Quantitation of beta-tubulin mRNA in mouse brain by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. Two types of riboprobes for quantitating mouse beta-tubulin mRNA were prepared; one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with the mouse brain RNA, yielding heat-stable hybrids. The truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of mice of 10 and 50 days old were quantitated.     

地高辛标记反意RNA探针检测脑组织切片生长抑素mRNA

本实验采用含大鼠生长抑素基因的ｐＳＰ６５ｃＤＮＡ质位通过转化至噬菌体内大量扩增,经提取,纯化后,用限制性内切酶进行酶切使质枝线性化,并将其作为模板,用地高辛（ＤｉｇｏｘｉｇｅｎｉｎＤｉｇ）作为标记物,体外转录合成生长抑素反意ＲＮＡ（ｃＲＮＡ）探针。实验动物选用ｗｉｓｔａｒ新生大鼠。冰冻切片,端、间脑切片经杂交前用Ｄｉｇ－ＵＴＰ标记的ｃＲＮＡ探针杂交,杂交后用抗Ｄｉｇ－碱性磷酸酶复合物进行酶联免疫反应。Ｘ－磷酸盐－ＮＢＴ显色。结果显示新生大鼠脑内生长抑素ｍＲＮＡ神经元着紫蓝色。杂交反应物集中于核周的胞浆及短小的突起内。胞核不着色。胞体轮廓清晰,周围背底浅淡。结果表明Ｄｉｇ标记ｃＲＮＡ探针不仅具备非同位素标记探针的优点而且能快速和准确检测组织细胞内ｍＲＮＡ的表达。     

Quantitation of specific mRNA by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure by a solution hybridization involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. For quantitating mouse beta-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with mouse brain total cellular RNA, yielding heat-stable hybrids. Both the truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of 10- and 50-day-old mice were quantitated to be 0.0056 and 0.0011% of total RNA, respectively.     

Follicular and luteal expression of insulin-like growth factors I and II and the type 1 IGF receptor in the bovine ovary.

The localization of mRNAs for insulin-like growth factors I (IGF-I) and II (IGF-II) and the type 1 IGF receptor (IGF-1R) in bovine follicles and corpora lutea was determined using in situ hybridization on sectioned ovaries collected from nonpregnant, cyclic Holstein cows in either the follicular (n = 3) or luteal (n = 5) phases of the cycle. Concentrations were measured as absorbance units of individual regions or follicles from autoradiographs. There was intense follicular expression of mRNAs encoding IGF-II and IGF-1R. For mRNA encoding IGF-II, expression was significantly higher in smaller follicles (< 5 mm diameter, P < 0.01) and, in this size range, expression was significantly greater in healthy compared with atretic follicles (P < 0.01). For mRNA encoding IGF-1R, there was no effect of size but concentrations were again significantly greater in healthy compared with atretic follicles of < 5 mm. In medium (5-10 mm) and large (> 10 mm) follicles, there was no effect of health for expression of either IGF-II or IGF-1R. mRNA encoding IGF-II was found exclusively in the theca, whereas mRNA encoding IGF-1R was confined to the granulosa layer. IGF-I expression was not detectable in 83% of the 53 follicles examined. In the remaining 17% of follicles, expression was very low and was unrelated to size or state of atresia. mRNAs encoding IGF-I, -II and IGF-1R were all present in the corpus luteum, whereas only those for IGF-II and IGF-1R were found in ovarian stroma. These data indicate that the insulin-like growth factors play a significant role in follicular and luteal development in the bovine ovary. Locally produced IGF-II is probably an important regulator of follicular growth, whereas most of the IGF-I present in follicular fluid is likely to be derived from the circulation.     

