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PubMed1996

Effects of long-term hydralazine treatment on myocardial structure and expression of myosin isogenes in cardiac pressure overload in rats.

The long-term effects of an arterial vasodilator, hydralazine, on myocardial structure and cardiac myosin isogene expression in pressure overload in rats were investigated. Portan rats were subjected to pressure overload by partial abdominal aortic constriction in order to study the effects of a 4-week treatment with hydralazine (15 mg/kg p.o.) on hemodynamics, ventricular structure, and ventricular total RNA, DNA, protein and myosin isoform expression pattern in sham (SO) and pressure-overloaded (p.o.) rats. P.o. increased the mean arterial pressure (MAP) and systolic blood pressure (SBP) and resulted in increased ventricular weight, LV wall thickness, total RNA and protein content; however, total DNA remained unchanged. The expression of fetal isogene beta-myosin heavy chain (beta-MHC) was markedly enhanced, whereas alpha-MHC was reduced. Hydralazine (15 mg/kg p.o.) normalized MAP and SBP, but did not modulate the hypertrophic changes of pressure-overloaded myocardium. It did not prevent an increase in ventricular total RNA and protein content and antithetical expression of myosin isoforms. Overall growth rate of rats was unaffected by hydralazine treatment. These results suggest that hydralazine has no direct effect in preventing the progression of cardiac hypertrophy and expression of beta-MHC, in spite of lowering blood pressure in pressure-overloaded rats. Thus, it further reaffirms that factors other than hemodynamics may play a pivotal role in the development of cardiac hypertrophy and induction of fetal isogene expression.

Analysis of Variance↗
PubMed1996

Long-term effects of restrictive culture conditions on Saccharomyces cerevisiae sec7 cells.

Long-term effects of restrictive conditions on the temperature-sensitive S. cerevisiae sec7 mutant were studied. By microscopic analysis no cell lysis could be detected of cells cultured for up to 19 days at 37 degrees C. The optical density as well as the cell number remained constant during the whole period under restrictive conditions. However, restrictive conditions decreased the incorporation of 35S-methionine into intracellular proteins in a reversible manner indicating that protein biosynthesis was inhibited whereas the cells remained alive. Northern blot experiments revealed that restrictive conditions did not markedly decrease the ratio of the mRNA levels to total RNA for the genes TEF1, TEF2, SUC2, and BGL2 up to 73 hours. However the content of total RNA decreased drastically with increasing incubation times at restrictive temperature. In spite of the reduced total RNA content, cells are capable of new synthesis of mRNA under restrictive conditions which was shown by incubation of the cells in the presence of actinomycin D--an inhibitor of the mRNA synthesis. Most of the cells which survived a long-term incubation at 37 degrees C are not able to divide and to form colonies immediately after their transfer to permissive conditions.

Fungal Proteins↗
PubMed1994

Tissue distribution and regulation of rat prolactin receptor gene expression. Quantitative analysis by polymerase chain reaction.

The rat prolactin receptor (PRLR) exists as two forms, short and long. We have developed a quantitative polymerase chain reaction (Q-PCR) in order to measure the absolute number of mRNA molecules encoding both forms of PRLR in 16 tissues of adult female rats at two stages of the estrous cycle (proestrus and diestrus I) and in the mammary gland of 20-day pregnant and 7-day lactating rats. Using this technique, it was possible to detect as few as 500 molecules of a target mRNA per micrograms of total RNA. All tissues examined expressed the two forms of receptor transcripts, ranging from 1.8 x 10(3) molecules/micrograms of total RNA in the skeletal muscle to 2.9 x 10(7) molecules/micrograms of total RNA in the ovary. Fourteen tissues expressed the long form mRNA predominantly, the thymus and the kidney expressed both forms equally, and the liver expressed the short form predominantly. In the liver, the level of mRNA expression of the short form was approximately 2-fold higher in proestrus than in diestrus. In the ovary, uterus, and cerebral cortex, the expression of the long form transcript was higher in proestrus than in diestrus: 4-fold in the ovary, 2.8-fold in the uterus, and 1.4-fold in the cerebral cortex. In contrast, the hypothalamus and the pituitary expressed 1.6-fold more long form transcript in diestrus than in proestrus. These results indicate that PRLR mRNA is ubiquitously but variably expressed in a tissue-specific manner and is clearly regulated by the hormonal environment associated with the stage of the estrous cycle, pregnancy, and lactation.

