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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↗
PubMed1979

Total synthesis of a tyrosine suppressor transfer RNA gene. XVII. Transcription, in vitro, of the synthetic gene and processing of the primary transcript to transfer RNA.

Primer- and promoter-dependent transcription of the synthesis gene had been studied. Primer-dependent transcription gave, as a major product, an end-to-end transcript which was strand-specific. The transcript was characterized rigorously by two-dimensional separation and analysis of the oligonucleotides formed on digestion with T1-RNase and pancreatic RNase and by nearest neighbor analyses of the oligonucleotides obtained when different alpha-32P-labeled ribonucleoside triphosphates were used as substrates. Minor products accompanying the major transcript were characterized similarly. The major transcript, when treated with an Escherichia coli S-100 extract, was processed to the tRNATyr with correct 5'- and 3'-ends. The nucleolytic cleavages occurring at the 3'-end were characterized. In promoter-dependent transcription, transcription of a restriction fragment containing phi80psu+III gene and the synthetic gene with and without the promoter were compared. Transcription of the synthetic gene was promoter-dependent and strand-specific, the initiation of transcription occurring at the same point as previously found in vivo. Although the synthetic gene contains only 16 base pairs corresponding to the natural sequence following the C-C-A end, processing of the transcript at the 3'-end occurred normally, the endonucleolytic cleavage being followed by exonucleolytic cleavages. The products of promoter-dependent transcription were completely characterized. An examination of the base modifications of the primary transcript during treatment of the latter with E. coli S-100 extract showed couplete modification of uridine to pseudouridine and partial methylation of uridine to ribosylthymine in TpsiCG sequence and partial formation of pseudouridine in the anticodon loop. However, hardly any formation of 2'-O-methylguanosine or of 2-methylthio-6-isopentenyl adenosine could be detected.

Base Sequence↗
PubMed2026

Alternative transcription of the mouse Gh gene identifies an immune-associated transcript with species-specific structural divergence.

Growth hormone (GH) in mice is primarily expressed in the anterior pituitary, although Gh expression has been reported in extrapituitary tissues, including immune organs. However, the structure of immune-associated Gh transcripts remains poorly characterized. To determine whether splenic Gh transcripts differ from pituitary Gh mRNA, 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses were performed. While 3' RACE showed a shared polyadenylation site, 5' RACE identified a novel exon located approximately 2 kb upstream of the conventional exon 1, generating a transcript (spl-Gh mRNA) with a distinct first exon but shared downstream exons with pituitary Gh mRNA (pit-Gh mRNA). RT-PCR analysis revealed that spl-Gh mRNA is predominantly expressed in immune tissues such as spleen and bone marrow, and its distribution did not correlate with Pit-1 mRNA expression. Quantitative RT-PCR further demonstrated that spl-Gh mRNA was expressed at levels comparable to those of pit-Gh mRNA in the mouse spleen, indicating that spl-Gh is one of the major Gh transcript forms in this tissue. Sequence analysis indicated that spl-Gh mRNA is predicted to retain coding potential for a GH protein. Comparative genomic analyses further demonstrated that genomic features associated with the spl-Gh transcriptional unit are conserved only in a subset of closely related Mus species. In contrast, although a spl-Gh-related transcript was detected in rat spleen, no properly spliced mouse-like transcript was identified under the present experimental conditions. The detected transcript exhibited intron retention and an in-frame stop codon, suggesting that it is unlikely to produce a functional GH protein. These findings identify a distinct immune-associated Gh transcript generated through alternative transcription of the mouse Gh gene and suggest that immune-associated Gh transcriptional mechanisms have undergone species-specific divergence among rodents. Together, these findings reveal previously unrecognized complexity in Gh gene regulation and highlight species-specific differences in immune-associated Gh transcripts.

Animals↗
PubMed1979

Control of bacteriophage lambda repressor establishment transcription: kinetics of l-strand transcription from the y-cII-oop-O-P region.

