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N Hernandez

Publications and source records attributed to N Hernandez.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of a trimeric complex containing Oct-1, SNAPc, and DNA.

The human small nuclear (sn) RNA promoters contain a proximal sequence element (PSE), which recruits the basal transcription factor SNAPc, and a distal sequence element characterized by an octamer sequence, which recruits the POU domain transcription factor Oct-1. The Oct-1 POU domain and SNAPc bind cooperatively to probes containing a PSE and an octamer sequence, and this effect contributes to efficient transcription in vitro. In vivo, however, Oct-1 regions outside of the POU domain can activate snRNA gene transcription. Here, we have examined whether the role of these regions is to contribute to cooperative binding with SNAPc. We find that they indeed improve cooperative binding, but most of the effect is nevertheless mediated by just the POU domain. This suggests that Oct-1 activates transcription of snRNA genes in at least two steps, recruitment of SNAPc mediated primarily by the POU domain, and a later step mediated by regions outside of the POU domain. We also show that a PSE-binding complex observed in nuclear extracts consists of Oct-1 and SNAPc. Although Oct-1 cannot bind effectively to the PSE probe on its own, in the complex it contacts DNA. Thus, in a nuclear extract, SNAPc can recruit Oct-1 to a probe to which Oct-1 cannot bind on its own.

Base Sequence↗
PubMed1997

Role for the amino-terminal region of human TBP in U6 snRNA transcription.

Basal transcription from the human RNA polymerase III U6 promoter depends on a TATA box that recruits the TATA box-binding protein (TBP) and a proximal sequence element that recruits the small nuclear RNA (snRNA)-activating protein complex (SNAPc). TBP consists of a conserved carboxyl-terminal domain that performs all known functions of the protein and a nonconserved amino-terminal region of unknown function. Here, the amino-terminal region is shown to down-regulate binding of TBP to the U6 TATA box, mediate cooperative binding with SNAPc to the U6 promoter, and enhance U6 transcription.

Amino Acid Sequence↗
PubMed1997

The largest subunit of human RNA polymerase III is closely related to the largest subunit of yeast and trypanosome RNA polymerase III.

In both yeast and mammalian systems, considerable progress has been made toward the characterization of the transcription factors required for transcription by RNA polymerase III. However, whereas in yeast all of the RNA polymerase III subunits have been cloned, relatively little is known about the enzyme itself in higher eukaryotes. For example, no higher eukaryotic sequence corresponding to the largest RNA polymerase III subunit is available. Here we describe the isolation of cDNAs that encode the largest subunit of human RNA polymerase III, as suggested by the observations that (1) antibodies directed against the cloned protein immunoprecipitate an active enzyme whose sensitivity to different concentrations of alpha-amanitin is that expected for human RNA polymerase III; and (2) depletion of transcription extracts with the same antibodies results in inhibition of transcription from an RNA polymerase III, but not from an RNA polymerase II, promoter. Sequence comparisons reveal that regions conserved in the RNA polymerase I, II, and III largest subunits characterized so far are also conserved in the human RNA polymerase III sequence, and thus probably perform similar functions for the human RNA polymerase III enzyme.

Amino Acid Sequence↗
PubMed1997

RNA-targeted activators, but not DNA-targeted activators, repress the synthesis of short transcripts at the human immunodeficiency virus type 1 long terminal repeat.

The human immunodeficiency virus type 1 (HIV-1) promoter directs the synthesis of two types of RNA molecules: full-length transcripts, whose synthesis is activated by the viral activator Tat, and short transcripts, whose synthesis is dependent on the inducer of short transcripts (IST), a bipartite DNA element located in large part downstream of the HIV-1 transcriptional start site. In the absence of Tat, short transcripts constitute the large majority of the RNA molecules synthesized from the HIV-1 promoter. In the presence of Tat, synthesis of the short transcripts is repressed and synthesis of the full-length transcripts is activated. Tat is unique among transcriptional activators in acting through an RNA target, the TAR element. However, Tat has been shown to activate transcription from a DNA target when fused to the appropriate DNA binding domain, raising the question of why Tat has been directed to the RNA. Here we have compared the abilities of Tat and other RNA- and DNA-bound activators to stimulate transcription from the HIV-1 promoter. We show that DNA-targeted activators, including DNA-targeted Tat, activate the synthesis of both short and long transcripts, while RNA-targeted Tat and another RNA-targeted activator activate the synthesis of full-length transcripts but specifically repress that of short transcripts. The unique ability of RNA-targeted activators to down-regulate short transcript synthesis suggests that Tat is directed to the RNA specifically for the purpose of repressing short transcripts.

Base Sequence↗
PubMed1997

Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes.

