Transcripts from amplified sequences of an inherited homogeneously staining region in chromosome 1 of the house mouse (Mus musculus).

Several populations of the house mouse, Mus musculus, are polymorphic for the presence or absence of an inherited homogeneously staining region (HSR) in chromosome 1. The HSR consists of highly amplified DNA sequences, present in low copy numbers in the HSR- genome. A cloned HSR-derived genomic sequence detected transcripts of about 1.3 and 4.5 kb on blots of poly(A)+ RNA from liver of HSR+ mice but not from that of HSR- mice. A cDNA library was established from RNA of HSR+ mice and screened with the HSR-derived genomic clone. Positive clones were isolated and shown to be complementary to the 1.3-kb RNA species and to amplified DNA sequences in the HSR+ genome. The combined sequence of four overlapping cloned cDNAs is 959 nucleotides long and includes an open reading frame encoding a putative protein of 208 amino acids. The pertinent gene is unidentified. No homologous sequence is stored in the EMBL data base. A stretch of 109 nucleotides at the 3' end of the 1.3-kb RNA homology region in the same genomic fragment, as indicated by hybridization data and sequence motifs resembling promoter elements. Thus, our data suggest that at least two genes or gene families are encoded in the HSR.

Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor.

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

Characterization of pre-rRNA components in ribosomal precursor particles from macronuclei of Tetrahymena thermophila.

Ribosomal precursor particles were extracted from purified macronuclei of Tetrahymena thermophila and separated in sucrose gradients. The RNA components of major particle fractions were isolated and analyzed by Northern blot hybridization using cloned rDNA fragments. A tentative scheme of preribosome maturation was established based on the RNA constituents and corresponding processing steps occurring in the different particle classes. The primary transcript of ribosomal genes, the unspliced precursor rRNA, was found in some experiments in the upper region (less than 40S) of sucrose gradients run for short times. This is in accordance with earlier results by others indicating that slowly sedimenting ribonucleoprotein (RNP) structures may exist as a transitory stage of preribosome formation. Usually, however, unspliced pre-rRNA was only found in 80S preribosomes, where splicing occurred as indicated by the presence of splice intermediates and products only in this fraction. In addition, the further processing of spliced pre-rRNA at three major sites in variable temporal order took place in the 80S preribosomes, i.e., (i) the cleavage at or near the 5'end of the 17S rRNA sequence, (ii) the central cleavage in the internal transcribed spacer (ITS2) between the 5.8S and 26S rRNA sequence, and (iii) the cleavage in the ITS1 at or near the 3' end of the 17S rRNA sequence. Only the latter event was found to result more or less immediately in the division of the 80S preribosomes into separate precursors (p40S and 60S) of the small and large ribosomal subunits. If the alternative pre-rRNA cleavage site in the ITS2 was used first the 80S preribosomes retained their integrity. The conversion of the p40S precursors into nuclear 40S subribosomal particles was correlated with the processing of pre-17S rRNA into 17S rRNA. In the 60S ribosomal precursor particles the processing of pre-26S rRNA, including the formation of precursors (ITS and 7S RNA) to 5.8S rRNA, occurred. A substantial proportion of 26S rRNA molecules isolated from these particles already contained the central hidden break as indicated by the presence of 26S rRNA alpha- and beta-subfragments. The major pre-rRNA processing by-products, IVS and ETS RNA, were partly associated with preribosomes and partly present as free RNAs in the supernatant of sucrose gradients. This indicates that they are liberated and degraded mainly outside the particles in which they are formed. In contrast, the initiation fragment (IF), a small promoter-proximal transcript, was exclusively associated with large particles.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of rRNA metabolism in Tetrahymena pyriformis. I. Nutritional shift-down.