The kinetics of lambda l-strand repressor establishment RNA synthesis were measured from the y-cII region of induced tof- prophage. The activity of the repressor is epistatic to the expression of gene tof coding for the antirepressor (Tof). The activity of Tof, is epistatic to the expression of repressor gene cI transcription from Prm and the expression of repressor establishment transcription from a site 600 to 800 nucleotides upstream from Prm. Three modes of l-strand rex-cI-tof-y-cII-oop transcription occur: (a) Prm promoted cI-rex mRNA synthesis from noninduced prophage, (b) coordinate lit and oop synthesis from induced tof+ prophage and (c) establishment transcription from induced tof- prophage. The synthesis or stability of oop RNA is much reduced from induced tof-, compared with tof+ prophage. The oop transcription from tof- prophage is not coordinate with RNA synthesis from the y-cII interval. The y-cII-(oop) portion of the establishment transcript appears more unstable than the translated downstream copy of genes rex-cI. The initiation of any repressor establishment transcription requires the products of lambda genes cIII, cII, P and Escherichia coli genes dnaB, dnaG, but not actual lambda DNA synthesis. This result demonstrates that common factors, i.e. replication gene products, are required for the initiation of establishment transcription, lambda replication and lit, oop RNA synthesis; and explains why cIII+ cI+ cII+ replication defective phage lysogenize poorly at low multiplicities of infection. The cIII and cII products were shown to act after an earlier replication initiation or activation event. Repressor establishment transcription and repressor mRNA synthesis from Prm (from induced cI- tof-, cIII- cI- tof- or cI- tof- cii- prophage) are amplified by gene dosage. The extent of lysogenization of E. coli by lambda cIII-, cII- or replication minus mutants, defective for initiation of establishment synthesis, is attributed to gene dosage dependent transcription from Prm. The mechanism by which Tof inhibits the initiation of establishment transcription does not appear to require repression of RNA synthesis from PL and PR. RNA synthesis from these promoters is blocked by renaturation of the repressor 5 min after induction, before establishment transcription is detected; however, establishment RNA synthesis measured between 12-13 min after induction, i.e. 7 min after renaturation of the repressor, is only partially reduced.

Coliphages↗
PubMed1979

Expression of the autonomous parvovirus H1 genome: evidence for a single transcriptional unit and multiple spliced polyadenylated transcripts.

We identified viral transcripts in parvovirus H1-infected rodent cells using the S1 nuclease mapping technique of Berk and Sharp (1977, 1978). The most abundant viral transcript, present in both nucleus and cytoplasm, is approximately 2.8 kb long and represents about 56% of the viral genome. Less abundant viral transcripts of 3.0, 1.45 and 1.30 kb, and possibly other minor viral transcripts, are also detected in nuclear and cytoplasmic fractions. In contrast, a prominent 4.7 kb viral transcript which corresponds to 95% of the viral DNA is found only in the nucleus; this finding suggests that the parvovirus genome may function as a single transcription unit. Virus-infected cells pretreated with cycloheximide accumulate all these viral transcripts. Analyses of RNA-DNA hybrids (isolated from neutral agarose gels) by electrophoresis on alkaline agarose gels indicate that the 4.7, 3.0 and 2.8 kb viral transcripts are "spliced" RNAs. The nuclear-specific 4.7 kb transcript appears to be encoded by two noncontiguous DNA segments of 2.2 and 2.6 kb. The 3.0 and 2.8 kb transcripts are apparently encoded by.a 2.6 kb segment of DNA and one or more much smaller noncontiguous DNA segments, one of which is approximately 170 nucleotides long.

Animals↗
PubMed1979

Transcription maps of mtDNAs of two strains of saccharomyces: transcription of strain-specific insertions; Complex RNA maturation and splicing.

We have developed a two-dimensional method for simultaneously mapping on the yeast mtDNA genome all the transcripts representing more than 0.01% of mtRNA. In two yeast strains, Saccharomyces carlsbergensis NCYC-74 and Saccharomyces cerevisiae KL14-4A, about 25 discrete transcripts were found apart from tRNAs. The mtDNAs of these strains differ by the absence (NCYC-74) or presence (KL 14-4A) of various large insertions located within genetically active regions. The transcripts can all be related to known loci on the genetic map. In nearly all cases the RNAs are much longer than required to specify the known protein product of the locus concerned. The organization of the transcripts is similar in the two strains except at the positions of the large insertions (500-3300 bp) in the oxi-3 and cob loci. The sequences of these insertions are present in RNA species larger than 25S, but are absent from smaller transcripts of the same regions. This is probably due to splicing, since the coding sequences for most of these smaller transcripts are noncontiguous. The smaller transcripts of other loci also seem to arise from processing of larger RNA species. The oxi-3 locus, containing the structural gene for cytochrome c oxidase subunit l, is transcribed in a very complex fashion that suggests differential splicing into partially overlapping transcripts. This may indicate that oxi-3 has additional genetic functions, including possible control of the biosynthesis of cytochrome c oxidase holoenzyme or its assembly into the mitochondrial inner membrane. As in the case of the eucaryote nucleus, the regulation of mitochondrial gene expression seems to occur more at the level of RNA processing than has been recognized thus far.