Animals↗
PubMed2004

Design of a real time quantitative PCR assay to assess global mRNA amplification of small size specimens for microarray hybridisation.

BACKGROUND: Low RNA yields from clinical samples are a limiting step for microarray technology. AIMS: To design an accurate real time quantitative polymerase chain reaction (PCR) assay to assess the crucial step of global mRNA amplification performed before microarray hybridisation, using less than 1 microg total RNA. METHODS: Three RNA extraction procedures were compared for small size samples. Total RNA was amplified from universal RNA or the BC-H1 breast cancer micrometastatic cell line using three different protocols. Real time quantitative PCR technology was used for accurate measurement of urokinase plasminogen activator receptor and cytokeratin 8 RNA amplification rates and ratios, using primer sets binding at various distances from the 3' end of transcripts. A 50 mer oligomeric array targeting 87 genes potentially involved in breast cancer metastatic progression was built and hybridised with amplified RNA. RESULTS: Eighteen nanograms of total RNA could be purified from 1000 BC-H1 micrometastatic cells. Amplification rates of 25,000 to 100,000 were achieved with as little as 10 ng of starting material. However, results were highly variable, depending on the amount of starting material, gene characteristics, sample quality, and protocols used. Oligomeric array hybridisation with 20 microg reference RNA resulted in specific and reproducible signals for 83% of the genes, whereas mRNA amplification from less than 400 ng of starting material resulted in selective detection of signals from highly expressed genes. CONCLUSIONS: Improvements in the design of global mRNA amplification procedures and oligomeric arrays are needed to extract informative gene expression data from clinical samples containing limited cell numbers.

Base Sequence↗
PubMed2000

Real-time RT-PCR fluorescent detection of tomato spotted wilt virus.

A real-time reverse transcription-polymerase chain reaction assay based on TaqMan chemistry was developed for the detection and quantification of tomato spotted wilt virus (TSWV). This method enabled sensitive, reproducible and specific detection of TSWV in 'leaf soak' and total RNA extracts from infected plants. TaqMan reliably detected TSWV in as little as 500 fg total RNA. The assay was 10-fold more sensitive than visualisation of ethidium bromide-stained bands following agarose gel electrophoresis. TSWV isolates from various crops and locations were detected with a cycle threshold of 20-26 in 1 ng total RNA extracted from fresh or freeze-dried leaves. A dilution series of in vitro transcripts from a cloned 628 base pair fragment of TSWV S RNA served as standard for quantification of viral template in infected leaf samples. The TaqMan assay detected reproducibly 1000 molecules of the target transcript.

Base Sequence↗
PubMed1994

Expression of retina-specific genes by mouse retinoblastoma cells.

PURPOSE: Two cell lines derived from ocular tumors of a transgenic mouse expressing the SV40 large T antigen have been established as models of human retinoblastoma. One line, TM, originated from a metastasis, and the other, TE, originated from the primary tumor. The authors compared these two lines with the normal adult mouse eye by analysis of the expression of five photoreceptor cell-specific proteins: IRBP, opsin, rod- and cone-specific transducins, and S-antigen. The authors sought to determine which of these proteins was expressed qualitatively and to examine semi-quantitatively for changes in the levels of expression in the cell lines. METHOD: Western blot analysis was used to detect photoreceptor-specific intracellular or secreted proteins. Total RNA was prepared from cultured cells or from mouse adult whole eye. Specific messenger levels in total RNA were determined either by northern hybridization analysis or by a semi-quantitative polymerase chain reaction (PCR), coupled to complementary DNA (cDNA) substrates prepared from total RNA. RESULTS: IRBP was present in the retinoblastoma cell lines and secreted into the medium. Neither S-antigen nor opsin were detectable by immunoblotting. IRBP and cone transducin mRNA were present in both cell lines. In contrast, opsin, rod transducin, and S-Antigen mRNAs were not detectable by PCR. beta-actin was present in the mRNA populations of whole eye and retinoblastoma. SV40 large T antigen mRNA was present only in retinoblastoma cells. CONCLUSIONS: IRBP and cone transducin expression in mouse retinoblastoma cells is independent of signaling provided directly or indirectly through large T antigen or Rb105 regulatory cascades. The pattern of photoreceptor-specific gene expression is similar to that seen in human retinoblastoma cell lines. These murine-derived cell lines may be useful as a tool to study IRBP and cone transducin expression in vitro and to determine early retinoblast expression patterns in the mouse.

