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PubMed1988

IS1-mediated mobility of the aerobactin system of pColV-K30 in Escherichia coli.

Genes determining the high affinity iron transport system mediated by the siderophore aerobactin are flanked in the enterobacterial plasmid pColV-K30 by inverted repeats of IS1 sequences, suggesting that the aerobactin genes are part of a transposon. To study this possibility, the entire region between the two IS1 sequences was cloned as an 18 kb HindIII-BamHI restriction fragment in pUC8 giving plasmid pMO1. A number of derivatives of pMO1, in which aerobactin genes were tagged with a kanamycin resistance gene, were prepared in order to assess the ability of both IS1s to promote the formation of cointegrates with pCJ105, an F derivative devoid of insertion sequences. Mating-out assays indicated that both flanking IS1s were active in cointegrate formation at detectable frequencies. In some cases, the cointegrates could be resolved, the final result being a transposition-like event for the entire aerobactin system.

DNA Transposable Elements↗
PubMed1989

Chloroplast ribosomal DNA organization in the chromophytic alga Olisthodiscus luteus.

There are almost no data describing chloroplast genome organization in chromophytic (chlorophyll a/c) plants. In this study chloroplast ribosomal operon placement and gene organization has been determined for the golden-brown alga Olisthodiscus luteus. Ribosomal RNA genes are located on the chloroplast DNA inverted repeat structure. Nucleotide sequence analysis, demonstrated that in contrast to the larger spacer regions in land plants, the 16S-23S rDNA spacer of O. luteus is only 265 bp in length. This spacer contains tRNA(Ile) and tRNA(Ala) genes which lack introns and are separated by only 3 bp. The sequences of the tRNA genes and 16S and 23S rDNA termini flanking the spacer were examined to determine homology between O. luteus, chlorophytic plant chloroplast DNA, and prokaryotes.

Autoradiography↗
PubMed1993

Characterization of two divergent adenovirus 31 strains.

Two divergent strains of adenovirus type 31 were analyzed by neutralization test and restriction endonuclease (RE) patterns in an effort to find the basis for their genetic variability. One strain, isolated from the throat of a child in Maryland during an upper respiratory illness in 1968, was partially neutralized by Ad 31 antisera (to 16-fold lower than homologous titer) while its own antiserum fully neutralized prototype Ad 31 virus, but shared only 9% of comigrating RE fragments with Ad 31 prototype (vs. 30% with Ad 18 prototype); however, PCR tests specific for the inverted terminal repeat (ITR) sequence of Ads 12 and 18 were negative. The other strain, recovered from a stool sample from an infant with diarrhea in Georgia in 1979, was inhibited by Ad 31 antiserum to within 4-fold homologous titer, but shared only 15% of comigrating fragments with Ad 31 prototype (vs. 91% with Ad 18 prototype); ITR-specific PCR tests with this virus were positive for Ad 12/Ad 18. These data suggest that both strains are from separate evolutionary lines of Ad 31 unrelated to all other isolates studied to date by RE analysis, and that the partial neutralization by prototype Ad 31 antisera might represent small mutations in the hexon gene.

Adenovirus Infections, Human↗
PubMed1987

Tn10 transposition and circle formation in vitro.

We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event. Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires Tn10 transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. Tn10 is composed of inverted repeats of insertion sequence IS10. Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.

Adenosine Triphosphatases↗
PubMed1990

Stem-loop structures at the 3' end of tobacco Rubisco large subunit mRNA.

There are two inverted repeat nucleotide (nt) sequences, each capable of forming a stem-loop structure (sls) at the 3' end of the tobacco Rubisco large subunit mRNA (rbcL). The smaller sls is followed by a larger sls. The in vivo functions of the 3' sls of the rbcL mRNA were characterized using the Escherichia coli system. S 1 mapping of the rbcL transcripts synthesized in E. coli revealed that the 3' end of a major transcript in the bacterial cell is almost identical to the 3' end of authentic chloroplast (cp) rbcL mRNA. This native 3' end is located 4 nt downstream from the larger sls for the cp mRNA and 6 nt for the bacterial transcript, respectively. Deletion experiments show that the larger sls is essential for producing the native 3' end of rbcL mRNA in E. coli. The sls do not function as an efficient transcription terminator but can stabilize upstream mRNA segments in vivo.

Base Sequence↗
PubMed1995

The human mdr1 (multidrug-resistance) gene harbours a long homopyrimidine.homopurine sequence next to a cluster of Alu repeated sequences in intron 14.

