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PubMed1991

Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam3.

We have sequenced HFL1 from D. melanogaster, the only cloned hobo element shown to have transposase activity. The 2959 bp HFL1 sequence predicts a 2.0 kb open reading frame (ORF1) with substantial amino acid similarity to the transposases of Activator (Ac) from maize (Zea mays) and Tam3 from snapdragon (Antirrhinum majus). Mutational analysis of a C-terminal region of ORF1 conserved with Ac and Tam3 indicates that it is essential for hobo transposase activity. This is an example of extensive amino acid sequence identity between short inverted repeat elements in different kingdoms. We discuss the possibility that the conservation of hobo, Ac, and Tam3 transposases represents an example of horizontal transmission of genetic information between plants and animals.

Amino Acid Sequence↗
PubMed1998

Cre-loxP-mediated DNA flip-flop in mammalian cells leading to alternate expression of retrovirally transduced genes.

While DNA excision by Cre-loxP homologous recombination has been exploited for mammalian genetic engineering, it has not been reported whether DNA inversion is achievable by the same mechanism in mammalian cells. To investigate whether Cre-loxP-mediated DNA inversion takes place in mammalian cells, a novel retroviral vector, NT(FF), was constructed. The vector carries a marker gene cassette consisting of the neo and tk genes linked tail-to-tail to each other and flanked by an inverted repeat of loxP sequences. In NT(FF)-transduced Rat2 cells, the marker gene cassette was inverted reversibly in a Cre-dependent manner, leading to DNA "flip-flop" associated with alternate expression of the neo and tk genes. This study provides the first example of Cre-loxP-mediated DNA inversion in mammalian cells facilitating regulation of retrovirally transduced genes, suggesting the usefulness of the system for genetic engineering.

Animals↗
PubMed1992

Sequence analysis of an aphid endosymbiont DNA fragment containing rpoB (beta-subunit of RNA polymerase) and portions of rplL and rpoC.

The aphid Schizaphis graminum is dependent on an association with a prokaryotic endosymbiont (Buchnera aphidicola). The nucleotide (nt) sequence of a 5040 base pair (bp) DNA fragment of B. aphidicola, homologous to the rplL-rpoB-rpoC portion of the Escherichia coli beta operon, was determined. The DNA coded for the terminal 35 amino acids of RplL (large ribosomal subunit protein L7/L12), the complete RpoB (beta-subunit of RNA polymerase), and the first 209 amino acids of RpoC (beta'-subunit of RNA polymerase). The deduced sequences of B. aphidicola RplL, RpoB, and RpoC were 71, 84, and 91% identical, respectively, to the homologous proteins of E. coli. The sequences of two portions of the intergenic region between rplL and rpoB were nearly identical in both B. aphidicola and E. coli. One sequence constituted an inverted repeat that could be an RNase III-messenger RNA processing site; the other sequence preceded RpoB. A compilation of the codon usage for RpoB, RpoC, and other B. aphidicola proteins indicated a major preference for A or T in the first and third positions, a result consistent with the low guanine plus cytosine (G + C) content of the DNA of this organism.

Amino Acid Sequence↗
PubMed1991

Characterization of insertion sequence IS892 and related elements from the cyanobacterium Anabaena sp. strain PCC 7120.

IS892, one of the several insertion sequence (IS) elements discovered in Anabaena sp. strain PCC 7120 (Y. Cai and C. P. Wolk, J. Bacteriol. 172:3138-3145, 1990), is 1,675 bp with 24-bp near-perfect inverted terminal repeats and has two open reading frames (ORFs) that could code for proteins of 233 and 137 amino acids. Upon insertion into target sites, this IS generates an 8-bp directly repeated target duplication. A 32-bp sequence in the region between ORF1 and ORF2 is similar to the sequence of the inverted termini. Similar inverted repeats are found within each of those three segments, and the sequences of these repeats bear some similarity to the 11-bp direct repeats flanking the 11-kb insertion interrupting the nifD gene of this strain (J. W. Golden, S. J. Robinson, and R. Haselkorn, Nature [London] 314:419-423, 1985). A sequence similar to that of a binding site for the Escherichia coli integration host factor is found about 120 bp from the left end of IS892. Partial nucleotide sequences of active IS elements IS892N and IS892T, members of the IS892 family from the same Anabaena strain, were shown to be very similar to the sequence of IS892.

Amino Acid Sequence↗
PubMed1988

Organization and nucleotide sequence of genes at both junctions between the two inverted repeats and the large single-copy region in the rice chloroplast genome.

We describe the isolation and organization, at the nucleotide sequence level, of genes located at the two junctions of the large single-copy region (LSCR) and the two inverted repeats (IRA and IRB) in the rice chloroplast genome. This is the first example where the two junctions are precisely located in a monocot. In rice, a ribosomal protein gene cluster, rpl23-rpl2-rps19, which codes for the ribosomal proteins L23 (rpl23), L2 (rpl2) and S19 (rps19), lies at the ends of the two IRs near the LSCR. The inverted repeats end 45 bp from the translation stop codon of rps19. The gene for the 32-kDa photosystem II protein, psbA, is located at the extremity of the LSCR near IRA, and transcribed towards IRA. The translation stop codon of psbA is 68 bp from the right-hand junction (JLA). Thus, JLA is located within the intergenic sequence of the two genes, rps19 and psbA. Around the left-hand junction (JLB), there is a typical ribosomal protein gene cluster, rpl23-rpl2-rps19-rpl22 (rpl22 for the ribosomal protein L22). The translation start codon of rpl22 is located in the LSCR 25 bp from JLB. Therefore, JLB is located within the intergenic sequence between rps19 and rpl22.