Erratum

"Mesenchymal-Epithelial Transition in the Developing Metanephric Kidney: Gene Expression Study by Differential Display," by Sergei Y. Plisov, Sergey V. Ivanov, Kiyoshi Yoshino, Lee F. Dove, Tatiana M. Plisova, Kathleen G. Higinbotham, Irina Karavanova, Michael Lerman, and Alan O. Perantoni The above article originally appeared in Volume 27, Number 1, the May 2000 issue, of genesis on pp. 22-31. The wrong affiliations were listed for two of the co-authors: Sergey V. Ivanov is affiliated with Intramural Research Support Program, SAIC-Frederick, Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland. Michael Lerman is affiliated with Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland. On page 24, left column, under "In situ mRNA Hybridization" in the "Results" section, the sentence, "To verify the results of DD and to determine in which cells of the developing kidney the differentially displayed genes were expressed we applied mRNA hybridization (ISH)," should read: "To verify the results of DD and to determine in which cells of the developing kidney the differentially displayed genes were expressed we applied in situ mRNA hybridization (ISH)." On page 27, the legend for Figure 2, should read: "In situ RNA hybridization with thin sections of 19 dpc fetal kidney. Labeled antisense RNA was in vitro transcribed from cloned cDNA fragments obtained after differential display." On page 30, right column, under "In situ Hybridization" in the "Methods" section, the sentence, "To generate sense or antisense probes, 5 &mgr;g of plasmids with cloned cDNA fragments were linearized either with NcoI or SpeI (Promega) restriction enzymes and transcribed with T7 or SP6 RNA polymerase in the presence of alpha-35S-dCTP," should read: "To generate sense or antisense probes, 5 &mgr;g of plasmids with cloned cDNA fragments were linearized either with NcoI or SpeI (Promega) restriction enzymes and transcribed with T7 or SP6 RNA polymerase in the presence of alpha-35S-CTP." The authors regret these errors.     

Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions

The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferoxamine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability.     

Localization of insulin-like growth factor-I mRNA in rat brain by in situ hybridization--relationship to IGF-I receptors

Recent evidence has demonstrated regional synthesis of insulin-like growth factor I (IGF-I) in rat brain, which is also known to contain widespread specific type I IGF receptors. In order to precisely define sites of IGF-I mRNA synthesis, and their relationship to IGF-I receptor sites, we have applied the techniques of in situ hybridization and in vitro receptor autoradiography in rat brain. Frozen sections of adult rat brain and liver were hybridized with 32P-labeled cDNA inserts for human IGF-I (780 base pairs) or a positive control transthyretin cDNA (1430 base pairs) probe, or a series of negative probes, followed by film or emulsion autoradiography. Receptor autoradiography was performed on similar sections using 125I-IGF-I in buffer, some chambers containing excess unlabeled IGF-I. Hybridization of IGF-I probe was clearly seen only in three major brain regions: the olfactory bulb, hippocampus and cerebellum, whereas transthyretin only hybridized to choroid plexus as expected, and other probes showed no hybridization. In olfactory bulb, hybridization was greatest in the internal granular and mitral cell layers, with lower levels in the glomerular layer, where IGF-I receptors were concentrated. In hippocampus, hybridization was to pyramidal cells of Ammon's horn in CA1 and CA2 layers and dentate gyrus, with some labeling in CA3. IGF-I receptors were most dense in CA2, CA3, CA4, and dentate gyrus. In cerebellum, hybridization was to the granule cell layer, with IGF-I receptors primarily in the adjacent molecular layer. We have clearly demonstrated precise sites of local IGF-I synthesis in adult rat brain, adjacent to, and sometimes overlapping sites of high density IGF-I receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antisense mRNA-Mediated Bacteriophage Resistance in Lactococcus lactis subsp. lactis

Resistance to a broad class of isometric bacteriophages that infect strains of Lactococcus lactis has been engineered into a dairy starter by expression of antisense mRNA targeted against a conserved bacteriophage gene. Maximum protection is obtained only when the entire 1,654-bp coding sequence for a 51-kDa protein is positioned in the antisense orientation with respect to a promoter sequence that functions in L. lactis subsp. lactis. Expression of the antisense mRNA results in more than 99% reduction of the total number of PFU. Plaques that do form are characterized by their relatively small size and irregular shape. A variety of truncated genes, including the open reading frame expressed in the sense orientation, fail to provide any significant measure of resistance as compared with that of the intact open reading frame. Southern hybridization with probes specific for the conserved region reveal that the [ill] plasmid constructs are maintained despite the presence of a large complement of other indigenous plasmids. Strains harboring the antisense mRNA plasmid construct grow and produce acid at a rate equivalent to that of the host strain alone, suggesting that antisense expression is not deleterious to normal cellular metabolism.     

