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PubMed1979

Diversity of mammary tumor viral genes within the genus Mus, the species Mus musculus, and the strain C3H.

Proviral sequences complementary to the C3H mouse mammary tumor virus RNA genome are present in the DNA of early occurring mammary tumors of C3H/HeN mice and are absent from apparently normal C3H/HeN tissues; these sequences are non-germ line transmitted in C3H/HeN mice and have been termed tumor-associated sequences; (W. Drohan et al., J. Virol. 21:986-995, 1977). We report here that tumor-associated sequences are present in the DNA of spontaneous mammary tumors that occur early in the life of several inbred, high-tumor-incidence mouse strains but are absent in mammary tumors that occur later in life in low- and moderate-tumor-incidence strains. These sequences are also absent in apparently normal organs tested from numerous laboratory mouse strains, feral mice, Mus musculus subspecies, and other Mus species. Sequences represented in tumor-associated sequence RNA, however, are present as endogenous provirus in GR mice (at approximately four copies per haploid genome) and in two of five substrains of C3H mice tested (at approximately one copy per haploid genome). The two substrains of C3H mice positive for endogenous tumor-associated sequence provirus were recently (circa 1930) separated from the negative substrains of C3H mice. The results may be explained by the unlikely chance segregation of proviral sequences or by the recent integration of viral genes (within the last few decades). Whereas radioactively labeled mouse mammary tumor virus 60-70S RNA or complementary DNA detected mouse mammary tumor virus-related proviral information in all laboratory mouse strains, feral mice, subspecies of M. musculus, and other species of Mus, the use of tumor-associated sequence RNA clearly revealed the genetic diversity that may exist between different colonies or substrains of "inbred" laboratory mice commonly used in cancer research.

Animals↗
PubMed1997

Insights into the interaction between tRNA and primer binding site from characterization of a unique HIV-1 virus which stably maintains dual PBS complementary to tRNA(Gly) and tRNA(His).

Previously our laboratory constructed an HIV-1 which stably maintained a primer binding site (PBS) complementary to tRNA(His) by mutating the region of the provirus within U5 postulated to interact with the anticodon of tRNA(His) (J. Wakefield, S-M Kang, and C. D. Morrow, 1996, J. Virol, 70, 966-975). From the analysis of the virus obtained after long-term culture, we identified an unusual proviral DNA in which the U5-PBS region contained a dual PBS complementary to tRNA(Gly) and tRNA(His), respectively, separated by a 21-nucleotide intervening sequence. To determine if this U5-PBS region containing the dual PBS would give rise to an infectious virus, the mutant U5-PBS containing the dual PBS was subcloned into an infectious HIV-1 proviral clone, pHXB2; the resultant proviral DNA was designated as pHXB2(Gly-His). Transfection of pHXB2(Gly-His) into cells gave rise to infectious virus. Analysis of the U5-PBS region revealed that the virus stably maintained the dual PBS rather than revert back to the wild-type PBS. In addition to genomes with the PBS complementary to tRNA(Gly) and tRNA(His), proviral genomes were identified after extended in vitro culture which contained dual PBS complementary to tRNA(Gly) and tRNA(Phe). To determine which PBS could be used for reverse transcription, we utilized an endogenous reverse transcription/PCR method which could discriminate (based on molecular size of the products) between the minus strand DNA initiated from the two PBSs. The results of this assay demonstrated that either the PBS complementary to tRNA(Gly) or tRNA(His) could be used for the initiation of reverse transcription. The results of our study highlight the complex interrelationship between U5-PBS and primer tRNA required for positioning the tRNA at the PBS and provides new insights into how the tRNA primer used to initiate reverse transcription is selected.

Animals↗
PubMed1989

Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein.

Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein. One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence. Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column. Binding affinities were in the millimolar-micromolar range. Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence. Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer. Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism. Proteolytic degradation was retarded by the binding of these peptides. Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract.

Adenosine Triphosphate↗
PubMedDate not supplied

Liposome-mediated transfer of vascular endothelial growth factor cDNA augments survival of random-pattern skin flaps in the rat.

Tissue engineering is an application for gene therapy that is in its infancy. We show that simple liposomal-mediated gene transfer could result in a potentially useful biological effect in the field of wound healing. cDNA encoding the 165 amino acid form of vascular endothelial growth factor complexed to commercially available liposomes was injected into rat skin 1 week before raising a random pattern 3 x 10 cm flap. The flap survival was enhanced by 14 percent, and was accomplished without accessing the arterial inflow of the territory. These results were statistically significant (p<0.002) and reproducible. No adverse effects were seen. Histological analysis of the angiogenesis localized much of the new vessel formation to the area around the hair follicles. Polymerase chain reaction amplification of extracted flap tissue confirmed the presence of the transgene.

