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At least 1,153 records · Page 64Linked to original sources
PubMed1987

Nucleotide sequence analysis of Tn4551: use of ermFS operon fusions to detect promoter activity in Bacteroides fragilis.

The Bacteroides pBI136 clindamycin resistance (Ccr) determinant from the composite transposon Tn4551 was cloned onto the shuttle plasmid pFD160, and the regions necessary for expression in Bacteroides fragilis were determined. These results suggested that transcriptional regulatory signals required for Ccr were located in the Tn4551 direct repeat sequence (DRS) adjacent to the resistance determinant. Analysis of the nucleotide sequence of this region revealed that the Ccr structural gene, 798 base pairs (bp), was located 17 bp from the terminus of the DRS and that this gene (ermFS) differed from ermF (pBF4) by one amino acid. The DRS element was found to be 1,155 bp and appeared to contain the ermFS transcription start signals. The DRS structure was typical of insertion sequence elements isolated from other bacterial species, and its termini were characterized by 25-bp regions of imperfect dyad symmetry. The DRS was dominated by a 978-bp open reading frame, which terminated in the left inverted repeat 27 bp from the ermFS start codon, and weak amino acid sequence homology was observed with the putative transposase of IS3. Promoter activity of the DRS in B. fragilis was demonstrated by in vitro construction of operon fusions with a promoterless ermFS gene followed by transformation of the recombinant plasmids with selection for resistance to clindamycin. The location of one DRS promoter was identified by using the ermFS fusions and then verified by in vitro mutagenesis of the site with single-stranded linkers. Northern blot (RNA blot) analysis of total RNA from B. fragilis strains containing pBI136 or ermFS recombinant plasmids confirmed the location of this promoter and indicated that it was used in vivo by Tn4551. A second DRS promoter, which activated ermFS transcription by readthrough of the large DRS open reading frame, was also identified by the Northern blot analysis. The bicistronic ermFS message was not observed in strains containing a complete copy of Tn4551, and the possibility of transcriptional regulation is discussed.

Amino Acid Sequence↗
PubMed1984

Defining the beginning and end of KpnI family segments.

Comparison of the sequences at the ends of several newly cloned and full length members of the monkey KpnI family with one another and with previously described monkey and human segments defines the nucleotide sequence at the two termini. No terminal repeats either direct or inverted are noted within full length family members which may or may not be immediately flanked by direct repeats. At the 3' terminus, several family members have polyadenylation signals followed by a d(A)-rich stretch. The genomic frequency of segments within the full length element increases markedly from the 5' to the 3' terminus, consistent with the cloning of various truncated family members. One such truncated version joined to a low copy number DNA segment is inserted in monkey alpha-satellite where the combination appears to have been amplified in conjunction with the satellite itself.

Animals↗
PubMed1998

Nested insertions of short mobile sequences in Drosophila P elements.

A potentially full-sized P element isolated from the genome of Drosophila ambigua by polymerase chain reaction amplification was completely sequenced. It has a length of 3329 bp and the termini are formed by 33 bp inverted repeats. Sequence comparisons show that it can be classified as a member of the T-type P element subfamily. The translational reading frames of all four exons are interrupted by stop codons and frameshift mutations. At the 3' end of exon 3 a 687 bp insertion sequence (IS-amb-P) is found that also occurs in the form of dispersed copies (IS-amb) in the genome in D. ambigua. At the interspecific level it shows homology to mobile sequences of other species of the obscura group. Although variable in length, these IS elements are characterized by conserved sections without coding function and by 14 bp inverted repeats, one at a terminal, the other at a subterminal position. In situ hybridization revealed that P elements in D. ambigua are restricted to only two euchromatic sites on chromosome elements A and E. This situation resembles that found in Drosophila guanche and Drosophila subobscura where P homologs are clustered at a single site on chromosome element E and where the section corresponding to exon 3 of P elements carries an IS element. The gene sik-hom, which is located at the 5' side of the D. guanche cluster of P homologs, was used as a marker to examine whether the P element sites on chromosome element E of D. guanche and D. ambigua are homologous. The results suggest that the nested insertions of IS elements into P elements must have occurred independently in the two different lineages.

Amino Acid Sequence↗
PubMed1988

Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis.

