaryotic transposons.

We describe here a family of foldback transposons found in the genome of the higher eucaryote, the sea urchin Strongylocentrotus purpuratus. Two major classes of TU elements have been identified by analysis of genomic DNA and TU element clones. One class consists of largely similar elements with long terminal inverted repeats (IVRs) containing outer and inner domains and sharing a common middle segment that can undergo deletions. Some of these elements contain insertions. The second class is highly heterogeneous, with many different middle segments nonhomologous to those of the first-class and variable-sized inverted repeats that contain only an outer domain. The middle and insertion segments of both classes carry sequences that also are found unassociated from the inverted repeats at many other genomic locations. We conclude that the TU elements are modular structures composed of inverted repeats plus other sequence domains that are themselves members of different families of dispersed repetitive sequences. Such modular elements may have a role in the dispersion and rearrangement of genomic DNA segments.

Animals↗
PubMed1991

The chromatin structure of centromeres from fission yeast: differentiation of the central core that correlates with function.

We have examined the chromatin structure of centromere regions from the fission yeast Schizosaccharomyces pombe. The large and complex centromere regions of the S. pombe chromosomes encompass many kilobase pairs of DNA and contain several classes of tandemly repeated DNA sequences. The repeated sequences are further organized into a large inverted repeat flanking a central core, a conserved structural feature among all three centromeres in S. pombe. The nucleosomal configuration of the centromere regions is nonuniform and highly varied. Most of the centromere-specific repeated DNA sequences are packaged into nucleosomes typical of bulk chromatin. However, the central core and core-associated repeated sequences from the centromere regions of chromosomes I (cen1) and II (cen2), when present in S. pombe, show an altered chromatin structure, with little or no evidence of regular nucleosomal packaging. The atypical chromatin organization of the cen2 central core is not due to transcription, as no transcripts from this region were detected. These same DNA sequences, however, are packaged into nucleosomes typical of bulk chromatin when present in a nonfunctional environment on a minichromosome in the budding yeast Saccharomyces cerevisiae. Because the cen2 central core sequences themselves do not preclude regular nucleosomal packaging, we speculate that in S. pombe they constitute a specialized site of kinetochore protein assembly. The atypical nucleosomal pattern of the cen2 central core remains constant during the cell cycle, with only minor differences observed for some sequences. We propose that the unusual chromatin organization of the core region forms the basis of a higher order structural differentiation that distinguishes the centromere from the chromosome arms and specifies the essential structure for centromere function.

Blotting, Northern↗