Animals↗
PubMed1994

Localization of diazepam-binding inhibitor and peripheral type benzodiazepine binding sites in the rat ovary.

Diazepam-binding inhibitor (DBI) is the precursor of a family of peptides, including an octadecaneuropeptide (ODN), that share with DBI the ability to specifically displace benzodiazepines (BZD) from their receptors. An association of ODN with the peripheral type BZD receptors (PBR) has been reported in the brain and a few peripheral tissues. In order to investigate whether DBI and PBR are present in ovarian tissue, we have localized DBI by means of immunocytochemistry, in situ hybridization and autoradiography of PBR in the rat ovary. Immunocytochemical localization was achieved by means of rabbit antibodies developed against rat ODN. Immunostaining was located in the cytoplasm of the theca interna, corpus luteum and interstitial gland cells, but not in the granulosa cells. Hybridization signal obtained following in situ hybridization with a [35S]-labelled single-stranded RNA probe complementary to DBI mRNA was observed in all the steroid-secreting cells, including granulosa cells of developing and mature follicles. Autoradiographic localization of PBR obtained by incubating ovary sections with [3H] PK11195, a ligand selective for PBR, revealed the presence of specific labelling in all the steroid-secreting cells. These results, which demonstrate for the first time that the ovarian steroid-secreting cells contain both PBR and its endogenous ligand, suggest that the BZD receptor might be involved in the regulation of ovarian function.

Animals↗
PubMed1995

Sleep-waking changes after c-fos antisense injections in the medial preoptic area.

The preoptic hypothalamus has been consistently implicated in the homeostatic regulation of sleep and waking. Recently, it was shown that periods of either spontaneous or forced wakefulness result in the induction in this region of the immediate-early gene c-fos. In this study, antisense oligonucleotides complementary to c-fos mRNA were employed to interfere with the expression of Fos protein. Injections in the rat medial preoptic area of c-fos antisense, but not of sense, oligonucleotides blocked the expression of Fos protein detected immunocytochemically. Rats receiving bilateral antisense injections showed a higher percentage of wakefulness the day after the injection than controls receiving sense or sham injections or antisense injections outside the preoptic area. These results suggest that blocking the expression of Fos protein in the preoptic hypothalamus may interfere with the homeostatic regulation of sleep and wakefulness.

Animals↗
PubMed2005

Identification and characterization of two antisense transcripts from the major immediate early region of rat cytomegalovirus.

We have identified and characterized two antisense transcripts from the rat cytomegalovirus (RCMV) major immediate early (MIE) region. These transcripts, designated IE-AS1 and IE-AS2, are complementary to part of the sense IE1 transcript. The IE-AS transcripts were first detected in peripheral blood leukocytes (PBL) of RCMV-infected rats at 7 days post-infection (pi) in the absence of IE1 transcription. Nevertheless, both the IE1 and IE-AS transcripts were found at the same time in the salivary glands of RCMV-infected rats at 7 and 120 days pi as well as in RCMV-infected rat embryo fibroblasts (REFs) at 48 h pi.

Amino Acid Sequence↗
PubMed1995

Inhibition of translation initiation on Escherichia coli gnd mRNA by formation of a long-range secondary structure involving the ribosome binding site and the internal complementary sequence.

Previous research has indicated that the growth rate-dependent regulation of Escherichia coli gnd expression involves the internal complementary sequence (ICS), a negative control site that lies within the 6-phosphogluconate dehydrogenase coding sequence. To determine whether the ICS acts as a transcriptional operator or attenuator, we measured beta-galactosidase-specific activities in strains carrying gnd-lac operon and protein fusions containing or lacking the ICS. Whereas the presence of the ICS repressed beta-galactosidase expression from a protein fusion by 5-fold during growth on acetate and by 2.5-fold during growth on glucose, it had no effect on beta-galactosidase expression from an operon fusion. In vitro ribosome binding experiments employing the primer extension inhibition (toeprint) assay demonstrated that the presence of the ICS in gnd mRNA reduces both the maximum extent and the rate of ternary complex formation. Moreover, the effects of deletions scanning the ICS on in vivo gene expression were highly correlated with the effects of the deletions on ribosome binding in vitro. In addition, the distal end of the ICS element was found to contribute more to ICS function than did the proximal portion, which contains the complement to the Shine-Dalgarno sequence. Finally, RNA structure mapping experiments indicated that the presence of the ICS in gnd mRNA reduces the access of the nucleotides of the ribosome binding site to the single-strand-specific chemical reagents dimethyl sulfate and kethoxal. Taken together, these data support the hypothesis that the role of the ICS in the growth rate-dependent regulation of gnd expression is to sequester the translation initiation region into a long-range mRNA secondary structure that blocks ribosome binding and thereby reduces the frequency of translation initiation.

