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PubMed2003

Structure of gene flanking regions and functional analysis of sequences upstream of the rat hsp70.1 stress gene.

We present structural and comparative analysis of the flanking regions of the rat hsp70.1 stress gene. Several repetitive sequences, microsatellites and short interspersed repetitive elements (SINEs) were found, as well as a significant gap in the 3' UTR, as compared to the orthologous mouse gene. We also show that the complex microsatellite region composed of partially overlapping inverted repeat and long homopurine-homopyrimidine sequence, which is localized 1.8 kbp upstream of the transcription start site, is capable to adopt non-B DNA structures (an H-DNA and a cruciform structure) in vitro. Functional analysis performed with the use of various fragments of the 5'end flanking regions ligated to the chloramphenicol acetyltransferase (CAT) reporter gene revealed a crucial role of cooperation between heat shock element (HSE) regulatory sequences, while none of the three HSEs alone is able to drive efficient heat induced transcription of the reporter gene. We also found that the microsatellite region does not influence transcription by itself, however, it abolishes the effect of the adjacent putative silencing element. To our knowledge, this is a first extensive structural and functional analysis of the promoter region of the mammalian heat inducible hsp70i gene localized distally to the hsp70-related spermatid-specific gene in the major histocompatibility complex III.

3' Flanking Region↗
PubMed1993

Molecular analysis of a gene encoding a serum-resistance-associated 76 kDa surface antigen of Haemophilus somnus.

Haemophilus somnus is a Gram-negative bacterial bovine pathogen which can cause disease or be carried asymptomatically. We previously showed that four serum-sensitive isolates from asymptomatic carriers lacked a 13.4 kb sequence of chromosomal DNA that was present in two virulent serum-resistant strains. We have since sequenced 5 kb of the 13.4 kb fragment from a serum-resistant strain, which contained an open reading frame (ORF) of at least 4.5 kb. From Western blot analysis, the ORF was shown to encode a 76 kDa protein (p76) that co-migrated with a 76 kDa H. somnus surface protein. Both the recombinant and natural p76 reacted with convalescent-phase serum from a cow in an experimental H. somnus abortion study. The translational start site for p76 was identified by deletion analysis of subclones of the 5 kb cloned sequence. The 4.5 kb ORF contained 1.2 kb tandem direct repeats (DRs), with 65% identity between the two repeats at the protein level. The 5' DR (DR1) included the start site for the 76 kDa protein, and DR2 had a flanking inverted repeat, suggestive of an insertion-sequence-like element.

Amino Acid Sequence↗
PubMed1994

Isolation and characterization of IS31831, a transposable element from Corynebacterium glutamicum.

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS1096 from Mycobacterium smegmatis. Both IS31831 and IS1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.

Amino Acid Sequence↗
PubMed2000

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA.

Approximately 6000 specific DNA deletion events occur during development of the somatic macro-nucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis -acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetra-hymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis -acting sequences outside the deleted DNA.

Animals↗
PubMed1998

The evolution of grass genome organisation and function.

New cloning technologies and more efficient DNA sequencing now permit comprehensive structural studies of complex eukaryotic genomes. Previous global investigations of genome organisation in plants had shown that abundant repetitive DNAs were intermixed with genes. However, the nature of the major repeats, their possible biological roles, their origins, and their precise patterns of organisation were not clearly defined. My laboratory has used large clones derived from homologous regions of the maize, sorghum and rice genomes to investigate the nature, functional properties and evolution of grass genome organisation. Unexpectedly simple patterns of genome composition and arrangement have been seen, and these appear to be similar in different grasses. Our detailed studies of the maize genome indicate that short (2-20 kb) blocks of gene-containing DNA alternate with large (2-200 kb) blocks of intermixed middle and highly repetitive DNAs. Most of the highly repetitive sequences, and many of the middle repetitive DNAs, are retrotransposons that have inserted within each other. These repetitive DNAs are usually methylated and mostly inactive, but they are homologous to transcripts found in many different tissues. The unmethylated DNA is composed primarily of genes interspersed with lower-copy-number retroelements and inverted-repeat transposable elements. Gene order and sequence are highly conserved, but the mobile DNAs between genes appear to be different due to their rapid evolution and their variable presence or locations in different grasses.

