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At least 1,243 records · Page 69Linked to original sources
PubMed1989

Repair of imidazole ring-opened purines in DNA: overproduction of the formamidopyrimidine-DNA glycosylase of Escherichia coli using plasmids containing the fpg+ gene.

The formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase) of Escherichia coli (E. coli) was overexpressed by cloning the fpg+ gene on a multicopy plasmid and placing this gene under the control of the lac promoter. The lac promoter contributed significantly to the overall expression of the fpg gene only after the deletion of an inverted repeat sequence located immediately upstream from the fpg promoter. The biological purpose of the inverted repeat sequence may be associated with the termination of an adjacent gene transcribed in the same direction as the fpg gene in E. coli. Cells harboring the fpg gene under the control of the lac promoter were able to produce the Fapy-DNA glycosylase as at least 17% of the total soluble proteins. Such strains allow the preparation of milligram quantities of pure protein for use in the study of its catalytic properties and three dimensional crystal structure.

DNA Damage↗
PubMed1988

The origin of conjugative IncP plasmid transfer: interaction with plasmid-encoded products and the nucleotide sequence at the relaxation site.

To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication we isolated and dissected the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. Essential features of oriT are conserved: symmetric sequence repeats, the nic site and a pair of potential promoter sites that allow for divergent transcription of two tra operons. The relaxation nick and the end of a 19 bp inverted repeat are interspaced by eight basepairs. The 5'-terminal nucleotide at the nick is modified by an alkali-resistant residue and the 3'-nucleotide is accessible to extension by DNA polymerase I. Transfer gene products essential for the formation of the initiation complex (relaxosome) of conjugative DNA synthesis map adjacent to oriT. Two of these products, TraJ and TraK confer specificity to their homologous oriT exclusively. Proteins TraJ and TraK are the only components of the RP4 and R751 transfer machinery which cannot be interchanged. TraJ and at least two additional plasmid-encoded products are necessary for specific relaxation. The purified TraJ protein of RP4 possesses oriT-binding ability. The recognition sequence contains a palindromic sequence located within the right arm of the 19 bp inverted repeat. The TraJ binding site and the nic site are located on one side of the DNA double helix. We presume that this nucleoprotein structure is the initial complex in the pathway to the assembly of functional relaxosomes.

Bacterial Proteins↗
PubMed1991

The ability of pseudorabies virus to grow in different hosts is affected by the duplication and translocation of sequences from the left end of the genome to the UL-US junction.

Pseudorabies virus is a herpesvirus which has a class 2 genome. However, under certain growth conditions it acquires a genome with class 3-like characteristics. In these variants, the leftmost sequences of the long (L) component of the viral genome have been duplicated and translocated to the right of the L component next to the short (S) component, resulting in an L component that is bracketed by inverted repeats. Consequently, the L component can invert and is found in two orientations relative to the S component. The translocation is accompanied invariably by a deletion of sequences that are normally present in the wild-type genome at the right end of the L component. The virion variants with an invertible L component have a growth advantage over wild-type virus in chicken embryo fibroblasts and chickens; they also have a growth disadvantage in mice or rabbit kidney cells. The changed growth characteristics of the variants reside entirely in the changed structure of the junction between the S and L components. Replacement of that region of the DNA with wild-type sequences restores the wild-type phenotype. To determine whether the modified growth characteristics of the variants are related to the translocation or to the deletion, mutants that have a deletion or that have a deletion as well as a translocation similar to those observed in the variants were constructed, and the growth characteristics of these mutants were determined. We show that the modified growth characteristics of the mutants with an invertible L component can be attributed to the translocation of the leftmost terminal sequences of the genome next to the inverted repeat; they are not related to the deletion of the sequences normally present at the right end of the L component. The translocation of the leftmost 325 bp of the genome is sufficient to confer upon the virus the modified cell-type-specific growth characteristics. Furthermore, the modified growth characteristics are contingent upon the presence of 68 bp spanning the internal junction between the L and S components.

