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PubMed1991

IS406 and IS407, two gene-activating insertion sequences for Pseudomonas cepacia.

We have determined the nucleotide sequences of IS406 (1368 bp) and IS407 (1236 bp), two insertion sequence (IS) elements isolated from Pseudomonas cepacia 249 on the basis of their abilities to activate the expression of the lac genes of Tn951. IS406 and IS407 when inserted into the lac promoter/operator region of Tn951 generated, respectively, duplications of 8 and 4 bp of target DNA. IS406 had 41-bp terminal inverted repeat (IR) sequences with eleven mismatches. IR-L (left) contained a 12-bp motif present at the ends of Tn2501. In other respects, IS406 was distinct from previously described bacterial IS elements listed in the GenBank and EMBL databases. IS407 had 49-bp terminal IRs with 18 mismatches. IR-R (right) contained an outwardly directed sigma 70-like promoter. IS407 was closely related to IS476 and ISR1 from Xanthomonas and Rhizobium sp., respectively.

Amino Acid Sequence↗
PubMed1998

Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator.

The nucleotide sequence of a 2-4 kb BamHI-SalI fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.

Anthranilate Synthase↗
PubMed1987

The herpes simplex virus origins of DNA synthesis in the S component are each contained in a transcribed open reading frame.

In the herpes simplex virus 1 genome, the origins of viral DNA synthesis are located in the unique sequences of the L component (Oril) and in the reiterated sequences of the S component (OriS) located between the 5' terminus of the alpha 4 gene and the 5' terminus of either the alpha 22 (left terminus of the S component) or the alpha 47 (right terminus of the S component) gene. Studies prompted by the finding that only one, but not both, OriS sequence is dispensable for growth in cell culture indicate that each OriS sequence is contained in an open reading frame designated as OriSORF. The transcription of OriSORF is initiated approximately 860 nucleotides upstream from that of the alpha 4 gene and 162 nucleotides downstream, but on the opposite strand from the transcription initiation site of the alpha 22 or alpha 47 genes within the inverted repeat c sequence. The OriSORF transcript is 3' coterminal with the mRNA of the alpha 4 gene, polyadenylated but not spliced, transported into the cytoplasm, and capable of directing the synthesis of a 330-amino-acid protein with a translated molecular weight of approximately 34,000. Transcription is cycloheximide but not phosphonoacetate sensitive and is therefore regulated as either a beta or a gamma 1 gene. The implications of the transcription of OriS and of possible functions of the product of OriSORF are discussed.

Animals↗
PubMed1983

Nucleotide sequence of the transforming region of human cytomegalovirus.

We report the nucleotide sequence of a BamHI-HindIII fragment of human cytomegalovirus containing the region (XbaI-HindIII) capable of transforming NIH3T3 cells. The single EcoRI site, shown to abolish transforming activity if cleaved, is present within an 8 base-pair inverted complementary repeat. Close to this sequence there is a small, potentially spliced, open reading frame possessing some of the signals involved in eukaryotic gene expression. Possible mechanisms of transformation, involving the inverted repeat sequence, are discussed.

Base Sequence↗
PubMed1984

Nucleic acid sequence and chromosome assignment of a wheat storage protein gene.

A cloned gliadin gene was isolated from a wheat genomic library, and 2.4 kb of its primary sequence determined. The gene, alpha-1Y, was found by Southern analysis to be located on chromosome 6A, and its derived amino acid sequence identifies it as a member of the A-gliadin subgroup of alpha-gliadins located on the short arm of that chromosome. alpha-1Y is apparently functional, and contains consensus TATA and CAAT boxes, and polyadenylation signals. This gliadin gene has no introns, and its noncoding flanking regions contain several short repeats and inverted sequences. The gene is contained in a 6.2 kb EcoRI genomic fragment whose apparent copy number varies in different wheat cultivars.

Amino Acid Sequence↗
PubMed2005

DNA-binding specificity of rice mariner-like transposases and interactions with Stowaway MITEs.

