SPARC/Osteonectin mRNA Is Induced in Blood Vessels Following Injury to the Adult Rat Cerebral Cortex

Recently we described the pattern of expression of the anti-adhesive glycoprotein SPARC/osteonectin in the developing and adult brain. SPARC mRNA was present in developing blood vessels during neurogenesis, but was not detected in the mature vasculature. We have now examined the effect of a lesion to the adult rat cerebral cortex on the expression of SPARC by in situ hybridization. SPARC mRNA was increased in the zone proximal to the wound at 3 to 10 days after cortical brain injury. During this period, SPARC was induced in mature blood vessels close to the lesion site and in blood vessels which develop following injury. These results suggest a role for SPARC in the process of angiogenesis following injury to the adult cerebral cortex.     

Localization and quantitative expression of mRNAs encoding the testis-specific histone TH2B,the phosphoprotein p19, the transition proteins 1 and 2 during pubertal development and throughout the spermatogenic cycle of the rat

Expression of the testis-specific histone TH2B, the phosphoprotein p19, and the transition proteins TP1 and TP2, was localized in the rat testis and quantified, using in situ hybridization of their mRNAs with radiolabeled probes and image analysis. In a first study, expression was assessed during testicular development between day 2 and day 65 postpartum. TH2B mRNAs appeared first in preleptotene spermatocytes (PL) on day 12 and in pachytene spermatocytes (PS) on day 18; p19 mRNAs were present in PS from day 18 onward, and TP1 and TP2 mRNAs were detected in round spermatids (RS) from day 32 onward. In the second trial, the expression of these four genes was studied throughout the cycle of spermatogenic epithelium in mature animals. TH2B mRNAs were localized in B spermatogonia at stage V, and in PL at stages VII and VIII but no longer in leptotene and zygotene spermatocytes. Thereafter, TH2B mRNAs were observed in PS from stages III–IV to XIII. The steady-state mRNA level per cell was high in PS with a maximum at stages IX–X. p19 mRNAs were present in PS from stages III–IV onward and in RS up to stages 1–2 of spermiogenesis. The maximum mRNA level per cell was observed in PS between stages VII and XIII. The presence of TP1 mRNAs was restricted to spermatids from steps 6 to 15–16 of spermiogenesis while TP2 mRNAs were detected in spermatids only between step 7 and step 13. The highest steady-state amounts of mRNAs were observed between step 7 and step 14 for TP1 and between step 10 and step 12 for TP2. Mol. Reprod. Dev. 51:22–35, 1998. © 1998 Wiley-Liss, Inc.     

Identification of bacterial diversity in the oyster Crassostrea gigas by fluorescent in situ hybridization and polymerase chain reaction

AIMS: To carry out a rapid and reliable identification of bacterial diversity in the oyster Crassostrea gigas from Todos Santos Bay, México, in the current study we applied the molecular techniques of fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). In order to reach this goal, genus and group-specific oligonucleotides targeted to 16S rDNA/rRNA were used. METHODS AND RESULTS: Oysters were collected and different tissues were analysed by means of culture-independent methodologies. In the digestive glands and gonads gamma-Proteobacteria and Gram-positive bacteria with a low G+C content, were identified as metabolically active by FISH. In the oyster gills a higher active diversity was observed, including Gram-positive bacteria with a low and high G+C content, members of the Cytophaga/Flavobacterium cluster and gamma-Proteobacteria. Consistent with FISH analysis, the amplification of 16S rDNA genes fragments with genus and group-specific oligonucleotides confirmed the presence of the same groups, as well as members of the alpha- and beta-Proteobacterias, Pseudomonas spp. and Bacillus spp. CONCLUSIONS: The combination of accurate and very easy-to-apply molecular methods allowed us to carry out a rapid screening of high bacterial diversity in oysters. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is the first report about bacterial diversity in oyster tissues analysed by FISH and PCR, without using culture-dependent methods and allowed us to determine the phylogenetic diversity of the bacterial communities present in oyster cultures, including bacteria with and without metabolic activity, as well as uncultivable cells, which are generally underestimated by traditional identification.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Sex determination of bovine embryos using the polymerase chain reaction

Abstract

One or two cell biopsies were obtained from 6‐7 days old bovine embryos. The sex of the embryos was determined with two different bovine Y‐chromosome‐specific primer pairs by using the polymerase chain reaction. These results were confirmed by karyotyping as well as in situ hybridization with an independent bovine Y‐chromosome‐specific sequence. The polymerase chain reaction was found to be a quick and accurate method of sex diagnosis of bovine preimplantation embryos.     

