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At least 19 recordsLinked to original sources

PubMed2026

Transcription Start Regions in PTU-intergenic regions drive cell cycle-dependent transcriptional activation events in Leishmania donovani.

Leishmania displays an unconventional mode of transcription, with long clusters of genes being transcribed polycistronically from Transcription Start Regions (TSRs), being processed into monocistronic units prior to translation. It has long been believed that transcription is constitutive: failure to identify consensus sequences across TSRs (except a GT-rich motif supporting transcription in Trypanosoma brucei) and absence of canonical eukaryotic transcription factors led to the conclusion that regulation is primarily post-transcriptional, with epigenetics playing a role in triggering transcription initiation. This study stems from our previous findings identifying a few genes to be activated in a cell cycle-dependent manner. Using nuclear run-on assays to analyze nascent transcripts of two chromosomes, chromosomes 2 and 14, we find that while most genes are constitutively transcribed, a subset of genes gets activated at specific cell cycle stages. Reporter assays reveal that this transcriptional activation is driven by the regions immediately upstream of the genes. Sequence analyses of these TSRs lying in polycistronic intergenic regions (PIRs) uncovered a 10-mer GT-rich motif, in synchrony with earlier findings in T. brucei identifying a GT-rich motif at bidirectional TSRs. We also identify a second 25-mer motif at these TSRs, and deletion analyses find this motif to be critical for regulating gene expression. The findings of this study reveal that transcriptional events in these unicellular parasites are more complex than believed thus far: not all transcriptional events are constitutive, polycistronic transcription is not the only mode of transcription, and cis-acting sequence elements regulate at least some transcriptional events in these parasites.IMPORTANCEEndemic to 90 countries, Leishmania parasites cause a spectrum of diseases called Leishmaniases. No vaccines for human use are available to date, and the drugs currently used to treat the disease are expensive, have toxic side effects, and have complex administration regimens, with emerging drug resistance compounding problems. Researchers continue to investigate Leishmania cellular processes, with the hope of uncovering new therapeutic target sites. Gene regulation in these parasites is unusual, being modulated by various mechanisms, including epigenetic modifications, gene dosage, and post-transcriptional processing. Transcription is typically polycistronic and constitutive, initiating from Transcription Start Regions (TSRs) lying upstream of the first gene in the polycistronic transcription unit (PTU). The work presented here reveals that a subset of genes is transcribed monocistronically in a cell cycle-dependent manner from Transcription Start Regions lying in the PTU-intergenic regions (PIRs), underscoring the complexities of gene regulation in these parasites.

Leishmania donovani↗

PubMed1995

Evolution of the noncoding regions in Drosophila mitochondrial DNA: two intergenic regions.

The sequences of the mitochondrial DNA (mtDNA) segment containing the two intergenic regions were determined for six species belonging to the Drosophila immigrans species group and compared to the corresponding segments of Drosophila species which had been studied previously. We found remarkable differences in the evolutionary rates of the two intergenic regions. The Intergenic I region, which lies between the tRNA(gln) and the tRNA(ile) genes, was found to be highly conserved in terms of both size (30 ntp) and nucleotide sequence among the species studied. In contrast, the sequences of the Intergenic II region, which lies between the tRNA(f-met) and the tRNA(ile) genes, showed considerable variation. The size of the Intergenic II region ranged from 0 to 88 ntp, and accurate alignment was possible only among sequences from geographical strains or very closely related species in the nasuta species subgroup. The observed differences in conservation of the two mtDNA intergenic regions are discussed in light of functional constraints on mtDNA sequences.

Animals↗

PubMed2002

Classification of common conserved sequences in mammalian intergenic regions.

Comparisons between orthologous intergenic regions of related genomes reveal numerous hits, i.e. pairs of relatively short highly similar sequences that evolved slowly, perhaps due to selective constraint. We analyzed and classified 2638 hits found within 100 pairs of complete, orthologous intergenic regions of human and murine genomes. We identified all common fragments of hits that align well with many other hits and constructed their classification. Our analysis revealed 20 abundant classes each containing 10 or more fragments. Fragments of the same class may perform the same function, e.g. bind a particular protein. Ten of the abundant classes apparently correspond to known functional consensuses, whereas others may represent novel conserved sites. Thus, large-scale comparative analysis of slowly evolving intergenic sequences can provide valuable insights into their function.

