Localization of lipoprotein lipase mRNA in selected rat tissues

Measurements of enzymatic activity have demonstrated that lipoprotein lipase (LPL), the principal enzyme responsible for hydrolysis of circulating triglyceride, is present in a number of tissues including brain, kidney, and adrenal gland. To determine the sites of synthesis of LPL in these tissues, in situ hybridization studies were performed using a non-sense 35S-labeled RNA probe produced from a 624-bp mouse LPL cDNA fragment. Control studies were performed with a sense RNA strand. Using 5-10-micron sections of 5-day-old rat brain, strong hybridization was found in pyramidal neurons of the hippocampus. Positive hybridization, indicating the presence of LPL mRNA, was also found in brain cortex and in the intermediate lobe of adult rat pituitary gland. Specific areas of adrenal and kidney medulla showed hybridization with the probe. LPL mRNA is, therefore, present in a number of specific regions of the body. LPL in these areas may not be important in regulating circulating levels of lipoproteins, but may be essential for cellular uptake, binding, and transfer of free fatty acids or other lipophilic substances.     

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.     

Localization of nerve growth factor receptor mRNA in the rat basal forebrain within situ hybridization histochemistry

1. In situ hybridization histochemistry was used to localize nerve growth factor receptor (NGFR) mRNA in the adult rat basal forebrain. 2. In emulsion-dipped sections 35S-labeled RNA antisense probes produced a high density of silver grains over cells located in the medial septum, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. 3. This distribution of NGFR mRNA overlaps with the distribution of NGFR protein localized using immunocytochemical techniques. 4. No hybridization signal was detected when sections were hybridized with a 35S-labeled RNA sense (control) probe. 5. We suggest that NGFRs are synthesized in these basal forebrain nuclei and transported to terminal areas where NGF is thought to be bound and internalized, an initial step in the many actions of this neurotrophic factor.     

Early changes of alpha B-crystallin mRNA in rat skeletal muscle to mechanical tension and denervation.

alpha B-Crystallin specifically decreases in atrophied rat soleus muscle with hindlimb suspension (HS). alpha B-Crystallin cDNA was cloned from rat heart cDNA library using oligonucleotide probe, and its complete coding and partial non-coding regions were sequenced. Northern blot analysis revealed that alpha B-crystallin mRNA in slow muscle decreases at 36 hour after HS but recovered at 24 hour after HS stopped. Denervation decreased the expression of alpha B-crystallin mRNA in slow muscle but increased it in fast muscles, which hardly expressed in normal condition. Passive tension increased the expression of alpha B-crystallin mRNA in both muscle types. Based upon these Northern blot analysis of alpha B-crystallin, nerve innervation and external load on muscle are essential regulatory factors on the expression of the mRNA of alpha B-crystallin in rat skeletal muscle.     

Expression of a sperm protein gene during spermatogenesis in mammalian testis: an in situ hybridization study.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Developmental and Regional Changes of mRNA for a Cerebellar Protein (Spot 35) in the Rat Brain

Spot 35 protein is a cerebellar Ca-binding protein. Because Southern blot analysis showed evidence for the nucleotide sequence of spot 35 protein cDNA in the rat genome, we applied cDNA to quantitate the mRNA of rat spot 35 protein. The size of this mRNA was about 1,900 nucleotides in length, and that of mRNA from bovine cerebellum was larger. We also examined the developmental changes and regional distribution of spot 35 protein mRNA in rat brains by dot-blot analysis using cDNA as a probe. During postnatal days 1-20, a rapid increase of mRNA levels was observed. Further, the level of mRNA for spot 35 protein was found in the cerebellum and was negligible in the cerebral cortex, striatum, and hippocampus.     

Expression and localization of luteinizing hormone receptor in the female mouse reproductive tract

The presence of the LH receptor (LHR) in nongonadal tissues of the reproductive tract has been reported, but localization studies have not been performed. Our objectives were to demonstrate the presence of LHR in the reproductive tract and to localize receptor expression. Reproductive age rats and mice were obtained and (125)I-hCG binding assays were performed on membrane preparations from the uterus, ovary, liver, and testis. In situ hybridizations were performed using (35)S-labeled antisense and sense RNA probes prepared from nucleotides 1-591 of the mouse LHR cDNA. Specific hCG binding was detected in membrane preparations from the ovary, uterus, and testis but not in the liver in both the rat and mouse. In the ovary, LHR mRNA was localized in theca cells, large follicles, and corpora lutea as expected. In the uterus, LHR mRNA was expressed in stromal cells of the endometrium and in the uterine serosa. Uterine smooth muscle cells had low levels of expression, and the endometrial epithelium was negative. In the oviduct, high levels of LHR expression were noted on the serosa and in subepithelial cells. Oviductal smooth muscle had low expression, and the epithelium was negative. We conclude that functional, nongonadal LHR are expressed in the mouse reproductive tract. The presence and localization of LHR expression in the mouse reproductive tract lay the foundation for transgenic models to address the physiologic role of these receptors.     

Expression of myelin basic protein gene in the developing rat brain as revealed by in situ hybridization

The developmental program controlling the expression of myelin basic protein (MBP) gene was studied in the rat using the technique of in situ hybridization. A 35S-labeled cDNA clone of mouse MBP encoding an amino acid sequence present in all four of the major forms of rodent MBP was used. The probe hybridized to the tracts of white matter with different intensities, depending on the age of the animal and the region of the brain examined. In the medulla oblongata, maximal hybridization was found in 5- and 7-day-old rats and was confined to the tectospinal tracts, fibers of the seventh cranial nerve, and the spinocerebellar tracts. By 12 days the amount of MBP mRNA had decreased in these areas. In the cerebrum, the greatest amount of MBP mRNA was observed in 17-day-old rats in the radiations of the corpus callosum. Thereafter, the levels decreased but could still be observed in the adult animals. Thus, using this technique, we have been able to demonstrate that the level of MBP-specific mRNA correlates closely with the development of myelin in different regions of the brain.     