RNA原位杂交技术的一些应用技巧

目的:检测基因在动物组织或细胞中的时空表达模式。方法:转录反义RNA探针;利用RNA原位杂交技术检测人和小鼠牙原基中若干基因的表达。结果与结论:通过优化条件,转录出完整的反义RNA探针,并成功地利用RNA原位杂交技术在组织中检测到基因的表达;分析了一些在RNA原位杂交的过程中可能碰到的问题及其解决方法。     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, ?B and ?C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.     

Localization of the ß-subunit of follicle stimulating hormone in cattle and sheep by in situ hybridization

The locus for the beta-subunit of the follicle stimulating hormone gene (FSHB) has been determined in both cattle and sheep by in situ hybridization of a bovine and an ovine cDNA probe, respectively, to metaphase chromosomes. Our results show that the FSHB locus is on cattle chromosome 15 in the region of bands q24-qter and in sheep on the cytogenetically homologous chromosome 15, also in the region q24-qter. The mapping of the FSHB gene in cattle together with the location of other genes (CAT, HBB and PTH) previously found to be syntenic in cattle and on human chromosome 11p, defines an evolutionarily conserved synteny. The localization of the FSHB gene to a cytogenetically homologous region in cattle and sheep is consistent with the hypothesis of extensively conserved chromosome structure in these two species.     

Quantitative determination of mRNA phenotypes by the polymerase chain reaction

A method for quantitative determination of specific cellular mRNA is described. The mRNA in a dilution series of total RNA was reverse transcribed by an oligo-dT primer and the cDNA was amplified by the polymerase chain reaction (PCR) using sets of specific primers. A 32P- or biotin-labeled specific probe was hybridized to the PCR products immobilized on nitrocellulose membrane. The intensity of the hybridization signals was evaluated for quantification of the PCR products. A standard curve was produced by the known amount of the in vitro transcribed cRNA, which contained the same sequence as the mRNA. The series of standard cRNA dilutions were reverse transcribed, amplified and hybridized in the same manner. The amount of the specific RNA was deduced by fitting to the standard curve. Two tissue specimens of intestinal tumors, evaluated on the basis of hybridization signals by three different methods, were shown to contain similar amounts of beta-actin mRNA. Furthermore, a Chinese hamster ovary (CHO) cell line transfected with platelet-derived growth factor (PDGF) beta-receptor cDNA was found to contain similar amounts of beta-actin mRNA as the untransfected CHO cell line. However, the transfected CHO cell line contained over 10(11) copies of the PDGF beta-receptor mRNA per microgram of total RNA, while the untransfected one showed no detectable RNA, indicating that the latter contained less than 10(6) copies per microgram of total RNA in this assay.     

Expression of CD52 mRNA in the rat embryo

CD52 is a leukocyte differentiation antigen first discovered in humans as expressed on the surface of lymphocytes, monocytes and eosinophils. The human CD52 is found on chromosome 1, and two alleles are both known to be reasonably common. A closely homologous gene has been identified in the cynomologous monkey and related genes have been found in mouse, rat and dog. The role of CD52 in lymphocyte is still unclear but the anti-CD52 antibodies named CAMPATH-1 antibodies are largely used for therapy where depletion of lymphocytes is required. In the past expression of the antigen on progenitors of leukocytes in bone marrow had been excluded, but recent work indicates CD52 is highly expressed on cells with colony-forming and NOD/SCID (non-obese diabetic-severe combined immunodeficiency)-engrafting capacities, both at the mRNA and membrane protein level. We have investigated CD52 expression during development in rat embryos by in situ hybridization. We report here that the antigen is highly expressed in the liver that is the major organ where multipotent hematopietic stem cells differentiate but also in the splancnopleuric mesoderm, at early stages of embryo differentiation, where hematopietic stem cells are suggested to arise. CD52⁺ cells were found in areas active in vasculogenesis at early embryo stages and in the walls of the vessels in the liver at mid gestation. CD52⁺ cells were also found to emerge among c-Kit positive cells.     

Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: Localization along the nephron and effect of corticosteroids

Summary To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO₃-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO₃-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO₃-ATPase in kidney membrane fractions. HCO₃-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO₃-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO₃-ATPase to vanadate indicates that it belongs to the F₀–F₁ class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO₃-ATPase activity at the sites of urinary acidification.     

Study on fibroin heavy chain of the silkworm Bombyx mori by fluorescence in situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized the Fib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified that Fib-H has only one locus, thus providing a temporary solution to the problem about its localization.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.