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PubMed1995

Two enhancer elements for DNA replication of pSC101, par and a palindromic binding sequence of the Rep protein.

The minimal replication origin (ori) of the plasmid pSC101 has been previously defined as an approximately 220-bp region by using plasmids defective in the par region, which is a cis-acting determinant of plasmid stability. This ori region contains the DnaA binding sequence, three repeated sequences (iterons), and an inverted repeat (IR) element (IR-1), one of the binding sites of an initiator protein, Rep (or RepA). In the present study, we show that plasmids containing par can replicate at a nearly normal copy number in the absence of IR-1 but still require a region (the downstream region) between the third iteron and IR-1. Because par is dispensable in plasmids retaining IR-1, par and IR-1 can compensate each other for efficient replication. The region from the DnaA box to the downstream region can support DNA replication at a reduced frequency, and it is designated "core-ori." Addition of either IR-1 or par to core-ori increases the copy number of the plasmid up to a nearly normal level. However, the IR-1 element must be located downstream of the third iteron (or upstream of the rep gene) to enhance replication of the plasmid, while the par region, to which DNA gyrase can bind, functions optimally regardless of its location. Furthermore, the enhancer activity of IR-1 is dependent on the helical phase of the DNA double helix, suggesting that the Rep protein bound to IR-1 stimulates the activation of ori via its interaction with another factor or factors capable of binding to individual loci within ori.

Bacterial Proteins↗

PubMed2005

Possible active origin of replication in the double stranded extended form of the left terminus of LuIII and its implication on the replication model of the parvovirus.

BACKGROUND: The palindromic termini of parvoviruses have proven to play an essential role as origins of replication at different stages during the replication of their viral genome. Sequences from the left-end telomere of MVM form a functional origin on one side of the dimer replicative form intermediate. In contrast, the right-end origin can operate in its closed replicative form hairpin configuration or as a fully duplex linear sequence derived from either arm of a palindromic tetramer intermediate. To study the possibility that the LuIII left hairpin has a function in replication, comparable to that described for MVM, the replication of a minigenome containing two copies of the LuIII left terminus (LuIII Lt-Lt) was studied. RESULTS: The data presented demonstrates that LuIII Lt-Lt was capable of replicating when NS1 helper functions were provided in trans. This extended hairpin, capable of acting as an origin of replication, lacks the arrangement of the specific domains present in the dimer duplex intermediate of MVM, the only active form of the left hairpin described for this parvovirus. CONCLUSIONS: These findings suggest that the left hairpin of LuIII has an active NS1 driven origin of replication at this terminus in the double stranded extended form. This difference between LuIII and MVM has great implications on the replication of these viruses. The presence of origins of replication at both the left and right termini in their natural hairpin form can explain the unique encapsidation pattern observed for LuIII hinting on the mechanism used by this virus for the replication of its viral genome.

Cloning, Molecular↗

PubMed1999

De novo inverted duplication 9p21pter involving telomeric repeated sequences.

We report on clinical and cytogenetic findings in a boy with partial 9p duplication, dup(9)(p21pter). Clinical manifestations included facial and hand anomalies and mental retardation. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were used to characterize further and confirm the conventional banding data. Investigation by FISH using whole chromosome 9 paint probe showed that the additional material was derived from chromosome 9. Using CGH, a region of gain was found in the chromosome segment 9p21pter. YACs and telomeric probes confirmed the duplicated region. Using the all-human telomeric sequences probe, intrachromosomal telomeric signal was noted on the short arm of the abnormal chromosome 9. Mechanism of formation of the duplication, including intrachromosomal telomeric sequences, is discussed.

Abnormalities, Multiple↗

PubMed2005

Transcriptional activation of the testis-specific histone H1t gene by RFX2 may require both proximal promoter X-box elements.