Changes of rates of rRNA transcription and accumulation as well as of stability of rRNA precursor fractions and mature cytoplasmic rRNAs were determined in Tetrahymena after shift-down to a non-nutrient buffer. During the initial period (4-6 hours) an enhanced degradation of pre-existing cytoplasmic rRNA and a successive reduction of rRNA transcription, processing and nucleo-cytoplasmic transport was detected. Thereafter, a "residual" rRNA metabolism at low turnover is maintained in the cells which has the following characteristics: (1) The rate of pre-rRNA transcription, as measured in vitro in isolated macronuclei, is about 3 to 5% of the rate in optimally growing cells, indicating regulatory processes at the level of initiation of the nucleolar RNA polymerases. The strong reduction of in vitro pre-rRNA synthesis can partially be reversed by pre-treatment of the starved cells with low concentrations of cycloheximide, but not with puromycin.--(2) The processing time of nuclear pre-rRNA is considerably prolonged and the introduction of the central hidden break into newly synthesized cytoplasmic 26S rRNA is strongly delayed, as shown by methionine pulse-chase experiments.--(3) The accumulation rate of cytoplasmic rRNA is 1 to 2% of the rate in optimally growing cells, as determined from the specific radioactivity of ATP at saturation with labelled exogeneous adenosine and the changes of the specific radioactivity of the AMP residues in rRNA as well as from the rRNA turnover rate.

In vitro synthesis and processing of pre-rRNA in isolated macronuclei from Tetrahymena.

Macronuclei were isolated from logarithmically growing Tetrahymena cells in isoosmotic medium containing the weak detergent n-octanol and were purified in sucrose gradients. Electron microscopy revealed good structural preservation including intact nuclear envelopes. Initial rates of [3H]UTP incorporation into these nuclei were relatively high (2-4 pmol UMP/micrograms DNA per min), and 70 to 80% of transcription was resistant to alpha-amanitin, which is similar to the percentage of nuclear label associated with the nucleoli in electron microscopic autoradiograms. The use of transcription initiation inhibitors indicated that elongation of in vivo initiated pre-rRNA chains had essentially occurred in vitro. The radioactivity profiles of in vitro synthesized RNA in gels exhibit a heterogeneous pattern with the exception of a small peak corresponding to the length of pre-rRNA molecules. Detailed analysis of the extent and specificity of pre-rRNA processing was performed by RNA transfer hybridizations using cloned rDNA fragments as probes. The results show that the early processing events, i.e., splicing, 5'terminal and central cleavage of pre-rRNA, proceed faithfully, but at reduced rates and efficiencies. Furthermore, processing of pre-17S rRNA at the 3'end, and pre-26S rRNA at the 5'end, including the formation of immediate 5.8S rRNA precursors (ITS and 7S RNA), occurred. In contrast to previous in vivo results, a central hidden break was also introduced into part of nuclear 26S rRNA molecules. In addition to the known intermediates and by-products of processing, a large number of distinct fragments due to non-random cleavages of rRNA precursors appeared during in vitro incubation of macronuclei. Most prominent were two novel small RNA fragments from the 5'terminal end of pre-rRNA which may be products of alternative processing sites in the external transcribed spacer. Another small promoter-proximal RNA which is present in substantial amounts in vivo, was not formed under in vitro processing conditions, but degraded rapidly. This is further support to the notion that this RNA species may represent a product of premature transcription termination.

Corticotropin-releasing hormone neurons in the paraventricular nucleus project to the external zone of the median eminence: a study combining retrograde labeling with immunocytochemistry.

Corticotropin-releasing hormone (CRH) is the major regulator of the pituitary-adrenal axis. CRH-immunoreactive perikarya are widely distributed in the central nervous system; however, only those which participate directly in the regulation of adrenocorticotropin are connected to the portal circulation in the external zone of the median eminence. The present study describes the identification of these hypophysiotropic neurons using retrograde labeling and CRH immunocytochemistry. Fluoro-Gold was injected peripherally then, 5 days later, the animals were treated with colchicine. Twenty-four hours later the animals were sacrificed, and their brains were immunostained for CRH with the indirect immunofluorescence technique. The results indicate that the vast majority of the Fluoro-Gold-accumulating and CRH-immunopositive perikarya (hypophysiotropic neurons) are located in the medial parvicellular subdivision of the paraventricular nucleus (PVN). However, not each CRH-immunoreactive neuron contains Fluoro-Gold, i.e. a small portion of these neurons project to areas of the brain other than the median eminence. The anterior, lateral and periventricular subdivisions of the PVN also contain hypophysiotropic CRH-immunoreactive perikarya, however, their number is much less than in the medial parvicellular subdivision. Scattered double-labeled cells are also present in the medial preoptic area and the dorsal hypothalamus, just behind the PVN. These results support previous observations that the PVN, particularly the medial parvicellular subdivision, is the predominant source of the hypophysiotropic CRH neurons.

