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PubMed1987

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2.

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.

Amino Acid Sequence↗
PubMed1991

Preproenkephalin messenger RNA-containing neurons in striatum of patients with symptomatic and presymptomatic Huntington's disease: an in situ hybridization study.

Previous studies have revealed a loss of enkephalin immunoreactivity in the terminals of striatal neurons projecting to the external globus pallidus in patients with early grades of Huntington's disease (HD). To assess the status of the perikarya of striatal enkephalinergic neurons, we performed in situ hybridization histochemistry with a radiolabeled RNA probe complementary to preproenkephalin messenger RNA. We studied postmortem brain tissue of 6 patients with symptomatic HD, 7 control subjects, and 2 presymptomatic carriers of the HD allele. There was a significant reduction in the areal density of striatal neurons expressing preproenkephalin messenger RNA in the patients with symptomatic HD, but the level of labeling in the remaining cells was not altered compared with the control subjects. In the specimens from presymptomatic individuals, there was no reduction of areal density of preproenkephalin messenger RNA-containing neurons in the striatum, despite the fact that loss of enkephalin immunoreactivity in the external globus pallidus had been previously demonstrated in the same brains. The results correlate with the previous demonstration of depleted enkephalin immunoreactive terminals in the external globus pallidus in patients with symptomatic HD. They also suggest that the early loss of enkephalin immunoreactivity observed in the external globus pallidus of presymptomatic carriers of the HD allele is not related to a generalized death of striatal enkephalinergic neurons early in the course of the disease.

Adolescent↗
PubMed1989

Characterization of striatal neurons expressing high levels of glutamic acid decarboxylase messenger RNA.

Two types of labelled cells are detected in sections of rat and mouse striata processed for in situ hybridization histochemistry with 35S-radiolabelled RNA probes complementary to the messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD), the synthesis enzyme for gamma-aminobutyric acid (GABA): numerous lightly, and fewer very densely labelled neurons. In order to determine whether the densely labelled cells correspond to the striatal somatostatinergic neurons with which they share morphological characteristics, the presence of GAD mRNA was examined in brain sections processed successively for dihydronicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, a marker of striatal somatostatinergic neurons, and in situ hybridization histochemistry. In addition, the distribution of GABAergic interneurons was analyzed with regard to striatal compartments (striosomes) indicated by patches of dense opiate binding sites. The results show that NADPH diaphorase activity and GAD mRNA do not co-exist in striatal neurons. Furthermore, in contrast to the somatostatinergic neurons which are almost exclusively located in the extrastriosomal matrix, densely labelled GAD cells were present both in the striosomes and the matrix, further suggesting that GABAergic and somatostatinergic neurons form two distinct interneuronal systems in the striatum of rats and mice.

Animals↗
PubMed1983

Translational control of IS10 transposition.

We present genetic evidence that insertion sequence IS10, the active element in transposon Tn10, can negatively control expression of its own transposase protein at the translational level. This control process is manifested in trans in a phenomenon called "multicopy inhibition": the presence of a multicopy plasmid containing IS10 inhibits transposition of a single copy chromosomal Tn10 element by reducing its ability to express transposition functions. Fusion analysis suggests that expression is reduced at the translational and not the transcriptional level. Only the outer 180 bp of IS10-Right are required on the plasmid for full inhibition. Plasmid-encoded transposase protein is not involved. The genetic structure of the essential plasmid region and the effects of point and deletion mutations on multicopy inhibition lead us to propose that inhibition of transposase translation occurs by direct pairing between the transposase messenger RNA and a small, complementary, regulatory RNA specified by the IS10-encoded pOUT promoter.

DNA Transposable Elements↗
PubMed1989

Replication of RNA by the DNA-dependent RNA polymerase of phage T7.

The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.

DNA-Directed RNA Polymerases↗
PubMed1991

Strand-specific detection of enteroviral RNA in myocardial tissue by in situ hybridization.