The rat testis-specific linker histone H1t gene is transcribed in pachytene primary spermatocytes during spermatogenesis. Our previous work using transgenic mice demonstrated that spermatocyte-specific transcription of the H1t gene is dependent upon a proximal promoter element designated the TE element. TE is composed of two adjacent and inverted imperfect repeat sequences designated TE1 and TE2 and both of these palindromic elements are similar in sequence to the X-box, a DNA consensus sequence that binds regulatory factor X (RFX). RFX2 is the major enriched protein derived from rat testis nuclear extracts when using the TE1 element as an affinity chromatography probe. Co-expression of RFX2 together with an H1t promoted reporter vector in transient expression assays activates the H1t promoter in the GC-2spd germinal cell line, and mutation of either X-box significantly represses activity. However, RFX2 partially reactivates the promoter when either of the X-box elements is independently mutated. In order to totally block reactivation by RFX2, it is necessary to mutate both X-boxes simultaneously. Therefore, RFX2 appears to be able to bind to either X-box independently to partially activate the promoter of the testis-specific histone H1t gene, but simultaneous binding of RFX2 to both X-box elements may be required for maximal promoter activation.

Animals↗

PubMed1993

DNA binding domains in Tn3 transposase.

Various segments of Tn3 transposase were fused individually to beta-galactosidase, and the resulting fusion proteins were examined for their DNA binding ability by a nitrocellulose filter binding assay. Analyses of a series of the fusion proteins revealed that the N-terminal segment of the transposase (amino acid positions 1-242; the transposase gene encodes 1004 residues in all) had specific DNA binding ability for the 38 bp terminal inverted repeat (IR) sequence, and the central segment (amino acid positions 243-632) had non-specific DNA binding ability. Further analyses of each of the two regions revealed that the N-terminal segment could be divided into at least two subsegments (amino acid positions 1-86 and 87-242), neither of which had specific DNA binding ability, but which both possessed non-specific DNA binding ability. The central segment included two subsegments (amino acid positions 243-289 and 439-505) with non-specific DNA binding ability. These results and other observations suggest that Tn3 transposase has several domains including those responsible for non-specific DNA binding, and a combination of two or more domains gives rise to specific DNA binding activity. The C-terminal segment of the transposase (amino acid positions 633-1004), which is very well conserved among transposases encoded by Tn3 family transposons, had no DNA binding ability. This segment may represent the main part of the catalytic domain responsible for the initiation step of transposition.

Amino Acid Sequence↗

PubMed1986

Characterization of inverted repeats from plasmid-like DNAs and the maize mitochondrial genome.

Integrated inverted repeat (IR) sequences similar to those of the S plasmids have been isolated from the genomes of the normal and S type male-sterile cytoplasms of maize mitochondria. The nucleotide sequences of both the IRs and their flanking regions have distinguished and characterized several different types of repeats. The repeats may be involved in the recombinational process that occurs continuously in the mitochondrial genome. One cloned fragment, derived from a fertile revertant and containing sequences similar to S-2, does not appear to act as a typical transposable element during reversion. Several of the flanking regions examined contain a small repeat of 34 base pairs, in which a nonanucleotide segment is found with similarity to the yeast mitochondrial promoter.

Chromosome Inversion↗

PubMedDate not supplied

The transposable element IS4712 prevents S-layer gene (sbsA) expression in Bacillus stearothermophilus and also affects the synthesis of altered surface layer proteins.

Cell surface (S)-layer protein synthesis in Bacillus stearothermophilus PV72/p6 is blocked when cells are grown at elevated temperature. From a culture exhibiting the S-layer-negative phenotype, the S-layer deficient mutant T5 (SbsA-) was isolated. Genetic analysis of the S-layer-encoding gene (sbsA) of mutant T5 revealed an insertion element (IS4712) integrated into the upstream regulatory region of the S-layer gene, thereby blocking sbsA transcription. The insertion element consists of 1371 base pairs which are flanked by two perfect inverted terminal repeats. Sequence similarity to other transposases of the IS4 family was detected. DNA-DNA hybridizations demonstrated that multiple homologues of IS4712 were also present within the genomes of several other thermophilic bacillus isolates. Attempts to isolate SbsA+ revertants failed. Instead, cells with altered surface proteins were detected. The synthesis of the altered S-layer proteins was correlated with the presence of IS4712 along with the occurrence of deletions in the sbsA coding region. Furthermore imprecise excision of IS4712 was detected. This work demonstrated that B. stearothermophilus is able to express at least four different S-layer proteins and that blocking of sbsA transcription by the insertion element IS4712 is associated with the expression of altered surface proteins.