Chloroplasts↗
PubMed1985

Initiation of replication of the Escherichia coli chromosomal origin reconstituted with purified enzymes.

A mixture of purified proteins has replaced a crude enzyme fraction capable of efficient replication of oriC-containing plasmids. The reconstituted enzyme system contains proteins which provide initiation, replication, and specificity functions required for dnaA-dependent replication specific for an oriC template. Replication can be separated into successive stages of RNA synthesis and DNA replication. Isolation of an intermediate no longer requiring RNA polymerase action requires the presence of dnaA protein, DNA gyrase, dnaB protein and dnaC protein. Intermediate formation likely involves binding of dnaA protein to a 9-bp sequence present 4 times as inverted repeats within the chromosomal origin sequence.

Bacterial Proteins↗
PubMed1996

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase.

The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the bed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD+-dependent dehydrogenase (bedD) required to convert benzene into catechol. Analysis of the nucleotide sequence upstream of the benzene dioxygenase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp sequence identical to the inverted repeat of IS26. In vitro translation analysis showed bedD to code for a polypeptide of ca. 39 kDa. Both the nucleotide and the deduced amino acid sequences show significant identity to sequences of glycerol dehydrogenases from Escherichia coli, Citrobacter freundii, and Bacillus stearothermophilus. A bedD mutant of P. putida ML2 in which the gene was disrupted by a kanamycin resistance cassette was unable to utilize benzene for growth. The bedD gene product was found to complement the todD mutation in P. putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenase. The dehydrogenase encoded by bedD) was overexpressed in Escherichia coli and purified. It was found to utilize NAD+ as an electron acceptor and exhibited higher substrate specificity for cis-benzene dihydrodiol and 1,2-propanediol compared with glycerol. Such a medium-chain dehydrogenase is the first to be reported for a Pseudomonas species, and its association with an aromatic ring-hydroxylating dioxygenase is unique among bacterial species capable of metabolizing aromatic hydrocarbons.

Amino Acid Sequence↗
PubMed2001

Inverted repeats as genetic elements for promoting DNA inverted duplication: implications in gene amplification.

Inverted repeats are important genetic elements for genome instability. In the current study we have investigated the role of inverted repeats in a DNA rearrangement reaction using a linear DNA substrate. We show that linear DNA substrates with terminal inverted repeats can efficiently transform Escherichia coli. The transformation products contain circular inverted dimers in which the DNA sequences between terminal inverted repeats are duplicated. In contrast to the recombination/rearrangement product of circular DNA substrates, which is exclusively one particular form of the inverted dimer, the rearrangement products of the linear DNA substrate consist of two isomeric forms of the inverted dimer. Escherichia coli mutants defective in RecBCD exhibit much reduced transformation efficiency, suggesting a role for RecBCD in the protection rather than destruction of these linear DNA substrates. These results suggest a model in which inverted repeats near the ends of a double-strand break can be processed by a helicase/exonuclease to form hairpin caps. Processing of hairpin capped DNA intermediates can then yield inverted duplications. Linear DNA substrates containing terminal inverted repeats can also be converted into inverted dimers in COS cells, suggesting conservation of this type of genome instability from bacteria to mammalian cells.

Animals↗
PubMed1998

A physical map of chromosome 7 of Candida albicans.

As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the SfiI restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Rel2 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.

Candida albicans↗
PubMed1985

Herpes simplex virus 1 reiterated S component sequences (c1) situated between the a sequence and alpha 4 gene are not essential for virus replication.

The herpes simplex virus 1 genome consists of two components, L and S, each containing unique sequences flanked by inverted repeats. Each of the 6.5-kilobase pair inverted repeats of the S component, designated a'c' and ca, contains an approximately 700-base pair sequence (designated c1) located between the a sequence and the 3' terminus of the alpha 4 gene. Like the a sequence, c1 consists of direct repeats and unique sequences. Its function is not known. To probe for its function, we constructed a plasmid containing a viral thymidine kinase (TK) gene inserted into the c1 sequence. The construct was recombined into the genome of a TK- virus by cotransfection with intact viral DNA and selection for TK+ virus. As predicted from previous studies (Knipe et al., Proc. Natl. Acad. Sci. U.S.A. 75:3896-3900, 1978), the TK gene was found to be present in both copies of the c1 sequence in the R3104 virus. To delete the c1 sequence we constructed a plasmid containing 4 kilobase pairs of pBR322 flanked by an a sequence and by structural sequences of the alpha 4 gene. In this instance the cells were transfected with the construct and R3104 DNA; the progeny of the transfection was plated in the presence of 5-bromo-2'-deoxyuridine, and the selection was for TK- virus (R3158). The pBR322 DNA sequences replaced the c1 at both termini of the S component in R3158 DNA, but a sequence homologous to c1 was present in proximity to the 3' terminus of the alpha 4 gene. The results indicate that the c1 region has no significant role in the replication of the virus in cell culture. The advantage of inserting the pBR322 sequence is that it permits efficient cloning of large herpes simplex virus 1 DNA fragments by simple ligation of digests and transformation of appropriate Escherichia coli strains. The effortless selection of recombinants carrying inserts in both copies of the c1 restates the usefulness of this technique for selection of insertion deletion recombinants and underscores the rapid emergence of sequence identity at both ends of the reiterated regions of the S component as previously reported (Knipe et al., Proc. Natl. Acad. Sci. U.S.A. 75:3896-3900, 1978).

