Labeled cRNA Probes Research Articles

LOCALIZATION OF P 2Y1 PURINOCEPTOR TRANSCRIPTS IN THE RAT PENIS AND URINARY BLADDER

Purpose The aim of this study was to determine the expression and localization of the P2Y 1 purinoceptor mRNA in rat penis and urinary bladder using reverse transcription polymerase chain reaction (RT-PCR), northern blotting and in situ hybridization (ISH). Materials and Methods RT-PCR: First strand cDNA was prepared from rat penis and urinary bladder dome total RNA and used for PCR with primers designed to amplify fragments of the P2Y 1 purinoceptor cDNA sequence. Northern blotting: PCR products were subcloned into the pGEM-5Zf(+) plasmid vector, sequenced and random primer labeled using 32 P. Labeled probe was hybridized. ISH: Digoxigenin labeled cRNA probes were synthesized by in vitro transcription. Results P2Y 1 purinoceptor mRNA was detected by RT-PCR analysis in both rat penis and urinary bladder. RNA blotting using a P2Y 1 purinoceptor cDNA probe revealed a single transcript of 4.2kb in both tissues. This band was the same size as that expressed by the heart, which contains high levels of P2Y 1 purinoceptor (Burnstock, G.: Physiological and pathological roles of purines: an update. Drug. Dev. Res., 28: 195, 1993). By ISH, P2Y 1 purinoceptor mRNA was localized in detrusor smooth muscle cells and blood vessels in urinary bladder. In penis, positive signals were detected in endothelial cells which line the lacunar space and blood vessels. No hybridization was seen in corpus cavernosum smooth muscle cells and urethra. Conclusion These results indicate that mRNAs for P2Y 1 purinoceptor are expressed in detrusor smooth muscle cells and blood vessels of rat urinary bladder. However, in penis, this receptor is expressed in endothelial cells which lines the lacunar space and blood vessels, but not expressed in corpus cavernosum smooth muscle cells and urethra.

Developmental Expression of Monocyte (MN) Early Growth Response-1 Gene(EGR-1) in Response to Lipopolysaccharide (LPS) and Nitric Oxide (NO) † 320

The maturation of monocyte function across gestational age is an important factor in the host response of preterm and term newborns to infectious and non-infectious agents. EGR-1 is essential in promoting cell differentiation of progenitor cells to macrophages. LPS stimulates MN immune function and EGR-1 expression. Nitric oxide modulates EGR-1 expression and induces MN differentiation. Investigating the developmental expression of EGR-1 in response to LPS and to NO is relevant to our study of development of monocyte immune responses. Freshly isolated cord blood MN and peripheral blood mononuclear cells (PBMC) were stimulated with 10μg/ml LPS for 2.5 hrs or preincubated with a NO donor,S-nitroso-N-acetylpenicillamine (SNAP, 0.5mM) for 30 min, then stimulated with LPS in cell culture. Northern analysis and slot blots of mRNA for EGR-1 were performed using a 32P labeled cRNA probe for human EGR-1. In Northern blots of PBMC mRNA, EGR-1 expression increased from baseline when preterm, term and adult cells were stimulated with LPS. In slot blots of MN mRNA, EGR-1 expression again increased with LPS stimulation. With SNAP pretreatment, EGR-1 expression in response to LPS was blunted in adult MN whereas expression was increased in term and preterm cells. These results were confirmed with densitometry scanning. The neonatal samples had a clear increase in EGR-1 expression when pretreated with the NO donor when compared to stimulation with LPS alone. Nitric oxide modulation of the effect of LPS on EGR-1 expression is developmentally regulated. Understanding the developmental aspects of monocyte responses to the oxidant stress in various disease states will be important in the pursuit of efficacious therapies for the newborn at varying levels of maturation.

An Improved Procedure for Extracting Nucleic Acids from Citrus Tissues for Diagnosis of Citrus Viroids.