Chromosome Mapping↗
PubMed1977

Transcription of yeast DNA by homologous RNA polymerases I and II: selective transcription of ribosomal genes by RNA polymerase I.

Purified yeast DNA was transcribed by homologous RNA polymerases I and II and Escherichia coli RNA polymerase. Transcripts synthesized in vitro were analyzed by molecular hybridization with complementary DNA (cDNA) synthesized from yeast poly(A)-containing mRNA with viral reverse transcriptase and ribosomal DNA labeled in vitro by nick translation with E. coli DNA polymerase I. RNA synthesized by polymerase I and II in the presence of Mn2+ contained sequences complementary to cDNA and rDNA at a frequency consistent with random transcription of the template. Similarly, E. coli RNA polymerase synthesized an apparently random transcript in the presence of either Mn2+ or Mg2+. In contrast to these results, RNA polymerase I but not polymerase II transcripts were markedly enriched in sequences complementary to rDNA when transcription was carried out in the presence of Mg2+. The observed enrichment was 15-30-fold higher than observed for polymerase II or E. coli polymerase transcripts and is consistent with the transcript being comprised of 6-10% ribosomal sequences. These data strongly suggest that RNA polymerase I plays a critical role in selective transcription of ribosomal cistrons.

DNA-Directed RNA Polymerases↗
PubMed1979

Electron microscopic analysis of transcription: mapping of initiation sites and direction of transcription.

An electron microscope technique is described that allows rapid characterization of transcription in vitro. DNA is transcribed with Escherichia coli RNA polymerase in vitro, and the RNA is hybridized to its template. Measurement of the resulting transcription R-loop molecules allows accurate mapping of transcription initiation sites (promoter sites) and analysis of the direction and rate of transcription and the level of transcription from each initiation site. The two major early promoters pR and pL of bacteriophage lambda have been mapped within 0.1-0.3 map units of the known positions and three additional sites have been confirmed. Six transcription initiation sites have been preliminarily mapped on plasmid pSF2124 DNA.

Bacteriophage lambda↗
PubMed1979

Macromolecular synthesis in cells infected by frog virus 3. XII. Viral regulatory proteins in transcriptional and post-transcriptional controls.

Using fluorophenylalanine (FPA) to interfere with functional viral protein synthesis, we have investigated the complex transcriptional and post-transcriptional controls that operate in cells infected with frog virus 3. Our previous data, obtained by polyacrylamide gel electrophoresis of viral RNAs and proteins, showed that the addition of FPA at the beginning of infection completely prevented the synthesis of late viral RNAs and late viral proteins and blocked the normal progressive decline in the rates of synthesis of two quantitatively different classes (class I and class II) of early proteins. These results indicated that the initiation of late RNA and late protein syntheses, as well as the post-transcriptional regulation of early protein synthesis, was under the control of virus-specific proteins (D. B. Willis, R. Goorha, M. Miles, and A. Granoff, J. Virol. 24:326-342, 1977). In this communication, we show that the viral protein required to "turn on" the synthesis of late RNAs and late (class III) proteins was made within 1 to 1.5 h postinfection (p.i.); when we added FPA after this time, we observed the synthesis of all of the late macromolecules. The data also suggest that another viral protein, separate from the "turn-on" protein, controlled the abundance of late RNAs. In addition, at least two separate proteins were involved in the post-transcriptional regulation of two classes of early proteins. When FPA addition was delayed until 2 h p.i., the rate of synthesis of class I proteins (which normally peaked at 2 h p.i.) was reduced by 6 h p.i. just as in a normal infection, but the rate of synthesis of class II proteins (which normally reached a maximum at 4 h p.i. before declining) was reduced only when we waited until 3 or 4 h p.i. to add FPA. These experiments corroborate and extend previous evidence for the existence of numerous viral regulatory proteins in the control of frog virus 3 gene expression at the transcriptional and post-transcriptional levels.

