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PubMed1982

P1 site-specific recombination: nucleotide sequence of the recombining sites.

Site-specific recombination between molecules of bacteriophage P1 DNA occurs at sites called loxP and requires the action of a protein that is the product of the P1 cre gene. Although recombination between two loxP sites is very efficient, recombination between loxP and a unique site in the bacterial chromosome (loxB) is inefficient and generates two hybrid lox sites called loxR and loxL. We present here the nucleotide sequences of all four lox sites. Analysis of these sequences indicates that (i) a region of extensive homology is not present at the loxP X loxB crossover point, in contrast to the 15-base pair common-core sequence in the bacteriophage lambda att sites, and (ii) the sites contain a region of dyad symmetry with 8- to 13-base pair inverted repeats separated by an 8- to 9-base pair sequence. The loxP X loxB crossover point falls in the sequence that separates the inverted repeats, and deletions that remove either the left or the right inverted repeat of loxP inactivate the site. These two observations are consistent with the conclusion that the region of dyad symmetry is important in los recombination. We have shown further that the loxP X loxP crossover point occurs in a 63-base pair sequence containing the loxP X loxB crossover point, suggesting that, despite the great difference in efficiencies of the two reactions, the crossover points may occur at the same place in both. Explanations for the different recombination properties of the various lox sites are discussed.

Base Sequence↗
PubMed2003

Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats.

Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNA degradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has potential as a powerful reverse-genetics tool in functional genomic programmes through transient, loss-of-function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40-60 base direct inverted-repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)-based vector carrying such inverted-repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real-time RT-PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted-repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.

Base Sequence↗
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↗
PubMed1999

The 11 kb FGA deletion responsible for congenital afibrinogenaemia is mediated by a short direct repeat in the fibrinogen gene cluster.

Congenital afibrinogenaemia is an autosomal recessive disorder characterised by the complete absence of detectable fibrinogen. We previously identified the first known causative mutations for this disorder in a non-consanguineous Swiss family. The four affected male individuals (two brothers and their first two cousins) were shown to have homozygous deletions of approximately 11 kb of the fibrinogen alpha chain (FGA) gene. Haplotype data suggested that the deletions occurred on three distinct ancestral chromosomes, implying that the FGA region of the fibrinogen locus is susceptible to deletion by a common mechanism, but the sequences responsible for the recombination remained to be identified. Here, we report the detailed characterisation of the deletion by nucleotide sequence analysis of all three deletion junctions and comparison with normal sequences. We found that all three deletions were identical to the base-pair and probably resulted from non-homologous (illegitimate) recombination. The centromeric and telomeric deletion junctions featured both a 7 bp direct repeat, AACTTTT, situated in FGA intron 1 and in the FGA-FGB intergenic sequence and a number of inverted repeats which could be involved in the generation of secondary structures. Analysis with closely linked flanking polymorphic markers revealed the existence of at least two haplotypes, further suggesting independent origins of the deletions in this family.

Adult↗
PubMed1992

Identification of a gene, rgg, which regulates expression of glucosyltransferase and influences the Spp phenotype of Streptococcus gordonii Challis.

Streptococcus gordonii Challis was previously shown to give rise to phase variants expressing high (Spp+) or low (Spp-) levels of extracellular glucosyltransferase (GTF) activity. Here, shotgun cloning of an S. gordonii Spp+ chromosomal digest resulted in a chimeric plasmid (pAM5010) able to complement the Spp- phenotype. In addition, introduction of pAM5010 into an Spp+ strain resulted in a 10-fold increase in GTF expression. Deletion analysis of pAM5010 identified a 1.2-kb DNA segment which exhibited the same functional properties as pAM5010. Nucleotide sequence analysis of this region revealed a gene approximately 1 kb in size. The gene was designated rgg. Disruption of the chromosomal rgg gene open reading frame in an Spp+ strain resulted in strain DS512, which displayed an Spp(-)-like phenotype and had 3% of wild-type GTF activity. A plasmid containing the rgg gene was able to complement the DS512 phenotype and significantly increase GTF expression above wild-type levels. Sequence analysis and other data showed that the S. gordonii GTF determinant, designated gtfG, is located 66 bp downstream of the rgg gene. The sequence also revealed interesting inverted repeats which may play a role in the regulation of gtfG. We conclude that rgg positively regulates the expression of GTF and influences expression of the Spp phenotype.

Amino Acid Sequence↗
PubMed2000

Highly conserved DNA sequence present in small plasmids from Selenomonas ruminantium.

Plasmid pJW1 from Selenomonas ruminantium subsp. lactilytica strain JW13 has been cloned in Escherichia coli vector pBluescriptSK(-) and completely sequenced. The plasmid is only 1410 bp with an overall GC content of 42.2%. Computer analysis of sequence data revealed a single open reading frame (ORF1, 146 amino acids, MW 16,525.5 Da) encoding a putative replication protein which is similar to the Rep protein of Ruminobacter amylophilus plasmid pRAO1. ORF1 is followed by a long AT-rich (75%) region and a region abundant in direct and inverted repeats. Comparison of DNA sequences revealed the presence of a short (<250 bp) DNA segment which is highly conserved between several small S. ruminantium plasmids including pJDB21.