Acetates↗
PubMed2004

An intronic mutation causes long QT syndrome.

OBJECTIVES: The purpose of this research was to determine whether an intronic variant (T1945+6C) in KCNH2 is a disease-causing mutation, and if expanded phenotyping criteria produce improved identification of long QT syndrome (LQTS) patients. BACKGROUND: Long QT syndrome is usually caused by mutations in conserved coding regions or invariant splice sites, yet no mutation is found in 30% to 50% of families. In one such family, we identified an intronic variant in KCNH2. Long QT syndrome diagnosis is hindered by reduced penetrance, as the long QT phenotype is absent on baseline electrocardiogram (ECG) in about 30%. METHODS: Fifty-two family members were phenotyped by baseline QTc, QTc maximum on serial ECGs (Ser QTc-max), and on exercise ECGs (Ex QTc-max) and by T-wave patterns. Linkage analysis tested association of the intronic change with phenotype. The consequences of T1945+6C on splicing was studied using a minigene system and on function by heterologous expression. RESULTS: Expanded phenotype/pedigree criteria identified 23 affected and 29 unaffected. Affected versus unaffected had baseline QTc 484 +/- 48 ms versus 422 +/- 20 ms, Ser QTc-max 508 +/- 48 ms versus 448 +/- 10 ms, Ex QTc-max 513 +/- 54 ms versus 444 +/- 11 ms, and LQT2 T waves in 87% versus 0%. Linkage analysis demonstrated a logarithm of odds score of 10.22. Splicing assay showed T1945+6C caused downstream intron retention. Complementary deoxyribonucleic acid with retained intron 7 failed to produce functional channels. CONCLUSIONS: T1945+6C is a disease-causing mutation. It alters KCNH2 splicing and cosegregates with the LQT2 phenotype. Expanded ECG criteria plus pedigree analysis provided accurate clinical diagnosis of all carriers including those with reduced penetrance. Intronic mutations may be responsible for LQTS in some families with otherwise negative mutation screening.

Adult↗
PubMed1991

Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma 2L subunit.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Amino Acid Sequence↗
PubMed1997

In situ hybridization study of cytokeratin 4, 13, 16 and 19 mRNAs in human developing junctional epithelium.

Cytokeratins (CKs) are now considered to be reliable markers for following the development and differentiation of epithelial tissue. We have investigated the pathway of differentiation in human developing junctional epithelium using monoclonal antibodies and two-dimensional gel electrophoresis of microdissected tissue to identify CK 19, CK 16, CK 14, CK 13, CK 6, CK 5, CK 4 in the junctional epithelium (JE) over partially erupted human teeth. The CK profile was similar to that of developing oral epithelia, suggesting that the junctional epithelium in teeth during eruption is of odontogenic origin. The present study used in situ hybridization to determine the distribution of the mRNAs of CKs 19, 16, 13 and 4 in human developing junctional epithelium and to examine the correlation between mRNAs and their encoded proteins. CK 19 mRNA was abundant in the basal cell layers of the primary junctional epithelium (PJE) but less concentrated in the suprabasal layers. CK16, 13 and 4 mRNAs were abundant in the basal cell layers of the PJE. The parabasal cell layers reacted intensely to the cRNA probe complementary to CK16 mRNA, as were the reactions in the suprabasal cell layers of the PJE for the CK 13 and 4 probes. Our results demonstrate that the PJE express the genes encoding for CKs 16 and 4 that have been revealed previously only by electrophoresis. They therefore confirm that the PJE is a well-differentiated stratified epithelium with a complex unique phenotype that produces CKs specific for basal cells (CK 19), CKs associated with hyperproliferation (CK 16), and finally those associated with stratification (CKs 4 and 13). Only synthesis of CK 19 protein and mRNA are strictly parallel. CKs 4 and 13 mRNAs are present in basal and suprasal cells, while their encoded proteins were not, except for CK 13 in suprabasal cell layers of PJE, where the amount of its mRNAs was coincident with the expression of the protein.