Simultaneous in situ detection of two mRNAs in the same cell using riboprobes labeled with biotin and 35S.

We used 35S-labeled and biotinylated cRNAs (riboprobes) to detect simultaneously two different mRNAs by in situ hybridization. In a first step we established the conditions under which each type of probe achieved the same high level of sensitivity. We then used these conditions to hybridize BHK cells infected with Theiler's virus, a murine picornavirus, with a mixture of a virus-specific biotinylated riboprobe and a 35S-labeled riboprobe specific for beta-actin mRNA. Both mRNAs could be detected in the same cell, although the sensitivity achieved by the radiolabeled probe was reduced by about 40% by the simultaneous hybridization with the biotinylated probe.     

Translocation of proteins into rat liver mitochondria. Existence of two different precursor polypeptides of liver fumarase and import of the precursor into mitochondria

Two different putative precursor polypeptides of rat liver fumarase were synthesized when RNA prepared from rat liver were translated in vitro using the rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P1) was synthesized as a larger molecular mass than the mature subunit of fumarase (45,000 daltons) by about 5,000 daltons and the other (P2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30 degrees C, P1 and the 35S-labeled mature size fumarase were associated with the mitochondria. Of these, the 35S-labeled mature size fumarase was resistant to externally added protease, but P1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P1, which was energy-dependently imported and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased proportionally to the duration of incubation, while the amount of P1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of the incubation. 2) Only P1 could bind with the mitochondrial outer membrane at 0 degrees C even in the presence of an uncoupler of the oxidative phosphorylation but P2 did not. 3) P1 bound to the mitochondrial outer membrane was imported into the matrix, when the mitochondria binding only P1 at 0 degrees C was reisolated and incubated at 30 degrees C in the presence of an energy-generating system. The specific receptor was involved in the binding of P1 to mitochondria, since a high concentration of NaCl did not interfere with the binding of P1 to the membrane and did not discharge P1 bound onto the membrane. It was shown that P1 formed an aggregate composed of 6 to 8 molecules and P2 was a dimer in the cell-free translation mixture and that P1 and P2 were enzymatically inactive. These results suggest that the precursor for the mitochondrial enzyme has a larger molecular weight than that of the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme.     

Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.     

(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles

We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle.     

The fetal rat binding protein for insulin-like growth factors is expressed in the choroid plexus and cerebrospinal fluid of adult rats

The biological effects of the insulin-like growth factors, IGF-I and IGF-II, on their receptors are modulated by IGF-binding proteins. Recently, we isolated a cDNA clone for one member of the family of IGF-binding proteins, BP-3A, a 30 kilodalton (kDa) protein synthesized by the BRL-3A rat liver cell line. BP-3A is related to but distinct from two other cloned IGF-binding proteins, the human amniotic fluid binding protein and the glycosylated binding subunit of the 150 kDa IGF-binding protein complex in serum. It is expressed in multiple nonneural tissues and in serum in the fetal rat and decreases after birth, similar to the developmental pattern of IGF-II expression. IGF-I, IGF-II, and their receptors are expressed in brain. The present study examines the expression of BP-3A in the rat central nervous system. By Northern blot analysis, BP-3A mRNA is present at high levels in brain stem, cerebral cortex, and hypothalamus from 21-day gestation rats and, like IGF-II mRNA, persists in adult rat brain. The site of BP-3A mRNA synthesis was localized by in situ hybridization to coronal sections of adult rat brain using 35S-labeled oligonucleotides, 48 bases in length, complementary and anticomplementary to the coding region of BP-3A. Specific hybridization of the BP-3A probe was observed exclusively to the choroid plexus extending from the level of the medial preoptic nucleus to the arcuate nucleus of the hypothalamus, similar to the previously reported preferential localization of IGF-II mRNA to the choroid plexus. Synthesis of BP-3A mRNA by choroid plexus suggested that BP-3A might be secreted into the cerebrospinal fluid. A 30 kDa IGF-binding protein was demonstrated in rat cerebrospinal fluid that is recognized by antibodies to BP-3A and, like purified BP-3A, has equal affinity for IGF-I and IGF-II. By analogy with other transport proteins synthesized by the choroid plexus, BP-3A may facilitate the secretion of IGF-II to the cerebrospinal fluid and modulate its biological actions at distant sites within the brain.     

Vasoactive intestinal peptide regulates cholesterol side-chain cleavage cytochrome P-450 (P-450scc) gene expression in granulosa cells from immature rat ovaries

Vasoactive intestinal peptide (VIP), a neuropeptide present in ovarian nerves, has been previously shown to induce synthesis of the side-chain cleavage cytochrome P-450 enzyme which catalyzes the conversion of cholesterol to pregnenolone (the rate-limiting step in progesterone synthesis). In the present study we demonstrate, by means of a bovine 3'-specific P-450scc cDNA probe, that this VIP effect is exerted at least partially at the level of gene expression in cultured granulosa cells that were isolated from estrogen-primed, immature rats. The size and level of the 2.0 kilobase P-450scc mRNA species was assessed by Northern blot analysis, while the translatability of this mRNA was assayed by immunoisolation of the 35S-labeled P-450scc precursor protein translated from total RNA of control and stimulated granulosa cells. FSH was much more effective than VIP at increasing P-450scc mRNA concentrations in cultured granulosa cells, whereas secretin treatment was ineffective. The results suggest that, like FSH, the stimulatory effect of the neuropeptide VIP on ovarian progesterone secretion involves regulation of P-450scc gene expression during functional maturation of the prepubertal ovary.