Mariner-like elements (MLEs) are DNA transposons found throughout the plant and animal kingdoms. A previous computational survey of the rice (Oryza sativa) genome sequence revealed 34 full length MLEs (Osmars) belonging to 25 distinct families. This survey, which also identified sequence similarities between the Osmar elements and the Stowaway superfamily of MITEs, led to the formulation of a hypothesis whereby Stowaways are mobilized by OSMAR transposases. Here we investigate the DNA-binding activities and specificities of two OSMAR transposases, OSMAR5 and OSMAR10. Like other mariner-like transposases, the OSMARs bind specifically to the terminal inverted repeat (TIR) sequences of their encoding transposons. OSMAR5 binds DNA through a bipartite N-terminal domain containing two functionally separable helix-turn-helix motifs, resembling the paired domain of Tc1-like transposases and PAX transcription factors in metazoans. Furthermore, binding of the OSMARs is not limited to their own TIRs; OSMAR5 transposase can also interact in vitro with TIRs from closely related Osmar elements and with consensus TIRs of several Stowaway families mined from the rice genome sequence. These results provide the first biochemical evidence for a functional relationship between Osmar elements and Stowaway MITEs and lead us to suggest that there is extensive cross-talk among related but distinct transposon families co-existing in a single eukaryote genome.

Base Sequence↗
PubMed1982

Sequence relationships between plasmids associated with conventional MLS resistance and zonal lincomycin resistance in Streptococcus pyogenes.

By using electron microscopy of self-annealed DNA and restriction enzyme analysis, we have compared the physical maps of two group A streptococcal plasmids associated with conventional MLS resistance (pEL1; 20 Md) and zonal lincomycin resistance (pSM10419; 15 Md). Of their monomeric molecules, about 40% and 60%, respectively, are occupied by identical non-tandem inverted repeats containing sequences specifying putative replication functions. Sequence homology also exists between their resistance determinants which are located in unique DNA. Moreover, homology between additional regions of unknown function is so extensive and restriction fragment arrangement so similar that, formally, pSM10419 can be considered a deletion variant of pEL1. The results suggest that MLS and zonal lincomycin resistance have the same biochemical basis (i.e. methylation of 23S ribosomal RNA) and differ only quantitatively in the inducible control systems.

Base Sequence↗
PubMed1997

Molecular cloning and characterization of a novel mosquitocidal protein gene from Bacillus thuringiensis subsp. fukuokaensis.

A novel mosquitocidal protein gene, cry20Aa, was cloned from Bacillus thuringiensis subsp. fukuokaensis (H-3a: 3d: 3e). The gene product, Cry20Aa, was naturally truncated and had a molecular mass of 86,138 Da. The Cry20Aa protein possessed five conserved sequence blocks, as do most other insecticidal Cry toxins. However, an amino acid comparison of Cry20Aa with other mosquitocidal toxins, including Cry4A, Cry4B, Cry10A, Cry11A, and Cry11B, demonstrated that Cry20Aa was quite different from other toxins except for the conserved blocks. The N terminus of Cry20Aa was, however, homologous to the N termini of Cry4A and Cry10A. Interestingly, an inverted repeat (IR1) sequence in the open reading frame of the cry20Aa gene caused incomplete expression of Cry20Aa. When this internal IR1 sequence was altered with no change of amino acid sequence, acrystalliferous B. thuringiensis cells transformed with cry20Aa gene dramatically produced crystal inclusions. However, the intact 86-kDa Cry20Aa protein is highly labile, and it is rapidly degraded to polypeptides of 56 and 43 kDa. To increase expression of the cry20Aa gene, the p20 chaperonelike protein and the cyt1Aa promoter were utilized. While p20 did not increase Cry20Aa expression or stability, chimeric constructs in which the cry20Aa gene was under control of the cyt1Aa promoter overexpressed the Cry20Aa protein in acrystalliferous B. thuringiensis. The expressed Cry20Aa protein showed larvicidal activity against Aedes aegypti and Culex quinquefasciatus. However, the mosquitocidal activity was low, probably due to rapid proteolysis to inactive 56- and 43-kDa proteins.