Chromosome Mapping↗
PubMed2002

Homology-dependent gene silencing and host defense in plants.

Analyses of transgene silencing phenomena in plants and other organisms have revealed the existence of epigenetic silencing mechanisms that are based on recognition of nucleic acid sequence homology at either the DNA or RNA level. Common triggers of homology-dependent gene silencing include inverted DNA repeats and double-stranded RNA, a versatile silencing molecule that can induce both degradation of homologous RNA in the cytoplasm and methylation of homologous DNA sequences in the nucleus. Inverted repeats might be frequently associated with silencing because they can potentially interact in cis and in trans to trigger DNA methylation via homologous DNA pairing, or they can be transcribed to produce double-stranded RNA. Homology-dependent gene silencing mechanisms are ideally suited for countering natural parasitic sequences such as transposable elements and viruses, which are usually present in multiple copies and/or produce double-stranded RNA during replication. These silencing mechanisms can thus be regarded as host defense strategies to foreign or invasive nucleic acids. The high content of transposable elements and, in some cases, endogenous viruses in many plant genomes suggests that host defenses do not always prevail over invasive sequences. During evolution, slightly faulty genome defense responses probably allowed transposable elements and viral sequences to accumulate gradually in host chromosomes and to invade host genes. Possible beneficial consequences of this "foreign" DNA buildup include the establishment of genome defense-derived epigenetic control mechanisms for regulating host gene expression and acquired hereditary immunity to some viruses.

Animals↗
PubMed1994

Isolation of an insertion sequence (IS1051) from Xanthomonas campestris pv. dieffenbachiae with potential use for strain identification and characterization.

A new insertion sequence was isolated from Xanthomonas campestris pv. dieffenbachiae. Sequence analysis showed that this element is 1,158 bp long and has 15-bp inverted repeat ends containing two mismatches. Comparison of this sequence with sequences in data bases revealed significant homology with Escherichia coli IS5. IS1051, which detected multiple restriction fragment length polymorphisms, was used as a probe to characterize strains from the pathovar dieffenbachiae.

Base Sequence↗
PubMed1994

Tc1 transposon-like sequences are widely distributed in salmonids.

Transposon-like elements flanked by inverted repeats, although common in invertebrates, have only recently been found in vertebrates. We report the presence of Tc1 transposon-like sequences in salmon, trout and charr species and find that these elements belong to several families that do not follow phylogenetic lines. As many as 15,000 copies reside in the Atlantic salmon haploid genome. The complete DNA sequence of one of these transposon-like elements (SALT1) is 1535 base-pairs long, including 35 nucleotide-long terminal inverted repeats. It contains a degenerate open reading frame (ORF) of 1273 nucleotides whose inferred amino acid sequence shares sequence similarity with the "D,D35E" family of transposases, particularly those from Caenorhabditis sp. and Drosophila sp. Southern blot analysis indicated that Tc1 transposon-like sequences are present in other lower vertebrates, including several fish species and amphibians, but the copy number can vary significantly in different lineages.

Amino Acid Sequence↗
PubMed1997

Centromeres, CENP-B and Tigger too.

The highly conserved centromere-associated protein CENP-B is a common feature of mammalian centromeres. Binding sites for CENP-B, so-called 'CENP-B boxes', are present in the otherwise unrelated centromeric satellite DNAs of humans, Mus musculus, Mus caroli, ferrets, giant pandas, tree shrews and gerbils, suggesting a role for CENP-B in centromere function. However, CENP-B and its binding sites are not detected at the centromeres of mammalian Y chromosomes and few, if any, binding sites seem present on African green monkey chromosomes. There is extensive sequence similarity between CENP-B and transposase proteins encoded by the pogo superfamily of transposable elements, which includes the human Tigger elements. Intriguingly, Tigger 2 has an almost perfect match to the CENP-B-binding site within its terminal inverted repeat. Comparison of the amino acid sequence of CENP-B with related proteins raises the possibility that CENP-B might share the ability to cause single-stranded DNA breaks. Such nicks could promote recombination, as has been suggested for the Charcot-Marie-Tooth disease duplication where a recombination hotspot exists close to a mariner-like element. We suggest that by promoting nicks adjacent to CENP-B boxes, CENP-B might facilitate the evolution and maintenance of satellite sequence arrays, rather than have a direct role in centromere function.