Animals↗
PubMed2004

Increased HIV-1 mucosal replication is associated with generalized mucosal cytokine activation.

The purpose of this study was to characterize intestinal mucosal cytokine profiles in subjects with HIV-1 infection and their relation to mucosal viral load (MVL). Intestinal mucosal cytokine mRNA (interleukin [IL]-2, interferon [IFN]-gamma, IL-12, IL-10, IL-1beta, tumor necrosis factor [TNF]-alpha, IL-6, and regulated upon activation, normal T-cell expressed and secreted [RANTES]) and HIV-1 RNA were quantified using real-time polymerase chain reaction (PCR). On the basis of MVL quantification, the HIV-1-infected subjects were divided into 3 groups: undetectable MVL (<50 copies/microg of tissue total RNA), low MVL (>50 but <5000 copies/microg of tissue total RNA), and high MVL (>5000 copies/microg of tissue total RNA). Compared with the control group, significant reductions in RANTES, IL-2, and IFNgamma expression were seen in the undetectable MVL group (P < 0.005). IL-6 was significantly increased in all the HIV groups (P < 0.005), and RANTES, IL-10, and IFNgamma were increased in the high MVL group (P < 0.005). Subjects with high MVL have generalized immune activation with increases in T helper (Th)1, Th2, and proinflammatory cytokines, whereas subjects with undetectable MVL have reduced expression of multiple cytokines. The pathologic basis for these observations is unclear but may relate to the success or failure of antiretroviral therapy in controlling mucosal viral replication.

Adult↗
PubMed1999

Gene expression in menisci from the knees of skeletally immature and mature female rabbits.

This study, using the sensitive molecular technique of semiquantitative reverse transcription-polymerase chain reaction, evaluated mRNA levels for several molecules in medial and lateral menisci from the knees of skeletally mature and immature rabbits. Total RNA was extracted from the medial and lateral menisci of New Zealand White rabbits with the TRIspin method. Total RNA and DNA were similar in the two menisci of both immature and mature rabbits. The total RNA was reverse-transcribed and analyzed by semiquantitative polymerase chain reaction using rabbit-specific primer sets; levels of mRNA for a subset of molecules differed between the medial and lateral menisci. These variations in mRNA levels were also influenced by the degree of skeletal maturity of the rabbits. For most of the genes, mRNA levels were generally higher in the medial than in the lateral meniscus. The medial meniscus from immature and mature rabbits had significantly increased levels of mRNA for molecules such as transforming growth factor-beta, cyclooxygenase-2, and tissue inhibitor of metalloprotease-1. In contrast, compared with mRNA in the lateral meniscus, that for types II and III collagen, biglycan, insulin-like growth factor-2, plasminogen activator inhibitor-1, and matrix metalloprotease-1 was significantly increased in the medial meniscus of mature rabbits only and that for versican and type-I collagen was significantly increased in the medial meniscus of immature rabbits only. Levels of mRNA for inducible nitric oxide synthase and basic fibroblast growth factor were similar in both menisci for both age groups. The present study demonstrates that regulation of mRNA levels in medial and lateral menisci is tissue-specific and influenced by the skeletal maturity of the animals.