Base Sequence↗
PubMed1996

Cloning and characterization of SNAP50, a subunit of the snRNA-activating protein complex SNAPc.

The human RNA polymerase II and III snRNA promoters share a common basal element, the proximal sequence element (PSE), which is recognized by a complex we refer to as the snRNA-activating protein complex (SNAPc). Biochemical purifications suggest that SNAPc is composed of at least four polypeptides of 43, 45, 50 and 190 kDa, as well as variable amounts of the TATA box binding protein, TBP. cDNAs encoding the 43 and 45 kDa subunits, SNAP43 and SNAP45, have been isolated, but there is no evidence that either of these subunits contacts DNA. Here we report the isolation of cDNAs encoding the 50 kDa subunit of SNAPc, SNAP50. The open reading frame predicts a 411 amino acid protein, which contains two potential zinc finger motifs. Depletions with anti-SNAP50 antibodies inhibit RNA polymerase II and III snRNA gene transcription in vitro. SNAP50 interacts with SNAP43 in co-immunoprecipitation experiments, but not with SNAP45 or TBP. UV cross-linking experiments suggest that SNAP50 contacts DNA in the SNAP complex. These results are consistent with the same core SNAP complex recognizing the PSEs of RNA polymerase II and III snRNA promoters, and provide an initial view of the architecture of the SNAP complex.

Amino Acid Sequence↗
PubMed1996

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Amino Acid Sequence↗
PubMed1996

Isolation alters striatal met-enkephalin immunoreactivity in rat pups.

Isolated preweanling rats emit ultrasonic vocalizations. Mu- and delta-opioid agonists quiet isolated pups; naltrexone, an opioid receptor blocker, prevents this quieting. A littermate companion is as effective as morphine in quieting vocalizations, and naltrexone also blocks companion quieting. We have now quantified methionine enkephalin (Met-ENK) immunoreactivity in the brains of 10-day-old Wistar rat pups taken directly from the home cage or kept either alone or with a companion for a brief or prolonged period. Met-ENK is an endogenous ligand that binds to the mu- and delta-opioid receptors. Striatal peptide levels were higher when pups were with a companion than when they were kept alone; the peptide level of pups in the home cage did not differ from either. Comparisons of pups in the brief (5 min) and prolonged (60 min) separation conditions showed significantly higher peptide levels following a brief period out of the nest than at the end of an hour. In hypothalamus, hippocampus, and frontal cortex neither social condition nor duration of separation significantly altered peptide quantity. Larger amounts of Met-ENK in pups provided with a companion could reflect an increase in posttranslational cleavage of the precursor molecule leading to stimulation of receptors that act to diminish USV. Reduced levels following 60 min out of the home cage might reflect depletion of the peptide following an initial release during the period when the pup's vocal response is most vociferous.

Animals↗
PubMed1996

The isolation and companion comfort responses of 7- and 3-day-old rat pups are modulated by drugs active at the opioid receptor.

Rat pups emit ultrasonic vocalizations (USVs) when isolated in a novel environment. In 10-day-olds, USV has been shown to be reduced by either the administration of 0.125 mg/kg morphine (MOR) or the presence of a littermate; these effects were both reversed by naltrexone (NLX), an opioid receptor blocker. The present study reports that the same dose of MOR produced NLX-antagonized quieting without sedation in 7- and 3-day-old pups; higher doses of MOR decreased USV but produced motor deficits as well. The 0.125 mg/kg dose of MOR is less effective in reducing USV in 3- and 7-day-olds; calling rates declined by no more than 42%, compared with 65% at 10 days of age. The presence of a companion also lowered the USV of 3- and 7-day-olds by a lesser amount (55-57%) than the 67% seen in 10-day-olds or the 90% decline when pups are 2 weeks old. This suggests that age-related changes in the opioid system may be relevant to the increased salience of a social companion that comes with maturity.

Age Factors↗
PubMed1996

Monoclonal antibodies directed against the amino-terminal domain of human TBP cross-react with TBP from other species.

The TATA box-binding protein (TBP) is a key transcription factor required for transcription by all three eukaryotic RNA polymerases. It consists of a conserved carboxy-terminal DNA binding domain and a highly divergent amino terminal domain. TBP and different sets of TBP-associated factors (TAFs) constitute at least four multisubunit complexes referred to as SL1, TFIID, TFIIIB, and SNAPC. SL1, TFIID, and TFIIIB are required for transcription by RNA polymerases I, II, and III, respectively, while the SNAP complex is involved in transcription of the small nuclear RNA (snRNA) genes by RNA polymerases II and III. TBP also associates with a number of basal transcription factors such as TFIIA and TFIIB, and with several regulatory factors such as VP16, E1A, and p53. Here we describe the characterization of a panel of monoclonal antibodies (MAbs) directed against the amino-terminal domain of human TBP. These MAbs recognize different TBP epitopes, some of which have been precisely defined. Different MAbs recognize different TBP-containing complexes and several of them crossreact with TBP from other species. These antibodies can be used to purify TBP-containing complexes in a functional form and should be useful to identify new protein-protein interactions involving TBP.