DNA, Viral↗
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↗
PubMed2003

Inhibition of Moloney murine leukemia virus integration using polyamides targeting the long-terminal repeat sequences.

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.

Animals↗
PubMed1977

Analysis of chromosomal integration and deletions of yeast plasmids.

Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA.

Chromosomes↗
PubMed1995

Analysis of 45 kb of DNA located at the left end of the chlorella virus PBCV-1 genome.

Forty-five kilobases of DNA, including the previously sequenced 2.2-kb inverted repeat region, located at the left termini of the 330-kb Chlorella virus PBCV-1 genome were sequenced and analyzed. Eighty-five complete open reading frames (ORFs) larger than 195 nucleotides were identified. Thirty-seven of the 85 ORFs, which are densely packed on both strands of the DNA, were considered major ORFs. Fifteen of the major ORFs have similarity to genes in the databases, including bacterial glycerophosphoryl diester phosphodiesterase, bacteriophage T4 endonuclease V, D-isomer specific 2-hydroxyacid dehydrogenases, and beta-alanine synthetase and bacterial nitrilases. Two major ORFs resemble the virus major capsid protein. Three major ORFs contain three or more ankyrin-like repeat elements and four ORFs encode proline-rich proteins.

Amino Acid Sequence↗
PubMed1984

Cytochrome oxidase subunit II gene of rice has an insertion sequence within the intron.

We have isolated and sequenced the cytochrome oxidase subunit II gene from rice (Oryza sativa L. var Labelle). The overall structural organization of this gene is very similar to that of the maize gene. This gene contains an intron in a position identical to the intron in the maize gene. However, the intron in the rice gene is longer than that of the maize gene largely due to a 461 bp insertion sequence, which has inverted repeats at its termini and is flanked by direct repeats, characteristic of transposable elements. Apart from this insertion sequence, the remainder of the intron sequence is strikingly homologous to that of maize (98.6% homology), suggesting a possible functional or structural role. The coding regions of the two genes exhibit 99.5% nucleotide sequence homology and their deduced amino acid sequences are identical. Similarly, the 3'-noncoding regions, except for several small insertions and deletions, show complete sequence homology. On the contrary, no sequence homology is detected in the 5'-noncoding regions.

Base Sequence↗
PubMed1991

Introduction of the glucocorticoid binding sequences into the expression vector p delta SVE-CAT and its effect on the CAT gene expression in mammalian cells.

We have demonstrated that synthetic oligonucleotide representing glucocorticoid responsive element (GRE I) of MMTV inserted into the enhancerless early promoter of SV40 in p delta SVE-CAT expression vector, enhances transient expression of chloramphenicol acetyltransferase gene in HeLa and hepatoma cells cultivated in the presence of dexamethasone. The following changes in the structure of the core sequences (GTTACAAACTGTTCT) of the synthesized GRE eliminated its enhancing ability: i, changes in the left end of the core sequences from GTTACAAAATGTTCT to TCTTCAAACTGTTCT or to TACTCAAACTGTTCT; ii, the increase of gap between TGTTCT and the inverted repeat of this sequence. The above changes did not eliminate specific binding of glucocorticoid receptor to the synthetic oligonucleotides studied.

Animals↗
PubMed1985

Sequence organization of repetitive elements in the flanking regions of the chloroplast ribosomal unit of Chlamydomonas reinhardii.

The flanking regions and the end of the chloroplast ribosomal unit of Chlamydomonas reinhardii have been sequenced. The upstream region of the ribosomal unit contains three open reading frames coding for 111, 117 and 124 amino acids, respectively. The latter polypeptide is partially related to the ribosomal protein L16 of E. coli. Two of the open reading frames overlap each other and are oriented in opposite direction. The region between these open reading frames and the 5' end of the 16S rRNA gene contains numerous short direct and inverted repeats which can be folded into large stem-loop structures. Sequence elements that resemble prokaryotic promoters are found in the same region. Several of the repeated elements are distributed throughout the non-coding regions of the chloroplast inverted repeat. Sequence comparison between the 5S rRNA and its gene does not reveal any significant sequence heterogeneity between the chloroplast 5S rRNA genes.

Base Sequence↗
PubMed1989

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.

Base Sequence↗