Amino Acid Sequence↗
PubMed1988

Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene.

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

Animals↗
PubMed2003

Genomic characterization of Rim2/Hipa elements reveals a CACTA-like transposon superfamily with unique features in the rice genome.

The availability of huge amounts of rice genome sequence now permits large-scale analysis of the structure and molecular characteristics of the previously identified transposase-encoding Rim2 (also called Hipa) element, which is transcriptionally activated by infection with the fungal pathogen Magnaporthe grisea and by treatment with the corresponding fungal elicitor. Based on genomic cloning and data mining from 230 Mb of rice genome sequence, 347 Rim2 elements, with an average size of 5.8 kb, were identified. This indicates that an estimated total of 600-700 Rim2 elements are present in the whole genome. Rim2 insertions occur non-randomly on the chromosomes, as visualized by fluorescence in situ hybridization. The elements harbor 16-bp terminal inverted repeats with the core sequence CACTG, 16-bp sub-terminal repeats, internal variable regions, 3-bp target sequence duplications in the flanking regions, and genes coding for Rim2 proteins (the putative transposase) and hydroxyproline-rich glycoproteins. High levels of insertion into genic regions are observed for members of this family, and the transposition history of the family can be deduced from the high level of shared sequences and analysis of repeat target sites of the elements. Phylogenetic analysis indicates that the putative RIM2 proteins fall into a subgroup distinct from the TNP2-like subgroup of transposases. Southern hybridization with genomic DNA from monocotyledonous and dicotyledonous plants demonstrates that the RIM2-coding sequence is unique to the Oryza genome. Our results demonstrate that the Rim2 elements from rice belong to a distinct superfamily of CACTA-like elements with evolutionary diversity.

Amino Acid Sequence↗
PubMed1997

Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.

The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.

Base Sequence↗
PubMed1983

Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics.

A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment.

Animals↗
PubMed1985

Crossover sites cix for inversion of the invertible DNA segment C on the bacteriophage P7 genome.

The bacteriophage P7 genome contains an invertible DNA segment called C which determines its host range. P7 C(+) phages produce plaques on Escherichia coli K12. The C segment consists of a 3-kb unique sequence and 0.62-kb inverted repeats of which one carries an internal 0.2-kb deletion. This deletion has been mapped within the right inverted repeat in the C(+) orientation. The crossover sites cix for inversion of the C segment do not map at the inside boundaries of the inverted repeats, as had been proposed. They are localized at the external ends of these repeats. Thus organization of the C segment in phage P7 is analogous to that in the related phage P1.

Base Sequence↗
PubMed1987

The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing.

The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization. We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion analysis and transposon mutagenesis. The nucleotide sequence of the oriT region contains 13 direct repeats of an 11 bp consensus sequence, 3 different pairs of 10 bp inverted repeats, and a segment that is extremely A-T rich. The direct repeats are within a region required for high frequency transfer and their sequence is such that their periodic alignment along the helix may induce curvature of the DNA. Analysis of Tn1725 insertions within the sequenced fragment of R46 revealed that, unlike most other transposons, transposition of Tn1725 can cause target sequence duplications of three different sizes.

Base Sequence↗
PubMed1988

Role of sequence amplification in the generation of nematode mitochondrial DNA polymorphism.

Romanomermis culicivorax, an obligate parasitic nematode of mosquitos, possesses an unusually large mitochondrial genome. Individuals are monomorphic for one of several mitochondrial DNA (mtDNA) size variants ranging from 26-32 kb. In this report, we demonstrate that the mitochondrial genome size differential in three isofemale lineages is due to the presence of mtDNA sequences amplified to different copy numbers within each mtDNA molecule. Restriction enzyme analysis and DNA sequencing studies reveal that each mitochondrial genome contains one of two 3.0 kb repeat types that differ by approximately 30 bp. This difference is primarily due to a short (23 bp) imperfect tandem duplication present within the larger of two polymorphic repeating units. The 3.0 kb reiterated DNA sequences are present as direct, tandem repeats and as inverted portions of the same sequence located elsewhere in the genome. Based on mtDNA analysis of an independently reared R. culicivorax culture, we conclude that events resulting in mitochondrial genome rearrangement occurred in natural field populations prior to propagation within the laboratory.

Animals↗
PubMed2003

On the normalization of RNA equilibrium free energy to the length of the sequence.

There is no universal definition of stability for RNA secondary structures. Here we present an approach that is based on normalization of the equilibrium free energy to the length of the sequence: a segment of RNA is said to be stable if the ratio of the equilibrium free energy to the length of the segment is greater than a certain threshold value. Discarding the segments whose normalized equilibrium free energies are smaller than the threshold allows us to view the secondary structure at different levels of stability. Confined to only highly stable structures, the algorithm for secondary structure prediction admits a number of simplifications that make it computationally tractable for large sequences and advantageous over most other methods on a genome-wide scale. This method was applied to the Caenorhabditis elegans genome to localize the regions that encode stable secondary structures. In particular, 36 of 56 previously reported micro-RNAs were localized to 4% of the genome. A fraction of long (>or=400 nt) stable inverted repeats in the genomic sequence of C.elegans was found. Their distribution is very uneven, and skewed towards the ends of chromosomes. This method can be used for genome-wide detection of transcription termination signals, putative micro-RNAs, and other regulatory elements that involve stable RNA secondary structures.