When detecting viroids in citrus tissues, contaminating polysaccharides and phenolic compounds often make it difficult to consistently extract nucleic acids. To extract the nucleic acids consistently, the method was improved as follows: the contaminants were removed by differential 2-butoxyethanol precipitation instead of a combination of 2-methoxyethanol extraction and cetyltrimethylammonium bromide precipitation. In contrast to the conventional method, the modified one consistently extracted some citrus viroids, i.e., citrus exocortis viroid (CEVd), group I citrus viroid (CVd-I) and hop stunt viroid-citrus isolate, which is a variant of group II citrus viroid (CVd-II), after they were sufficiently subjected to sequential polyacrylamide gel electrophoresis (sPAGE) and dot blot hybridization using digoxigenin (DIG)-labeled cRNA probes. A viroid-like RNA presumed to be a group III citrus viroid (CVd-III) was detected in nucleic acids extracted from Japanese citrus samples by sPAGE. The cDNA fragments of the viroid-like RNA were cloned and sequenced. The nucleotide sequences of the cDNAs were completely identical to those reported previously for CVd-IIIa and CVd-IIIb abroad. CVd-III was detected from several other Japanese citrus samples using the DIG-labeled cRNA probe prepared from the cloned cDNA. Many of these samples were also co-infected with other citrus viroids.

Atlas of the neurons that express mRNA for the long form of the prolactin receptor in the forebrain of the female rat.

Prolactin has a variety of important physiological effects on peripheral tissue and on the brain. The behavioral effects of prolactin include the induction of maternal behavior and increased food intake. Prolactin acts via its cognate receptors which have two forms, a short and a long form. The long form of the receptor is predominant in the preoptic area-hypothalamus and is positioned to support maternal behavior since this form is regulated across pregnancy and lactation (Nagano and Kelly [1994] J. Biol. Chem. 269:13337-13345; Sugiyama et al. [1994] J. Endocrinol. 141:325-333). By using in situ hybridization with [33P] labelled cRNA probe specific for the long form of the receptor mRNA(L-PRL mRNA) we have mapped, in brains from 2- and 21-day-old pregnant females, the neuroanatomical distribution of neurons expressing the long form of the receptor. Many neurons with high expression of L-PRL mRNA were located in the anteroventral periventricular nucleus, the medial preoptic area (MPO), specific subdivisions of the paraventricular and supraoptic nuclei, and in the arcuate and ventromedial nuclei. Labelled neurons were also found in limbic system structures such as the bed nucleus of stria terminalis (BST) and the medial nucleus of the amygdala, in a few thalamic nuclei, and in the central gray. All cells throughout the choroid plexus expressed high levels of L-PRL mRNA. The levels of L-PRL mRNA were higher in females on day 21 of pregnancy in the MPO and in the choroid plexus, than in females on day 2 of pregnancy; levels in the ventromedial nucleus of the hypothalamus (VMH) were unchanged across pregnancy. The neuroanatomical distribution of neurons expressing L-PRL mRNA may have special relevance for the mediation of maternal behavior, lactation, sexual behavior, and feeding.

Downregulation of IGF-I mRNA expression during postnatal pancreatic development and overexpression after subtotal pancreatectomy and acute pancreatitis in the rat pancreas.