Aging↗
PubMed1998

Influence of pregnancy on gene expression in rabbit articular cartilage.

OBJECTIVE: Articular cartilage is known to be influenced by estrogen and the pregnancy-associated hormone, relaxin, in vitro. Such observations have raised the possibility that articular cartilage in females may be subjected to unique regulatory influences by such hormones in vivo. The purpose of this study was to evaluate mRNA levels for several relevant molecules in the articular cartilage of pregnant and non-pregnant rabbits. DESIGN: Total RNA was extracted from New Zealand White rabbit knee articular cartilage using the TRIspin method. The total RNA was reverse transcribed and analysed by the sensitive molecular technique of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rabbit specific primer sets. RESULTS: Total RNA yield from articular cartilage from primigravida rabbits was reduced to 65% of age-matched control values (P = 0.0003); however the yield from multiparous animals was not significantly depressed. In both cases, DNA yields were not affected by pregnancy. There was a general tendency for depressed mRNA levels for most genes investigated in cartilage from pregnant animals. Articular cartilage from multiparous rabbits showed a significant decrease in mRNA levels for relevant molecules such as type II collagen, biglycan, collagenase and tissue inhibitors of metalloproteinases (TIMP)-1, as well as necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS) and cyclo-oxygenase 2 (COX-2). Transcripts for collagenase and lumican were significantly lower in cartilage from primigravida rabbits. Transforming growth factor beta 1 (TGF-beta 1) transcript levels were significantly decreased in both pregnant groups. In contrast, basic fibroblast growth factor (bFGF) and insulin-like growth factor-2 (IGF-2) mRNA levels were significantly decreased in cartilage from primigravida rabbits, whereas transcripts for these molecules were upregulated in the cartilage of multiparous rabbits. CONCLUSIONS: The present study demonstrates that regulation of RNA levels in articular cartilage during pregnancy is complex and is influenced by the parity and/or the skeletal maturity of the animals.

Animals↗
PubMed1978

A nuclear extract of Xenopus laevis oocytes that accurately transcribes 5S RNA genes.

Xenopus 5S RNA genes in recombinant form with the plasmid pMB9 are transcribed accurately when added to a supernatant fraction obtained from disrupted nuclei of Xenopus laevis oocytes. After an initial 30 min lag period, the rate of synthesis of 5S RNA is constant for at least an hour and synthesis is still detected after 18 hr. As much as 40% of the total RNA synthesized from the recombinant DNA used in these experiments can be 5S RNA. The coding strand of the 5S RNA genes is transcribed at a rate 10 to 15 times greater than the noncoding strand. Plasmid and spacer DNA, however, are also transcribed. What fraction of total RNA synthesized is 5S RNA is strongly affected by DNA concentration, ionic strength and MgCl2 concentration. Inhibition of transcription by intermediate concentrations of alpha-amanitin demonstrates that RNA polymerase III transcribes at least 90% of all RNA synthesized. Adenovirus 2 DNA is also transcribed in the nuclear supernatant by RNA polymerase III. Approximately 15% of the total RNA synthesized migrates in an acrylamide gel as a band of 5.5S RNA and has been identified as virus-associated RNA1 by its oligonucleotide fingerprint.

Animals↗
PubMed2000

Quantitative RT-PCR analysis of multiple genes encoding putative metronidazole nitroreductases from Helicobacter pylori.