Animals↗

PubMed1992

Evolution of the primate beta-globin gene region: nucleotide sequence of the delta-beta-globin intergenic region of gorilla and phylogenetic relationships between African apes and man.

A 6.0-kb DNA fragment from Gorilla gorilla including the 5' part of the beta-globin gene and about 4.5 kb of its upstream flanking region was cloned and sequenced. The sequence was compared to the human, chimpanzee, and macaque delta-beta intergenic region. This analysis reveals four tandemly repeated sequences (RS), at the same location in the four species, showing a variable number of repeats generating both intraspecific (polymorphism) and interspecific variability. These tandem arrays delimit five regions of unique sequence called IG for intergenic. The divergence for these IG sequences is 1.85 +/- 0.22% between human and gorilla, which is not significantly different from the value estimated in the same region between chimpanzee and human (1.62 +/- 0.21%). The CpG and TpA dinucleotides are avoided. CpGs evolve faster than other sequence sites but do not confuse phylogenetic inferences by producing parallel mutations in different lineages. About 75% of CpG doublets have become TpG or CpA since the common ancestor, in agreement with the methylation/deamination pattern. Comparison of this intergenic region gives information on branching order within Hominoidea. Parsimony and distance-based methods when applied to the delta-beta intergenic region provide evidence (although not statistically significant) that human and chimpanzee are more closely related to each other than to gorilla. CpG sites are indeed rich in information by carrying substitutions along the short internal branch. Combining these results with those on the psi eta-delta intergenic region, shows in a statistically significant way that chimpanzee is the closest relative of human.

Animals↗

PubMed2006

Mutational analysis of the varicella-zoster virus ORF62/63 intergenic region.

The varicella-zoster virus (VZV) ORF62/63 intergenic region was cloned between the Renilla and firefly luciferase genes, which acted as reporters of ORF62 and ORF63 transcription, and recombinant viruses were generated that carried these reporter cassettes along with the intact native sequences in the repeat regions of the VZV genome. In order to investigate the potential contributions of cellular transregulatory proteins to ORF62 and ORF63 transcription, recombinant reporter viruses with mutations of consensus binding sites for six proteins within the intergenic region were also created. The reporter viruses were used to evaluate ORF62 and ORF63 transcription during VZV replication in cultured fibroblasts and in skin xenografts in SCIDhu mice in vivo. Mutations in putative binding sites for heat shock factor 1 (HSF-1), nuclear factor 1 (NF-1), and one of two cyclic AMP-responsive elements (CRE) reduced ORF62 reporter transcription in fibroblasts, while mutations in binding sites for HSF-1, NF-1, and octamer binding proteins (Oct-1) increased ORF62 reporter transcription in skin. Mutations in one CRE and the NF-1 site altered ORF63 transcription in fibroblasts, while mutation of the Oct-1 binding site increased ORF63 reporter transcription in skin. The effect of each of these mutations implies that the intact binding site sequence regulates native ORF62 and ORF63 transcription. Mutation of the only NF-kappaB/Rel binding site had no effect on ORF62 or ORF63 transcription in vitro or in vivo. The segment of the ORF62/63 intergenic region proximal to ORF63 was most important for ORF63 transcription, but mutagenesis also altered ORF62 transcription, indicating that this region functions as a bidirectional promoter. This first analysis of the ORF62/63 intergenic region in the context of VZV replication indicates that it is a dual promoter and that cellular transregulatory factors affect the transcription of these key VZV regulatory genes.

Animals↗

PubMed2006

Contrasting patterns of sequence divergence and base composition between Drosophila introns and intergenic regions.

Two non-coding DNA classes, introns and intergenic regions, of Drosophila melanogaster exhibit contrasting evolutionary patterns. GC content is significantly higher in intergenic regions and affects their degree of nucleotide variability. Divergence is positively correlated with recombination rate in intergenic regions, but not in introns. We argue that these differences are due to different selective constraints rather than mutational or recombinational mechanisms.