Animals↗
PubMed1982

Generation of authentic 3' termini of an H2A mRNA in vivo is dependent on a short inverted DNA repeat and on spacer sequences.

We have determined what sequences are required to generate the authentic 3' termini of a sea urchin H2A histone mRNA. We have constructed a series of deletion and insertion mutants in the cloned histone repeat unit h22 of Psammechinus miliaris and have analyzed the transcripts of both wild-type and mutant DNAs produced in the frog oocyte. The protein-coding sequences of the H2A gene can be removed without any deleterious effects on transcription initiation or termination. A 12 bp deletion, which removes a highly conserved inverted DNA repeat immediately preceding the H2A mRNA 3' terminus, elicits read-through of the polymerase into the spacer DNA further downstream. However, the inverted repeat and the sequence coding for the 3' terminus of the mRNA are by themselves not sufficient to generate faithful 3' ends. Our data suggest that spacer sequences downstream of the 3' mRNA terminus are required as well.

Animals↗
PubMed1989

The small subunit of ribulose-1,5-bisphosphate carboxylase is plastid-encoded in the chlorophyll c-containing alga Cryptomonas phi.

The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) is located in the large single-copy region of the plastid genome of the chlorophyll c-containing alga Cryptomonas phi. The coding sequence is 417 base pairs long, encoding a protein of 139 amino acids, considerably longer than most other small subunit proteins. It is found 83 base pairs downstream from the gene for the large subunit and is cotranscribed with it. An 18 base pair perfect inverted repeat is located 8 base pairs beyond the termination codon. Sequence analysis shows the gene to be more closely related to cyanobacterial and cyanelle small-subunit genes than to those of green algae or land plants. This is the first reported sequence of a Rubisco small-subunit gene which is plastid-encoded and it exhibits a number of unique features. The derived amino acid sequence shows extensive similarity to a partial amino acid sequence from a brown alga, indicating that this gene will be of major interest as a probe for the small subunit genes in other algae and for determining possible evolutionary ancestors of algal plastids.

Amino Acid Sequence↗
PubMed1993

A new middle repetitive sequence of Nicotiana plumbaginifolia genome.

A middle repetitive sequence NPR18 was isolated from Nicotiana plumbaginifolia nuclear genome [8]. Sequences homologous to the repeat are dispersed through genomes of several Nicotiana species. Computer-assisted data analysis of NPR18 primary sequence reveals several features attributed to mobile genetic elements: an AT content higher than average for nuclear DNA of genus Nicotiana plants; a number of direct and inverted repeats. Some of the repeats displayed homology to the terminal and subterminal repeats of Ac/Ds-like plant elements.

Base Sequence↗
PubMed1994

Nucleotide sequence analysis and potential environmental distribution of a ferric pseudobactin receptor gene of Pseudomonas sp. strain M114.

The nucleotide sequence of the Pseudomonas sp. strain M114 pbuA gene, encoding the outer membrane receptor for ferric pseudobactin M114, has been determined. The region sequenced spans 2788 bases of plasmid pCUP3, within which the receptor gene had previously been localised. A single open reading frame, potentially encoding 826 amino acids and including a leader peptide of 44 amino acids, is evident and is followed by an inverted repeat segment, which may act as a transcriptional terminator. A 20 bp region of DNA, having significant homology with the E. coli Fur-binding consensus sequence, is located upstream of the open reading frame. PbuA displays characteristics in common with other outer membrane proteins and displays strong homology with the TonB boxes of both E. coli and Pseudomonas receptors. More extensive homologies were found with the PupA receptor of P. putida WCS358 and the FhuE and BtuB receptors of E. coli. It is suggested that areas exhibiting the least homology between these receptors may represent ferric siderophore-specific recognition sites of the PbuA protein. The deduced amino acid sequence of pbuA was compared with that of pupX, encoding the outer membrane receptor for ferric pseudobactin B10, of Pseudomonas sp. strain B 10. A direct alignment of the two proteins gave an identity score of 92.5%. The distribution of PbuA-like receptors among Pseudomonas isolates was investigated by DNA-DNA hybridisation analysis. The results suggest that a PbuA-like receptor may be widely distributed among Pseudomonas rhizosphere isolates.