Amino Acid Sequence↗
PubMed1989

Complete foldback transposable elements encode a novel protein found in Drosophila melanogaster.

An apparently complete foldback (FB) transposable element homologous to FB white-crimson (FBwc) was analyzed. A complete FB element could encode one or more proteins required for regulation of FB transposition. The central DNA region (the loop) and the junctions between the loop and the inverted terminal repeats were sequenced. Three open reading frames (ORFs) are present in the loop, and a novel 308 bp inverted repeat is present at the junctions. No significant homologies were found when the DNA sequences of the loop region and the novel inverted repeat were screened against the Gene data bank. Antibodies were prepared in guinea-pigs against a peptide present near the amino terminus of ORF1, the longest ORF. A 71,000 dalton protein was isolated from an extract of Drosophila melanogaster early-stage embryos on an anti-ORF1 peptide-affinity column. Immunohistochemical studies of adult flies demonstrate localization of this protein in egg chambers.

Amino Acid Sequence↗
PubMed1998

Viral sequences enable efficient and tissue-specific expression of transgenes in Xenopus.

Expression of transgenes within a single generation by direct DNA injection into vertebrate embryos has been plagued by inefficient and nonuniform gene expression. We report a novel strategy for efficient and stable expression of transgenes driven by both ubiquitous and tissue-specific promoters by direct DNA injection into developing Xenopus laevis embryos. This strategy involves flanking expression cassettes of interest with inverted terminal repeat sequences (ITRs) from adeno-associated virus. Our results suggest that the ITR strategy may be generally applicable to other systems, such as zebra fish and embryonic stem cells, and may enable tissue-specific expression of transgenes in problematic contexts.

Actins↗
PubMed1995

Two copies of a DNA element, 'Wendy', in the chloroplast chromosome of Chlamydomonas reinhardtii between rearranged gene clusters.

We have characterized two copies of a 2.4 kb DNA element that we call 'Wendy', in the chloroplast chromosome of Chlamydomonas reinhardtii. The two copies of Wendy reside in different single-copy regions at opposite positions in the chloroplast genome. Like many mobile DNA elements, both copies of Wendy are bordered by inverted repeats and contain several additional degenerate copies of these repeat sequences in direct or inverted orientation. In addition, four basepairs are repeated in direct orientation. Two major open reading frames (ORFs) are predicted from the DNA sequence of Wendy I. These ORFs are co-transcribed from a promoter inside the element. The deduced amino acid sequence of the larger of these ORFs shares some weak similarities with sequence motifs of transposases and integrases of other mobile elements. Wendy II appears to be altered relative to Wendy I by point mutations and small deletions and insertions which destroy the ORFs. The leader sequence of the Wendy transcript is nearly identical with the leader sequence of the rbcL transcript of C. reinhardtii, but not of C. moewusii (where the complete Wendy was also undetectable). Furthermore, both copies of Wendy are bracketed by gene clusters that are separated in C. reinhardtii but are contiguous in C. moewusii where they exist in an inverted orientation compared with C. reinhardtii. Wendy was not found in any of the completely sequenced chloroplast genomes of rice, tobacco, pine, Euglena or Marchantia, nor in any other GenBank entry. Our results suggest that Wendy has invaded C. reinhardtii after divergence from other species. Subsequent Wendy-dependent illegitimate homologous or site-specific recombination events or both may have contributed to scrambling of the C. reinhardtii chloroplast genome relative to genomes of other species.

Amino Acid Sequence↗
PubMed1989

An autonomously replicating sequence of pSRI plasmid is effective in two yeast species, Zygosaccharomyces rouxii and Saccharomyces cerevisiae.