In order to identify specific DNA sequences useful as 'genetic landmarks' in the construction of a complete map of the human mdr1 (multidrug-resistance) gene, we investigated the introns in the central region. In intron 14, we identified a long stretch of a homopyrimidine.homopurine sequence most probably adopting an unconventional DNA conformation, followed by a cluster of three Alu repeated sequences in an inverted orientation. Here, we describe the structure, formation and nucleotide sequence of these DNA elements.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Identification of a DNA fragment in the genome of Bordetella pertussis carrying repeated DNA sequences also present in other Bordetella species.

A DNA fragment (3.8 kbp) which hybridized to repeated sequences in the genome of Bordetella species has been cloned from Bordetella pertussis chromosomal DNA. Eleven subclones were constructed from this fragment. They exhibited distinct inter-species hybridization patterns in genomic blots of each of the Bordetella used in the study. One subclone revealed intraspecies variability among B. pertussis strains and another did not hybridize to B. parapertussis. The 3.8 kbp DNA fragment possesses a middle sequence surrounded by repeated sequences organized in an opposite symmetrical orientation. The external inverted repeats of it hybridized to a 680 bp DNA sequence which is located about 800 bp upstream of the pertussis toxin operon. The novel structural organization of the 3.8 kbp fragment suggests the possibility that this DNA segment is an IS-like element which may have an important function in the expression of virulence determinants in Bordetella bacteria.

Blotting, Southern↗
PubMed2004

Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors.

Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors.

Adenoviridae↗
PubMed1999

Gene structure and polymorphism of an invertebrate nitric oxide synthase gene.

Nitric oxide synthases (NOSs) are ubiquitous in living organisms. However, little is known about the evolution of this large gene family. The first inducible NOS to be described from an invertebrate regulates malaria parasite (Plasmodium spp.) development in the mosquito Anopheles stephensi. This single copy gene shows the highest homology to the vertebrate neuronal isoforms, followed by decreasing homology to endothelial and inducible isoforms. The open reading frame of 1247 amino acids is encoded by 19 exons, which span approximately 33 kilobases. More than 50% of the mosquito exons, distributed around the putative heme, calmodulin, and FAD/NADPH cofactor-binding domains, are conserved with those of the three human genes. Repetitive elements identified within the larger introns include a polymorphic dinucleotide repeat, two tandem repeats, and a putative miniature inverted repeat transposable element. Sequence analysis and primer extension indicate that the upstream promoter is 'TATA-less' with multiple transcription start sites within approximately 250 base pairs of the initiation methionine. Transcription factor binding sites in the 5'-flanking sequence demonstrate a bipartite distribution of lipopolysaccharide- and inflammatory cytokine-responsive elements that is strikingly similar to that described for vertebrate inducible NOS gene promoters.

Animals↗
PubMed1993

Isolation of a repeated sequence from the genome of Bordetella parapertussis with characteristics of an insertion sequence element.

A repeated DNA sequence in the genome of Bordetella parapertussis was identified by means of heteroduplex formation and S1 nuclease treatment. Cloning and sequencing of the repeated sequence, revealed that it comprised 1306 basepairs, contained inverted repeats at its termini, and two large open reading frames. This insertion sequence element was designated IS1001, and was found in about 20 copies in 10 B. parapertussis strains analyzed. Identical hybridization patterns were observed in B. parapertussis when IS1001 was used as a probe. B. pertussis strains did not hybridize to IS1001.

Amino Acid Sequence↗
PubMed2003

Free DNA ends are essential for concatemerization of synthetic double-stranded adeno-associated virus vector genomes transfected into mouse hepatocytes in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in vivo. In hepatocyte nuclei, the incoming single-stranded (ss) vector genomes are converted into various forms of double-stranded (ds) genomes including extrachromosomal linear and circular monomers and concatemers, and a small portion of the vector genomes integrate into chromosomes. The mechanism of genome conversion is not well understood. In the present study, we analyzed the role of inverted terminal repeat (ITR) sequences of ds circular or linear rAAV vector intermediates in concatemerization. We synthesized supercoiled ds circular monomers with a double-D ITR (DDITR) (C+), and ds linear monomers with an ITR at each end (L+), and their control molecules, C- and L-, which lack the ITR-derived sequences, and transfected mouse hepatocytes with these molecules in vivo to assess their capacity for concatemerization. The transfected L+ or L-, but not C+ or C- molecules, concatemerized in vivo irrespective of the presence or absence of the ITRs. In addition, our results suggested that transfected C+ or C- species were not efficient substrates for integration. Based on these observations, we propose a model whereby ds linear molecules with free DNA ends, but not circular molecules, play an important role in rAAV vector genome concatemerization.