5' Untranslated Regions↗

PubMed2026

Functional analysis of stem-loop structures within the SARS-CoV-2 5' untranslated region using a plasmid-based reporter system.

The 5' untranslated region (5'UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains highly conserved stem-loop structures that regulate viral gene expression. This study investigated the functional contributions of selected 5'UTR stem-loop elements to reporter gene expression using a plasmid-based mammalian expression system. Five constructs were tested using a non-integrating plasmid: the wild-type (WT) 5'UTR fused to GFP under the CMV promoter, and four deletion variants (ΔB, ΔC, ΔD, and ΔE) corresponding to deletions of stem-loop 4 (SL4), SL4.5, SL5, and SL5a, respectively. Following transfection into HEK293 cells, GFP fluorescence was quantified using a fluorescence microplate reader, and relative GFP transcript abundance was assessed by RT-qPCR. Deletion of SL4 (ΔB) resulted in marked reduction in both fluorescence and relative transcript abundance compared to WT construct, indicating substantially reduced reporter gene expression. In contrast, deletion of SL4.5, SL5, or SL5a did not produce the pronounced reduction observed for ΔB, although descriptive RT-qPCR analysis indicated differences in relative transcript abundance among these variants. Statistical analysis of fluorescence data demonstrated significant differences among constructs (one-way ANOVA, p < 0.05). Because the reporter assay was based on plasmid expression, the observed differences likely reflect combined contributions from transcription, transcript abundance, RNA stability, and translation rather than translation alone. These findings demonstrate that the SL4 region contributes substantially to reporter gene expression in this experimental system, whereas the remaining stem-loop regions examined exert comparatively modest effects. This study provides additional insight into the functional organization of the SARS-CoV-2 5'UTR and establishes a framework for future investigations aimed at distinguished the transcriptional, post-transcriptional, and translational contributions of individual RNA structural elements.

5' Untranslated Regions↗

PubMed1985

Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans.

We have sequenced 11 representative mutations of the unc-54 myosin heavy chain gene of Caenorhabditis elegans that affect the synthesis, assembly or enzymatic activity of the encoded myosin heavy chain. Six of the sequenced unc-54 mutations cause premature termination of protein synthesis. Four mutations (e1092, e1115, e1213, e1328) were ochre mutations, one mutation (e903) was a frameshift, which caused premature termination at a nearby UGA terminator, and one mutation (e190) was a deletion that altered the reading frame and caused termination at an ochre codon. Two mutations (e675 and s291) were inphase deletions, which resulted in a shortened myosin rod segment. These aberrant myosins fail to assemble into normal thick filaments. The sequence alterations of the missense mutations (e1152, s74, s95) indicated amino acid residues that are critical for myosin function. The mutation e1152 causes the production of a myosin heavy chain that fails to assemble into thick filaments. It had two adjacent amino acid substitutions at the extreme amino terminus of the rod, indicating a role for subfragment-2 in thick filament assembly. Mutants homozygous for s74 or s95 are very slow-moving, although they make myosin heavy chains that assemble normally. The encoded amino acid substitutions of s95 and s74 are in the 23 X 10(3) Mr and 50 X 10(3) Mr domains of the myosin head, flanking the ATP binding site. The sequenced mutations are distributed throughout the gene in the order predicted from genetic fine-structure mapping experiments. Seven of eight point mutations isolated following ethylmethane sulphonate mutagenesis were G X C to A X T transitions. A single X-ray-induced allele proved to be a deletion of two adjacent thymidine residues. The three deletion mutations were found in a region of the myosin rod with numerous direct and inverted nucleotide sequence repeats, but their origin cannot be accounted for by homologous recombination. Instead, a comparison of the deletion junctions suggests that the deletions arose by a site-specific mechanism.