Amino Acid Sequence↗
PubMed1991

A common ancestor DNA motif for invertebrate and vertebrate hormone response elements.

The ecdysone-responsive DNA sequence of the Drosophila hsp27 gene promoter contains four direct and inverted repeats reminiscent of those that compose the vertebrate palindromic estrogen response element (ERE) and the thyroid hormone/retinoic acid response element (TRE/RRE). Interestingly, a 3 bp substitution in the wild-type Hsp27 ecdysone response element (EcdRE) increases both its similarity with the vertebrate ERE and TRE/RRE and its capacity to confer ecdysone responsiveness to a heterologous promoter. Remarkably, increasing the spacing between the inverted repeats of this strong EcdRE by two nucleotides converts it into an ERE. Inversely, decreasing the spacing between the two inverted repeats of the vertebrate consensus palindromic ERE, from three to one nucleotide, converts it into a functional EcdRE. Thus, the only difference between an invertebrate EcdRE and a vertebrate palindromic ERE or TRE/RRE is in the spacing between the conserved inverted repeated motifs forming these palindromic HREs. The finding that the sequence motif 5'-GGTCA-3' present in the vertebrate ERE and TRE/RRE is also a functionally important characteristic of an invertebrate HRE, suggests that a common ancestor regulatory DNA sequence gave rise to all HREs known so far. We discuss the possibility that this progenitor motif is the GGTCA sequence.

Animals↗
PubMed1988

Plasminogen activator inhibitor 2. Isolation and characterization of the promoter region of the gene.

We have isolated the promoter region and part of the coding region of the human plasminogen activator inhibitor 2 gene. The first two exons (65 and 177 base pairs, respectively) are separated by a 3.6 kilo base pair intron. More than 2 kilo base pair of the promoter region was sequenced and analyzed. The promoter region is characterized by: a) a TATAAA box located 31 base pairs upstream of the transcription initiation site; b) a 123 base pair stretch (-598 to -720) that is homologous (greater than 90% identity) to the inverted sequence of -1268 to -1380; c) a 24 base pair inverted repeat at -276 to -299 and three direct repeats; and d) several sequences that are homologous (up to 1 mismatch) to the cAMP responsive element or to binding sites for the transcription factors AP1 and AP2.

Base Sequence↗
PubMed1981

An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA.

On purification of human adenovirus type 12 (Ad12) by equilibrium sedimentation in CsCl density gradients, two bands of particles, Ad12-3 and Ad12-3a, are observed. The particles from band Ad12-3a contain a recombinant of human host cell DNA and of Ad12 DNA. The human cell DNA sequences contain repetitive DNA recurring 200 to 500 times in cellular DNA. Ad12 DNA and the recombinant genomes exhibit the same or similar lengths. This finding suggests that a constant amount of DNA is packaged into complete Ad12 particles. On cleavage of KB cellular DNA with EcoRI, BamHI, HinfI, Msp I, Mbo I Pst I, or Bgl II, the (32)P-labeled cellular DNA from Ad12-3a particles hybridizes on Southern blots to distinct bands of KB DNA. There is also less-specific background hybridization that is not observed in the control. The cellular DNA from Ad12-3a particles is not methylated, whereas the same cellular sequences in KB cell DNA appear to be extensively methylated. On denaturation and renaturation, the recombinant DNA molecules are converted to molecules half as long as Ad12 DNA, as determined by gel electrophoresis and electron microscopy. The recombinant DNA molecules were terminally labeled by exonuclease III treatment and subsequent refilling of the depleted segments with [(32)P]dNTPs by using DNA polymerase I (Klenow fragment). When these molecules were cleaved with EcoRI, BamHI, Msp I, or Pst I, only one terminal DNA fragment was found to be labeled. The results of partial digestion experiments using Msp I, HinfI, or Mbo I are consistent with a model in which 700-1150 base pairs from the left terminus of Ad12 DNA are linked to host cell DNA containing repetitious sequences, and this structure is symmetrically duplicated as a large inverted repeat of the type ABCDD'C'B'A'. The Ad12 DNA sequences are flanking the entire molecule, which consists mainly of human KB cell DNA. The recombinants appear to be stable on serial passage of the virus preparation for many years, although variations in the sequence of the recombinants occur. These symmetric recombinant (SYREC) molecules suggest a way to use adenovirus DNA as a eukaryotic vector. Their occurrence provides further evidence for the generation of virus-host DNA recombinants and may help elucidate the role this interaction may have in adenovirus replication and oncogenesis.