Amino Acid Sequence↗
PubMed1996

Mutations in the carboxy-terminal domain of TBP affect the synthesis of human immunodeficiency virus type 1 full-length and short transcripts similarly.

The human immunodeficiency virus type 1 promoter generates two types of RNA molecules, full-length transcripts and short transcripts. Synthesis of the short transcripts depends on the inducer of short transcripts (IST), an element located downstream of the start site. In the presence of the viral activator Tat, the synthesis of full-length transcripts is up-regulated while that of short transcripts is down-regulated. Full-length and short transcripts are probably generated by different types of transcription complexes. The first is IST independent, capable of efficient elongation, and up-regulated by Tat. The second is IST dependent, incapable of efficient elongation, and down-regulated by Tat. We have used an in vivo assay to assess the role of TBP in human immunodeficiency virus type I transcription and to test the effect of mutations in TBP on synthesis of full-length and short transcripts. We find that TBP bound to the TATA box is required for the synthesis of short and full-length transcripts as well as for Tat activation and that both yeast TBP and the carboxy-terminal domain of human TBP can replace full-length human TBP for these processes. Mutations in TBP affect the synthesis of short and full-length transcripts as well as Tat activation similarly, and these effects correlate with the previously described effects of these mutations on binding of TBP to the TBP-associated factor TAFII250 in vitro. Together, these results suggest that if short and full-length transcripts are generated by variant transcription complexes, these complexes use TBP similarly, probably as part of the TFIID complex.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1996

RNA polymerase III transcription from the human U6 and adenovirus type 2 VAI promoters has different requirements for human BRF, a subunit of human TFIIIB.

Mammalian TFIIIB can be separated into two fractions required for transcription of the adenovirus type 2 VAI gene, which have been designated 0.38M-TFIIIB and 0.48M-TFIIIB. While 0.48M-TFIIIB has not been characterized, 0.38M-TFIIIB corresponds to a TBP-containing complex. We describe here the purification of this complex, which consists of TBP and a closely associated polypeptide of 88 kDa, and the isolation of a cDNA corresponding to the 88-kDa polypeptide. The predicted protein sequence reveals that the 88-kDa polypeptide corresponds to a human homolog of the Saccharomyces cerevisiae BRF protein, a subunit of yeast TFIIIB. Human BRF (hBRF) probably corresponds to TFIIIB90, a protein previously cloned by Wang and Roeder (Proc. Natl. Acad. Sci. USA 92:7026-7030, 1995), although its predicted amino acid sequence differs from that reported for TFIIIB90 over a stretch of 67 amino acids as a result of frameshifts. Immunodepletion of more than 90 to 95% of the hBRF present in a transcription extract severely debilitates transcription from the tRNA-type VAI promoter but does not affect transcription from the TATA box-containing human U6 promoter, suggesting that the 0.38M-TFIIIB complex, and perhaps hBRF as well, is not required for U6 transcription.

Adenoviruses, Human↗
PubMed1996

The Oct-1 POU-specific domain can stimulate small nuclear RNA gene transcription by stabilizing the basal transcription complex SNAPc.

The RNA polymerase II and III human small nuclear RNA promoters have a common basal element, the proximal sequence element, which binds the TATA box-binding protein-containing complex SNAPc. They also contain an enhancer characterized by a highly conserved octamer sequence, which constitutes a binding site for the broadly expressed POU domain transcription factor Oct-1. The POU domain is a bipartite DNA-binding domain consisting of a POU-homeo (POUH) domain and a POU-specific (POUs) domain joined by a flexible linker. Here, we show that the Oct-1 POU domain but not the related Pit-1 POU domain can facilitate the binding of SNAPc to the proximal sequence element, and activate transcription. The effect is probably mediated by protein-protein contacts, and 1 of 30 amino acid differences between the Oct-1 and Pit-1 POUs domains is the key determinant for the differential interaction with SNAPc and the ability to activate transcription. These results show that a function that is the hallmark of activation domains, namely, recruitment of a basal transcription complex resulting in activation of transcription, can be performed by a DNA-binding domain. In this case, subtle changes between activator DNA-binding domains, as subtle as a single amino acid difference, can profoundly affect interaction with the basal transcription machinery.

Amino Acid Sequence↗
PubMed1995

A TBP-TAF complex required for transcription of human snRNA genes by RNA polymerase II and III.