Animals↗

PubMed1987

Regulation of equine herpesvirus type 1 gene expression: characterization of immediate early, early, and late transcription.

The regulation of equine herpesvirus type 1 (EHV-1) transcription was examined in infected rabbit kidney cells using metabolic inhibitors. In order to map EHV-1 immediate early, early, and late transcripts, viral RNA was 32P-labeled in vivo and hybridized to EHV-1 DNA restriction fragments immobilized on nitrocellulose filters. Immediate early viral RNA was mapped to one region of the viral genome within the inverted repeat DNA sequences (map units 0.78-0.83 and 0.95-1.0). Northern blot hybridization analysis using a 32P-labeled cloned DNA probe from this region identified a single immediate early viral transcript (approximately 6 kb). Transcription of early and late genes was not restricted to any specific region on the viral genome as indicated by the ability of 32P-labeled early and late RNA to hybridize to EHV-1 restriction endonuclease fragments from both the long and short components of EHV-1 DNA. Additional experiments performed without the use of metabolic inhibitors confirmed that EHV-1 transcription is temporally regulated. The characterization of EHV-1 transcription during productive infection will serve as a reference for the analysis of viral transcripts in oncogenically transformed and persistently infected cells.

Animals↗

PubMed1986

Three classes of homologous Bacillus thuringiensis crystal-protein genes.

Four homologous genes encoding insecticidal crystal proteins from Bacillus thuringiensis have been cloned in Escherichia coli. Differences in lengths of HindIII restriction fragments containing the 5' ends of the genes allowed the identification of three classes termed the '4.5- and 5.3- and 6.6-kb-class genes'. A survey of 24 strains from subspecies kurstaki and thuringiensis revealed strains containing one, two, or three of these classes of crystal protein genes. The 4.5- and 6.6-kb-class genes encode polypeptides of Mr 133,500 and 133,330, respectively, while the two 5.3-kb-class genes encode a ca. 130-kDa polypeptide. The polypeptide composition of crystals from the B. thuringiensis strains agreed with the composition predicted from the content of the three classes of genes. The representative genes from each class were isolated from B. thuringiensis plasmids of different sizes. An analysis of plasmid DNA flanking three of these genes revealed a complex pattern of two different inverted repeat (IR) sequences, IR2150 and IR1750. Four variant forms of IR1750 were found. The IR elements were located near deletions and rearrangements adjacent to crystal protein genes and may account for the diversity of plasmids carrying crystal protein genes in other subspecies of B. thuringiensis.

Bacillus thuringiensis↗

PubMed1996

Expression of the bacteriophage T4 DNA terminase genes 16 and 17 yields multiple proteins.

The products of the bacteriophage T4 terminase genes 16 and 17 are known to mediate cutting and packaging of concatemeric vegetative DNA. We show here that the larger of these genes, 17, yields multiple protein species. The complex expression of the T4 terminase genes includes overlapping transcripts, probably initiated from multiple promoters, RNA processing at certain preferred sites and translation initiation from multiple ribosome binding sites (RBS). Translation initiation from these RBS may be modulated by inverted repeat (IR) sequences whose folding can be predicted to differ in different RNA species. In T4 infected bacteria, genes 16 and 17 are probably co-transcribed from several near-consensus late promoters upstream from gene 16, and processed at multiple sites. Additional 5' ends of late transcripts are located downstream from a near-consensus late promoter inside gene 17 and further downstream, unrelated to any known promoter consensus sequence. The gene 17 transcripts that are initiated or cleaved internally contain RBS for shorter open reading frames (ORFs) in the same frame as full-length gene product (gp) 17 of 70 kDa. The truncated proteins, a 59-kDa gp17' and a 45-kDa gp17", are synthesized from cloned gene 17 segments in which the first gene 17 RBS is deleted. Expression of gene 17 is different in BL21(DE3) or W3110[pACT7] host bacteria. The gp17' and gp17" proteins are predicted to contain one or more of the ATPase motifs that are common among large subunits of other phage terminases. They lack a predicted single stranded (ss) DNA binding motif that is unique the large terminase proteins in T4 gp17, and that has been implicated in recognizing ssDNA regions in replicating and recombining T4DNA destined to be packaged. We hypothesize that a truncated gene 17' is an evolutionary precursor of the full-size T4 gene 17. Its function may have been maintained to allow processive packaging from double stranded (ds) DNA ends.