Animals↗
PubMed1996

Mode of selectivity in cyclic AMP receptor protein-dependent promoters in Escherichia coli.

Escherichia coli cAMP receptor protein (CRP) controls more than 20 genes. There are significant differences in the promoter regions in these genes. Thus, an elucidation of the mechanism of CRP action requires knowledge about the mode of selectivity in these promoters. An earlier study [Heyduk, T., & Lee, J. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 81, 1744-8] indicates that the CRP(cAMP)1 conformer exhibits the highest affinity for the lac PI site in the lac operon. It is conceivable that the CRP conformer that binds with the highest affinity to these other sites may not be CRP(cAMP)1. To investigate this possibility, the binding of CRP to nine CRP binding sites was studied as a function of cAMP concentration. The CRP binding sites employed in this investigation were chosen to represent the primary promoter sites from class I (lac site PI) and class II (sites PI of gal and crp) as well as secondary CRP binding sites (crp site PII and cat PII) to further understand the molecular mechanism of CRP in controlling the transcription of these bacterial genes. The affinity of CRP for three synthetic CRP binding sites was also examined to explore the contribution of the inverted repeat region and sequences surrounding the recognition motifs. The synthetic sequences are gallac which contains the lac recognition motifs in the background of gal, modified cat PII which contains an 8-base pair spacer between the recognition motifs rather than the 7-base pair sequence naturally found in cat PII, and a random sequence that has no known similarity to any CRP binding site found in nature. The apparent affinities of these sequences for CRP were quantitatively determined to be biphasic in their cAMP dependence. The CRP(cAMP)1 conformer was found to have the highest affinity for all of the DNA sequences examined. No specific affinity was observed for these sequences with free CRP and CRP(cAMP)2. The affinity of CRP for DNA was sequence-dependent and increased in the following order: random < cat site PII, modified cat site PII, crp sites PI and PII < gal site PI < lac site PI < gallac. These results indicate that the entire CRP binding site sequence and its natural variability provide information to CRP. These promoter sites which appear to have different mechanisms at the molecular level are transcriptionally controlled by the same CRP conformer, CRP(cAMP)1. Thus, the regulation of transcription by CRP is more subtle than choosing different conformational forms of CRP. Using "physiological" concentrations of various components, a computer simulation study was conducted to illustrate the possible consequences of the thermodynamic parameters determined in this study. It is evident that the promoters of protein systems regulating the transport and metabolism of carbohydrates are responsive to low cAMP concentrations. However, the promoter for controlling the expression of CRP is highly regulated by the fluctuation of cAMP concentration.

Base Sequence↗
PubMed2000

Survey of mariner-like elements in the housefly, Musca domestica.

The presence of mariner-like elements in four strains of the housefly, Musca domestica, was surveyed by PCR. Using the inverted terminal repeat (ITR) sequences of the Mos 1 element as primers, DNAs were successfully amplified from all strains of the housefly. Southern blot analysis indicated that these amplified DNAs were repetitive sequences in the genome of M. domestica. Sequence analyses of cloned PCR products showed that they were 45% identical to the Mos 1 element. These fragments appeared to be nonfunctional, because they contained no intact open reading frame (ORF) capable of encoding transposase. We conclude that these DNAs are degraded mariner-like elements (MLEs) in M. domestica. Because these endogenous MLEs in M. domestica do not encode any functional proteins, they probably would not affect the behavior of mariner-based vectors if such were introduced into this species as transformation vectors.

Animals↗
PubMed1997

Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii.

