Sense RNA Probes Research Articles

Analysis of HLA-G mRNA in human placental and extraplacental membrane cells by in situ hybridization.

Trophoblast cells arising from the implanted blastocyst form the fetal component of the maternal-fetal interface throughout pregnancy. Previous in situ hybridization studies have shown that some subpopulations of these cells, cytotrophoblast cells within and exterior to placental villi, contain class I HLA mRNA. Those studies, performed under moderate conditions of stringency, did not determine which member(s) of the class I HLA gene family was transcribed. In this study, in situ hybridization experiments were conducted under conditions of high stringency using biotinylated antisense and sense RNA probes specific for HLA-G and HLA-E. HLA-G mRNA was identified in first trimester cytotrophoblast cells and in term chorionic membrane cytotrophoblast cells, but was low to undetectable in syncytiotrophoblast of both early and late gestation placentas. Placental villous mesenchymal cells in first trimester but not term placentas contained HLA-G transcripts. HLA-E mRNA was clearly identified only in small round cells present in first trimester decidua and term membranes. These experiments provide the first direct evidence for transcription of the HLA-G gene by cytotrophoblast cells in situ. Expression of this nonpolymorphic gene in place of HLA-A,B,C by trophoblast cells exposed to maternal blood and tissues may allow the juxtaposition of genetically disparate cells required for human pregnancy.

Elevated 32-kDa LBP and low laminin mRNA expression in developing mouse cerebrum

Abstract Several laminin receptors have been identified, originally a high-affinity 67-kDa laminin binding protein (’LBP-67′), and later galactosyltransferase and the low- affinity but functionally potent integrin receptors. At- tempts at obtaining cDNA for LBP-67, although unsuccessful, have given rise to a full-length cDNA coding for an interesting 32-kDa protein, tentatively referred to as ‘32-kDa LBP’, whose relationship to LBP-67 is unclear. Since no information is available on the in vivo expression of 32-kDa LBP mRNA nor of the three laminin chains during CNS development, appropriate 35S-antisense and -sense RNA probes were applied to developing mouse cerebral wall at embryonic day (E)10–16, birth and 1–3 weeks after birth. Expression was examined using Northern blot analysis and in situ hybridization. The 32-kDa LBP mRNA was found to be elevated during the embryonic and perinatal period, and then rapidly declined. At the cellular level, 32-kDa LBP mRNA was distributed throughout the embryonic cerebral wall and became concentrated during the perinatal period in the proliferative ventricular zone and in the cortical plate. By comparison, laminin B1, B2, and A chain mRNA expression was relatively low at all times examined, in keeping with the punctate distribution of laminin antigenicity previously observed by others in developing brain parenchyma. Whereas the functional characterization of 32-kDa LBP and the nature of its laminin and proposed nonlaminin ligands is incomplete, the elevated and unique distribution of 32-kDa LBP mRNA raises interesting questions of the role of 32-kDa LBP mRNA in CNS development.

Analysis of IL-1 and TNF-alpha gene expression in human rheumatoid synoviocytes and normal monocytes by in situ hybridization.

Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.

Detection of deletions, insertions and single nucleotide substitutions in cloned β-globin genes and new polymorphic nucleotide substitutions in β-globin genes in a Japanese population using ribonuclease cleavage at mismatches in RNA: DNA duplexes

The applicability of ribonuclease cleavage at mismatches in RNA:DNA duplexes (RNase cleavage method) for determining nucleotide variant rates has been examined in a Japanese population. DNA segments of various lengths obtained from 4 different regions of a normal and 3 thalassemic cloned human β-globin genes were inserted into transcription vectors. Sense and antisence RNA probes uniformly labeled with 32P were prepared. When RNA probes of 771 nucleotides (nt) or less were hybridized with cloned DNAs and the resulting duplexes were treated with a mixture of RNases A and T1, the length of products agreed with theoretical values. Twelve possible mismatches were examined. Since both sense and antisense probes were used, uncleavable mismatches such as G:T and G:G which were made from one combination of RNA and DNA strands could be converted to the cleavable C:A and C:C mismatches, respectively, by using the opposite combination. Deletions and insertions of 1 (G), 4 (TTCT), 5 (ATTTT) and 10 (ATTTTATTTT) nt were easily detected. A polymorphic substitution of T to C position 666 of the second intervening sequence (IVS2-666) of the β-globin gene was detected using genomic DNAs from cell lines established from the peripheral B lymphocytes of 59 unrelated Japanese from Hiroshima or those amplified by polymerase chain reaction (PCR). The frequency of the gene with C at the IVS2-666 (allele C) was 0.48 and that of the gene with T (allele T) was 0.52. The associations of the 2 alleles were in agreement with Hardy-Weinberg proportions. No contradiction to Mendelian inheritance was observed in the results obtained from 11 family studies. Two new polymorphic substitutions of C to A and A to T were detected at nucleotide positions 1789 and 1945 from the capping site, respectively, using genomic DNAs amplified by PCR. The feasibility of the RNase cleavage method combined with PCR for large-scale screening of variation in chromosomal DNA is discussed.

Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization

In situ hybridizations using 35S-labelled antisense and sense RNA probes of rPLII, rPLP-A and rPLP-B were carried out on developing rat placenta to determine which cell types synthesized each specific mRNA. Cellular localization of the sites of synthesis of these placental RNAs would help to decide whether these proteins were functioning in the mother of the fetus. The cells of the basal zone are known to have access only to the maternal blood supply, while the labyrinth region is supplied by both maternal and fetal blood vessels. The data in this paper show that at day 12 of pregnancy the rPLII mRNA is synthesized in the primary and secondary giant cells. At later days, hybridization is seen in both the giant cells of the basal zone, and cells in the labyrinth, suggesting that rPLII has a function not only in the mother, but also in the fetus. The rPLP-A mRNA is synthesized in both the giant cells and the cytotrophoblasts of the basal zone. No hybridization is seen to any cells in the labyrinth, even at the later days when it appears that all cytotrophoblasts synthesize rPLP-A mRNA. The rPLP-B mRNA is synthesized exclusively by the cytophoblasts of the fetal placenta. Like rPLP-A, all these cells synthesize this mRNA in the late term placenta. The synthesis of the rPLP-A and rPLP-B mRNAs in cells which have access only to the maternal circulation suggest that they have a role in the mother.

Expression and cellular localization of substance P/neurokinin A and neurokinin B mRNAs in the rat retina

The mammalian tachykinin peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) are encoded by distinct mRNAs derived from separate preprotachykinin (PPT) genes. The SP/NKA-encoding PPT gene generates three mRNAs by alternative RNA processing: alpha-PPT mRNA, which encodes SP only, and beta- and gamma-PPT mRNAs, which encode both SP and NKA. The NKB-encoding PPT gene generates mRNAs that produce NKB. The distribution and cellular localization of SP, NKA and NKB mRNAs in the rat retina were studied by RNA blot and in situ hybridization techniques. Blot hybridization analysis of retinal RNA extracts with [32P]-labeled RNA probes complementary to SP/NKA and NKB mRNAs demonstrated single bands of hybridization at 1300 and 900 bases, respectively. Solution hybridization-nuclease protection experiments showed multiple SP/NKA-encoding transcripts with relative levels of gamma-PPT mRNA greater than beta-PPT mRNA much greater than alpha-PPT mRNA. In situ hybridization histochemistry with [35S]-labeled antisense RNAs demonstrated that SP/NKA-encoding transcripts are expressed in small-to-medium somata located in the proximal inner nuclear, inner plexiform, and ganglion cell layers, whereas NKB-encoding transcripts are expressed in small-to-medium somata located only in the ganglion cell layer. In this layer, cells containing NKB mRNAs are more numerous than those containing SP/NKA mRNAs. Only background labeling was observed in sections incubated with sense RNA probes, pretreated with RNase A prior to hybridization or incubated in hybridization buffer without the labeled probe. Immunohistochemical studies with a monoclonal antibody directed to the conserved COOH-terminal sequence of the tachykinin peptides revealed tachykinin-like immunoreactive somata with similar size and distribution to those containing SP/NKA- and NKB-encoding transcripts. These results indicate that both SP/NKA and NKB mRNAs are present in the rat retina and that the PPT genes are differentially expressed in specific cell populations. The size and distribution of these cells suggest that they are amacrine and displaced amacrine cells, however, the possibility that tachykinins are present also in ganglion cells in the rat retina cannot be ruled out.

Possible Recruitment of Osteoblastic Precursor Cells from Hypertrophic Chondrocytes during Initial Osteogenesis in Cartilaginous Limbs of Young Rats

The appearance of the bone phenotype during rat embryogenesis was studied by in situ hybridization using a cDNA clone to osteopontin. Radiolabeled sense and antisense RNA probes were prepared from the osteopontin cDNA by in vitro transcription. The probes were used to hybridize paraffin sections of the cartilaginous diaphysis from embryonic rats at day 17 of gestation. The hybridization pattern was analyzed by auto radiography. Hybridization with the antisense probe gave patterns of silver grain labeling, indicating the presence of osteopontin mRNA among the hypertrophic chondrocytes. No silver grains could be detected in the corresponding region following hybridization of consecutive sections with the sense probe, showing the specificity of the technique being used. Whether these results indicate that the osteopontin gene is transiently expressed by hypertrophic chondrocytes or that osteopontin is an early marker for osteoblastic procursor cells will have to be explored further.

Expression of myelin protein genes in Schwann cells

The expression of myelin protein genes in Schwann cells has been studied in situ hybridization. 35S-UTP-labelled, antisense and sense RNA probes to the major protein Po, myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and proteolipid protein (PLP) were employed with paraffin-embedded sections, teased fibres and dissociated Schwann cells from sciatic nerves of rats. Teased fibres were also prepared from cervical sympathetic trunks. Po mRNA was strongly expressed in the mid-internodal perinuclear area of Schwann cell cytoplasm. The degree of signal appeared to be related to fibre size. MBP mRNA showed a diffuse pattern along the Schwann cell internode with a marked increase in grains at the paranodal cytoplasm, particularly in larger fibres. This distribution suggests that the paranodal area is a major site of insertion of MBP into myelin membrane. The expression of MAG and PLP mRNA was markedly lower than Po and MBP. Both mRNAs were localized in the perinuclear cytoplasm and showed a dependence on fibre size. No significant signal was present in Schwann cells associated with unmyelinated axons. In addition to providing data on the cellular expression of myelin protein genes, these studies have shown that teased fibres are invaluable in allowing the localization of low abundance mRNAs.