Insulin-like growth factors (IGFs) are important peptides involved in the regulation of cell growth and differentiation in many tissues. The ontogeny of IGF-I was examined in pancreata from 19-day rat fetuses, newborns and 5-, 11-, 26- and 70-day-old rats. For the regeneration studies two models were used: (i) 90% pancreatectomy was carried out and the rats were killed at 1, 2, 3 and 6 days after resection; (ii) acute pancreatitis was induced with caerulein (12 micrograms/body weight three times a day every 8 h for 2 days) and the rats were killed at 1, 2, 5, 7 and 9 days after the first injection. Total RNA was extracted by the guanidinium isothiocyanate method and Northern blots were performed using total RNA and labeled cRNA probes. Abundance of the different mRNA transcripts was estimated by densitometric scanning and normalized to the abundance of 18 S rRNA for each time point. Northern blot analysis during ontogeny showed four (0.8-1.2, 1.9, 4.7 and 7.5 kb) major transcripts in the rat pancreas and liver. Total IGF-I mRNA was 40-fold higher in the adult liver than in the adult pancreas. Moreover, in the liver, IGF-I mRNA levels were higher in the adult than in the fetus, whereas in the pancreas, the highest levels were observed around birth. During the first 3 days after pancreatectomy, a peak of maximal expression was observed after the second day. Densitometric analysis of each IGF-I mRNA species showed concomitant increases in all transcripts. After 6 days, all transcripts had returned to near-control values. IGF-I mRNA expression 2 days after pancreatectomy was 3.5-fold higher than in the newborn. During the first 2 days of acute pancreatitis induction, overexpression of IGF-I mRNA was observed. However, soon after the second day of caerulein treatment, the 7.5 kb transcripts remained elevated whereas those of the others regressed toward control values. Our results show that IGF-I mRNA is overexpressed in both models of pancreatic regeneration but downregulated in the normal adult pancreas.

QUANTITATIVE CONCORDANCE BETWEEN GLOBIN mRNAs AND SYNTHESIS OF FETAL HEMOGLOBIN (HbF) AND ADULT HEMOGLOBIN (HbA) DURING HYPOXEMIC STRESS ERYTHROPOIESIS. † 1293

Increased production of HbF can be considered a marker for hypoxemia, since increased levels of HbF synthesis have been found in newborn infants following maternal hypoxemia, placental insufficiency, and diabetes, as well as in infants with broncho-pulmonary dysplasia and children with congenital cyanotic heart disease. To examine if a quantitative relationship exists between the mRNAs encoding globins and their translation into proteins during hypoxemic erythropoietic stress, the relative amounts of the mRNAs and the relative amounts of the α,β,γ-globin synthesis in samples obtained from cord blood from infants of insulin-dependent diabetic mothers, intrauterine growth-retarded infants of preeclamptic mothers and older infants with cyanotic heart disease were measured and compared. The synthesis of globins in reticulocytes was measured by the incorporation of [3H]-leucine followed by the separation and quantitation of the polypeptides on a C4-reverse phase HPLC. The relative proportions of the mRNAs of globins were determined by RNase protection assay. Total RNA isolated from blood (50μl) was incubated with [32P]-labelled cRNA probes ofα,β,γ-globins, followed by RNase A/T1 digestion, separation of the protected fragments and densitometry of the radioactive bands by phosphorimaging. The study population showed an increase in HbF synthesis, compared to controls. There was a strong correlation (r2=0.993; n=16) between the ratio of mRNAs encoding HbF and HbA and the ratio of de novo synthesis of HbF and HbA. Therefore, under conditions of stress erythropoiesis there is a close correlation between quantitation of globin mRNAs and HbF& HbA synthesis. As well, globin mRNA quantitation could replace the usual method of HbA & HbF synthesis determination.

Haploid transcripts persist in mature human spermatozoa.

Mammalian spermiogenesis is marked by the morphological and functional differentiation of round haploid spermatids into mature spermatozoa. A molecular restructuring of the chromatin accompanies this process facilitated by the transition proteins and protamines which compact and condense the genetic material within the developing spermatid. Previous studies from this laboratory have demonstrated that human protamines PRM1, PRM2 and transition protein TNP2 transcripts are associated with round and elongating spermatids. Extending this investigation, we examined the occurrence of these transcripts in mature spermatozoa by in-situ hybridization analysis using [35S]-labelled cRNA probes. These results demonstrate that PRM1, PRM2 and TNP2 haploid-specific transcripts are present in mature spermatozoa. Quantitative analysis of the localized signal also indicates that the PRM1, PRM2 and TNP2 transcripts persist at a similar ratio to that previously described for these transcripts in human testes, i.e. PRM2 > PRM1 approximately equal to TNP2. The persistence of these transcripts in mature spermatozoa warrants further investigation.

Distribution of angiotensin II type-2 receptor (AT2) mRNA expression in the adult rat brain.