Adolescent↗
PubMed2002

Increased concentrations of radioisotopically-labeled complementary ribonucleic acid probe, dextran sulfate, and dithiothreitol in the hybridization buffer can improve results of in situ hybridization histochemistry.

The goal of the present studies was to optimize mRNA detection with radioisotopic in situ hybridization histochemistry (ISHH). Test experiments performed on sections of rat brain tissue used computer-assisted image analysis to compare autoradiographic signals resulting when varying concentrations of (35)S-labeled cRNA probes, dextran sulfate (DS), and dithiothreitol (DTT) were used for ISHH. We found that greatly enhanced corrected signal density (total density of signal area minus background density) was obtained using concentrations of probe and/or DS that were several-fold higher than those widely recommended in published ISHH procedures (probe concentration >4 x 10(4) cpm/microl; DS concentration >10%). Extended hybridization reaction (>16 hr) also significantly augmented the corrected signal density. Finally, nonspecific probe binding was greatly reduced and corrected signal density enhanced by including 750-1000 mM, rather than the widely used 10-200 mM DTT, in the hybridization buffer. These observations indicate that the low efficiency of hybridization and the formation of high background may largely compromise the sensitivity of routine ISHH procedures. We suggest that the new method using increased concentrations of (35)S-labeled cRNA probe, DS, and DTT will be especially important for the cellular localization of rare mRNA species.

Animals↗
PubMed2002

A novel sensitive microarray approach for differential screening using probes labelled with two different radioelements.

We have developed a novel microarray approach for differential screening using probes labelled with two different radioelements. The complementary DNAs from the reverse transcription of mRNAs from two different biological samples were labelled with radioelements of significantly different energies (3H and 35S or 33P). Radioactive images corresponding to the expressed genes were acquired with a MicroImager, a real time, high resolution digital autoradiography system. An algorithm was used to process the data such that the initially acquired radioactive image was filtered into two subimages, each representative of the hybridisation result specific for one probe. The simultaneous screening of gene expression in two different biological samples requires <100 ng mRNA without any amplification. In such conditions, the technique is sensitive enough to directly quantify the amount of mRNA even when present in small amounts: 10(7) molecules in the probe as assessed with an added control sequence and 2 x 10(5) molecules with an endogenous tyrosine hydroxylase mRNA. This novel technique of double radioactive labelling on a microarray is thus suitable for the comparison of gene expression in two different biological samples available in only small quantities. Consequently, it has great potential for various biological fields, such as neuroscience.

Animals↗
PubMed2001

Teneurin-2 is expressed in tissues that regulate limb and somite pattern formation and is induced in vitro and in situ by FGF8.

Teneurin-2 is a member of a novel family of transmembrane proteins characterized to date in fish, birds, mammals, and Drosophila (e.g., the pair-rule gene product Ten-m). We have shown that teneurin-2 is expressed by neurons in the developing avian visual system in a pattern complementary to the expression of teneurin-1 and that recombinant teneurin-2 induces morphologic changes in neuronal cells in culture (Rubin et al., 1999). Here we have used cRNA probes to two newly identified splice variants and a teneurin-2-specific antibody to determine whether teneurin-2 is also expressed outside the nervous system. Both reverse transcriptase-polymerase chain reaction and in situ hybridization indicate that the three splice variants known so far are coexpressed at sites of pattern formation during development. Teneurin-2 mRNAs and protein are found in the developing limbs, somites, and craniofacial mesenchyme. In addition to expression of teneurin-2 by the apical ectodermal ridge, teneurin-2 transcripts also appear transiently at sites of tendon development. Teneurin-2 expression patterns were strikingly similar to those of fibroblast growth factor 8 (FGF8). In agreement with the overlapping expression pattern, FGF8-coated beads implanted into chicken limb buds induced the ectopic expression of teneurin-2 and soluble FGF8 induced teneurin-2 in limb explant cultures. Thus, teneurin-2 could act downstream of FGF8 during morphogenesis.

Alternative Splicing↗
PubMed2003

Down-regulation of GABA-transporter function by hippocampal translation products: its possible role in epilepsy.