Expression of the proto-oncogene fos (c-fos) by preimplantation blastocysts of the pig.

Blastocyst material was obtained from 25 pigs during the period 10 to 33 days post coitum, and fixed thin sections of tissue were hybridized in situ to sense and antisense fos RNA probes synthesized using the expression vector Bluescribe M13. Indirect immunofluorescence using antisera to a synthetic peptide fragment of c-fos was used to confirm the tissue distribution of oncogene-encoded proteins, which were shown by immunoprecipitation to have Mrs of 55,000 and 40,000, which are the known Mrs of the fos gene product and an associated nucleoprotein, respectively. Northern and slot blots were used to assess the distribution of c-fos mRNA and the size of the fos transcript was found to be 2.3 kbases. C-fos was expressed in trophectoderm from blastocysts early in pregnancy but declined with increasing blastocyst development so that it was virtually absent by day 19 of gestation. High levels of fos proto-oncogene expression were, however, retained in the allantoic membranes up to at least day 19 of pregnancy. The expression of the fos protein could be prolonged in trophectodermal cells in monolayer culture by addition of conditioned medium from blastocysts cultured for 2 h, suggesting the presence of a growth-factor-like substance.

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization.

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.

Localization of c-myc expression during oogenesis and embryonic development in Xenopus laevis.

The expression of the proto-oncogene c-myc during oogenesis and embryonic development was followed by in situ hybridization using a cytological protocol adapted to amphibian embryos. The c-myc RNA was highly expressed in the cytoplasm of young oocytes and was further diluted during oocyte growth without specific localization. From the neurula stage on, new myc transcripts were detected and the whole embryo appeared positive with antisense myc RNA probes relative to control sense RNA probes. In addition, a spatial localization of high levels of the transcript was also observed in specific areas of the developing embryo, including the epidermis, gill buds, optic vesicles and lens placodes. These observations might indicate a specific role of the c-myc gene during the differentiation of these tissues. Alternatively, this high level of myc expression might prevent such tissues from entering into terminal differentiation during the growth of the embryo.

Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization.

A method using flow cytometry and fluorescent in situ hybridization (ISH) to detect RNA in cells is described. L1210 murine leukemia cells were fixed with 1% formaldehyde in HEPES buffered Hank's balanced salt solution (HH) followed by 70% ethanol. Endogenous RNAses were blocked by diethylpyrocarbonate treatment. Single-stranded sense and antisense RNA probes, labeled with biotin-11-UTP, were transcribed from a 2.1 kb 28S ribosomal RNA (rRNA) gene fragment subcloned into the pGEM2 plasmid. For good results, it was essential that the probes were degraded to 100-150 nucleotides before use. Hybridization was performed at 45 degrees C in 50% formamide, 5 x SSC, 0.5% SDS. Hybrids were detected with streptavidin-FITC by flow cytometry. Antisense rRNA probe signal was 100 times higher than the background. The hybrids were largely resistant to RNAse and melted at high temperature. The sense probe also gave a signal (5 times background), which was not RNAse resistant and was attributed to the presence of internal inverted repeats in the ribosomal RNA. When sufficient background reduction can be achieved, it is expected that as few as ten mRNA molecules per cell can be detected with the fluorescent in situ hybridization method.

Analysis of Aleutian Disease of Mink Parvowvirus Infection Using Strand-Specific Hybridization Probes

A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned into a bacteriophage SP6 based transcription vector and used to produce radiolabeled viral RNA transcripts corresponding to either 'plus' or 'minus' sense. The radiolabeled transcripts were reacted against Southern blots of whole cell DNA from ADV infected cell cultures as hybridization probes. The 'plus' sense RNA probe hybridized both to duplex replicative forms (RFs) as well as to single-stranded virion DNA (SS DNA), which is 'minus' in sense. In contrast, the 'minus' sense RNA probe reacted preferentially with the duplex RFs. When these probes were tested against DNA extracted from mink infected with the virulent ADV-Utah I strain, RFs were detected at 10 days after infection in mesenteric lymph node, liver, spleen and gut, but only in gut and mesenteric lymph node at 43 days. SS DNA was noted in these tissues at 10, 43 and 60 days, and was more abundant than RFs. Only SS DNA at very low levels was observed in bone marrow cells. Serum contained large amounts of SS DNA (probably in virions) at 10 days, less at 43 days, and no detectable DNA at 60 days. These findings suggest that ADV replication may have occurred in the gut as well as lymphoreticular tissues, and that bone marrow was not a major site of ADV replication.