Tyrosine phosphorylation in rat spinal cord after sciatic nerve transection.

Immunocytochemistry using antibodies against phosphotyrosine was employed to identify changes in tyrosine phosphorylation in the rat spinal cord consequent to sciatic nerve injury. Increased immunostaining in the spinal gray matter, dorsal columns and gracile nucleus on the side of the lesion became evident after 3 days and was more pronounced with longer survival times up to 3 weeks (the longest survival tested). This increase was most prominent in the fourth lumbar segment (the focus of termination of sciatic nerve afferents). Immunostaining was ain astroglial cells and their processes in the dorsal horn; stained microglia were also seen. Immunopositivity also increased in glial cells surrounding motoneurons at the same levels. These changes suggest that a diffusible growth factor released centrally by injured nerve fibers activates tyrosine phosphorylation in glial cells via receptor tyrosine kinases.

Interference of biocytin with opioid-evoked hyperpolarization and membrane properties of rat spinal substantia gelatinosa neurons.

In our laboratory, preliminary whole-cell, tight seal recordings of rat spinal substantia gelatinosa neurons including biocytin in the patch pipette yielded a significantly smaller proportion of neurons hyperpolarized by selective opioid agonists compared with recordings without biocytin. Therefore, we investigated the effects of biocytin inclusion on opioid responses and other membrane properties during whole-cell, tight seal recordings of these neurons. The percentage of neurons hyperpolarized by mu-, delta(1)-, and kappa-selective opioids was significantly reduced when 1% but not < or =0.2% biocytin was included in the recording pipette, compared with neurons recorded without biocytin. However, a significantly higher proportion of neurons fired spontaneous action potentials with either 0.05-0.2 or 1% biocytin compared to no biocytin. Resting membrane potential, input impedance and the proportion of neurons displaying transient outward rectification were each significantly altered for neurons recorded with 1% but not 0.05-0.2% biocytin. These effects may be due to a relatively specific blockade of diverse potassium channel types. Because efficient labeling can be achieved with 0.1% biocytin with whole-cell recording, higher concentrations are contraindicated.

Differential contribution of TRPV1 to thermal responses and tissue injury-induced sensitization of dorsal horn neurons in laminae I and V in the mouse.

Our previous recordings from dorsal root ganglion and spinal lamina V neurons from TRPV1-mutant mice showed dramatic decreases in responses to temperatures near the activation threshold of this channel (43-49 degrees C). Somewhat unexpectedly, we only observed behavioral deficits in these mice at higher temperatures (50-58 degrees C). In the present study, we tested the hypothesis that the noxious heat-evoked pain behavior that persists in TRPV1-mutant mice reflects residual responsiveness of neurons in the superficial, but not deep, dorsal horn. To this end, we performed in vivo extracellular recordings of spinal nociresponsive neurons in laminae I and V in wild type (WT) and TRPV1 mutant mice. Neurons in WT and mutant mice from both laminae did not differ in their spontaneous activity or evoked responses to mechanical or cold stimuli. By contrast, most lamina I neurons from mutant mice responded to noxious heat with significantly higher thresholds than in WT mice. In contrast, lamina V neurons from mutant mice were virtually unresponsive to noxious heat before and after topical mustard oil-induced tissue injury. Interestingly, lamina I neurons in mutant mice displayed thermal sensitization following tissue injury, comparable in magnitude, but of shorter duration, than in WT mice. We conclude that TRPV1 is necessary for noxious heat-evoked responses of lamina V neurons, both before and after tissue injury. It is also an essential contributor to the normal activation threshold of lamina I neurons to noxious heat and for the full duration of thermal sensitization of lamina I neurons following injury. Finally, our results suggest that the processing of noxious thermal messages by neurons in lamina I involves convergent inputs from a heterogeneous population of primary afferent thermal nociceptors.