Amino Acid Sequence↗
PubMed1990

Identification of the replication terminator protein binding sites in the terminus region of the Bacillus subtilis chromosome and stoichiometry of the binding.

DNase I footprinting of the interaction between the replication terminator protein (RTP) of Bacillus subtilis and the inverted repeat region (IRR) at the chromosome terminus, to which it binds to block the clockwise replication fork, showed that two major regions of 41 base pairs (bp) were protected from cleavage. These regions corresponded approximately to the imperfect inverted repeats (IRI and IRII) identified previously. Band retardation analyses of the interaction between RTP and portions of the IRR established that each inverted repeat (IRI or IRII) contained two RTP binding sites. By sedimentation equilibrium in the ultracentrifuge, RTP was found to exist as a dimer of 29 kDa at neutral pH and concentrations above 0.2 g/l. Quantitative studies of the RTP-IRR interaction using [3H]RTP and [32P]IRR showed that the fully saturated complex contained eight RTP monomers per IRR. It is concluded that a dimer of RTP binds to each of the four sites in IRR. The apparent dissociation constant for the interaction was estimated (in the presence of 50% glycerol) to be 1.2 x 10(-11) M (dimer of RTP). Glycerol was found to have a marked effect on the affinity of RTP for the IRR and on the relative amounts of the interaction complexes formed; in the absence of glycerol the dissociation constant was approximately 50-fold higher and there was pronounced co-operative binding of RTP dimers to adjacent sites in each inverted repeat. Examination of the DNA sequence in IRI and IRII identified two 8 bp direct repeats in each. The regions protected from DNase I cleavage in each inverted repeat and the protection afforded by a core sequence spanning just one of the 8 bp direct repeats were consistent with each 8 bp repeat representing a recognition sequence for the RTP dimer. A model describing the binding of RTP to the IRR is presented.

Bacillus subtilis↗
PubMed1990

Sequence analysis of the rice dwarf phytoreovirus segment S3 transcript encoding for the major structural core protein of 114 kDa.

The primary structure of rice dwarf phytoreovirus (RDV) genome segment S3 was determined. RDV S3 consists of 3195 nucleotides. A 14-bp segment-specific inverted repeat is located immediately adjacent to the conserved terminal sequence (5'GGCAAA---UGAU3'). A single long open reading frame encoding for 1019 amino acids with an Mr of 114,289 is also identified. In order to investigate the localization of the predicted polypeptide, we determined the amino acid sequence of the 26-kDa peptide fragment obtained from the structural core protein digested by Staphylococcus aureus V8 protease. The sequence of the fragment was found in the translational product presumed from the nucleotide sequence of RDV S3, indicating that RDV S3 encodes the major structural core protein of 114 kDa.

Amino Acid Sequence↗
PubMed1990

Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction.

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.

Base Sequence↗
PubMed1989

The Tc3 family of transposable genetic elements in Caenorhabditis elegans.

We describe genetic and molecular properties of Tc3, a family of transposable elements in Caenorhabditis elegans. About 15 Tc3 elements are present in the genomes of several different wild-type varieties of C. elegans, but Tc3 transposition and excision are not detected in these strains. Tc3 transposition and excision occur at high frequencies, however, in strain TR679, a mutant identified because of its highly active Tc1 elements. In TR679, Tc3 is responsible for several spontaneous mutations affecting the unc-22 gene. Tc3-induced mutations are unstable, and revertants result from precise or nearly precise excision of Tc3. Although Tc3 is very active in TR679, it is not detectably active in several other mutator mutants, all of which exhibit high levels of Tc1 activity. Tc3 is 2.5 kilobases long, and except for sequences near its inverted repeat termini, it is unrelated to Tc1. The termini of Tc3 are inverted repeats of at least 70 base pairs; the terminal 8 nucleotides of Tc3 are identical to 8 of the terminal 9 nucleotides of Tc1.