Animals↗

PubMed1988

Evolution of avian coronavirus IBV: sequence of the matrix glycoprotein gene and intergenic region of several serotypes.

We have sequenced 200 to 240 bases of the matrix (M) glycoprotein gene of 23 strains of infectious bronchitis virus (IBV) representing the A (D207), B (D3896), C (D3128), D (D212), Massachusetts (Mass), UK11 and UK12 serotypes. The bases examined code for the external, hydrophilic region and the first membrane-embedded hydrophobic region of M, both regions comprising approximately 20 amino acids. As predicted from protein Mr studies the A/D and B/C serotypes had two and one potential glycosylation sites respectively. This variation appeared to derive from a combination of base substitutions and deletions/insertions. The glycosylation sequence Asn-Cys-Thr was highly conserved. Overall, the exposed part of M exhibited a fourfold greater extent of amino acid variation than did the membrane-embedded sequence. The transcription-associated homology region sequence (CUUAACAA) in the 5' intergenic region was identical in all strains but there was considerable variation as to its location. The M gene of UK12 appeared to have evolved from a group A-like M gene by a two stage process involving a base substitution in the intergenic region which generated a new AUG translation start codon followed by deletion of the original AUG. Isolate UK11 closely resembled Mass strains in the intergenic region but was dissimilar from all strains in the protein coding region. The M sequences of serotypes B and C were identical and those of the A and D serotypes very similar. These results are discussed in relation to recent sequencing of part of the spike glycoprotein gene of some of these strains and the discovery of in vitro recombination of murine hepatitis coronavirus.

Amino Acid Sequence↗

PubMed1996

The structurally diverse intergenic regions of respiratory syncytial virus do not modulate sequential transcription by a dicistronic minigenome.

The first nine genes of respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus, are separated by intergenic regions which range in size from 1 to 52 nucleotides for strain A2 and lack obvious consensus elements except that each ends in an A (genome sense). Their significance for gene expression was investigated by using RSV-CAT-LUC RNA, a helper-dependent cDNA-encoded dicistronic analog of RSV genomic RNA in which the viral genes were replaced by a negative-sense copy of the translational open reading frame (ORF) encoding chloramphenicol acetyltransferase (CAT) as the upstream, leader-proximal gene and that encoding luciferase (LUC) as the downstream gene. These foreign ORFs were flanked by the RSV gene-start (GS) and gene-end (GE) transcription signals and separated by the naturally occurring G/F intergenic region. The RSV-CAT-LUC minigenome was synthesized in vitro and transfected into RSV-infected cells, and synthesis of the CAT and LUC mRNAs was monitored by enzyme assay and Northern (RNA) blot hybridization. Surprisingly, substitution of each of the other naturally occurring RSV intergenic regions in turn did not significantly alter the absolute or relative amounts of the two mRNAs. Substitution of a nonnatural 10-nucleotide intergenic region, or elimination of the intergenic region altogether, also had little effect on the level of expression of the two genes. Four of the minigenome variants containing naturally occurring intergenic regions were modified further by replacing part of the LUC ORF with a second copy of the CAT ORF, so that each of the two mRNAs would hybridize equally with a CAT-specific probe and their relative molar amounts could be determined. The level of expression of the downstream gene was 0.30 to 0.36 that of the upstream one. This determined the magnitude of RSV transcriptional polarity across a gene pair and confirmed that this value was very similar among the various intergenic regions. Minigenome transcription also yielded a CAT-LUC readthrough mRNA at a level 0.10 to 0.13 that of the LUC mRNA. In summary, the structurally diverse RSV intergenic regions do not appear to play a role in modulating RSV gene expression.

Base Sequence↗

PubMed2004

Variations in intergenic region sequences of Human respiratory syncytial virus clinical isolates: analysis of effects on transcriptional regulation.