The autonomously replicating sequences (ARSs) of pSR1, a cryptic circular DNA plasmid detected in a strain of Zygosaccharomyces rouxii, were delimited by subcloning and deletion analysis and by the isolation of nucleotide substitution mutations. A 30 base-pair (bp) sequence from inverted repeat 1 (IR1) and presumably the same region from IR2 of pSR1 functions as an ARS in the native host, Z. rouxii, and in a heterologous host, Saccharomyces cerevisiae. Thus, pSR1 has two ARSs per molecule, either of which is sufficient for replication of the plasmid molecule in both hosts. These hosts, however, respond differently to nucleotide substitutions in the 30 bp sequence, suggesting that the sequences required for ARS function in the two organisms are not exactly the same. In addition, a 137 bp sequence that overlaps the 30 bp sequence by 11 bp also functions as an ARS in Z. rouxii but not in S. cerevisiae. However, this 137 bp sequence enhances the stability of plasmids carrying the pSR1 ARS in S. cerevisiae. The 30 bp and 137 bp sequences each contain a single copy of the 11 bp ARS consensus sequence, which is essential for ARS function in S. cerevisiae. Small insertions between the 11 bp overlapping region and the 11 bp ARS consensus sequence showed that a proper distance between these two 11 bp sequences is essential for the ARS function of the 30 bp sequence. Point mutations that inactivate ARS function show that the ARS consensus sequence, as well as a short A:T segment in the overlapping sequence, is required for the ARS function of the 30 bp sequence.

Base Sequence↗
PubMed1996

Nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3.

We have determined the complete nucleotide sequences of adeno-associated virus 3 (AAV-3) and generated an infectious clone. The single-stranded DNA genome of AAV-3 is 4726 nucleotides in length. The positive strand contains two large open reading frames; the left open reading frame encodes the nonstructural proteins and the right open reading frame encodes the structural proteins. The coding regions are flanked by identical inverted terminal repeat sequences containing palindromes. AAV-3 has little homology with the autonomous parvoviruses or erythroviruses but has 82% overall sequence homology with AAV-2. At the amino acid level there was 88% homology with AAV-2 nonstructural (Rep) proteins and 87% homology with AAV-2 capsid proteins. In addition, AAV-3 differed importantly from AAV-2 in the lack of a typical promoter sequence (TATA box) at p40 and the presence of the consensus sequence for adenovirus-related transcription factor E4F binding within the upstream region of the p5 promoter. These results suggest that AAV-3 not only consists of serologically distinct structural proteins but that viral propagation also may be controlled by different gene regulatory elements at the transcription level. The infectious clone confirmed the sequence and may be useful for developing new vectors for gene therapy.

Amino Acid Sequence↗
PubMed2005

The puf operon of the purple sulfur bacterium Amoebobacter purpureus: structure, transcription and phylogenetic analysis.

The puf operon, encoding photosynthetic reaction center and light-harvesting genes, of the purple sulfur phototrophic bacterium Amoebobacter purpureus was cloned and sequenced. This revealed an unusual operon structure of the genes pufB1 A1 LMCB2 A2 B3 A3. The sequence represents the second complete puf operon available for Chromatiaceae. So far, additional sets of light-harvesting 1 (LH1) genes, pufB2 A2 and pufB3 A3 in the region downstream of pufC have only been described for Allochromatium vinosum. Along with reports of multiple LH1 polypeptides found in some Ectothiorhodospiraceae by direct protein sequencing, our results indicate that multiple LH1 genes may occur frequently in phototrophic gamma-proteobacteria. Phylogenetic analyses suggested a coevolution of the core puf genes pufB1 A1 LM. Separate analysis of the LH1 alpha and beta polypeptides revealed a high intraspecies relatedness for the secondary LH1beta polypeptides, possibly caused by functional constraints. In contrast, LH1alpha subunits of Amb. purpureus and Alc. vinosum are closely related (85% sequence identity) which could reflect horizontal gene transfer. RNA analyses suggested co-transcription of all puf genes in Amb. purpureus as a 5.5 kb primary transcript which appears to be more stable than the puf operon primary transcripts of purple non-sulfur bacteria. The 5' end of the transcript mapped to a putative promoter, which contains a -35 region located in an inverted repeat DNA sequence.

