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PubMed1992

Protein D is differentially expressed and regulated in the rat epididymis.

We report the use of a sensitive and specific enyzme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) to study the expression of protein D, a major androgen-regulated sperm-binding glycoprotein at the protein and mRNA level in different anatomical regions of the rat epididymis. The concentration of protein D in the caput, corpus and cauda region of the epididymis was 10.2 +/- 0.67, 7.3 +/- 0.61 and 22.8 +/- 1.34 ng/micrograms total protein, respectively. The total RNA extracted from the caput, corpus and cauda regions of the rat epididymis was amplified by PCR with oligonucleotide primers specific for the 5' and 3' portion of protein D cDNA. Compared to the caput and cauda region, a significant reduction (greater than 82 +/- 3%) in the expression of protein D mRNA levels was observed for corpus epididymal RNA. This data demonstrates regional differences in the concentration of protein D and suggests that protein D expression may be regulated at the level of mRNA within the corpus epididymidis.

Animals↗
PubMed1973

Transcription of simian virus 40. 3. Mapping of "early" and "late" species of RNA.

To determine the orientation of transcription of the E and L strands of DNA from simian virus 40 (SV40), we used linear DNA prepared by cleavage of superhelical viral DNA by endonuclease R.R(1) from Escherichia coli as a primer.template for DNA polymerase. The resulting molecules, which were labeled only at the 3' end of each DNA strand, were then cleaved with Hemophilus parainfluenzae endonuclease Hpa I. The ensuing four DNA fragments, whose locations on the viral genome are known, were separated by electrophoresis, denatured, and hybridized to asymmetric SV40 complementary RNA. From the pattern of hybridization of the fragments containing the labeled 3' ends, we conclude that transcription of SV40 proceeds in a clockwise direction on the L strand and in a counterclockwise direction on the E strand as drawn on the conventional SV40 map. To map the "early" and "late" regions of the viral genome, we extracted RNA from lytically infected cells and hybridized it to the separated strands of the four fragments of (32)P-labeled SV40 DNA. Early after infection, RNA complementary to part of the E strand of the contiguous fragments A and C was detected. Late polysomal RNA was complementary to part of the L strand sequences of fragments A and C and to the total L strand sequence of fragments B and D.

Animals↗
PubMed1969

Formation of viral ribonucleic acid and virus in cells that are permissive or nonpermissive for murine encephalomyelitis virus (GDVII).

GDVII virus growth in BHK-21 cells, a permissive host for the virus, resembled productive infections with other picornaviruses. Virus yields ranged from 100 to 600 plaque-forming units (PFU)/cell. Virus replication in HeLa cells, a nonpermissive host for GDVII virus, was characterized by virus yields of only 0.1 to 5 PFU/cell. Similar low yields of virus have been obtained from HeLa cells at all multiplicities of input up to 6,000 per cell. The progeny particles from HeLa cells were, like the infecting particles, restricted in the HeLa cell host. Despite the great difference in final yields of virus from BHK-21 and HeLa cells, the times when maximal yields were reached were similar. GDVII virus stock grown in BHK-21 cells was designated HeLa(-). A variant of GDVII virus which is capable of extensive growth in HeLa cells was obtained. This variant, designated HeLa(+) GDVII virus, was passaged serially in HeLa cells. Virus yields of 50 to 150 infective virus particles per cell were obtained from infection of HeLa cells with HeLa(+) GDVII virus. The major species of HeLa(+) virus-specific ribonucleic acid (RNA) produced was single stranded and sedimented with an S value of 35S. The rate of accumulation of HeLa(+) virus-specific RNA in HeLa cell cultures was about four times that of HeLa(-) RNA. The amount of virus-specific HeLa(+) RNA formed in HeLa cells was several-fold greater than that of HeLa(-) RNA. With HeLa(-) parent GDVII virus undergoing productive replication in BHK-21 cells or abortive replication in HeLa cells, the major species of virus-specific RNA produced was single stranded and sedimented with an approximate S value of 35S. The amount of HeLa(-) virus-specific RNA extracted from BHK-21 cells was several-fold greater than the amount obtained from HeLa cells.

Centrifugation, Density Gradient↗
PubMed1995

An improved method for isolating RNA from coccidian oocysts.

A method, based on the disruption of eimerian oocysts in a French pressure cell in the presence of guanidine isothiocyanate, has been developed to isolate large quantities of high quality total RNA efficiently. This procedure results in a 12.5-fold greater number of oocysts broken, and a 22-fold greater yield of total RNA than from disruption by conventional grinding in liquid N2. In addition, the RNA isolated by the French pressure cell method was of equal or superior quality when compared to RNA isolated by grinding. This procedure provides a significant improvement in RNA extraction from eimerian oocysts and will greatly facilitate the study of gene expression in this important group of parasites.

Animals↗
PubMed1996

Evidence for immunoglobulin heavy chain variable region gene replacement in a patient with B cell chronic lymphocytic leukemia.