Nuclear genes coding for carbonic anhydrase, a major mitochondrial constituent in Chlamydomonas reinhardtii grown under limited CO2, were characterized. Two genes, ca1 and ca2, were found within 7 kb of genomic DNA, organized 'head to head' in a large inverted repeat. The DNA sequences for the two genes were very similar, even in the promoter regions and in introns, indicating that the repeat is a result of a recent duplication. To study gene regulation, elements from the upstream region of ca1 were fused to the arylsulphatase reporter gene. After transformation, the expression of arylsulphatase was regulated similarly to the endogenous ca1/ca2 genes, even when the promoter was trimmed down to 194 nt. Expression could not be detected when 5% CO2 was bubbled into the growth medium, but was induced within hours after transfer to air. The ca1 promoter was not induced in low light, but at intermediate light levels its activity was dependent on the irradiance. O2 concentration had no effect on the promoter activity, indicating that photorespiratory metabolites are not triggering the response. The availability of cells transformed with a CO2-regulated reporter gene should facilitate further studies on the metabolic adaptations that occur in some green algae in response to the external CO2 level.

Animals↗
PubMed2001

P instability factor: an active maize transposon system associated with the amplification of Tourist-like MITEs and a new superfamily of transposases.

Miniature inverted-repeat transposable elements (MITEs) are widespread and abundant in both plant and animal genomes. Despite the discovery and characterization of many MITE families, their origin and transposition mechanism are still poorly understood, largely because MITEs are nonautonomous elements with no coding capacity. The starting point for this study was P instability factor (PIF), an active DNA transposable element family from maize that was first identified following multiple mutagenic insertions into exactly the same site in intron 2 of the maize anthocyanin regulatory gene R. In this study we report the isolation of a maize Tourist-like MITE family called miniature PIF (mPIF) that shares several features with PIF elements, including identical terminal inverted repeats, similar subterminal sequences, and an unusual but striking preference for an extended 9-bp target site. These shared features indicate that mPIF and PIF elements were amplified by the same or a closely related transposase. This transposase was identified through the isolation of several PIF elements and the identification of one element (called PIFa) that cosegregated with PIF activity. PIFa encodes a putative protein with homologs in Arabidopsis, rice, sorghum, nematodes, and a fungus. Our data suggest that PIFa and these PIF-like elements belong to a new eukaryotic DNA transposon superfamily that is distantly related to the bacterial IS5 group and are responsible for the origin and spread of Tourist-like MITEs.

Base Sequence↗
PubMed1996

A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography.

The extensive testing of adeno-associated virus (AAV) as a vector for human gene therapy has been hampered by low efficiency of the current packaging system, which is based on transient transfection with plasmid DNAs and infection with adenovirus in permissive cells. In an effort to resolve this problem, HeLa cell-based packaging cell lines were established. These packaging cells carry multiple copies of the AAV genome lacking the inverted terminal repeat (ITR) sequences. The AAV genes were silent in these cells but inducibly expressed by adenovirus infection. When the AAV vector plasmid containing the neoR gene flanked by the ITRs was also integrated into these cells, efficient production of the recombinant AAV particles occurred after adenovirus infection. AAV vector particles in cell lysates could be concentrated by sulfonated cellulose column chromatography. Using the packaging cells and the column chromatography technique, it is possible to prepare AAV vectors with the titer of higher than 10(8) cfu/ml or 5 x 10(10) particles/ml. This new strategy should be useful for testing AAV vectors in vivo.

Cellulose↗
PubMed1997

HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep-) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep- hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells.

Animals↗
PubMed1999

Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans.

Previous work has suggested that de novo methylation of plant nuclear genes can be triggered by an RNA-DNA interaction. To test whether transcription of a promoter would induce de novo methylation and silencing of unlinked genes driven by the same promoter, a chimeric 'gene' consisting of a nopaline synthase promoter (NOSpro) positioned downstream of the cauliflower mosaic virus 35S promoter (35Spro) and flanked at the 3' end by a NOS terminator (NOSter) was constructed and introduced into the genome of a plant that normally expresses an unmethylated NOSpro-neomycinphosphotransferase (nptII) gene. Transformants were tested for kanamycin resistance and NOSpro RNA synthesis. Most produced a full-length polyadenylated NOSpro RNA, which did not induce silencing or methylation at the NOSpro-nptII target gene. One, however, contained truncated non-polyadenylated NOSpro RNA; in this plant, the NOSpro-nptII gene became silenced and methylated in the NOSpro region. Molecular analysis of the NOSpro silencing locus revealed two incomplete copies of the 35Spro-NOSpro gene arranged as an inverted repeat with NOSpro sequences at the center. Reducing NOSpro transcription by crossing a 35Spro-silencing locus partially reactivated nptII gene expression and decreased NOSpro methylation at the target locus, thus implicating aberrant NOSpro RNA in this trans-silencing phenomenon.

Amino Acid Oxidoreductases↗