Adenoviruses, Human↗
PubMed1986

Activation of rat and human c-raf(-1) by rearrangement.

Activated c-raf(-1) gene was found in three transformants obtained by transfecting DNAs from rat hepatocellular carcinoma, metastasis of human colon cancer in mesocolon and normal mucosa from a different colon cancer patient. Rat and human activated c-raf(-1) genes were cloned into cosmid vectors; restriction enzyme mapping revealed both activated c-raf(-1) genes to have rearrangement in the center of the normal form of the gene, and the upstream sequences were replaced by unrelated sequences. Using genomic DNA fragments located immediately downstream of the recombination points, the activations of all these c-raf(-1) were shown to have occurred during the transfection process. The recombination points in both the rat and human clones isolated were located in the intron between exons 7 and 8, and nucleotide sequencing around these recombination points showed there to be an inverted repeat which could be involved in inducing in vitro recombination. Nucleotide sequencing of rat and human c-raf(-1) cDNAs revealed the upstream sequences, recombined to the 3' half of c-raf(-1), to be expressed as fusion mRNAs; the production of fused proteins was predicted from a long open reading frame, which is in-frame with the kinase domain encoded from the 3' half of the c-raf(-1) gene. There is a cysteine clustering region in an N-terminal region of the c-raf(-1) product deduced from the nucleotide sequence, and this cysteine clustering region was found to be highly homologous to that present in an N-terminal region of protein kinase C, although, in the latter cysteine clusters are present in duplicate. From analogy with the activation mechanism of protein kinase C, the N-terminal region of serine/threonine kinase coded by the c-raf(-1) gene is suggested to be a regulatory part of the enzyme activity, and it proposed that the replacement or truncation of this regulatory part could be the mechanism whereby c-raf(-1) is activated.

Amino Acid Sequence↗
PubMed1996

[Sequencing of right terminal fragment of canine adenovirus type 1 vaccine strain DNA].

Current studies show that adenoviruses are more advantageous as expression vectors than other viruses. Canine adenovirus type 1 (CAV-1) vaccine strain, CLL (cannaught laboratory limited), as vectors will play more important role for the prophylaxis of zoonosis than human adenoviruses. Studies on molecular biology of CAV-1 or CLL are necessary for construction of vectors of CLL. In this paper, the right terminal of CLL DNA cleaved by Hind II was sequenced. Authors found that it contained a 238 bp inverted terminal repeats (ITR). There are three copies of 40 bp short repeat sequences in the ITR. According to the DNA structural model of human adenovirus type 2, the 40 bp short repeats are the sites that are combined with nuclear factor 1 (NF-1) that is an initiation factor of DNA replication of virus, so they are essential structure of CLL DNA.

Adenoviruses, Canine↗
PubMed2001

Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated gus transgenes in Arabidopsis.

The transgene locus KH15, which is highly susceptible to silencing in Arabidopsis thaliana, contains two inversely repeated beta-glucuronidase (gus) genes separated by a palindromic sequence and has a low GUS activity, was found to be heavily methylated in the gus coding sequence and in the center of the inverted repeat. The locus KHsb67, which is less prone to silencing, was found to be less densely methylated in the non-repetitive region that separates the inversely repeated gus genes. After the removal of one of the gus genes by Cre-mediated recombination, methylation in both loci decreased or was totally lost. Despite the presence of a 732-bp palindromic sequence in the deletion line derived from KH15, this sequence was not methylated. Whereas the KH15 locus triggers methylation of homologous gus genes when placed in trans to them, the deletion derivative did not, suggesting that the capacity for cross-talk was severely affected by disruption of the palindromic arrangement. This result suggests that the transcribed palindromic sequences are required to maintain the methylation of both symmetrically and non-symmetrically arranged cytosines.