Effect of puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.

Characterization of an authentic intermediate in the self-splicing process of ribosomal precursor RNA in macronuclei of Tetrahymena thermophila.

We have characterized a 1.5 kb RNA species in T. thermophila macronuclei previously found in vivo and including intron sequences linked to the 3' exon. This IVS-3' exon RNA could be detected in gels as a discrete molecule only after denaturation of nuclear RNA. After addition of 32P-GTP, as splicing cofactor in a nuclear in vitro system, the IVS-3' exon RNA was labeled at its 5' terminus, as was the by-product of splicing, the excised IVS RNA. The time course of labeling indicates that the IVS-3' exon RNA acts like a reaction intermediate and specifically a kinetic precursor to IVS RNA. Partial nuclease digestions showed that the IVS-3' exon RNA and the IVS RNA have the same 5' terminal sequence. In addition the IVS-3' exon RNA can release the 15-mer oligonucleotide cleaved off during circularization of IVS RNA under conditions of high temperature. Taken together, the structural, functional, and kinetic properties of the IVS-3' exon RNA strongly suggest that it represents a previously postulated in vivo intermediate in the splicing pathway.

Synchronizing temperatures and rRNA metabolism in Tetrahymena pyriformis.

1. In Tetrahymena pyriformis pre-rRNA is synthesized and efficiently processed and translocated into the cytoplasm at both a supraoptimal (34 degrees C) and a suboptimal (8 degrees C) synchronizing temperature. 2. At the heat shock temperature (34 degrees C) no substantial differences in the kinetics of the main intracellular events of rRNA-metabolism compared to the optimal growth temperature (28 degrees C) were found. 3. The high temperature, however, induces a strong retardation of uptake of [3H]adenosine into the cells and a reduction of the cellular ATP pool size. 4. At the cold shock temperature (8 degrees C) the rates of transcription and nucleocytoplasmic transport of rRNA as well as of nucleotide pool equilibration are reduced to a similar extent (25-30% of the optimal rates at 28 degrees C). 5. The results are discussed and compared with the effects of sub- and supraoptimal temperatures on rRNA synthesis and processing in mammalian cells.

Presymptomatic diagnosis in families with adenomatous polyposis using highly polymorphic dinucleotide CA repeat markers flanking the APC gene.

A panel of five multiallelic and highly informative dinucleotide CA repeat markers flanking the APC gene was used for presymptomatic diagnosis of familial adenomatous polyposis coli (FAP). Marker regions were amplified by PCR. DNA fragments were separated by electrophoresis in denaturing polyacrylamide gels and visualised by ethidium bromide staining. Two or more markers were found to be informative in all nine families tested, and all 23 persons at risk could be diagnosed as affected or unaffected by the disease gene, the probability being > 99.9% in 14 cases from six families in which flanking markers were informative. We found no indication for locus heterogeneity of the disease in our sample. The polyposis phenotype and its extracolonic manifestations co-segregated with a distinct haplotype determined by the markers flanking the APC gene. In one family with no remaining living affected members, we could infer the high risk haplotype from genotyping of first degree relatives. The segregation of this haplotype is consistent with the occurrence of CHRPEs in the progeny. In a sporadic case we made use of the typical early extracolonic manifestations of the disease (osteomas, desmoids) to identify the high risk haplotype. We conclude from our experience that indirect genotyping of FAP with this particular panel of closely linked and highly polymorphic microsatellite markers is a rapid, efficient, and highly reliable method for presymptomatic diagnosis of FAP.

The intervening sequence in the 26S rRNA coding region of T. thermophila is transcribed within the largest stable precursor for rRNA.