Animals↗
PubMed2026

Acidic transcription factors position the genome at nuclear speckles through transcription-dependent and -independent mechanisms.

A small fraction of the genome reproducibly positions near nuclear speckles (NSs), increasing the expression and/or splicing efficiency of NS-associated genes. How specific genomic regions in mammalian cells are targeted to NSs remains unclear. Here, we demonstrate the establishment of genome-wide NS association without active transcription. We show that DNA sequences derived from NS-associated regions, when integrated as transgenes, are autonomously targeted to NSs. By systematically dissecting one such genomic locus, the COL1A1-SGCA locus, we identified redundant NS-targeting cis-regulatory elements, including an ∼600-bp fragment with 17 binding motifs for 8 transcription factors (TFs). Four NS-targeting TFs within this fragment contain acidic activation domains (AADs) that provide both chromatin-context and transcription-dependent NS targeting, properties that appear to be common among several other tested AADs. A subset of acidic activator TFs contains an additional, transcription-independent NS-targeting activity. Our findings establish diverse and partially redundant NS-targeting activities, which may facilitate dynamic gene positioning at the NS periphery for context-specific transcriptional responses.

Transcription, Genetic↗
PubMed1979

Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression.

The mechanism by which prolactin, a peptide hormone, regulates casein gene expression has been studied in mammary gland organ culture. After prolactin addition, a 2-4 fold increase in the rate of casein mRNA transcription was observed within 1 hr and maintained for at least 24 hr. This increased rate of transcription is not sufficient to account for the mass accumulation of casein mRNA. The half-life of casein mRNA is also increased 17-25 fold in the presence of prolactin. This change in casein mRNA half-life, coupled with a 2-4 fold increase in the rate of transcription, can account for the normal accumulation of casein mRNA observed after prolactin addition. This hormone-induced change in casein mRNA half-life appeared to be selective, since prolactin was found to exert only a slight effect (1-4 fold) on the half-life of poly(A) RNA determined under identical pulse-chase conditions. The hormonal regulation of casein gene expression thus does not app-ar to be an "all or none" process occurring only at the transcriptional or post-transcriptional levels, but rather may involve a coordinated response at several levels to permit the efficient expression of specialized differentiated functions.

Animals↗
PubMed1977

Characterization of early simian virus 40 transcriptional complexes: late transcription in the absence of detectable DNA replication.

Isolation of early viral transcriptional complexes and incorporation in vitro of radiolabeled precursors into nascent RNA has permitted an analysis of early simian virus 40 (SV40) transcription. Under conditions such that viral DNA replication was undetectable, both early and late SV40 RNA were synthesized. This finding provides evidence that viral DNA replication is not an absolute requirement for late transcription and supports earlier observations that late viral RNA is synthesized in SV40-infected nonpermissive mouse cells. The majority of the early viral transcriptional activity can be solubilized, indicating that a substantial portion of this RNA is transcribed from free rather than integrated templates. Sedimentation analysis of the transcriptional complexes resulted in the detection of two separate peaks of activity, suggesting the possibility of two distinct types of early SV40 templates.

Chromosome Mapping↗
PubMed1977

The sv40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription.

A small fraction of the SV40 chromatin isolated from infected monkey cell cultures by the Triton method contains active RNA polymerase which had initiated transcription in vivo. This viral transcription complex (VTC) was utilized to answer the question of whether proteins dissociate from chromatin during transcription in vitro. 3H-RNA was synthesized by the VTC under conditions such that over half the label was in transcripts which were longer than half the length of the SV40 genome. Virtually all of the 3H-RNA remained associated with the SV40 chromatin, causing an increase in sedimentation rate from 55S to 78S. The density of the VTC-3H-RNA complex indicated that less than 5% of the original protein dissociated from the SV40 DNA which served as a template for transcription. We conclude that SV40 chromatin can be transcribed while the proteins remain associated with the DNA.