Bacteriophage T4↗

PubMedDate not supplied

Interferon-regulated viral replication in chronically HIV1-infected promonocytic U937 cells.

To further document the role of interferons in the restriction of HIV1 replication in cells of the monocyte/macrophage lineage, the antiviral effects of alpha-interferon (IFN alpha) were studied in chronically HIV 1-infected promonocytic cells U937, in which IFN alpha was endogenously produced or to which recombinant IFN alpha was added. Protein analysis performed after immunoprecipitation of culture media revealed that the addition of anti-IFN alpha antibody led to an increase in the production of viral particles, whereas addition of IFN alpha caused its decrease in a dose-dependent manner, indicating that IFN alpha mainly affects viral assembly and virion release. However, the treatment of HIV 1-infected cells with IFN alpha did not cause an increase in the amount of intracellular viral proteins, suggesting that this cytokine might act on other steps in the viral life cycle. No decrease in the level of the 3 main types of viral RNA was observed by Northern blot analysis, indicating that proviral transcription was not restricted by IFN alpha. Furthermore, cotransfection experiments with TAR(trans-activation responsive)-element-containing expression plasmids demonstrated that viral replication appears to be restricted at either a post-transcriptional and/or a translational level. These experiments suggest that the double-stranded (ds) inverted repeat TAR sequence present in HIV1 leader RNA may inhibit viral protein synthesis by phosphorylating the dsRNA-activated protein kinase induced by IFN. These results provide an impetus for achieving antiretroviral therapy based on the constitutive expression of IFN in monocytes and macrophages.

Cell Line↗

PubMed1997

A new hobo, Ac, Tam3 transposable element, hopper, from Bactrocera dorsalis is distantly related to hobo and Ac.

A new transposable element from the hobo, Ac, Tam3 transposon family was isolated as a genomic clone from the oriental fruit fly, Bactrocera dorsalis. It is approximately 3.1 kb in length with 19-bp inverted terminal repeat sequences having a single mismatch. Though sharing several amino acid sequence identities with other hAT elements, it is distantly related to both hobo and Ac. Among hAT elements thus far described in insects, it is apparently the most distantly related to hobo.

Amino Acid Sequence↗

PubMed1998

Analysis of the genomic organisation of a small chromosome of Leishmania braziliensis M2903 reveals two genes encoding GTP-binding proteins, one of which belongs to a new G-protein family and is an antigen.

Leishmania braziliensis M2903 contains a highly amplified small chromosome. This work is aimed at resolving its structural organization and determining whether this unusual chromosome contains specific genes encoding proteins with important functions in disease pathology or drug resistance. Our results show that the M2903 250-kb small chromosome contains LD1 sequences and has an inverted repeat structure. The LD1 sequences and two cDNAs (cDNA2 and cDNA53) were mapped on a cosmid contig, and the two cDNAs and the corresponding genomic fragments from the small chromosome were sequenced. The gene encoding cDNA2 predicts a putative GTP-binding protein with homology to other GTP-binding proteins only in the G-1 domain region; however, four other conserved motifs can be recognized. Sequence similarity to cDNA53 is located in at least five chromosomes, and its small chromosome copy is a pseudogene. An open reading frame downstream of the cDNA53 pseudogene predicts another GTP-binding protein that belongs to a new G-protein family with an unusual conserved GTP-binding domain and a newly characterized conserved sequence motif. A portion of this GTP-binding protein gene was studied previously in L. aethiopica as a recombinant antigen that reacts with human antibodies.

Amino Acid Sequence↗

PubMed2000

Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12).