Radioactively labeled cRNA probes were used for in situ hybridization histochemistry to establish a detailed map of the sites of expression of the recently cloned angiotensin II, type 2 (AT2) receptor mRNA in the adult rat brain. The distribution of the AT2 receptor mRNA was consistent with that of the AT2 binding sites, which were previously established by autoradiographic binding studies. Thus, high AT2 receptor mRNA expression was observed in the lateral septum, in several thalamic nuclei, in the subthalamic nucleus, in the locus coeruleus, and in the inferior olive. Due to the superior resolution and sensitivity of in situ hybridization, AT2 receptor expression was localized at the cellular level, and some additional brain nuclei expressing AT2 receptor mRNA have been identified. These include the red nucleus, the pedunculopontine tegmental nucleus, the bed nucleus of the supraoptic decussation, the paragenual nucleus, and numerous brainstem nuclei. Several brain nuclei, such as the motor hypoglossal nucleus and the cerebellar nuclei, where AT2 receptor binding had previously been identified in young animals only, showed a high expression of the AT2 receptor mRNA in the adult rat. No correlation was found between the expression of the AT2 and the type 1 (AT1) receptor mRNAs. A combination of the in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemistry shows that the AT2 receptor in the lateral septum showed that the AT2 receptor was not detected in GFAP immunoreactive astroglial cells, therefore indicating that AT2 is neuronal rather than glial in this brain region.

QUANTITATIVE CONCORDANCE BETWEEN GLOBIN mRNA AND SYNTHESIS OF FETAL AND ADULT HEMOGLOBIN DURING HEMOGLOBIN SWITCHOVER IN PERINATAL PERIOD. • 1725

During the perinatal period the switchover from fetal (HbF) to adult hemoglobin (HbA) synthesis in humans, γ-globin levels decline as the proportion of β-globin increases under the influence of a developmental clock. In order to examine if a quantitative relationship existed between the mRNAs encoding globins and their translation into proteins during the period of switchover, the relative amounts of the mRNAs and the relative amounts ofα, β, γ-globin synthesis in cord blood samples were measured and compared. The synthesis of globins in reticulocytes was measured by the incorporation of [3H]-leucine followed by the separation and quantitation of the polypeptides on a C4-reverse phase HPLC equipped with an integrator. The relative proportions of the mRNAs of globins were determined by RNase protection assay. Total RNA isolated from cord blood was incubated with [32P]-labelled cRNA probes (of equivalent specific activity) of α, β, γ-globins overnight at 50°C, followed by RNAse A (10 μg/ml) /T1 (200 U/ml) digestion at 25°C, separation of the protected fragments on 6% urea-polyacrylamide gels and densitometry of the radioactive bands by phosphorimaging. A comparison cord blood samples from 18 newborn infants of different gestational ages (27-40 weeks; birth weight: 850-4035 g) with no congenital anomalies, revealed a strong and significant correlation (r2= 0.934) between the ratio of mRNAs encoding HbF and HbA and the ratio of de novo synthesis of HbF and HbA. There was a linear relationship between the proportions of mRNAs encoding HbF and HbA with the proportional synthesis of HbF and HbA throughout the gestational age studied. This information can be useful for studies furthering the understanding of the mechanisms regulating globin gene expression.

Connexin 32 mRNA levels in the rat supraoptic nucleus: up-regulation prior to parturition and during lactation.

Toward the end of pregnancy, mammalian mothers undergo a cascade of hormonal, neural and somatic events that culminate in birth and lactation. Milk ejection, the final step in lactation, is regulated by high-frequency, synchronized firing of oxytocinergic neurons in the magnocellular nuclei of the hypothalamus. This synchronization of neural activity may be regulated by an increase in the cell-to-cell coupling of magnocellular neurons by connexins. Direct intercellular channels between neurons are formed by the connexin 32 protein. The present study examined the pattern of connexin 32 mRNA levels using semiquantitative in situ hybridization histochemistry with isotopically labeled cRNA probes complementary to connexin 32 and connexin 43, found in neurons/oligodendrocytes and astrocytes, respectively. Connexin 32 mRNA levels were relatively low in the supraoptic nucleus of virgin females and were dramatically up-regulated during late pregnancy. Immediately after birth, connexin 32 mRNA levels dropped and were similar to levels in virgins. On the 13th day of lactation when dye-coupling between magnocellular neurons is high, connexin 32 message levels were again very high. Levels of the astrocytic connexin 43 were no different in lactating and virgin animals. These results demonstrate that there are two peaks of connexin 32 expression, one immediately preceding parturition and one during lactation. So far, only the second peak of connexin 32 is known to be related to an increase in dye-coupling of magnocellular neurons in the supraoptic nucleus, suggesting that in this case, the elevation of connexin 32 message levels leads to the subsequent increase in intercellular coupling.