Animals↗
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↗
PubMed1997

Molecular characterization of the bet genes encoding glycine betaine synthesis in Sinorhizobium meliloti 102F34.

As a first step towards the elucidation of the molecular mechanisms responsible for the utilization of choline and glycine betaine (betaine) either as carbon and nitrogen sources or as osmoprotectants in Sinorhizobium meliloti, we selected a Tn5 mutant, LTS23-1020, which failed to grow on choline but grew on betaine. The mutant was deficient in choline dehydrogenase (CDH) activity, failed to oxidize [methyl-14C]choline to [methyl-14C]betaine, and did not use choline, but still used betaine, as an osmoprotectant. The Tn5 mutation in LTS23-1020 was complemented by plasmid pCHO34, isolated from a genomic bank of S. meliloti 102F34. Subcloning and DNA sequencing showed that pCHO34 harbours two ORFs which showed 60% and 57% identity with the Escherichia coli betB gene encoding betaine-aldehyde dehydrogenase (BADH) and betA gene encoding CDH, respectively. In addition to the homology with E. coli genes, the deduced sequence of the sinorhizobial BADH protein displays consensus sequences also found in plant BADHs. The deduced sequence of the sinorhizobial CDH protein shares only 21% identical residues with choline oxidase from Arthrobacter globiformis. The structural organization of the betBA genes in S. meliloti differs from that described in E. coli: (i) the two ORFs are separated by a 210 bp sequence containing inverted repeats resembling a putative rho-independent transcription terminator, and (ii) no sequence homologous to betT (high-affinity choline transport system) or betI (regulator) was found in the vicinity of the sinorhizobial betBA genes. Evidence is also presented that the S. meliloti betBA genes are not located on the megaplasmids.

Alcohol Oxidoreductases↗
PubMed1989

Organization of complex transposon Tn2610 carrying two copies of tnpA and tnpR.

Transposon Tn2610 has two elements of 3.5 kilobase pairs as inverted repeats, one set at each end. This unique terminal element contained the transposition genes tnpA and tnpR. Only the tnpA gene in the left element was functional for transposition, whereas both tnpR genes were active. Possible evolutionary relationships among class II transposable elements are proposed on the basis of the genetic and structural organization of Tn2610.

Anti-Bacterial Agents↗
PubMed2001

Organization and transcriptional analysis of a six-gene cluster around the rplK-rplA operon of Corynebacterium glutamicum encoding the ribosomal proteins L11 and L1.

A cluster of six genes, tRNA(Trp)-secE-nusG-rplK-rplA-pkwR, was cloned and sequenced from a Corynebacterium glutamicum cosmid library and shown to be contiguous in the C. glutamicum genome. These genes encode a tryptophanyl tRNA, the protein translocase component SecE, the antiterminator protein NusG, and the ribosomal proteins L11 and L1 in addition to PkwR, a putative regulatory protein of the LacI-GalR family. S1 nuclease mapping analysis revealed that nusG and rplK are expressed as separate transcriptional units and rplK and rplA are cotranscribed as a single mRNA. A 19-nucleotide inverted repeat that appears to correspond to a transcriptional terminator was located in the 3' region downstream from nusG. Northern analysis with different probes confirmed the S1 mapping results and showed that the secE-rplA four-gene region gives rise to four transcripts. secE was transcribed as a 0.5-kb monocistronic mRNA, nusG formed two transcripts of 1.4 and 1.0 kb from different initiation sites, and the two ribosomal protein genes rplK and rplA were cotranscribed as a single mRNA of 1.6 kb. A consensus L1 protein binding sequence was identified in the leader region of the rplK-rplA transcript, suggesting that expression of the rplK-rplA cluster was regulated by autogenous regulation exerted by the L1 protein at the translation level. The promoters of the nusG and rplK-rplA genes were subcloned in a novel corynebacterial promoter-probe vector and shown to confer strong expression of the reporter gene.