We studied the transcription of the intervening sequence in the 26S rRNA coding region of the extrachromosomal rDNA molecules in the macronucleus of T. thermophila by hybridization of purified nuclear rRNA precursors or cytoplasmic 26S rRNA to purified native rDNA or specific rDNA restriction fragments. Examination of R loop hybrids in the electron microscope and analyses of S1-protected rDNA fragments in alkaline agarose gels showed that mature 26S rRNA, nuclear pre-26S rRNA and a fraction of the pre-rRNA molecules containing both the sequences for 17S and 26S rRNA all lack the region corresponding to the intervening sequence. The rest of the pre-rRNA molecules, however, hybridize in a colinear fashion to the whole coding region, and thus must contain the intervening sequence. We can conclude from these results that the intervening sequence is transcribed within the primary transcription product of the rDNA, and that the post-transcriptional removal of the intervening RNA sequence is a very early processing event in the organism.

Complex endonucleolytic cleavage pattern during early events in the processing of pre-rRNA in the lower eukaryote, Tetrahymena thermophila.

We have analysed nuclear RNA from T. thermophila by RNA transfer hybridization using cloned rDNA fragments. A very high number of in vivo intermediates and by-products of rRNA processing were identified. These include putative intermediates of the splicing process and alternative products resulting from temporal variability in various endonucleolytic cleavages. In addition, four small RNA species including only transcribed spacer sequences were detected. These are (1) the IVS RNA (approximately 400 bases), the by-product of the splicing process, (2) a fragment from the internal transcribed spacer (approximately 360 bases), possibly resulting from 3'-end processing of pre-17S rRNA, (3) a fragment comprising most or all of the external transcribed spacer (approximately 600 bases) obviously representing the major by-product of 5'-end processing, and, in addition, (4) a small fragment from the initiation region (approximately 230 bases) which might be a product of premature transcription termination.

Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons.

Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J. Neurophysiol. 80: 2954-2962, 1998. Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the mu-opioid agonist (-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO) on the membrane potential and conductance of substantia gelatinosa (SG) neurons. We observed that bath application of 1-5 microM DAMGO caused a robust and repeatable hyperpolarization in membrane potential (Vm) and decrease in neuronal input resistance (RN) in 60% (27/45) of hamster neurons and 39% (9/23) of rat neurons, but significantly only when ATP (2 mM) and guanosine 5'-triphosphate (GTP; 100 microM) were included in the patch pipette internal solution. An ED50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Because G-protein mediation of opioid effects has been shown in other systems, we tested if the nucleotide requirement for opioid hyperpolarization in SG neurons was due to G-protein activation. GTP was replaced with the nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S; 100 microM), which enabled DAMGO to activate a nonreversible membrane hyperpolarization. Further, intracellular application of guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S; 500 microM), which blocks G-protein activation, abolished the effects of DAMGO. We conclude that spinal SG neurons are particularly susceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate mu-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activated hyperpolarization in SG neurons observed in previous sharp electrode experiments and the more recent failures to observe these effects with whole cell patch techniques.

Introduction of hidden breaks during rRNA maturation and ageing in Tetrahymena pyriformis.

The stability of Tetrahymena pyriformis cytoplasmic rRNAs and nuclear rRNA precursors has been studied by polyacrylamide gel electrophoresis under partly and completely denaturing conditions. Cytoplasmic 17-S rRNA (Mr = 0.66 X 10(6) consists of a continuous polynucleotide chain throughout its lifetime, whereas the bulk of 26-S rRNA (Mr = 1.2m X 10(6) dissociates upon denaturation. Two large fragments (F1, F2) of somewhat different molecular weights (Mr 0.63 X 10(6) and 0.58 X 10(6) and the small 5.8-S rRNA fragment (Mr about 50 000) are regularly observed. Some additional distinct minor fragments (F3-F6) are noted under certain preparative conditions, suggestive of artifactual origin. The following conclusions were made from the data obtained . (a) Newly synthesized 26-S rRNA molecules do not contain the 'central' hidden break (separating F1 and F2) until about 15 min after their appearance in the cytoplasm; however, they release during denaturation the 5.8-S and/or a short-lived 7-S fragment (Mr about 75 000) which might represent a direct precursor to the 5.8-S rRNA. (b) The immediate nuclear precursor to the 26-S rRNA (Mr 1.39 X 10(6) releases a small fragment of similar size (7 S). (c) The largest stable transcription product of the rDNA (pre-rRNA) does not contain any hidden break.