Sequences at the beginnings and ends of Human respiratory syncytial virus (HRSV) genes are necessary for efficient initiation and termination of transcription. The gene start sequences are well conserved and contain signals required for initiation, while the semi-conserved sequences at the gene ends direct transcriptional termination with varying efficiencies. The intergenic regions, which lie between the gene ends and the downstream gene start sequences, are not conserved in length or sequence, and certain positions have been reported to play a role in transcriptional regulation. We have previously shown that the gene end sequences in HRSV subgroup A clinical isolates are variable and that variations found at certain gene ends decreased transcriptional termination and downstream mRNA expression. Here, we have extended this work to examine variation in the intergenic regions between the genes of clinical isolates. We determined the sequences of the eight intergenic regions and the M2/L overlap from clinical isolates from the US and UK and found that all of these regions contained variations from the prototype A2 strain. The amount of variation observed was disparate among the different intergenic regions and did not correlate with length. The effects of selected variant sequences on transcription were examined in the context of subgenomic replicons. While some changes in the intergenic regions had minor effects, certain sequence variations significantly altered transcription termination or initiation. A single nucleotide deletion in the M/SH intergenic region decreased initiation at the SH gene start seven-fold, while changes in the F/M2 intergenic region were found that in some cases increased and in others decreased termination at the F gene end. The P/M intergenic region was the most variable, but none of the changes examined affected either termination at the P gene end or initiation of the downstream M gene start. These results show that in HRSV clinical isolates the intergenic region sequences are variable and that changes in these regions have the potential to affect transcriptional control at the gene junctions.

Base Sequence↗

PubMed1995

Effects of 3' untranslated and intergenic regions on gene expression in Trypanosoma cruzi.

The effects of 3' untranslated regions (UTR) and intergenic regions (INT), from various Trypanosoma cruzi stage-specific and constitutive genes, on the expression of the reporter firefly luciferase gene (luc), were studied using stable episomal transformation. The 3' UTR influenced luciferase expression by changing the steady-state level and/or the translation efficiency of luc mRNA. Glycoprotein 72 gene (gp72), glycoprotein 85 gene (gp85) or amastin gene (ama) 3' UTR decreased the luc mRNA level 6- to 14-fold, compared to the glyceraldehyde 1-phosphate dehydrogenase gene (gapdh) 3' UTR, in epimastigotes. Luciferase activity decreased in parallel with the luc mRNA level in transformants utilizing the gp85 or ama 3' UTR, whereas luc mRNA containing the gp72 3' UTR showed approximately 5-fold higher translation efficiency than luc mRNA containing a minimal 3' UTR. In amastigotes, the inhibitory effect of the ama 3' UTR observed in other life cycle stages was abolished and luciferase expression was stimulated 16-fold. The overall stage-specific difference mediated by the ama 3' UTR, between epimastigotes and amastigotes, was approximately 100-fold. INT, which was expected to influence polyadenylation efficiency, of gapdh, gp72, or heat shock protein 60 gene inserted after gapdh 3' UTR increased luc mRNA 2- to 8-fold, whereas gp85 INT slightly decreased luc mRNA. By separating effects attributable to the 3' UTR and INT, this study shows the effects of 3' UTR on RNA levels and translational efficiency in T. cruzi.

Animals↗

PubMed1991

rDNA intergenic region from Arabidopsis thaliana. Structural analysis, intraspecific variation and functional implications.

The ribosomal gene intergenic region from Arabidopsis thaliana contains four clusters of mutually unrelated repeated sequences. By comparison with the respective regions in two other Brassicaceae, Raphanus and Sinapis, the putative promoter sequence for RNA polymerase I was located. The homologies suggest that the RNA polymerase I promoter in Brassicaceae ranges further upstream than in animals. Upstream duplications of at least a part of the promoter region were found to be located between individual blocks of the largest internal repeat family ("A" repeats), which is made up of multiple repeats of two closely related sequences 21 or 20 bp in length. Overall structural similarities of the A. thaliana rDNA intergenic region with those from wheat and from Xenopus laevis are discussed. We also present data on the range of intraspecific length heterogeneities found in the central EcoRI fragment of the intergenic region and on the frequencies with which specific length variants occur in the genome. To determine the nature of the length heterogeneities, we sequenced the central EcoRI fragments from four independently isolated genomic clones. Three levels of rearrangements were detected. Length variation can be caused by duplication of a whole A repeat block, or, most frequently, by insertion and/or deletion of one or a few A repeat units. Surprisingly, single base mutations are extremely rare, which hints at some mechanism of homogenization which might be acting on the intergenic region. A possible function of the described sequences in transcriptional regulation is discussed and will be the aim of further investigations.