In this report we describe the development and application of single-stranded RNA probes for strand-specific detection of enterovirus RNA in infected heart tissue by in situ hybridization. For synthesis of RNA probes a full-length reverse-transcribed, recombinant CVB3 cDNA was inserted into the transcription vector pSPT18. Run-off transcripts of plus-strand and minus-strand orientation were produced using either T7 or SP6 RNA polymerase. Binding specificity and sensitivity of the radioactively labelled RNA probes were determined by slot-blot hybridization. Due to the high degree of genetic identity among enteroviruses, the in vitro transcribed CVB3 RNA probes hybridized with various enterovirus serotypes, including group A and B coxsackieviruses and echoviruses, which are commonly implicated in human viral heart disease. Strand-specific in situ hybridization led to detection of viral plus-strand or minus-strand RNA in infected cell cultures and in myocardial tissue sections of infected mice. In consecutive sections either viral genomic plus-strand RNA or complementary minus-strand RNA were localized in the same infected myocardial cells. In situ hybridization with enterovirus-specific and highly sensitive single-stranded RNA probes is of particular interest for the diagnosis of myocardial infections and for studies concerning viral RNA replication.

Animals↗
PubMed1997

Thalamic-projecting preprocholecystokinin messenger RNA-expressing neurons in the dorsal column nuclei of the rat.

This study aimed at investigating the expression of preprocholecystokinin messenger RNA among thalamic-projecting neurons in the dorsal column nuclei of the rat. Thalamic-projecting neurons were identified by injection of cholera toxin subunit b into the ventroposterolateral nucleus. Following immunohistochemical detection of retrogradely transported tracer substance, the expression of preprocholecystokinin messenger RNA in the projection neurons of the dorsal column nuclei was detected by in situ hybridization, using autoradiographic visualization of a 35S-labeled RNA probe complementary to preprocholecystokinin messenger RNA. Many preprocholecystokinin-expressing neurons were seen in the dorsal column nuclei. A large proportion of these neurons were also labeled with cholera toxin. The double-labeled neurons, as well as neurons single-labeled with preprocholecystokinin messenger RNA or cholera toxin, were preferentially found within the middle region of the dorsal column nuclei, located just caudal to the obex. These findings demonstrate that neurons in the dorsal column nuclei express preprocholecystokinin messenger RNA, and show that these neurons provide a peptidergic projection from the dorsal column nuclei to the ventroposterolateral nucleus of the thalamus. These observations suggest that cholecystokinin may be involved in the transmission of somatosensory (tactile) information from the dorsal column nuclei to the thalamus.

Animals↗
PubMed1997

Expression of brain-derived neurotrophic factor and its receptor mRNA in the vestibuloauditory system of the bullfrog.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin which has been suggested to play a crucial role in the development and maintenance of the inner ear. In the present study, we investigated the expression of mRNAs of BDNF and its high-affinity receptor trkB in the vestibuloauditory system of the adult bullfrog. In situ hybridization was performed using riboprobes transcribed from Xenopus BDNF and trkB cDNA clones. BDNF mRNA was expressed in the sensory epithelia of the ampullary cristae, utricular and saccular maculae, lagena, and amphibian and basilar papillae. Strong hybridization for BDNF mRNA was also found in neuron somata of the vestibuloauditory nuclear complex. trkB mRNA was detected in the sensory epithelia of all vestibular and auditory endorgans. High levels of both BDNF and trkB mRNAs were found in vestibuloauditory ganglion cells. These results support the hypothesis that BDNF participates in the maintenance of vestibuloauditory neurons and may be important for the trophic regulation of vestibular and auditory sensory epithelia in this animal model.

Animals↗
PubMed1998

Evidence for specific RNA/protein interactions in the differential segment of the W sex chromosome in the amphibian Pleurodeles waltl.

Pleurodeles exhibits a ZZ/ZW system of GSD (genotype sex determination). However, the Z and W sex chromosomes appear to be morphologically identical. A short RNA sequence is described that was specifically bound to lampbrush loops in the differential segment of the sexual bivalent IV. The distribution of these labeled loops in experimentally produced ZZ and WW females enabled us to demonstrate that such labeled loops were perfectly correlated with the W chromosome. Therefore, this RNA sequence constitutes an excellent marker for the W differential segment. Furthermore, analysis of the labeled loops under various experimental conditions suggested that their labeling is caused by specific interactions between this RNA sequence and lampbrush loop-associated proteins (RNA/protein interactions). North-western assays revealed that nuclear polypeptide(s) of 65 kDa could be responsible for such binding.

Animals↗
PubMed1986

Structure and replication of the genome of the hepatitis delta virus.