Monitoring Prunus Necrotic Ringspot Virus Infection by Hybridization with a cRNA Probe after in vitro Micrografting

In vitro micrografting was tested as a technique for inoculating peach [Prunus persica (L.) Batsch] shoot cultures with Prunus necrotic ringspot virus (PNRSV). Cultured `Suncrest' shoots derived from a naturally infected tree (as indicated by ELISA testing) maintained virus in vitro, with virus concentrations in growing tips and folded leaves being several times those of fully expanded leaves. Infected shoots served as graft bases and source of the virus. Grafted tips were derived from `Suncrest' trees that had tested negative for the virus. Leaf samples were collected from the tips following grafting and analyzed for the presence of virus by slot-blot hybridization with a (DIG)-labeled cRNA probe derived from PNRSV RNA 3. Rates of successful grafting ranged from 55% to 73% in three trials and PNRSV was found in all tips analyzed. Virus concentrations approximated those found in source shoots, suggesting that in vitro micrografting should be useful for screening transformed peach shoots for coat protein-mediated resistance to PNRSV. Chemical name used: digoxigenin (DIG).

Coordinate Expression of the PRM1, PRM2, and TNP2 Multigene Locus in Human Testis

Maintenance of the transcriptionally inert state of the mature human spermatozoon requires the expression of the various members of the human protamine gene cluster prior to the final stages of spermatogenesis. During this process, known as spermiogenesis, round spermatids morphologically differentiate into mature spermatozoa. The expression of the PRM1, PRM2, and TNP2 genes facilitates the compaction and condensation of the genetic material within the developing spermatid. To understand better the coordinate control governing this transformation, we have examined the localization and distribution of the human protamines PRM1 and PRM2 and transition protein TNP2 transcripts during human spermatogenesis. The stage-specific expression of these transcripts was determined by in situ hybridization analysis using [alpha-35S]-labeled cRNA probes. PRM1, PRM2, and TNP2 transcripts were abundant in association with round and elongating spermatids, located in the adluminal region of the seminiferous epithelium. They were not observed in association with spermatogonia, spermatocytes, Sertoli cells, or interstitial cells. These data indicate that the human PRM1, PRM2, and TNP2 transcripts are expressed postmeiotically in round and elongating spermatids. The quantitative evaluation of each transcript was determined as a function of the relative optical density per unit area. In all cases examined, the relative level of each transcript was consistent with the following pattern, PRM2 > PRM1 congruent to TNP2.

Differential opioid gene expression in neurons and neuroendocrine cells of rat and human gastrointestinal tract

There is immunocytochemical evidence that peptides derived from all three opioid precursors are present in intrinsic neurons of the gastrointestinal (GI) tract of human and rodents (1,2,3). During development, opioids show a non-neuronal and non-endocrine expression in many tissues and the expression of opioids in immune cells is under discussion. Therefore, the aim of this study was to evaluate the presence, relative abundance and distribution of the mRNAs encoding the opioid precursors proenkephalin (PENK), prodynorphin (PDYN) and proopiomelanocortin (POMC) in the human and rat GI tract using in situ hybridization histochemistry with 35S labeled cRNA probes. PENK mRNA is expressed at high levels in myenteric neurons while the labelling for PDYN mRNA was weaker in intrinsic neurons. In addition, PDYN mRNA was detected in a subpopulation of epithelial cells in the small and large intestine. POMC gene expression in the intestine of both species was below detection level. There was no evidence for expression of opioids in gut immune cells. The expression of PENK mRNA and PDYN mRNA in intrinsic neurons supports the concept of opioid modulation of gut motility. Epithelial expression of PRO-DYN mRNA suggests a role in autocrine and paracrine function of the intestinal epithelium.