Animals↗

PubMed2004

Growth and virulence alterations of equine herpesvirus 1 by insertion of a green fluorescent protein gene in the intergenic region between ORFs 62 and 63.

Nucleotide sequences of the intergenic region between ORF 62 and ORF 63 of equine herpesvirus 1 (EHV-1) isolates were analyzed. The sequences of this region consisted of variable and conserved domains among EHV-1 isolates. An EHV-1 mutant, Ab4-GFP, was constructed by inserting a green fluorescent protein (GFP) expression cassette flanked by lox P at both ends into the intergenic region between ORF 62 and ORF 63. Another mutant, Ab4-loxP, which contains one lox P site, was constructed by excision of the GFP cassette from the Ab4-GFP virus genome by cre enzyme. The recombinant Ab4-GFP formed smaller plaques than the wild type in MDBK cells. Virus production also decreased for Ab4-GFP in multistep growth analyses. Virulence of Ab4-GFP in both mice and hamsters was weaker than that of the wild type. Ab4-loxP exhibited properties similar to those of the wild type. These results suggest that the intergenic region between ORF 62 and ORF 63 plays various roles in the virus growth.

Animals↗

PubMed2002

Analysis of similarity within 142 pairs of orthologous intergenic regions of Caenorhabditis elegans and Caenorhabditis briggsae.

Patterns of similarity between genomes of related species reflect the distribution of selective constraint within DNA. We analyzed alignments of 142 orthologous intergenic regions of Caenorhabditis elegans and Caenorhabditis briggsae and found a mosaic pattern with regions of high similarity (phylogenetic footprints) interspersed with non-alignable sequences. Footprints cover approximately 20% of intergenic regions, often occur in clumps and are rare within 5' UTRs but common within 3' UTRs. The footprints have a higher ratio of transitions to transversions than expected at random and a higher GC content than the rest of the intergenic region. The number of footprints and the GC content of footprints within an intergenic region are higher when genes are oriented so that their 5' ends form the boundaries of the intergenic region. Overall, the patterns and characteristics identified here, along with other comparative and experimental studies, suggest that many footprints have a regulatory function, although other types of function are also possible. These conclusions may be quite general across eukaryotes, and the characteristics of conserved regulatory elements determined from genomic comparisons can be useful in prediction of regulation sites within individual DNA sequences.

Animals↗

PubMed1988

The role of insertions/deletions in the evolution of the intergenic region between psbA and trnH in the chloroplast genome.

TrnH and the intergenic region between trnH and psbA of the chloroplast genomes of alfalfa (Medicago sativa), Fabaceae, and petunia (Petunia hybrida), Solanaceae, were sequenced and compared to published sequences of that region from other members of those families. A striking feature of these comparisons is the occurrence of insertions/deletions between short, nearly perfect AT-rich direct repeats. The directionality of these mutations in the petunia, tobacco and Nicotiana debneyi lineages within the Solanaceae cannot be discerned. However, we present several alternative hypotheses that are consistent with Goodspeed's 1954 evolutionary treatment of the genus Nicotiana and family Solanaceae. Within the Fabaceae, the major size differences in the intergenic region between alfalfa, pea and soybean are due to insertions/deletions between direct repeats. The alfalfa intergenic region has an inverted repeat stem-loop structure of 210 bases directly 5' to trnH. This structure is an insert relative to the liver-wort. Marchantia polymorpha. Portions of the insert are found also in pea and soybean as well as in published sequences from other dicots representing diverse orders: petunia, tobacco, N. debneyi (Scrophulariales), spinach (Caryophyllales), and Brassica napus (Capparales). Some of the regions of the insert that are missing in these plants appear to have resulted from deletions of sequences between different imperfect direct repeats within, or 5' to and within the insert. Other deletions are not flanked by repeated sequences. A short insert flanked by imperfect direct repeats in B. napus occurs just within the longer alfalfa insert suggesting that both alfalfa and B. napus have remnants of an even longer insert relative to M. polymorpha. From these analyses we hypothesize the insertion of a stem-loop structure into an M. polymorpha-like ancestral land plant, followed by deletions of sequences, often between different imperfect direct repeats within and upstream of the insert, leading to the psbA-trnH intergenic sequences represented by the present-day plants examined.