Arabidopsis↗
PubMed1993

Promoter, spliced leader, and coding sequence for BICP4, the largest of the immediate-early proteins of bovine herpesvirus 1.

We report the complete nucleotide sequence of the bovine herpesvirus 1 (BHV-1) immediate-early gene encoding BICP4, the homolog of the ICP4 protein of herpes simplex virus. Combined with previous mapping studies, the sequence analysis revealed that the transcript for BICP4 consisted of a noncoding leader RNA (exon 1; 0.35 kb) separated by an intron (0.46 kb) from the main body (exon 2; 4.1 kb). The open reading frame for BICP4 (1343 amino acid residues) started 27 nt after the splice site and extended across exon 2 for most of its length, BICP4 contained two domains of high homology (regions 2 and 4), which had been recognized earlier to be most conserved in the ICP4 homologs of alpha-herpesviruses and to be functionally important. These domains were flanked by three regions of lower but still discernible homology. Unique features of BICP4 were two large clusters of glutamic acid residues near the end of region 3, and the displacement of a polyserine tract to region 5, which in all other ICP4 homologs residues near the end of region 1. Transient expression assays showed that BICP4 repressed its own promoter and activated other herpes-virus genes. The 8.1-kb sequence summarized here completes analysis of the inverted repeats of the BHV-1 genome; it includes a segment (2.5 kb) upstream of the BICP4 gene apparently devoid of coding sequences but containing numerous scattered transcription signals.

Alphaherpesvirinae↗
PubMed1999

A subtelomeric satellite DNA family isolated from the genome of the dioecious plant Silene latifolia.

In an ongoing effort to trace the evolution of the sex chromosomes of Silene latifolia, we have searched for the existence of repetitive sequences specific to these chromosomes in the genome of this species by direct isolation from low-melting agarose gels of satellite DNA bands generated by digestion with restriction enzymes. Five monomeric units belonging to a highly repetitive family isolated from Silene latifolia, the SacI family, have been cloned and characterized. The consensus sequence of the repetitive units is 313 bp in length (however, high variability exists for monomer length variants) and 52.9% in AT. Repeating units are tandemly arranged at the subtelomeric regions of the chromosomes in this species. The sequence does not possess direct or inverted sequences of significant length, but short direct repeats are scattered throughout the monomer sequence. Several short sequence motives resemble degenerate monomers of the telomere repeat sequence of plants (TTTAGGG), confirming a tight association between this subtelomeric satellite DNA and the telomere repeats. Our approach in this work confirms that SacI satellite DNA sequences are among the most abundant in the genome of S. latifolia and, on the other hand, that satellite DNA sequences specific of sex chromosomes are absent in this species. This agrees with a sex determination system less cytogenetically diverged from a bisexual state than the system present in other plant species, such as R. acetosa, or at least a lesser degree of differentiation between the sex chromosomes of S. latifolia and the autosomes.

Base Sequence↗
PubMed1989

New tandem repeat region in the non-transcribed spacer of human ribosomal RNA gene.

A new repetitive DNA region was identified in the non-transcribed spacer of human rDNA, namely a long (4.6 kb) sequence motif (Xbal element) was present in two copies. The repeating unit composed of two parts. One of them consisted of unique nucleotide sequences, interrupted by some simple sequences. The other, about 3.1 kb long one assembled only from highly repeated simple sequences. The unique sequence region contained two, inverted copies of the human AluI type repetitive DNA family. The authors suggest that the XbaI elements may flank the tandem arrays of human rRNA genes as terminal repeats and they might function both as the origin of rDNA replication and/or site of homologous recombination.

Base Sequence↗