Chemiluminescent detection of potato and pome fruit viroids by digoxigenin-labeled dot blot and tissue blot hybridization

A chemiluminescent molecular hybridization protocol was compared to 32P autoradiography for detecting potato spindle tuber viroid (PSTVd) and apple scar skin group viroids (ASSVd). Labeled cRNA probes for PSTVd and ASSVd were synthesized by SP6 RNA polymerase transcription using digoxigenin-11-UTP or α-[ 32P]UTP. Dot blot hybridization of purified viroids and sap extracts from infected plants showed that chemiluminescent detection using digoxigenin-labeled probes was as sensitive as autoradiography using 32P probes. A minimum of 2.0–2.5 pg purified viroid was detected. ASSVd could be detected in as little as 0.4 ng of total nucleic acid extract from infected tissue or in sap extracts diluted to 10 −3 with healthy extracts. Tissue blots of PSTVd-infected potato tubers and tomato roots, stems and leaves and ASSVd-infected apple fruit, stems and petioles, gave positive reactions when hybridized with the digoxigenin probe. No reaction with similar tissues from healthy plants was observed.

Production of Labeled cRNA Probes without Vector Cloning: Application to Localization of Basic Fibroblast Growth Factor and Tyrosine Hydroxylase mRNAs by in Situ Hybridization Histochemistry

We describe an approach for generating labeled, single-stranded cRNA probes, using the polymerase chain reaction with primers containing RNA polymerase promoter sequences. Transcription reactions using the amplified products and RNA polymerases yield, for the most part, full-length products. cRNA probes for basic fibroblast growth factor and tyrosine hydroxylase which have incorporated 35 S-labeled nucleotides were used successfully for in situ hybridization histochemistry. This method provides a significantly faster, less work-intensive procedure for obtaining labeled single-stranded RNA useful for nucleic acid hybridization studies.

Neurofilament mRNA is reduced in Parkinson's disease substantia nigra pars compacta neurons.

Lewy bodies are filamentous neuronal inclusions characteristic of Parkinson's disease, and neurofilament triplet proteins are the major components of the filaments in Lewy bodies. Since the neurofilament proteins found in Lewy bodies are abnormally phosphorylated and partially degraded, the formation of Lewy bodies may be due to the defective metabolism of these proteins, and this could lead to impairments in the structure and function of neurofilament rich neuronal processes (i.e., large caliber axons). To gain further insights into the metabolism of neurofilaments in Parkinson's disease, we evaluated neurofilament mRNA levels by semi-quantitative in situ hybridization histochemistry in postmortem tissues from Parkinson's disease and control subjects. Substantia nigra pars compacta neurons were examined with digoxigenin-UTP labeled cRNA probes to the heavy and light neurofilament mRNAs. The relative abundance of these mRNAs was measured by videodensitometric image analysis of chromogenic reaction product. Using this approach, we demonstrated that the levels of both heavy and light neurofilament mRNAs were reduced in Parkinson's disease substantia nigra pars compacta neurons. Additionally, the levels of heavy neurofilament mRNA were lowest in Lewy body containing neurons in the Parkinson's disease cases. These results suggest that the formation of neurofilament-rich Lewy bodies in substantia nigra pars compacta neurons is associated with reduced levels of the heavy and light neurofilament mRNAs in Parkinson's disease. Thus, it is possible that the accumulation of abnormal neurofilament proteins in Lewy bodies and diminished neurofilament mRNAs contribute to the degeneration of substantia nigra pars compacta neurons in Parkinson's disease.