The TATA-box-binding protein TBP exists in the cell complexed with different sets of TBP-associated factors (TAFs). In general, each of these TBP-TAF complexes is dedicated to transcription by a single RNA polymerase. Thus, SL1, TFIID and TFIIIB are required for transcription by polymerases I, II and III, respectively. Here we characterize a fourth TBP-TAF complex called SNAPc. Unlike the other TBP-TAF complexes, SNAPc is implicated in transcription by two types of polymerases; it is required for transcription of both the RNA polymerase II and III small-nuclear RNA genes and binds specifically to the proximal sequence element PSE, a non-TATA-box basal promoter element common to these two types of genes. In addition to TBP, SNAPc is composed of at least three TAFs, SNAP43, SNAP45 and SNAP50. The predicted amino-acid sequence of SNAP43 reveals that it corresponds to a new protein.

Amino Acid Sequence↗
PubMed1995

Successful treatment of New World cutaneous leishmaniasis with a combination of topical paromomycin/methylbenzethonium chloride and injectable meglumine antimonate.

Colombian patients with New World cutaneous leishmaniasis were treated with a combination of a topical formulation (15% paromomycin sulfate/5% methylbenzethonium chloride, twice a day) and parenteral meglumine antimonate (20 mg of antimony [Sb]/kg.d]). Cohort 1 received topical therapy for 10 days and Sb for 7 days; 18 (90%) of the 20 patients were cured (follow-up, 12 months). Other clinical data suggested that neither the topical formulation alone nor the 7-day regimen of Sb alone would have cured many patients. In a subsequent cohort, which received topical therapy for 10 days and Sb for 3 days, the cure rate was 42% (eight of 19 patients). In Colombian cohorts (historical controls) treated with Sb alone for 10-15 days, the cure rate was 31%-36%. Side effects in cohort 1 patients consisted of local reactions to the topical formulation: burning and pruritus in 25% of patients and vesicle formation in 15% of patients. This is the first report that a regimen partially composed of topical antimicrobial agents can be highly effective for treatment of New World cutaneous leishmaniasis.

Administration, Topical↗
PubMed1993

GM1 ganglioside reduces glutamate toxicity to cortical cells. Lowered LDH release and preserved membrane integrity.

As an in vitro model of CNS excitatory amino acid (EAA) injury, rat cortical neuronal cultures were challenged with glutamate (0.5 or 10 mM) and the levels of released lactate dehydrogenase (LDH) were monitored at 1 h, 1, 2, and 7 d. LDH release is correlated with levels of plasma membrane damage. GM1 has been shown to be continuously distributed on the outer surface of CNS cellular membranes. By staining for the distribution of endogenous GM1 ganglioside using cholera toxin/antitoxin immunohistochemistry, we were able to assess morphologically cellular plasma membrane integrity after damage. We used these two measures (LDH and GM1 localization) to study the neuroprotective effects of exogenous GM1 ganglioside to further elucidate its mechanism. Cortical cultures derived from 15-d rat fetuses were subjected to the glutamate challenge for 30 min. Parallel cultures were either pre- or post-treated with 80 microM of GM1. Exposure to 10 mM glutamate caused a highly significant increase in LDH release at 1-48 h. Pretreatment with GM1 reduced the release, whereas posttreatment reduced the LDH release even more. Plasma membrane changes observed by the GM1 immunohistochemistry reflected the LDH release data. All cultures treated with GM1 evidenced substantial structural integrity (continuous staining of GM1 along perikarya and processes) as compared to untreated cultures. These data support our hypothesis that GM1 treatment (pre- and post-) reduces plasma membrane damage.

Animals↗
PubMed1993

Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex activates transcription from snRNA promoters through the PSE.

In the human small nuclear RNA (snRNA) promoters, the presence of a TATA box recognized by the TATA box-binding protein (TBP) determines the selection of RNA polymerase III over RNA polymerase II. The RNA polymerase II snRNA promoters are, therefore, good candidates for TBP-independent promoters. We show here, however, that TBP activates transcription from RNA polymerase II snRNA promoters through a non-TATA box element, the snRNA proximal sequence element (PSE), as part of a new snRNA-activating protein complex (SNAPc). In contrast to the previously identified TBP-containing complexes SL1, TFIID, and TFIIIB, which appear dedicated to transcription by a single RNA polymerase, SNAPc is also essential for RNA polymerase III transcription from the U6 snRNA promoter. The U6 initiation complex appears to contain two forms of TBP, one bound to the TATA box and one bound to the PSE as a part of SNAPc, suggesting that multiple TBP molecules can have different functions within a single promoter.

Animals↗