The nucleotide sequence of the protective antigen (PA) gene from Bacillus anthracis and the 5' and 3' flanking sequences were determined. PA is one of three proteins comprising anthrax toxin; and its nucleotide sequence is the first to be reported from B. anthracis. The open reading frame (ORF) is 2319 bp long, of which 2205 bp encode the 735 amino acids of the secreted protein. This region is preceded by 29 codons, which appear to encode a signal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located at the putative -10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located 7 bp upstream from the ATG start codon. The codon usage for the PA gene reflected its high A + T (69%) base composition and differed from those of genes for bacterial proteins from most other sequences examined. The TAA translation stop codon was followed by an inverted repeat forming a potential termination signal. In addition, a 192-codon ORF of unknown significance, theoretically encoding a 21.6-kDa protein, preceded the 5' end of the PA gene.

Amino Acid Sequence↗
PubMed1994

Hypertransposing derivatives of the streptomycete insertion sequence IS493.

Transposons derived from the Streptomyces lividans insertion sequence IS493 are useful for the genetic analysis and manipulation of a number of Streptomyces spp. Tn5099-10, an IS493 derivative that contains a spontaneous deletion terminating in the left inverted repeat (IR-L), transposed at a 1000-fold higher frequency in Streptomyces griseofuscus, and at a tenfold higher frequency in Streptomyces fradiae, than the IS493 derivatives, Tn5096 and Tn5099. The IR-L from Tn5099-10 was used to construct a cassette which hypertransposes from plasmids containing the transposon genes, ORFA and ORFB, outside of the inverted repeats. The target sequences of two Tn5099-10 insertions conformed to the consensus target sequence of the other IS493 derivatives, gNCaNTgNNy (where lower-case letters indicate that other nt have been observed at this position and N is any nt). Transposition mutant libraries of S. griseofuscus and S. fradiae can be easily prepared in broth culture by using the hypertransposing elements and a temperature-sensitive delivery plasmid.

Base Sequence↗
PubMed2006

The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

Bacterial Proteins↗
PubMed1987

Isolation and characterization of a 1.7-kb transposable element from a mutator line of maize.

We have cloned and sequenced a 1.7-kb Mu element from a Mutator line of maize and compared its structure to Mu1, a 1.4-kb element. With the exception of a 385-bp block of DNA present in the 1.7-kb element, these transposable elements are structurally similar, sharing terminally inverted and internal direct repeated sequences. Derivation of 1.4-kb elements from the 1.7-kb class via deletion of internal sequence is suggested by the finding that a portion of the extra DNA in Mu1.7 is part of a truncated direct repeat sequence in the 1.4-kb element. An abundant poly(A)+ RNA homologous to a portion of this extra DNA is present in several tissues of both Mutator and non-Mutator lines. Analysis of transcripts from an unstable mutant bronze 1 (bz) allele containing a Mu1.7 element inserted in an exon of the gene detects three species of poly(A)+ RNA that hybridize to a Bz1 (Bronze) gene probe: the largest contains the entire Mu1.7 element in the Bz1 gene transcript; another appears to be a spliced, chimeric transcript; the smallest is normal size Bz1 mRNA. The latter is most likely encoded by the normal-size alleles detected by Southern analysis of tissue expressing purple pigment, suggesting that normal gene function is restored by excision of the Mu1.7 element.

Base Sequence↗
PubMed1993

Proposed structure for the DNA-binding domain of the helix-loop-helix family of eukaryotic gene regulatory proteins.

A modelled tertiary structure for the dimeric HLH domain of the E47 protein is presented. Structural information was obtained from the aligned sequences of > 40 members of the HLH family. The information was used to model each monomer as an alpha-helical hairpin, with knobs-into-holes packing of side-chains as found in antiparallel coiled-coil. The dimer forms a four-helix bundle with additional knobs-into-holes packing at the dimer interface. The size and electrostatic properties of core-forming residues are all accounted for in the model. The model does not violate any known properties of protein structure. The monomers are related by two-fold rotational symmetry, in agreement with the observed DNA-binding sites which are imperfect inverted repeats. The N-terminal basic region, in which DNA binding and base specificity reside, forms the first part of helix 1. A prediction based on the model structure is that the HLH domains do not bind to DNA in its B form but require a partially unwound conformation in order to enter the major groove.

Amino Acid Sequence↗
PubMed1990

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa.