HMG‐CoA reductase mRNA in the post‐implantation rat embryo studied by in situ hybridization

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) is the rate limiting step in the mevalonate pathway that produces isoprenoids and cholesterol. Inhibitors of HMG-CoA reductase are teratogenic in vivo and induce neural tube defects in rat embryo culture, effects which appear unrelated to cholesterol deficiency. This study is the first to localize HMG-CoA reductase mRNA by in situ hybridization (ISH). Expression of reductase mRNA was examined in post-implantation rat embryos, and for control purposes in rat liver and UT-1 cells, using a digoxigenin-11 (dig-11) labelled cRNA probe. Eighteen-day fetal liver showed heavy but patchy hybridization, and adult rat liver showed strong hybridization only on some periportal hepatocytes, which was absent in livers of fasted animals. UT-1 cells stimulated to overexpress HMG-CoA reductase mRNA were strongly positive with the same probe. Control hybridizations with sense strand RNA probe, or with cRNA probe on pre-RNased tissue were negative. Strong hybridization signal for HMG-CoA reductase mRNA was observed in all tissues of the post-implantation rat embryo, from egg cylinder to 30 somite stages (7 to 12 days). Heavy signal was noted in primitive ectoderm and neural tube. The wide embryonic and extraembryonic distribution and abundance of HMG-CoA reductase mRNA may reflect developmental requirements for products of the mevalonate pathway, e.g., isoprenoids for post-translational farnesylation of p21ras.

Synthesis of digoxigenin-labeled cRNA probes for nonisotopic in situ hybridization using reverse transcription polymerase chain reaction.

Nonisotopic methods of mRNA in situ hybridization have distinct advantages over isotopic techniques. Nonisotopically labeled probes are stable and nontoxic, have short detection times, demonstrate excellent spatial resolution of their signals and have sensitivities comparable to radiolabeled probes. We developed a simple method of generating nonisotopically labeled cRNA probes which is based on the reverse transcription polymerase chain reaction (RT-PCR) and used it to synthesize a panel of probes for various murine extracellular matrix genes. Engelbreth-Holm-Swarm (EHS) tumor RNA was reverse transcribed and PCR was used to amplify defined regions of multiple extracellular matrix protein genes from the resulting first strand cDNAs. Bacteriophage promoters which had been incorporated into the PCR products were then used to generate digoxigenin-conjugated antisense and sense cRNAs. The antisense probes were employed to detect the specific extracellular matrix protein mRNAs in the EHS tumor by in situ hybridization. This technique provides a rapid and efficient alternative to conventional transcription systems which use plasmid vectors for the synthesis of digoxigenin-labeled cRNA probes.

Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex

In situ hybridization histochemistry with labeled cRNA probes complementary to monkey brain derived neurotrophic factor (BDNF) mRNAs has been used to study the cellular localization and expression of this neurotrophin in the prefrontal cerebral cortex of fetal and adult monkeys. Expression could not be detected in prefrontal cortex before the 121st fetal day. Thereafter, in fetal life and in adulthood BDNF mRNA could be detected primarily in large, putative pyramidal cells of layers III and VI throughout the prefrontal cortex. The temporal course and cellular localization of BDNF expression suggests its association with the development and stabilization of specific connections in regions of cortex that display marked functional plasticity.

Activation of the gene encoding the glycolytic enzyme β-enolase during early myogenesis precedes an increased expression during fetal muscle development

We define the spatial and temporal patterns of expression of the gene encoding the glycolytic enzyme, β-enolase, during mouse ontogenesis. Transcripts were detected by in situ hybridization using 35S labelled cRNA probes. The β-enolase gene is expressed only in striated muscles. It is first detected in the embryo, in the cardiac tube and in newly formed myotomes. In the muscle masses of the limb, β gene expression occurs at a low level in primary fibers, and subsequently greatly increases at a time which corresponds to the onset of innervation and secondary fiber formation. Later in development, it becomes undetectable in slow-twitch fibers. Our results demonstrate the multistep regulation of the β-enolase gene. The regulation of this muscle-specific gene in somites is discussed in terms of the myogenic sequences of the MyoD family shown to be present when it is activated.