IL-12 is a heterodimeric cytokine that is known to induce tumor regression and long-term antitumor immunity. Recombinant adeno-associated virus (rAAV) vectors are advantageous for gene therapy in that they lack pathogenicity in humans, infect dividing as well as nondividing cells, and show a broad range of infectivity. We constructed an rAAV vector expressing interleukin-12 (IL-12) for cancer immunotherapy studies in a mouse model by inserting murine IL-12 (mIL-12) p35 and p40 cDNAs into the plasmid pRep4 and inserting the encephalomyocarditis virus internal ribosomal entry site between the p35 and p40 cDNAs. The mIL-12 expression cassette containing the Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal was subcloned into the AAV plasmid p008Sub/NeoR, which contains two AAV inverted terminal repeat sequences and the NeoR gene driven by the thymidine kinase promoter. rAAV virions (10(4) infectious particles/ml) were generated by cotransfection of rAAV-mIL-12 and a helper plasmid (pAAV/Ad) into 293 cells previously infected with adenovirus 5. After infection of D6 fibroblasts with rAAV-mIL-12, G418-resistant clones were isolated. Each of the 1D D6 clones isolated produced up to 5.2 ng/10(6) cells/48 hours of mIL-12 as determined by enzyme-linked immunosorbent assay. Induction of interferon-gamma, enhanced lymphocyte proliferation, and cytotoxicity assays confirmed biologically functional IL-12 production by the vector. This is the first report indicating that an rAAV vector expresses mIL-12, which can be used to model the effects of mIL-12 alone and/or in combination with other antitumor agents.

Animals↗

PubMed1996

A cell-free system of Tn3 transposition and transposition immunity.

BACKGROUND: Tn3 is a bacterial transposon, which encodes transposase required for its transposition. Tn3 has terminal inverted repeat (IR) sequences of 38 bp in length, whose inner region, called the B domain, is bound by transposase. Tn3 confers transposition immunity, a phenomenon in which Tn3 transposes to a target replicon with Tn3 much less frequently than to a target replicon with no Tn3. RESULTS: To understand transposition and transposition immunity at the molecular level, we constructed a cell-free system using a plasmid as the target. Transpositional recombination occurred in a cell extract containing transposase between the target and a donor plasmid carrying mini-Tn3 at a high frequency. The reaction required ATP, Mg2+, dNTPs and 2% polyvinyl alcohol, and was inhibited by inhibitors for DNA synthesis and DNA gyrase. In this system, when a plasmid with the IR sequence was used as the target, the frequency of transposition was significantly decreased, demonstrating that the transposition immunity conferred by Tn3 is reproduced in vitro. Preincubation of the target in the cell extract increased the level of transposition immunity. On the other hand, mutations within the B domain in the IR sequence of the target abolished transposition immunity. CONCLUSIONS: Transposition of Tn3 and transposition immunity could be reproduced in vitro. The results demonstrate that the binding of transposase to domain B of the IR sequence in the target replicon is responsible for transposition immunity. We propose that the transposition immunity results from conversion of the normal synaptic complex formed between the donor and target molecules to another complex which is inactive for transposition, due to the interaction between transposases binding to the IR sequences in the donor and target molecules.

Base Sequence↗

PubMed1975

Transposition of R factor genes to bacteriophage lambda.

Transpositions of segments of R factor (antibiotic resistance plasmids) to bacteriophage lambda have been selected and characterized. Cells of Escherichia coli harboring R factors that determine kanamycin resistance were infected with phage lambda, and lambdakan transducing lines were obtained. Each of the three examined is unusual when compared to lambda transducing phages containing E. coli chromosomal genes: the kan insertions (a) occur at several sites, each well removed from the integration region POP', (b) are not associated with deletion of lambda phage DNA, and (c) are separable from the lambda genome during transduction or during lytic growth. Two insertions from the same R factor contain 1.5 kilobase sequences repeated in inverted order. The properties of the lambdakan phage suggest that R factors contain systems capable of mediating genetic exchange in the absence of extensive DNA homology. It is suggested that such systems of exchange may have played important roles in R factor evolution.

Chromosome Mapping↗