Excision of the transposon Tn5 from sites of insertion in plasmid DNA was shown to occur at high frequency in Pseudomonas aeruginosa. Plasmids with Tn5 insertions conjugated poorly into P. aeruginosa and adversely affected growth compared to the respective parental plasmids. The kanamycin-resistance phenotype of Tn5 was expressed poorly in P. aeruginosa and kanamycin-sensitive strains were common during the manipulation of the P. aeruginosa transconjugants. Examination of plasmid DNA isolated from kanamycin-sensitive P. aeruginosa transconjugants revealed excision of Tn5 sequences. A plasmid containing a selectable marker (mercury resistance) inactivated by a Tn5 insertion was constructed, and Tn5 excised precisely, permitting the expression of the mercury-resistance marker at high frequency (10(-3) in P. aeruginosa and at the expected low frequency (10(-7] in Escherichia coli. The recombinational mechanism that promotes frequent Tn5 excision in P. aeruginosa operated in the absence of the P. aeruginosa recA gene product. Fragments of Tn5 were also examined for excision and instability in P. aeruginosa. A plasmid containing the terminal 485 bp of inverted repeat sequences from Tn5, but lacking the transposase or kanamycin-resistance genes, also showed precise excision of Tn5 DNA at high frequency (10(-2] in P. aeruginosa. Unlike plasmids containing a complete Tn5 insertion, this plasmid transferred to P. aeruginosa at normal frequencies and growth of the host was not severely impaired. In contrast, plasmids containing either IS50 element transferred to P. aeruginosa at greatly reduced frequencies, and transconjugants containing the IS50R element (which contains the active transposase gene) were small and especially difficult to maintain. P. aeruginosa transconjugants harbouring a plasmid containing only the DNA between the IS50 elements (which included the kanamycin-resistance gene) were of normal size and stably maintained.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1991

Tec2, a second transposon-like element demonstrating developmentally programmed excision in Euplotes crassus.

The analysis of a repetitive DNA interruption of the micronuclear precursor to a 0.85-kb macronuclear gene in the hypotrich Euplotes crassus has led to the identification of a second transposon-like element named Tec2. Two copies of this element, one inserted into the other, compose the interruption. The Tec2 element resembles the previously characterized Tec1 element in overall size, copy number, length, and extreme terminal sequence of its inverted repeats and in the apparent use of a 5'-TA-3' target site. In addition, extrachromosomal circular forms of Tec2 appear in DNA isolated from cells undergoing macronuclear development at the same time and with the same conformation as extrachromosomal circular forms of Tec1. These similarities suggest that the Tec1 and Tec2 elements may be under the same type of regulation during macronuclear development.

Animals↗
PubMed1989

Integration of an aberrant retrotransposon in Saccharomyces cerevisiae.

We describe an atypical composite Ty1 element that apparently resulted from the concurrent integration of two complete elements. A portion of the central region of one of these elements was inverted between two long terminal repeats. Inversions of this type have been detected among unintegrated retroviral circles. It now appears that such intermediates can be incorporated into the genome.

Base Sequence↗
PubMed2007

Methods for transient assay of gene function in floral tissues.

BACKGROUND: There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue. RESULTS: Two constructs, one expressing an inverted repeat of the Antirrhinum majus (Antirrhinum) chalcone synthase gene (CHS) and the other an inverted repeat of the Antirrhinum transcription factor gene Rosea1, were shown to effectively induce CHS and Rosea1 gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing in vitro. A high-throughput vector expressing the Antirrhinum CHS gene attached to an inverted repeat of the nos terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient Agrobacterium-mediated transformation of petal tissue of tobacco and petunia flowers in situ or detached was also achieved, using expression of the reporter genes GUS and GFP to visualise transgene expression. CONCLUSION: We demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via Agrobacterium infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to insert the gene sequence of interest. These techniques should allow analysis of gene function in a much broader range of flower species.

Journal Article↗
PubMed1988

Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae.

DNA segments from Neisseria gonorrhoeae, cloned and propagated in Escherichia coli, were tested for the ability to competitively inhibit gonococcal transformation. The nucleotide sequences of active segments were determined and compared; these sequences contained the sequence 5' GCCGTCTGAA 3' in common. Subcloning studies confirmed the identity of this sequence as the gonococcal DNA recognition site. The three instances of the recognition sequence isolated from N. gonorrhoeae chromosomal DNA contain the sequence in the immediate neighborhood of its inverted repeat. Because a single copy of the sequence functions as a recognition site, the inverted duplication is not required for specific binding. The dyad symmetric arrangements of the chromosomal recognition sequences may form stable stem-loop structures that can function as terminators or attenuators of transcription. These inverted repeats are located at the boundaries of long open reading frames. The recognition sequence also constitutes part of two other probable terminators of gonococcal genes. We conclude that the signal for recognition of transforming DNA by gonococci is a frequent component of transcriptional terminator sequences. This regulatory function might account for the origin and maintenance of recognition sequences in the chromosomes of Gram-negative transformable bacteria.

