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PubMed1991

Ammonium transport in Escherichia coli: localization and nucleotide sequence of the amtA gene.

Escherichia coli expresses a concentrative ammonium (methylammonium) transport system which is strongly repressed under conditions of nitrogen excess. We have previously reported the cloning of a structural gene (amtA) for this transporter by complementation. In this study, a 3.4 kb HindIII-BamHI fragment containing amtA was cloned into the pBluescript KS(+) vector, and unidirectional nested deletions from each end of this 3.4 kb fragment were generated by exonuclease III digestion. The deletions were analysed by complementation of the structural gene mutation produced by Tn10 insertion. This allowed amtA to be localized within a 1.4 kb region which spans the site of the mutation. By application of the Sanger dideoxy method, we sequenced the region containing amtA. The gene contains an open reading frame which encodes a protein with a predicted molecular mass of 27 kDa. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by an inverted repeat which might function as a simple transcription terminator. Hydropathic analysis of the inferred amino acid sequence of the gene product predicts that amtA encodes a cytoplasmic component of the ammonium transport system.

Amino Acid Sequence↗
PubMed1992

Characterization of the minimal origin required for replication of the streptococcal plasmid pIP501 in Bacillus subtilis.

By using deletional analysis the origin of replication, oriR, of the streptococcal plasmid pIP501 in Bacillus subtilis has been mapped at a position immediately downstream of the repR gene. Determination of both the right and left border of oriR allowed the definition of a sequence of a maximum of 52 nucleotides which theoretically constitutes the minimal origin of replication. Recently, the start point of leading-strand synthesis of the closely related plasmid pAM beta 1 has been mapped at a position which is located exactly in the middle of this sequence (Bruand et al., 1991). The function of oriR did not depend on its location downstream of the repR gene. Translocation of oriR-containing fragments to other regions of the plasmid proved to be possible. The smallest translocated fragment that still reconstituted autonomous replication was 72bp in size. This fragment was also active in directing the replication of an Escherichia coli plasmid in B. subtilis when the RepR protein was supplied in trans from a repR gene integrated into the host chromosome. The transformation efficiency of plasmids carrying translocated oriR fragments showed a certain dependence on the fragment length and orientation. The DNA sequence of oriR included an inverted repeat, both branches of which appeared to be essential for oriR function. The repeats of oriR shared sequence similarity with a repeat located upstream of promoter pII, which has been suggested to be involved in autoregulation of repR expression.

Amino Acid Sequence↗
PubMed2003

Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection.

BACKGROUND: Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. RESULTS: In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23-25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenic plants 6 weeks after viral inoculation. CONCLUSIONS: The ihprolC-PP197 transgene confers systemic resistance to PPV disease in N. benthamiana. Local infection is unaffected. This transgene and/or similar constructs could be used to confer PPV resistance to fruit trees where systemic disease causes economic damage.

Gene Expression Regulation, Plant↗
PubMed1998

Genetic organization and DNA sequence of early region 4 of bovine adenovirus type 3.

We have identified and sequenced 3614 nucleotides located at the extreme right-end of the bovine adenovirus type 3 (BAV3) genome from map units 89.5-100. Analysis of the sequence revealed an inverted terminal repeat (ITR) of 195 bp, and identified five open reading frames (ORFs) designated ORF1, ORF2, ORF3, ORF4 and ORF5. When compared with known E4 ORFs of other adenoviruses, ORFs 1, 2 and 4, which code for proteins of 143, 69 and 143 amino acids respectively, were found to be unique to BAV3. ORFs 3 and 5, which code for proteins of 268 and 219 amino acids respectively, showed partial homology to the E4 34 kDa protein of human adenovirus 2. Nucleotide sequence analysis also identified two potential TATA boxes upstream of ORF1 and a potential polyadenylation signal downstream of ORF5 suggesting that E4 transcripts may be 3' co-terminal.

Adenoviridae↗
PubMed1992

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3.

The genetic environment of plasmid-borne blaTEM mutant genes, encoding nine distinct TEM-type extended-spectrum beta-lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine blaTEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes blaTEM-1 or -2. A 6.6 kb DNA fragment of pCFF04 containing blaTEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that blaTEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with blaTEM-3.

Amino Acid Sequence↗
PubMed2003

Alternative transcription initiation sites and polyadenylation sites are recruited during Mu suppression at the rf2a locus of maize.