Cell Line↗
PubMed1977

Characterization of simian virus 40 tsA58 transcriptional intermediates at restrictive temperatures: relationship between DNA replication and transcription.

When nuclei from simian virus 40 (SV40)-infected cells are lysed with Sarkosyl and the chromatin is pelleted, the supernatant fluid contains a nucleoprotein complex capable of synthesizing viral RNA (Laub and Aloni, Virology 75:346-354, 1976; Gariglio and Mousset, FEBS Lett. 56:149-155, 1975). The level of activity of the RNA polymerase in the complex increased during infection in parallel with the amount of viral DNA that had been synthesized. If cells infected at 33 degrees C with the SV40 mutant tsA 58 were shifted to the nonpermissive temperature of 40 degrees C at any time between 18 and 48 h postinfection, no viral DNA replication was detected after 45 min and no new rounds of synthesis were initiated after 20 to 30 min. However, after this shift, polymerase activity associated with the nucleo-protein complex did continue to increase for 5 h, at which time it reached a plateau. There was an increase of RNA synthesized from both the early (E) and late (L) SV40 DNA strands, and there was a threefold increase in the ratio of early-to-late RNA species after the shift. In comparable experiments with cells infected with wild-type virions, no increase in polymerase activity occurred because of the temperature change alone. At 33 degrees C, the relative amount of RNA transcribed from the wild-type E-strand was less than tsA 58 at 33 degrees C and did not increase after a shift to 40 degrees C. The tsA 58 transcriptional complexes extracted from cells grown at 33 degrees C sedimented heterogeneously in sucrose gradients, with a peak near 26S. There were no detectable alterations in the sedimentation properties of the complexes when tsA 58-infected cells were shifted to 40 degrees C for 2 h. We conclude that continued synthesis of viral DNA is not an obligatory prerequisite for maintenance of late viral transcription nor is the sedimentation of the transcriptional complex at 26S related to actively replicating DNA molecules serving as templates for transcription. Further, an increase in late transcription can occur under conditions where reinitiation of viral DNA synthesis is prevented. The increase in the synthesis of early and late RNA at the restrictive temperature without concurrent DNA synthesis is discussed in relationship to the function of the A gene product.

Cell Line↗
PubMed2005

SUMO modification of the Ets-related transcription factor ERM inhibits its transcriptional activity.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

Adenoviridae↗
PubMed2026

Genome-wide survey of spliceosomal snRNA transcripts across hundreds of human biosamples reveals abundant transcription but low maturation level of snRNA variants.

Small nuclear RNAs (snRNAs) are essential components of the spliceosome and are encoded by large, multicopy gene families. However, their genome-wide identification and quantification have remained challenging due to high sequence similarity among family members. To address this, we utilized RAMPAGE (Rapid Amplification of cDNA Ends) data from the ENCODE project to comprehensively profile nascent transcription of spliceosomal snRNAs across 115 human biosamples. We identified 74 expressed snRNA variants, characterized by canonical promoter features including bidirectional transcription flanking a positioned nucleosome, active histone modifications, and evolutionary conservation- features largely absent from unexpressed variants. These transcriptional events were corroborated by total RNA-seq and Bru-seq data, yet the majority of these variants showed extremely low levels in small RNA-seq, indicating post-transcriptional bottlenecks for snRNA processing and maturation. Our findings reveal new layers of regulation in snRNA variant expression and suggest that selective post-transcriptional processing plays a critical role in shaping the functional snRNA repertoire and its contribution to splicing regulation.

Journal Article↗
PubMed1979

Termination of transcription by Escherichia coli RNA polymerase: influence of secondary structure of RNA transcripts on rho-independent and rho-dependent termination.

The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with attendant weakening of secondary structure, abolished the rho-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. rho-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major rho-dependent termination sites on the T3 DNA-at 8 and 15%. The termination activity of rho in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of rho action but rho was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased rho-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated rho-mediated ATP hydrolysis. The rho-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive rho isolated from rho 15 mutant of E. coli, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G.C-rich regions in RNA stem-loop structures or RNA.DNA hybrids are essential for rho-independent termination, whereas rho-dependent termination requires weakly paired cytosine residues for its action.

Adenosine Triphosphatases↗