Base Sequence↗

PubMed2003

Genomic variability among enteric pathogens: the case of the mutS-rpoS intergenic region.

The mutS-rpoS intergenic region of enteric bacteria ranges in size from 88 bp in Yersinia to > 12000 bp in Salmonella. We interpret this expansion as the result of the horizontal transfer of segments of DNA from diverse origins. Both comparative genomic analysis and selective sequencing of a variety of Escherichia coli pathogens have provided additional evidence for reassortment of segments within this region.

Adenosine Triphosphatases↗

PubMed2004

Compact intergenic regions of the pufferfish genome facilitate isolation of gene promoters: characterization of Fugu 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (fPapss2) gene promoter function in transgenic Xenopus.

The highly compact nature of the pufferfish (Fugu rubripes) genome renders it a useful tool not only for annotating coding regions within vertebrate genomes, but also for the identification of sequences important to gene regulation. Indeed, owing to this compaction it will be feasible in many instances to initiate analyses using entire intergenic regions when mapping gene promoters; a strategy that is very rarely feasible with the expanded genomes of other species. Stemming from our interest in studying promoters expressed in chondrocytes, we selected for study the intergenic region upstream of Fugu 3'-phosphoadenosine 5'-phosphosulfate synthase 2, fPapss2, a gene required for the normal development of cartilage extracellular matrix. Functional characterization of the entire fPapss2 5' intergenic region was carried out by monitoring expression of the enhanced green fluorescent protein (EGFP) gene reporter in the developing cartilage of transgenic Xenopus laevis. By evaluating a series of 5' intergenic region deletions we defined a minimal fPapss2 sequence of approximately 300 bp that was essential for EGFP expression in tadpole cartilage. This functional analysis of an entire Fugu intergenic region, combined with the efficiency of Xenopus transgenesis, serves as a model for the rapid characterization of evolutionarily-conserved regulatory regions of other pufferfish genes.

Animals↗

PubMed2001

Selective constraint in intergenic regions of human and mouse genomes.

We aligned and analyzed 100 pairs of complete, orthologous intergenic regions from the human and mouse genomes (average length approximately 12 000 nucleotides). The alignments alternate between highly similar segments and dissimilar segments, indicating a wide variation of selective constraint. The average number of selectively constrained nucleotides within a mammalian intergenic region is at least 2000. This is threefold higher than within a nematode intergenic region and at least twofold higher than the number of selectively constrained nucleotides coding for an average protein. Because mammals possess only two- to threefold more proteins than Caenorhabditis elegans, the higher complexity of mammals might be primarily because of the functioning of intergenic DNA.

Animals↗

PubMed1990

Detection of length heterogeneity in the ribosomal DNA of Pythium ultimum by PCR amplification of the intergenic region.

About one-half of the ribosomal repeat unit of two isolates of Pythium ultimum was amplified by means of the polymerase chain reaction using one primer pair. The amplified region includes a small part of the large subunit ribosomal RNA gene, about half of the small subunit ribosomal RNA gene, and the entire intergenic region. The intergenic region of both isolates of P. ultimum has length heterogeneity due to the presence of subrepeat arrays (Klassen and Buchko 1990). PCR amplification of the heterogeneous target DNA resulted in sets of fragments which accurately reflect the heterogeneity in the target DNA, although there is a preferential amplification of the smaller targets. PCR product sizes ranged from 4.6 to 5.8 kb.

Base Sequence↗