Assignment of porcine cyclin-dependent kinase 4 (CDK4) and oncogene c-mos (MOS) by nonradioactive nonfluorescence in situ hybridization

Two pig genes, cyclin-dependent kinase 4 (CDK4) and the oncogene c-mos (MOS) were mapped by means of nonradioactive nonfluorescence in situ hybridization. Our approach was based on the detection of hybridized biotinylated probe by peroxidase conjugated extravidin and the reaction of peroxidase with its substrate diaminobenzidine (DAB) resulting in a dark precipitate. To increase the sensitivity of the method in single-copy gene mapping, two amplifications of the peroxidase signal were used: immunological amplification by biotinylated antiavidin, and peroxidase-catalysed deposition of biotinylated tyramide. Using this method, two 2-kb-long probes for the porcine genes CDK4 and MOS were mapped to pig chromosomes 5p12 and 4q14-15, respectively. Non-radioactive nonfluorescence in situ hybridization described here is a method of choice for gene mapping of short probes.     

Glucose transporter 8 expression and translocation are critical for murine blastocyst survival

Glucose transporter (GLUT) 8 is an insulin-responsive facilitative glucose transporter expressed predominantly in the murine blastocyst. To determine the physiologic role of GLUT8, two-cell embryos were cultured to a blastocyst stage in antisense or sense oligonucleotides to GLUT8. Apoptosis was assessed using the TUNEL techniques and recorded as the percentage of TUNEL-positive nuclei/total nuclei. Embryos cultured in GLUT8 antisense experienced increased TUNEL-positive nuclei, whereas sense embryos did not. Embryos cultured in a control AS oligonucleotide, specific for heat shock protein 70-2, showed a rate of apoptosis similar to sense. To determine the outcome of these apoptotic embryos, blastocysts exposed to sense vs. antisense were transferred back into foster mice and the pregnancy continued until Day 14.5, at which time the uteri were examined for normal gestational sacs and resorptions. Embryos exposed to GLUT8 antisense experienced higher rates of resorptions and lower normal pregnancy rates compared to embryos cultured in GLUT8 sense. To examine the insulin growth factor (IGF)-1/insulin intracellular signaling pathways involved in GLUT8 translocation, IGF-1 receptor (IGF-1R) expression was decreased in the blastocysts with antisense oligonucleotides. Using confocal immunofluorescent microscopy, GLUT8 translocation in response to insulin was observed. Exposure to insulin in the embryos exposed to IGF-1R sense induced translocation of GLUT8 from intracellular compartments to the plasma membrane. Blastocysts exposed to IGF-1R antisense, however, failed to demonstrate any change in the intracellular location of GLUT8 with insulin treatment. The IGF-1R antisense embryos also displayed significantly greater TUNEL staining compared to sense embryos. These data suggest that GLUT8 expression and translocation in response to insulin are critical for blastocyst survival.     

Simultaneous determination of polymorphism in N-acetyltransferase 1 and 2 genes by reverse line blot hybridization

Polymorphism in N-acetyltransferases NAT1 and NAT2 may contribute to differences in cancer susceptibility of subjects exposed to alkylating compounds. We developed a robust method for simultaneous determination of these NAT polymorphisms: Reverse line blot (RLB) hybridization, based on PCR followed by allele-specific oligo hybridization. On a membrane, allele-specific oligonucleotide probes of the NAT genes (NAT1*4, *3, *10, *11 and NAT2*4, *5, *6, *7, *12) were applied in lines. After separate amplification of the NAT genes, simultaneous hybridization of these products in lines perpendicular to the lines with oligonucleotide probes was performed, followed by nonradioactive detection. This resulted in hybridization patterns, representing the NAT genotype of an individual. RLB hybridizations were conducted on DNA from 240 Dutch Caucasian participants in an ongoing case-control study on colorectal adenoma (including 126 polyp-free control subjects). Results were in complete agreement with those obtained by commonly used methods, i.e., allele-specific PCR and PCR-RFLP. Allele-frequencies in the polyp-free control group were similar to those described in the literature. RLB hybridization is, however, considerably faster and cheaper than the common assays. Moreover, expansion with allelic variants of other genes is relatively easy, which makes RLB hybridization very useful for multiplex analysis of numerous polymorphisms in epidemiological studies.