The genetically epileptic mouse strain (El) is used as a model for human temporal lobe epilepsy. To address the question of whether altered function of the neuronal GABA transporter GAT1 is involved in the pathology of epilepsy of El mice, we expressed in Xenopus oocytes cloned GAT1 of mouse brain by injection of complementary ribonucleic acid (cRNA) and co-injected messenger ribonucleic acid (mRNA) isolated from the hippocampus of non-epileptic control mother strain (ddY) mice and from El mice. GABA transporter activity was investigated by measurements of [(3)H]-GABA uptake as well as by steady-state and transient current measurements under voltage clamp.Co-injection of hippocampal mRNA into oocytes reduced GAT1-mediated transport. This effect was more pronounced for mRNA from ddY mice than for that from El mice that never experienced seizures, El(-), and being absent for mRNA from El mice that have had high seizure experience, El(+). The pronounced inhibition of GABA transport after injection of mRNA from the ddY strain results from reduced expression of functional GAT1, but to about one third also from a reduced GABA translocation rate. The reduced translocation can be attributed to a reduced forward rate of a step associated with extracellular Na(+) binding. If the results can be applied to the mouse brain, we may hypothesise that in ddY mice some GAT down-regulating factor translated from hippocampal mRNA may be involved to keep GAT1 activity low, and hence GABA concentration in synaptic cleft high. In El(-) mice such regulatory mechanism may be reduced or counteracted by another unknown factor present in El(-) brain. The repeated seizure experience in El(+) mice enhances this compensatory effect.

Animals↗
PubMed2004

Expression of regulatory genes during differentiation of thalamic nuclei in mouse and monkey.

Expression patterns of genes implicated in development of the thalamus were examined in mice and monkeys, using in situ hybridization with RNA probes complementary to Cad6, Dlx1, Dlx2, Dlx5, Gbx2, Id2, and Lef1 cDNAs. Expression patterns were related to the evolving cytoarchitecture in mice at birth (P0) and in adulthood, and in fetal monkeys early and late in the period of gestation when thalamic nuclei are becoming histologically differentiated out of a series of pronuclear masses. At the earlier developmental stage, each gene was expressed in a pattern that appeared to be pronucleus-specific and maintained a nucleus-specific pattern into adulthood, with the possible exception of Gbx2. Each gene displayed a unique expression pattern in the dorsal thalamus, ventral thalamus, and epithalamus, and no gene was expressed throughout all three divisions or in every nucleus of a division. With the exception of Dlx2, whose expression disappeared at the later time point, all continued to be expressed into adulthood at higher levels and with identical patterns. Despite late appearance of gamma-aminobutyric acid (GABA)ergic cells in the dorsal lateral geniculate nucleus of mice, no Dlx genes, which promote formation of a GABAergic phenotype elsewhere, were detected in dorsal thalamus. Each thalamic nucleus was distinguished by expression of a combination of genes, and homologous nuclei in mouse and monkey exhibited the same combination. The presence of a centre médian nucleus and four pulvinar nuclei in monkeys was marked by patterns of expression not found in mice. The centre médian nucleus was marked by high expression of Id2, which was expressed only weakly in very few nuclei of mice.

Animals↗
PubMed2005

Expression of the glucocorticoid-induced receptor mRNA in rat brain.

The glucocorticoid-induced receptor (GIR) is an orphan G-protein-coupled receptor awaiting pharmacological characterization. GIR was originally identified in murine thymoma cells, and shows a widespread, yet not completely complementary distribution in mouse and human brain. Expression of the mouse GIR gene is modulated by dexamethasone in the brain and periphery, suggesting that GIR function is directly responsive to glucocorticoid signals. The rat GIR was cloned from rat prefrontal cortex by our group and was shown to be up-regulated following chronic amphetamine. The physiological role of GIR in the rat is not known at present. In order to gain a clearer understanding of the potential functions of GIR in the rat, we performed a detailed mapping of GIR mRNA expression in the rat brain. GIR mRNA showed widespread distribution in forebrain limbic and thalamic structures, and a more restricted distribution in hindbrain areas such as the spinal trigeminal nucleus and the median raphe nucleus. Areas with moderate to high levels of GIR include olfactory regions such as the nucleus of olfactory tract, hippocampus, various thalamic nuclei, cortical layers, and some hypothalamic nuclei. In comparison with previous studies, significant regional differences exist in GIR distribution in mouse and rat brain, particularly in the thalamus, striatum and in hippocampus at a cellular level. Overall, the expression of GIR in rat brain more closely approaches that seen previously in human than mouse, suggesting that rat models may be more informative for understanding the role of GIR in glucocorticoid physiology and glucocorticoid-related disease states. GIR mRNA distribution in the rat indicates a potential role of this receptor in the control of feeding and ingestive behavior, regulation of stress and emotional behavior, learning and memory, and, drug reinforcement and reward.

Animals↗