Amino Acid Sequence↗
PubMed1998

Ikirara, a novel transposon family from the malaria vector mosquito, Anopheles gambiae.

Members of a novel transposon family, Ikirara, were found in the genome of the malaria vector Anopheles gambiae. They are most abundant in A. gambiae sensu stricto, but related sequences were found in all four other tested members of this species complex. No relatives were found in A. funestus or A. stephensi. Ikirara1, the first isolated family member, was found between two of the tandem Vitellogenin (Vg) genes. Because it was found at this location in G3 and only one of nine other A. gambiae s.s. strains examined, and because its 216 bp inverted terminal repeats are 100% identical, transposition to this locus may have been recent. Ikirara1 inverted repeat terminal sequences are similar to those of DNA to DNA transposons of the mariner/Tc1 and hAT superfamilies. Also similar to mariner/Tc1 elements, insertion of Ikirara1 apparently created a duplication of the dinucleotide TA at the target site.

Animals↗
PubMed1999

Cotranscription of the rpl5-rps14-cob gene cluster in pea mitochondria.

In pea mitochondria the rpl5, rpsl4 and cob ORFs are clustered in a unique genomic environment and are cotranscribed into a 4.7-kb primary transcript and several other polycistronic RNAs with sizes between 4.0 and 2.3 kb. All of the larger RNAs terminate at a common 3' end, 52 nucleotides downstream of the cob gene. Transcription is initiated at a promoter about 1.3 kb upstream of the rpl5 start codon. The promoter sequence 5'-AATAAGAGA-3' corresponds to the highly conserved 5'-CRTAAGAGA-3' motif often found in promoters in dicot plants. Functional analysis in a homologous in vitro transcription system showed the pea rpl5 promoter to be active, despite the presence of an altered base in first position of the promoter motif. In Oenothera, in contrast to pea, transcription of the rpl5 gene is driven by a promoter motif that conforms perfectly to the consensus sequence. Double inverted repeats located in the 3' flanking regions of the rpsl4 and cob ORFs in pea were investigated with respect to their possible role in defining transcript termini and their potential function in controlling exo- and endonucleolytic processing or transcript stabilization.

Base Sequence↗
PubMed1998

[Cloning of large imperfect palindromes in circular and linear vectors].

Data on comparative analysis of cloning of large imperfect artificial palindromes and one natural palindrome in the circular pUC19 and linear pN15L vectors, constructed on the basis of temperate N15 bacteriophage minireplicon are presented. The artificial palindromes consisted of the two head-to-head oriented 5:5-kb lambda bacteriophage DNA fragments interrupted by a short sequence of varied size. Natural palindrome was represented by the 12.5-kb BamHI fragment of Tetrahymena pyriformis ribosomal genes cluster. Integration of some artificial palindromes and a natural palindrome into a circular vector resulted in a considerable decrease of its copy number. This was assumed to result from cruciform formation mediated by supercoiling of a circular vector DNA. Thus, a linear is vector preferable in cloning of inverted repeated DNA sequences.

Animals↗
PubMed1983

Lambda phage DNA sequences affecting the packaging process.

Our previous work identified a minimal region of bacteriophage lambda DNA that is necessary for packaging into phage particles. It consists of 40 bp of the right arm and 45 bp of the left arm [Miwa and Matsubara, Gene 20 (1982)267-279]. A part of this region, 22 bp of the right arm and 38 bp of the left arm, is sufficient for cutting at cos lambda (the minimal sequence for cos lambda cutting). An 84-bp region to its right contains a binding site for lambda terminase, a complex of Nu1 and A gene products. This second region, which we called the enhancing region for packaging, stimulates cutting at cos lambda as well as packaging. This region is not active if it is physically separated from the minimal packaging region. The enhancing region has 15-bp inverted repeats. These sequences are conserved in the corresponding region of bacteriophage phi 80, which has the same packaging specificity as lambda.

Bacteriophage lambda↗