Base Sequence↗
PubMed1999

Specific interaction of the RNA-binding domain of the bacillus subtilis transcriptional antiterminator GlcT with its RNA target, RAT.

Expression of the Bacillus subtilis ptsGHI operon is controlled by transcriptional antitermination mediated by the antiterminator protein GlcT. The antiterminator is inactivated in the absence of glucose, presumably by phosphorylation. A conditional terminator in the ptsG mRNA leader region has been identified. Mutations in this terminator resulted in constitutive expression of the operon. The terminator is overlapped by an inverted repeat (called ribonucleic-antiterminator, RAT) which is thought to form a stem-loop structure upon binding of the antiterminator protein GlcT. The N-terminal 60 amino acid residues of GlcT are able to bind to the RAT and prevent transcriptional termination in vivo. Sequence-specific interaction between the RNA-binding domain and the RAT was demonstrated by surface plasmon resonance analysis. Mutations affecting the RNA-binding domain were isolated and will be discussed with respect to their consequences for dimerization and RNA binding.

Amino Acid Sequence↗
PubMed2004

Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus.

Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.

Animals↗
PubMed1991

Analysis of the complex transcription termination region of the Escherichia coli rrnB gene.

The complex terminator region of the Escherichia coli rrnB gene was analyzed by subcloning the terminators T1 and T2 and the inverted repeats IR1 and IR2 individually, or in various combinations, in a normal or inverted orientation into a terminator probe vector. The in vivo terminating efficiency was assayed by measuring the galactokinase activity encoded by the downstream galK gene. Termination efficiencies of all fragments were compared in two constructs, differing in the presence or absence of readthrough translation over the investigated terminator signal. The following main conclusions were drawn. (a) T1 and T2 are both efficient terminators in isolated forms. (b) IR1 and IR2 have some terminating effect (much lower than the proper terminators), especially in the inverted orientation. Their presence modifies the effect of the proper terminators in a quite unpredictable way, especially if these regions are translated. (c) The terminators are not symmetrical; in the inverted orientation T1 is practically inactive and T2 termination is reduced. (d) Translation radically decreases the efficiency of the terminators. (e) Several sequences in the rrnB gene, upstream of the terminator region (one in the 16S RNA and one in the 5S RNA coding region), are very efficient in vivo terminators in the inverted orientation.

Base Sequence↗
PubMed1981

XbaI, PstI, and BglII restriction enzyme maps of the two orientations of the varicella-zoster virus genome.

Cleavage of varicella-zoster virus DNA with the restriction endonucleases PstI, XbaI, and BglII resulted in 18, 22, and 20 fragments, respectively. Based on the molecular weights and molarities of these fragments, a molecular weight of 84 x 10(6) could be calculated for the varicella-zoster virus genome. In both the XbaI and the BglII patterns, four 0.5 M fragments were identified. The arrangement of the fragments was determined by molecular hybridization techniques, and the terminal fragments were identified by lambda exonuclease digestion. The 0.5 M fragments, of which two were located at the same terminus of the genome, contained repeated sequences: one terminally and one inverted internally. These results were in agreement with the existence of two equimolar subpopulations of the varicella-zoster virus genome, differing in the relative orientation of a short region of unique sequences. This region was bounded by the repeated sequences. From the molecular weights of the submolar fragments, a maximal molecular weight of 5 x 10(6) for the repeated region and a minimal molecular weight of 3.5 x 10(6) for the short unique sequence could be calculated.

Bacterial Proteins↗
PubMed1993

Mobilization of quiet, endogenous Tc3 transposons of Caenorhabditis elegans by forced expression of Tc3 transposase.

The commonly studied Caenorhabditis elegans strain Bristol N2 contains approximately 15 copies per genome of the transposon Tc3. However, Tc3 is not active in Bristol N2. Tc3 contains one major open reading frame (Tc3A). We have fused this open reading frame to an inducible promoter and expressed it in a transgenic Bristol N2 line. Tc3A expression resulted in frequent excision and transposition of endogenous Tc3 elements. This shows that the Bristol N2 genome contains Tc3 transposons that are cis proficient for transposition, but are immobile because Tc3A is absent. We demonstrate that recombinant Tc3A binds specifically to the terminal nucleotides of the Tc3 inverted repeat, indicating that Tc3A is the Tc3 transposase. Activation of Tc3 transposition in vivo was accompanied by the appearance of extrachromosomal, linear copies of Tc3. These may be intermediates in Tc3 transposition.

Animals↗