Metronidazole (Mtz), a pro-drug, requires reductive activation by ferredoxin-like electron carrier proteins to kill bacteria and Mtz resistance is associated with a decrease or deficiency of Mtz nitroreductase activities in a target cell. Several genes encoding ferredoxin-like or -linked proteins such as pyruvate oxidoreductase (POR), ferredoxin oxidoreductase (FOR), ferredoxin (FdxA), ferredoxin-like protein (FdxB), flavodoxin (FldA) and oxygen insensitive nitroreductase (RdxA) have been identified from the complete genomic sequence of Helicobacter pylori. To understand the roles of these genes in H. pylori Mtz resistance, the gene expression for the proteins was examined using a method optimized for quantitative reverse transcription polymerase chain reaction (RT-PCR). The RT-PCR products of FOR and RdxA were significantly decreased in the total RNA prepared from H. pylori cultured in the presence of Mtz as compared to the total RNA prepared from H. pylori cultured without Mtz in the media. A slight decrease, however, in band intensity of the RT-PCR products of the POR and, to a lesser extent, FdxB was obtained in the presence of Mtz. In contrast, the RT-PCR products of the FdxA, FldA, and GalE (UDP-galactose 4-epimerase; a control gene) were unchanged in total RNA prepared from H. pylori cultured with or without Mtz in the culture media. These results suggest that Mtz resistance may also be acquired by decreasing the transcription of some genes involved in Mtz reductive activation, in addition to the mutation in some individual genes such as rdxA.

Base Sequence↗
PubMed1992

The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-beta.

Hepatocyte growth factor (HGF) has been detected in non-parenchymal cells but not in hepatocytes. We performed Northern blot analysis of total RNA extracted from rat hepatocytes, Kupffer cells, endothelial cells and fat-storing (Ito-) cells. Total RNA was extracted from fat-storing cells at different times after isolation and from cells treated with different amounts of transforming growth factor beta. The RNA was hybridized with HGF, fibronectin-, and alpha-actin-specific cDNA probes, consecutively. We found an abundant amount of HGF mRNA in freshly isolated fat-storing cells, but not in other liver cells. The amount of the HGF transcripts decreases significantly in FSC during the time of culture, while fibronectin gene expression increases and alpha-actin gene expression as well. TGF-beta dramatically inhibits HGF gene expression, but causes an enhanced fibronectin mRNA level. Northern blot hybridisation of total RNA from CCl4-chronically damaged liver with HGF cDNA shows a significant increase of HGF mRNA during development of liver fibrosis. We suggest that in damaged liver either non-parenchymal cells, others than FSC, became able to express the HGF in vivo, or other mediators overcome the inhibitory effect of TGF-beta.

Actins↗
PubMed1997

Effect of chronic hypokalemia on H(+)-K(+)-ATPase expression in rat colon.

Although the kidney plays the major role in the regulation of systemic K+ homeostasis, the colon also participates substantively in K+ balance. The colon is capable of both K+ absorption and secretion, the magnitude of which can be modulated in response to dietary K+ intake. The H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) has been proposed as a possible mediator of K+ absorption in distal colon, but inhibitor profiles obtained in recent studies suggest that two, and perhaps more, distinct H(+)-K(+)-ATPase activities may be present in mammalian distal colon. We have developed highly specific probes for the catalytic alpha-subunits of colonic and gastric H(+)-K(+)-ATPase, alpha 1-Na(+)-K(+)-ATPase, and beta-actin, which were used in Northern analysis of total RNA from whole distal colon and stomach obtained from one of three experimental groups of rats: 1) controls, 2) chronic dietary K+ depletion, and 3) chronic metabolic acidosis. The probe for the colonic but not the gastric H(+)-K(+)-ATPase alpha-isoform hybridized to distal colon total RNA in all groups. A significant increase in colonic H(+)-K(+)-ATPase mRNA abundance was observed in response to chronic dietary K+ depletion but not to chronic metabolic acidosis. The alpha 1-isoform of Na(+)-K(+)-ATPase, which is also expressed in distal colon, did not respond consistently to either chronic dietary K+ depletion or chronic metabolic acidosis. The gastric probe did not hybridize to total RNA from distal colon but, as expected, hybridized to total stomach RNA. However, the abundance of gastric H(+)-K(+)-ATPase or Na(+)-K(+)-ATPase in stomach was not altered consistently by either chronic dietary K+ depletion or metabolic acidosis. Under the conditions of this study, it appears that the mRNA encoding the colonic alpha-isoform is upregulated by chronic dietary K+ restriction, a condition shown previously to increase K+ absorption in the distal colon.