Even in the absence of excisional loss of the associated Mu transposons, some Mu-induced mutant alleles of maize can lose their capacity to condition a mutant phenotype. Three of five Mu-derived rf2a alleles are susceptible to such Mu suppression. The suppressible rf2a-m9437 allele has a novel Mu transposon insertion (Mu10) in its 5' untranslated region (UTR). The suppressible rf2a-m9390 allele has a Mu1 insertion in its 5' UTR. During suppression, alternative transcription initiation sites flanking the Mu1 transposon yield functional transcripts. The suppressible rf2a-m8110 allele has an rcy/Mu7 insertion in its 3' UTR. Suppression of this allele occurs via a previously unreported mechanism; sequences in the terminal inverted repeats of rcy/Mu7 function as alternative polyadenylation sites such that the suppressed rf2a-m8110 allele yields functional rf2a transcripts. No significant differences were observed in the nucleotide compositions of these alternative polyadenylation sites as compared with 94 other polyadenylation sites from maize genes.

Base Sequence↗
PubMed2002

Cloning and analysis of the telomere and terminal inverted repeat of the linear chromosome of Streptomyces griseus.

Cloning and sequencing of the telomere of Streptomyces griseus revealed five palindromic sequences in the terminal 116 nucleotides, all of which can make a hairpin loop structure. However, the end sequence cannot form the foldback secondary structure that is common in Streptomyces telomeres and is suggested to be necessary for terminal replication. Both inside ends of the terminal inverted repeat (TIR) were also cloned and sequenced. The results confirmed the size of the TIR to be 24 kb and identified two almost identical open reading frames that might have been involved in the formation of the TIR.

Base Sequence↗
PubMedDate not supplied

Cloning and characterization of the natural lactose operator.

A 55-bp DNA segment carrying the wild-type lactose operator sequence has been cloned. Its sequence is: (Formula: see text). With the exceptions of the bases at positions 19 and 41, 26 and 34, and 28 and 32, the sequence is a perfect inverted repeat about base pair 30. This segment was obtained from the wild-type lactose promoter and operator region of lambda h80dlac phage DNA by a combination of in vitro and in vivo steps. Up to four direct-repeat copies of this segment have been cloned in plasmid pMB9 and pBR325. Repressor affinity for this 55-bp fragment does not differ significantly from that for a 40-bp synthetic operator fragment cloned previously, even though the 55-bp fragment contains the complete set of sequence symmetries associated with the natural operator, whereas the 40-bp fragment does not. An improved procedure for operator purification is described: this was used to prepare 14 mg of the 55-bp fragment over a 2-month period.

Bacteriophage lambda↗
PubMed1994

Nitrite and nitrate regulation at the promoters of two Escherichia coli operons encoding nitrite reductase: identification of common target heptamers for both NarP- and NarL-dependent regulation.

Expression from both the Escherichia coli nir and nrf promoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium. The nir promoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite. The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 69 1/2 bp upstream of the transcript startpoint. The nrf promoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate. Mutational analysis of the nrf promoter has been exploited to corroborate the location of the -10 hexamer and the FNR-binding site, and to find the sites essential for nitrite-dependent activation and nitrate-dependent repression. Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at the nir promoter, but centred 74 1/2 bp upstream from the transcript start. NarL-dependent repression by nitrate is due to two heptamer sequences that flank the FNR-binding sequence. We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from site to site.

Bacterial Proteins↗
PubMed1986

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli.

The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430 omega APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430 omega APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).

Bacillus thuringiensis↗
PubMed1997

Nht1, a transposable element cloned from a dispensable chromosome in Nectria haematococca.

Certain isolates of the plant-pathogenic fungus Nectria haematococca mating population VI (MPVI) contain dispensable chromosomes that are unstable during sexual reproduction. Several of these chromosomes carry genes for phytoalexin detoxification and thus contribute to the pathogenic potential of this organism. A repeated DNA sequence, Nht1, was cloned from one of these dispensable chromosomes in N. haematococca MPVI. One copy of the repeated element (Nht1A) was completely sequenced. It is 2,198 bp long and it possesses incomplete inverted terminal repeats (ITRs) at each end. Nht1B, a partially sequenced copy of Nht1, has complete ITRs. Nht1A appears to contain 2 introns and encodes a protein of 550 amino acids that is highly similar to the protein encoded by the Fusarium oxysporum transposon, Fot1. Due to the presence of ITRs, its repeated nature, and its similarity to Fot1, we conclude that Nht1 is a transposable element. Within North American N. Haematococca MPVI populations, Nht1 is distributed discontinuously. Its copy number in different field isolates varies from zero to approximately 100 copies per genome. The Nht1A source isolate is estimated to contain nine to 11 copies of Nht1; at least six are on the chromosome from which Nht1A was cloned.

Amino Acid Sequence↗
PubMed1998

Cloning, expression, and catabolite repression of a gene encoding beta-galactosidase of Bacillus megaterium ATCC 14581.