Algorithms↗
PubMed1989

Cloning and characterization of the structural gene for the class 2 protein of Neisseria meningitidis.

The class 2 protein of Neisseria meningitidis is the major outer membrane protein and a porin. A lambda gt11 bank of meningococcal chromosomal DNA was screened with monoclonal antibodies against gonococcal protein IB that cross-react with meningococcal class 2 protein. Three independent immunoreactive clones were isolated. DNA sequence analysis indicated that these clones included regions encoding the N-terminal portion of the class 2 protein. An oligonucleotide of 21 bases that was complementary to this sequence was synthesized and used as a probe for a second screening of the lambda gt11 bank. One of the positive clones isolated contained the complete gene, including the ribosome binding site, but lacked the promoter region. On the basis of the DNA sequence, a protein of 360 amino acids was predicted. Comparison of the predicted protein sequence with that of gonococcal protein I showed little homology in six regions constituting 29% of the total amino acids, while the remainder of the coding frame showed 81% homology of amino acid residues. The DNA homology in the immediate 5' and 3' noncoding sequences was very striking. Following the putative transcription terminator, the 3' DNA sequence contained a complex pattern of direct and inverted repeats having some similarity to the 3' sequence of the gonococcal protein IB gene and very close homology to a sequence located in the pilS1 region (R. Haas and T. F. Meyer, Cell 44:107, 1986), an area of the gonococcal genome containing silent pilin genes.

Amino Acid Sequence↗
PubMed2003

Identification and chromosomal localization of mariner-like elements in the cabbage moth Mamestra brassicae (Lepidoptera).

A complete mariner-like element has been identified in the lepidopteran Mamestra brassicae. This element, called Mbmar, represents a new type of mariner transposon. It has a transposase similar to that of other insect mariner coding sequences but its inverted terminal repeats differ from typical mariner ones. This observation is unique since generally both mariner coding region and ITRs are evolutionarily conserved in insects. Mbmar is detectable by FISH only in the heterochromatic regions of both the sex chromosomes.

Animals↗
PubMed1995

A sequence analysis of the genomic regions involved in the rearrangements between TPM3 and NTRK1 genes producing TRK oncogenes in papillary thyroid carcinomas.

Papillary thyroid carcinomas have frequently been found to display oncogenic rearrangements of the NTRK1 gene, which encodes the high-affinity nerve growth factor receptor. Replacement of its extracellular domain by sequences coding for the 221 amino-terminal residues of the TPM3 gene was responsible for the oncogenic NTRK1 activation in three of eight of these tumors. In all of them, the illegitimate recombination involved the 611-bp NTRK1 intron placed upstream of the transmembrane domain and the TPM3 intron located between exons 7 and 8. Therefore, due to the splicing mechanism, all of the TPM3/NTRK1 gene fusions encoded an invariable transcript and the same chimeric protein of 70 kDa, which was constitutively phosphorylated on tyrosine. In two of the three tumors the simultaneous presence of the reciprocal products of the TPM3/NTRK1 recombination, 5'TPM3-3'NTRK1 and 5'NTRK1-3'TPM3 sequences, respectively, and the previously demonstrated localization of both genes on the long arm of chromosome 1 lead us to suggest that an intrachromosomal inversion could be responsible for their recombination. In an attempt to understand the molecular basis that predisposes NTRK1 and TPM3 genes to be a recurrent target of illegitimate recombination, we have determined the nucleotide sequence around the breakpoints of the recombination products in all three patients as well as those of the corresponding regions from the normal TPM3 and NTRK1 genes. In these regions, a search for common features usually involved in illegitimate recombination in mammalian cells revealed the presence of some recombinogenic elements as well as pal-indromes, direct and inverted repeats, and Alu family sequences.

3T3 Cells↗
PubMed1986

Enhanced rate of conversion or recombination of markers within a region of unique sequence in the herpes simplex virus genome.

Insertion mutants of herpes simplex virus type 1, containing a second copy of the sequences of BamHI fragment L (map coordinates 0.706 to 0.744) inserted in inverted orientation into the thymidine kinase gene (at map coordinate 0.315), have been further characterized. We reported previously that, as a result of intramolecular or intermolecular recombination between copies of the BamHI-L sequence at the normal locus and inserted locus, a high proportion of progeny genomes exhibited either inversions of the unique sequence flanked by these inverted repeats or other rearrangements. Now we report that a genetic marker (syn-1 or syn-1+) originally present only in the inserted copy of BamHI fragment L appears in progeny at both the normal and inserted loci, and vice versa, at high frequency. Because these phenomena have not been observed with other insertion mutants containing duplications of other sequences from unique regions of the genome, we conclude that BamHI fragment L contains an element that enhances the rate of homologous recombination in adjacent sequences, resulting in genome rearrangements and gene conversion-like events.

Base Sequence↗