Acidosis↗
PubMed1976

Estrogen-induced changes of ribonucleic acid in the rat uterus.

The incorporation of [3H]uridine into uterine RNA of immature rats was studied up to 6 h after a single injection of estradiol. Under these experimental conditions, estradiol progressively increased the incorporation of the radioactive precursor into the total RNA. This increase could not be explained by variations in the uptake of [3H]uridine by the tissue. The total RNA and the fractions obtained by differential thermal extraction were analysed by gel electrophoresis. One hour after hormone treatment, a similar increase of incorporation of the labelled precursor in the different RNA species was observed. After a long period of time, [3H]uridine was preferentially incorporated into rRNA as compared to HnRNA and to heterogenous cytoplasmic RNA. Experiments which involved the use of low doses of actinomycin D sufficient to inhibit any rRNA synthesis, confirmed the relatively slight increase in precursor incorporation into non-ribosomal RNA. The distribution of the radioactivity incorporated into the 3 fractions of RNA, obtained by thermal extraction (2 nuclear fractions and 1 cytoplasmic), suggested an increase in the rate of transcription and transport of the RNA during hormonal treatment. The polyA-containing uterine RNA was isolated on a column of oligo(dT)-cellulose and subsequently studied by electrophoresis. There is no preferential incorporation of precursor into the polyA-containing RNA when compared with the total RNA. However, the polyA-containing RNA constitutes the only part of the non-ribosomal RNA whose synthesis continues to increase throughout the period of hormone treatment.

Animals↗
PubMed1982

The transcription of various types of ribonucleic acid by hepatocytes isolated from rats of various ages.

The synthesis of various types of ribonucleic acid (RNA) isolated from 6- to 3-month-old female Fischer F344 rats was compared. The rate of RNA synthesis by freshly prepared hepatocytes was determined by dividing the amount of [3H]orotic acid incorporated into RNA as uridine-5'-monophosphate by the specific activity of the uridine-5'-triphosphate pool. The rate of total RNA synthesis by hepatocytes from 19-month-old rats was 40% less than the rate for hepatocytes from 12-month-old rats. No significant difference in the rate of total RNA synthesis was observed between 19 and 30 months of age. The percentage of [3H]orotic acid incorporated into poly(A) + RNA by 30-month-old rats was approximately 50% less than that observed for hepatocytes isolated from 6-month-old rats. The percentage of [3H]orotic acid incorporated into poly(A)-RNA as ribosomal RNA (38S, 18S, and 5S RNAs) or transfer RNA was similar for 12- and 30-month-old rats. The rate of poly(A) + RNA synthesis by hepatocytes isolated from 30-month-old rats was 65% less than that observed for hepatocytes from 6-month-old rats. In contrast to total RNA synthesis, the rate of poly(A) + RNA synthesis for the 30-month-old rats was significantly less than the rate for 19-month-old rats.

Age Factors↗
PubMed1997

Tissue-specific regulation of renal and cardiac atrial natriuretic factor gene expression in deoxycorticosterone acetate-salt rats.