A gene encoding beta-galactosidase, designated mbgA, was isolated from Bacillus megaterium ATCC 14581. Chromosomal beta-galactosidase production could be dramatically induced by lactose but not by isopropyl-beta-D-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of mbgA in the B. megaterium chromosome resulted in loss of lactose-inducible beta-galactosidase production. A 27-bp inverted repeat was found to overlap the mbgA promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s).

Bacillus megaterium↗
PubMedDate not supplied

Gene-for-genes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes.

Six plasmid-borne avirulence (avr) genes were previously cloned from strain XcmH of the cotton pathogen, Xanthomonas campestris pv. malvacearum. We have now localized all six avr genes on the cloned fragments by subcloning and Tn5-gusA insertional mutagenesis. None of these avr genes appeared to exhibit exclusively gene-for-gene patterns of interactions with cotton R genes, and avrB4 was demonstrated to confer avr gene-for-R genes (plural) avirulence to X. c. pv. malvacearum on congenic cotton lines carrying either of two different resistance loci, B1 or B4. Furthermore, the B1 locus appeared to confer R gene-for-avr genes resistance to cotton against isogenic X. c. pv. malvacearum strains carrying any one of three avr genes: avrB4, avrb6, or avrB102. Restriction enzyme, Southern blot hybridization, and DNA sequence analyses showed that the XcmH avr genes are all highly similar to each other, to avrBs3 and avrBsP from the pepper pathogen X. c. pv. vesicatoria, and to the host-specific virulence gene pthA from the citrus pathogen X. citri. The XcmH avr genes differed primarily in the multiplicity of a tandemly repeated 102-base pair motif within the central portions of the genes, repeated from 14 to 23 times in members of this gene family. The complete nucleotide sequence of avrb6 revealed that it is 97% identical in DNA sequence to avrB4, avrBs3, avrBsP, and pthA and that 62-bp inverted terminal repeats mark the boundaries of homology between avrb6 and all members of this Xanthomonas virulence/avirulence gene family sequenced to date. The terminal 38 bp of both inverted repeats are highly similar to the 38-bp consensus terminal sequence of the Tn3 family of transposons. Up to 11 members of the avr gene family appear to be present in North American strains of X. c. pv. malvacearum, including XcmH. The high level of homology observed among these avr genes and their presence in multiple copies may explain the gene-for-genes interactions and also the observed high frequencies (10(-3) to 10(-4) per locus) of X. c. pv. malvacearum race change mutations. Five spontaneous race change mutants of XcmH suffered avr locus deletions, strongly indicating intergenic recombination as the primary mechanism for generating new races in X. c. pv. malvacearum.

Amino Acid Sequence↗
PubMed1992

Rapid identification and differentiation of the vaccine strain Rac H from EHV 1 field isolates using a non-radioactive DNA probe.

A method for rapid differentiation between the EHV 1 live vaccine strain Rac H and field isolates is described. Total DNA was isolated from virus-infected small scale cell cultures. DNA fragments digested with restriction endonuclease BamHI were separated, transferred and immobilized on filter membranes. A Digoxigenin-labeled probe derived from EHV 1 was used for hybridization. This probe hybridized specifically to sequences of the inverted terminal repeat region which in case of Rac H include a deletion of 0.8 kb. By comparing the different migration patterns after blot hybridization it could be shown that in 65 isolates from cases of abortion the live vaccine strain Rac H was not involved.

Abortion, Veterinary↗
PubMed2000

Sequence analysis of transposable elements in the sea squirt, Ciona intestinalis.

A systematic search of 1 Mb of genomic sequences from the sea squirt, Ciona intestinalis, revealed the presence of six families of transposable elements. The Cigr-1 retrotransposon contains identical 245-bp long terminal repeats (LTRs) and a 3,630-bp open reading frame (ORF) encoding translation products in the same order as the domains characteristic of gypsy/Ty3-type LTR retrotransposons. The closest homologs of the reverse transcriptase domain were in gypsy elements from Drosophila and the sushi element from the pufferfish. However, the capsid-nucleocapsid region shows the clearest homology to an echinoderm element, Tgr1. Database searches also indicated two classes of non-LTR retrotransposon, named Cili-1 and Cili-2. The Cili-1 sequences show matches to regions of the ORF2 product of mammalian L1 elements. The Cili-2 sequences possess similarity to the RNaseH domain of Lian-Aa1, a mosquito non-LTR retrotransposon. The most abundant element was a short interspersed nucleotide element named Cics-1 with a copy number estimated at 40,000. Cics-1 consists of two conserved domains separated by an A-rich stretch. The 172-bp 5' domain is related to tRNA sequences, whereas the 110-bp 3' domain is unique. Cics-1 is unusual, not just in its modular structure, but also in its lack of a 3' poly(A) tail or direct flanking repeats. A second abundant element, Cimi-1, has an A+T-rich 193-bp consensus sequence and 30-bp terminal inverted repeats (TIRs) and is usually flanked by A+T-rich 2-4-bp putative target site duplications-characteristics of miniature inverted-repeat transposable elements found in plants and insects. A single 2,444-bp foldback element was found, possessing long TIRs containing an A+T-rich internal domain, an array of subrepeats, and a flanking domain at the TIR ends; this is the first example of a chordate foldback element. This study provides the first systematic characterization of the families of transposable elements in a lower chordate.