The hepatitis delta virus can be found in the serum and liver of some hepatitis B virus patients. We now report that the RNA genome of serum-derived delta virus is single-stranded and circular. Livers of infected chimpanzees or woodchucks contained as many as 300,000 copies of genomic strand RNA per average cell, and at least some of this RNA had a circular conformation. Also present in the livers were RNA species complementary to the virion RNA. The genomic RNA was 5-22 times more abundant than this antigenomic strand. Some of the antigenomic RNA was complexed with genomic RNA, as evidenced by the fact that at least 34% of the antigenomic RNA was resistant to digestion with either RNase A in 0.3 M NaCl or S1 nuclease. Some of the antigenomic RNA was in a circular conformation. These and other findings showed that the structure and replication of hepatitis delta virus are in many ways similar to those of the previously described plant viroids, virusoids, and satellite RNAs.

Animals↗
PubMed1991

Antisense oligonucleotides from the stage-specific myeloid zinc finger gene MZF-1 inhibit granulopoiesis in vitro.

Zinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony-stimulating factor-driven granulocyte colony formation in vitro.

Adult↗
PubMed1981

In vitro replication of bacteriophage GA RNA. Subunit structure and catalytic properties of GA replicase.

An RNA replicase of GA phage, one of the Group II RNA phages, was isolated and purified to a homogeneous state. By SDS polyacrylamide gel analysis, the purified GA replicase was found to contain four different subunits, numbered I, II, III, and IV, the molecular weights of which were 74,000, 60,000, 47,000, and 36,000, respectively. Three of them, I, III, and IV, proved to be host-coded proteins, ribosomal protein S1 (I), and elongation factors Tu (III) and Ts (IV) of protein biosynthesis, respectively. On a phosphocellulose column, the RNA replicase was separated into two components: One composed of subunits I and II, and the other composed of subunits III and IV. Each component alone had no replicase activity. However, when the two components were combined at 0 degree C, 60% of the replicase activity was restored within 10 min. The purified GA replicase catalyzed the GA phage RNA-directed synthesis of template-size RNA. However, the maximum level of product RNA synthesized was less than 20% of the amount of template RNA added. RNA-RNA hybridization experiments indicated that the product RNA included only the RNA strand complementary to the template RNA, and not the viral strand.

Catalysis↗
PubMed1996

Functional analysis of the Enterococcus faecalis plasmid pAD1-encoded stability determinant par.

The molecular organization and functional characteristics of the PAD1 replicon-encoded par stability determinant were examined. par encodes two convergently transcribed RNAS of approximately 210 and 65 nucleotides designated RNA I and RNA II, respectively. The sequence of RNA II is largely complementary to RNA I, suggesting that RNA II could regulate RNA I function as an anti-sense RNA. Results of functional studies are consistent with a role for par as a post-segregational killing system, the first to be identified in Gram-positive bacteria, with RNA I encoding the toxin and RNA II the antidote. These results include: (i) destabilization of par-containing replicons in the presence of a second complete par or the RNA II coding sequence in the same cell; (ii) par-dependent stabilization of a highly unstable vector at the expense of host-cell growth rate; and (iii) protection of cells from the toxic effects of overexpression of RNA I by RNA II supplied in trans.

Base Sequence↗
PubMed1988

A homologous in vitro system to analyze transcription of a mouse immunoglobulin mu heavy-chain gene.

In order to investigate the molecular mechanisms of the regulation of immunoglobulin (Ig) gene transcription, a cell-free system was developed in which a cloned mouse Ig mu heavy-chain gene was transcribed using nuclear extracts prepared from a mouse B cell hybridoma line. To monitor transcription, an RNA.RNA hybridization assay was developed in which a 32P-labeled, SP6-synthesized RNA probe complementary to Ig mu RNA was hybridized to unlabeled RNA transcribed in the nuclear extract. Accurate initiation of transcription, which resulted in the protection of the RNA probe from digestion with nuclease S1, was detected by the separation of the products on denaturing polyacrylamide gels, followed by autoradiography. Using this assay, an in-vitro-synthesized RNA was detected. The 5' end of the in-vitro-transcribed Ig mu RNA maps exactly to the same position as the 5' end of the corresponding in vivo mRNA and its formation was sensitive to the addition of low levels of alpha-amanitin (1 microgram/ml), indicating transcription by RNA polymerase II. It was shown by competition experiments with oligonucleotides containing the 'decamer recognition site' that this sequence interacts with (a) decamer-binding factor(s) and plays a positive role in transcription. The competition effects of the decamer-containing oligonucleotide appeared to be restricted to the decamer motif present in the promoter region. No effects of the enhancer region were detectable in vitro. Little or no transcriptional activity was found in transcription experiments using the Ig mu promoter and nuclear extracts prepared from HeLa cells. This suggests that tissue-specific factors involved in Ig mu heavy-chain gene transcription are present in the mouse B cell extracts.