Atrial natriuretic factor (ANF) is expressed in several noncardiac tissues where it may have an autocrine or paracrine function. Such function may be expected of locally synthesized ANF in the renal parenchyma. Previous investigations of the existence of ANF mRNA in the renal parenchyma have yielded conflicting results. The investigations reported here were designed to detect and measure ANF mRNA in normal rats and in rats subjected to a deoxycorticosterone acetate (DOCA)-salt treatment schedule known to strongly activate cardiac ANF gene expression. The expression of the renal ANF gene was measured using a newly developed quantitative competitive reverse transcription-polymerase chain reaction (QC-RT-PCR). This method uses an internal competitor that serves as an internal standard and makes the procedure independent of measurement relative to housekeeping genes. It was found that renal ANF mRNA levels were 10(7) times lower than those found in left or right atria, but immunoreactive (ir) renal ANF concentration by specific radioimmunoassay was 10(4) times lower than that of atrial irANF levels. Reverse-phase high-performance liquid chromatography analysis revealed that more than 99% of renal irANF is processed ANF(99-126). This finding suggests that most of the irANF measured in kidney extracts likely originates from atrial sources. Left atrial ANF mRNA levels after 1 week of DOCA-salt treatment was significantly higher than that of control rats ([21.06+/-2.99] x 10(-l5) mol/microg total RNAversus [8.59 +/-1.26] x 10(-5) mol/microg total RNA, P<.05). However, renal ANF mRNA levels in DOCA-salt rats were significantly decreased compared with those of control rats ([1.64+/-0.34] x 10(-22) mol/microg total RNA versus [3.96+/-0.61]x 10(-22) mol/microg total RNA, P<.05). These results indicate that (1) renal ANF mRNA can be consistently and specifically demonstrated after reverse transcription and PCR amplification; (2) renal and cardiac ANF synthesis are regulated in a tissue-specific, opposite manner during DOCA-salt treatment; and (3) the finding that renal ANF mRNA is downregulated by DOCA-salt treatment together with previous findings suggest the need for further investigation into the role of renal ANF mRNA downregulation in the pathogenetic mechanism that leads to volume expansion and hypertension after chronic DOCA-salt treatment.

Animals↗
PubMed1985

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VI. Evidence for posttranscriptional modification of 6-thioguanosine residues in RNA from L5178Y cells treated with 6-mercaptopurine.

Mammalian cells incorporate 6-thioguanosine into their nucleic acids when grown in the presence of 6-mercaptopurine. 35S-labeled total RNA was prepared from L5178Y murine lymphoma cells grown in vitro in the presence of 6-[35S]mercaptopurine. Base analyses of this RNA suggested that 6-thioguanosine residues in RNA molecules undergo posttranscriptional modification. Thus, enzymatic peak-shifting analyses using anion-exchange high-performance liquid chromatography were applied to the hydrolysis products released from total RNA preparations by digestion with nuclease P1 or nuclease P1 plus nucleotide pyrophosphatase. At least eight 35S-labeled, phosphatase-sensitive compounds structurally different from [35S]6thioGMP were found in nuclease P1 digests. Four of these compounds were susceptible to cleavage with nucleotide pyrophosphatase, thus indicating that they contained phosphoric acid anhydride bonds. Individual RNA species were not separately examined, the radiochromatographic data, however, which were obtained from digests of total RNA preparations, present evidence that 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate exist as 5'-terminal starting nucleotides (in tRNA and rRNA) and that 6-thioguanosine becomes incorporated into the highly modified dinucleoside triphosphate structures (caps) which commonly block the 5'-termini of eukaryotic poly(A)+ mRNA-molecules.

Animals↗
PubMed1998

The obesity gene in swine: sequence and expression of porcine leptin.

Leptin is a 16-kDa protein secreted by adipocytes that has been proposed to regulate feed intake in mice, rats, and humans. The present study was designed to characterize porcine leptin structure and expression. Successful RT-PCR resulted in development of a cDNA clone to the full length coding region of porcine leptin. Sequence data demonstrate 85% base homology to rodent, 88% to human, and a 92% homology to the bovine sequence. For assessment of porcine leptin gene expression, total RNA was extracted from the subcutaneous adipose tissue of genetically selected high backfat pigs and from contemporary crossbred swine. Total RNA derived from genetically selected high fat pigs contained 113% higher (P < .05) concentrations of porcine leptin mRNA than total RNA derived from contemporary crossbred pigs. Western blotting was used to evaluate serum levels of porcine leptin in genetically selected high backfat and contemporary, crossbred pigs. Relative levels of porcine leptin in sera from obese swine were approximately 306% higher (P < .05) than levels present in sera from contemporary, crossbred swine. These data indicate that leptin is expressed in pigs, the expressed protein is secreted into the bloodstream, and obese swine express higher levels of leptin mRNA and protein than nonobese swine at similar body weight.

Adipose Tissue↗