Amino Acid Sequence↗
PubMed2000

IS1294, a DNA element that transposes by RC transposition.

IS1294, found on the ColD-like resistance plasmid pUB2380, is IS91-like. It is an active 1.7-kb insertion sequence that lacks terminal inverted repeats, displays insertion-site specificity, and does not generate direct repeats of the target site. The element has one large open reading frame, tnp(1294), encoding a transposase of 351 amino acids, related to members of the REP family of replication proteins used by RC-plasmids of gram-positive bacteria. IS1294 transposes using rolling-circle replication, initiated at one end of the element, oriIS, and terminated at the other, terIS. oriIS and terIS are highly conserved among like IS elements. oriIS resembles the leading strand replication origins of RC-plasmids; terIS resembles a rho-independent transcription terminator. IS1294 mediates not only its own transposition, but also sequences adjacent to terIS. A transposition model for IS1294 and related elements, involving rolling-circle replication and single-strand DNA intermediates, is presented.

Amino Acid Sequence↗
PubMed1999

Long inverted repeats are an at-risk motif for recombination in mammalian cells.

Certain DNA sequence motifs and structures can promote genomic instability. We have explored instability induced in mouse cells by long inverted repeats (LIRs). A cassette was constructed containing a herpes simplex virus thymidine kinase (tk) gene into which was inserted an LIR composed of two inverted copies of a 1.1-kb yeast URA3 gene sequence separated by a 200-bp spacer sequence. The tk gene was introduced into the genome of mouse Ltk(-) fibroblasts either by itself or in conjunction with a closely linked tk gene that was disrupted by an 8-bp XhoI linker insertion; rates of intrachromosomal homologous recombination between the markers were determined. Recombination between the two tk alleles was stimulated 5-fold by the LIR, as compared to a long direct repeat (LDR) insert, resulting in nearly 10(-5) events per cell per generation. Of the tk(+) segregants recovered from LIR-containing cell lines, 14% arose from gene conversions that eliminated the LIR, as compared to 3% of the tk(+) segregants from LDR cell lines, corresponding to a >20-fold increase in deletions at the LIR hotspot. Thus, an LIR, which is a common motif in mammalian genomes, is at risk for the stimulation of homologous recombination and possibly other genetic rearrangements.

Animals↗
PubMed1994

Homo- and heterodimers of the retinoid X receptor (RXR) activated transcription in yeast.

The polymorphic nature of sequences which act as retinoic acid response elements (RAREs and RXREs) in transactivation assays in mammalian cells, suggests that elements consisting of a direct repetition of a half site motif, separated by 1 to 5 base pairs (DR1 to DR5), are targets for retinoic acid (RA) signalling. In a previous report we showed that in yeast cells, heterodimers of the retinoic acid receptors RAR alpha and RXR alpha were required for efficient transcription of a reporter gene containing a DR5 element [Heery et al., (1993); Proc. Natl. Acad. Sci. USA, 90: 4281-4285]. Here we report that DR1 to DR5 elements containing a direct repeat of the 5'-AGGTCA-3' motif, and an inverted repeat of the same sequence with no spacer (IR0), behave as RAREs in yeast cells coexpressing RAR alpha and RXR alpha, albeit with different efficacies. Heterodimer activity was strongest on a DR5 reporter gene, and the strength of activation of the reporter series (DR5 > DR1 > DR3 > DR2 = IR0 = DR4) correlated with the ability of the heterodimer to bind to the corresponding sequences in vitro. Significantly, a reporter containing a DR1 element was selectively and efficiently activated in yeast cells expressing only RXR alpha. This activity was dependent on the induction by 9-cis retinoic acid of an activation function (AF-2) located in the RXR alpha ligand binding domain. In addition, a strong synergistic activity of RXR alpha was observed on a reporter containing the putative RXR element (RXRE) from the rat CRBPII gene promoter. Thus, RXR alpha can function independently as a transcription factor, in the absence of RARs or other heteromeric partners. Similarly, homodimers of RAR alpha selectively stimulated the transcription of a DR5 reporter in a ligand-dependent manner, but less efficiently than RAR alpha/RXR alpha heterodimers.

Base Sequence↗