Amanitins↗
PubMed1989

In situ hybridization for quantitative assay of infectious hepatitis A virus.

A method of in situ hybridization using single-stranded RNA probes of opposite polarity for quantitative enumeration of hepatitis A virus (HAV) in infected cells has been developed. Kinetic experiments showed that foci of infected cells appeared as early as day 2 postinfection. The absence of foci in cells examined immediately after virus adsorption indicated that foci detected subsequently were related to viral replication. Foci were detected by hybridization with RNA probes complementary to HAV genomic RNA but not with RNA probes identical to HAV genomic RNA. The number of foci observed was linearly related to the HAV dose inoculated. Focus formation was reduced when a virus inoculum was pretreated with guinea pig anti-HAV hyperimmune serum but not when it was pretreated with preimmune serum. The high resolution of hybridization signals and relative rapidity of the test indicated that this technique will be useful for measuring serum neutralizing antibodies and for quantitative assay of infectious HAV.

Animals↗
PubMed1979

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.

Cell-Free System↗
PubMed1996

Expression of the met receptor tyrosine kinase in muscle progenitor cells in somites and limbs is absent in Splotch mice.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates proliferation, dissociation, migration and morphogenesis of cells in culture. To investigate a possible role for HGF/SF and its receptor, the Met tyrosine kinase, in embryonic development, we have analyzed their expression in mouse embryos from day 7.5 of gestation by whole-mount in situ hybridization. Met expression is first detected in the ventral portion of somites at day 9.25 of gestation (22 somite embryo) at the level of fore limb buds. As somites mature, met expression is detected in caudal somites, and is confined to the lateral and media] tips of the dermomyotome and dermomyotome/myotome respectively. In contrast, HGF/SF is expressed exclusively in the mesodermal core of the limb bud. As the dermomyotome elongates ventrolaterally, the met-expressing cells at the lateral tip appear to detach from the somite, invade the limb bud and localize at the dorsal and ventral limb sides in close proximity to HGF/SF-expressing cells. At later stages, both met- and HGF/SF-expressing cells appear to migrate distally and localize to the digit forming area of the developing hand plate. Met expression in the lateral dermomyotome and limb bud coincides with expression of Pax-3, a marker for migrating muscle precursor cells in the somite and limb. Splotch-2H and Splotch-delayed mice, which harbor mutations in Pax-3, show major disruptions in early limb muscle development. Significantly, no met-expressing cells were observed in the limbs of homozygous Splotch-2H and Splotch-delayed animals, whereas HGF/SF expression was not affected. The restricted expression of met to a sub-population of Pax-3-expressing cells in the lateral tip of the dermomyotome, demonstrates that met represents a unique molecular marker for this migratory cell population. From these observations, together with the biological activities of HGF/SF, we propose that in homozygous Splotch embryos the failure of muscle precursors to migrate into and populate the limb bud results from a loss of met expression in the cells at the ventrolateral edge of the somitic dermomyotome.

Animals↗
PubMed1998

Inhibitory effects of modified oligonucleotides complementary to the leader RNA on the multiplication of mouse hepatitis virus.

Phosphorothioate oligonucleotides (PS-oligo) and PS-oligos with cholesterol conjugates (ChPS-oligo) complementary to the leader RNA of strain JHM of mouse hepatitis virus (JHMV) were more effective inhibitors of viral multiplication than natural oligodeoxynucleotides (PO-oligo) in JHMV-infected DBT cells. PS- and ChPS-oligos were 1,000 times more potent than unmodified PO-oligo. No significant difference was observed in the inhibitory efficiency between PS-oligo and ChPS-oligo. Sequence-dependent inhibition of viral multiplication was shown at low concentrations (0.001-0.1 M) of antisense PS-oligo and ChPS-oligo. Phosphorothioate oligodeoxycytidine, PS-(dC)20, and PS-(dC)20 with cholesterol conjugates, and PS- and ChPS-oligo which have no significant homology to the JHMV sequences, showed inhibitory effects on JHMV multiplication at concentrations higher than 0.5 M. These results showed that PS-oligo and ChPS-oligo were more potent than PO-oligo in the inhibition of JHMV multiplication, and that PS-oligo and ChPS-oligo may inhibit JHMV multiplication by two different mechanisms, that is by sequence-dependent and sequence-independent manners.

5' Untranslated Regions↗