Base Sequence↗
PubMed2006

Plasticity of the P junc promoter of ISEc11, a new insertion sequence of the IS1111 family.

We describe identification and functional characterization of ISEc11, a new insertion sequence that is widespread in enteroinvasive E. coli (EIEC), in which it is always present on the virulence plasmid (pINV) and very frequently also present on the chromosome. ISEc11 is flanked by subterminal 13-bp inverted repeats (IRs) and is bounded by 3-bp terminal sequences, and it transposes with target specificity without generating duplication of the target site. ISEc11 is characterized by an atypical transposase containing the DEDD motif of the Piv/MooV family of DNA recombinases, and it is closely related to the IS1111 family. Transposition occurs by formation of minicircles through joining of the abutted ends and results in assembly of a junction promoter (P juncC) containing a -10 box in the interstitial sequence and a -35 box upstream of the right IR. A natural variant of ISEc11 (ISEc11p), found on EIEC pINV plasmids, contains a perfect duplication of the outermost 39 bp of the right end. Upon circularization, ISEc11p forms a junction promoter (P juncP) which, despite carrying -10 and -35 boxes identical to those of P juncC, exhibits 30-fold-greater strength in vivo. The discovery of only one starting point in primer extension experiments rules out the possibility that there are alternative promoter sites within the 39-bp duplication. Analysis of in vitro-generated transcripts confirmed that at limiting RNA polymerase concentrations, the activity of P juncP is 20-fold higher than the activity of P juncC. These observations suggest that the 39-bp duplication might host cis-acting elements that facilitate the binding of RNA polymerase to the promoter.

Base Sequence↗
PubMed1980

Human cytomegalovirus DNA: physical maps for restriction endonucleases BglII, hindIII and XbaI.

It is proposed that the genome of human cytomegalovirus (HCMV) consists of two unique sequences, L and S, bounded by two sets of redundant sequences (P. Sheldrick et al. unpublished data). In this arrangement the terminal sequences (TR1 and TR8) are repeated in an intenal inverted form (IR1 and IR8) and delimit L and S. After restriction endonuclease cleavage of the DNA, four o.5 M and four 0.25 M fragments are found, indicating that HCMV DNA preparations consist of four equimolar populations differing only in the relative orientation of the L and S components. Cleavage of the CMV DNA with the restriction endonucleases BglII, HindIII and XbaI results in 32, 27 and 21 fragments, respectively. The arrangement of these fragments has been determined using molecular hybridization techniques, identification of terminal fragments and the identification of linkage groups by double-digestion. In this report the physical maps for the restriction endonucleases BglII, HindIII and XbaI are presented.

Base Sequence↗
PubMed1990

Cloning of the human parvovirus B19 genome and structural analysis of its palindromic termini.

We describe the molecular cloning of the entire 5.6-kb single-stranded DNA genome of the human parvovirus B19 in bacterial plasmids. Stable amplification of the recombinant plasmid DNA was achieved in Escherichia coli JC8111 but not in HB101 cells. Sequence analysis of the cloned DNA shows that the terminal 383 nucleotides at each end of the genome are identical inverted repeats. The distal 365 nucleotides of the repeat represent an imperfect palindrome which presumably folds over to form a hairpin structure. The sequence of the hairpin occurs in two distinct configurations which are related in that one is the inverted complement of the other. Such alternative configurations of the terminal hairpins have been found for all parvoviruses analyzed so far and are referred to as flip and flop.

Base Sequence↗