Immunoglobulin heavy chain variable (V) gene replacement is an unusual recombinatorial event characterized by rearrangement of a germline V gene to a preformed VDJ gene complex. This phenomenon has occasionally been implicated in the emergence of clonal subpopulations during the course of acute lymphoblastic leukemia; it has also been found in murine precursor B cell lines. V gene replacement has never been described in lymphoproliferative disorders corresponding to more differentiated stages of B cell ontogeny. The present communication provides evidence for the operation of the same mechanism in B cell chronic lymphocytic leukemia (B-CLL). Genomic DNA and total cellular RNA extracted from peripheral blood mononuclear cells of a 48-year-old female patient diagnosed as having typical B-CLL were subjected to polymerase chain reaction (PCR) amplification aiming to detect rearranged clonal heavy and light chain variable genes (VH and VL, respectively). PCR consistently gave two VH amplification products, both at the DNA and the RNA level; similar analysis for the VL region revealed the presence of a single rearranged VK gene. Direct sequence analysis of the PCR products revealed that, except for a number of silent mutations, the single rearranged VK gene was identical to the germline A1-A17 VK gene. The two rearranged VH gene segments belong to the VHl and VHIII gene families and are closely homologous, respectively, to the germline gene segments V1-18 and V3-30, which have been shown to be used by autoantibodies. Both rearranged VH genes showed identical in-frame D-N-JH junctions and JH gene usage (JH5b), whereas the VH-N-D junctions were different. The above findings indicate that, during the course of the disease of our patient, VH gene replacement took place giving rise to two different clonally related subpopulations. This raises the intriguing possibility that the recombinase machinery, which governs Ig recombinatorial processes, might be operative even at more advanced stages in B cell ontogeny.

Amino Acid Sequence↗
PubMed1994

Identification of type VI collagen in the trabecular meshwork and expression of its mRNA by trabecular cells.

To investigate the nature of the 140 kDa glycoprotein in the trabecular meshwork, polypeptides were extracted with either urea/sodium dodecyl sulfate (SDS)/beta-2-mercaptoethanol (BME) or guanidine hydrochloride followed by pepsin digestion. After electrophoresis and immunoblotting with anti-type-VI-collagen antibodies, a single fraction of molecular weight 140 kDa was identified in the urea/SDS/BME extracts. Pepsin solubilization revealed two immunoreactive fractions (molecular weights 75 and 85 kDa) that comigrated with purified, pepsin-solubilized type VI collagen. By using the polymerase chain reaction (PCR) and primers specific for the alpha 2(VI) chain of type VI collagen, a single PCR product was obtained, which corresponded to the expected size of 137 base pairs, from the total RNA extracted from the trabecular meshwork ex vivo. Southern hybridization with the antisense oligonucleotide probe of the alpha 2(VI) chain confirmed that the amplified sequence was specific. The results show that the trabecular meshwork contains a significant amount of type VI collagen and that trabecular cells express the mRNA coding for the alpha 2(VI) chain of this glycoprotein. The presence of type VI collagen in the trabecular meshwork is implicated in cell-extracellular matrix interactions at this site, and its abnormal accumulation in glaucomatous and aging eyes probably signifies a defect in the function of the trabecular cells in these eyes.

Amino Acid Sequence↗
PubMed1985

Tissue-specific regulation of two functional malic enzyme mRNAs by triiodothyronine.

Rat liver malic enzyme (ME) synthesis is known to be regulated by 3,5,3'-triiodo-L-thyronine (T3). Hybridization of 32P-labeled ME cDNA with RNA extracted from normal and T3-induced livers (15 or 50 micrograms/100 g body weight for 10 days) showed an increase in the ME mRNA concentration by approximately 11-fold in T3-treated rats. ME activity and ME mass were stimulated to the same degree as ME mRNA. Northern blot analysis of either total or poly(A+) RNA revealed two distinct ME mRNAs (21 and 27 S) which were equally induced by T3 treatment. Both mRNAs were shown by in vitro translation assay to program the synthesis of the same immunoprecipitable protein corresponding to full-sized ME. From all the above, we concluded that both messages code for active enzyme. ME activity and ME mRNA were also detected in nonhepatic tissues for which different responses to T3 induction were observed without direct correlation with their respective content of T3 nuclear receptor. Increases in ME activity and level of hybridizable ME mRNA were seen 48 h after a single administration of T3 (200 micrograms/100 g body weight) in liver, kidney, and heart (10.3- and 15.5-, 1.7- and 2.6-, and 1.72- and 3.4-fold above basal values, respectively). Lower levels of induction could already be detected after 24 h, liver being the most stimulated tissue. ME was not affected in brain, lung, testis, and spleen. Northern blot analysis showed that both ME mRNAs are present in all tissues tested, although in different relative proportions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Infectivity of RNA from inactivated poliovirus.

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.

Animals↗
PubMed1977

Genome of infectious bronchitis virus.

Techniques are described for the growth and rapid purification of the avian coronavirus infectious bronchitis virus (IBV). Purified IBV has a sedimentation coefficient of 320S and a buoyant density of 1.22 g/ml in sucrose-deuterium oxide equilibrium gradients. IBV RNA extracted by proteinase K in the presence of sodium dodecyl sulfate and further purified by phenol extraction and gradient centrifugation is single stranded and has a sedimentation coefficient of 64S, as determined by isokinetic gradient centrifugation. Analysis on sucrose gradients under both aqueous and denaturing conditions together with agarose gel electrophoresis in the presence of the chaotropic agent methylmercuric hydroxide gave a value of 8 X 10(6) for the moleclar weight of IBV RNA. This value was confirmed by RNase T1 fingerprinting, which also indicated that IBV RNA is haploid. No evidence was found of subunit structure in IBV RNA. From these results together with the recently reported observation that IBV RNA is infectious and contains a tract of polyadenylic acid (Lomniczi, J. Gen. Virol., in press), we conclude that the genome of the coronaviruses is a single continuous chain of about 23,000 mononucleotides that is of messenger polarity.

Base Sequence↗
PubMed2000

Cardiac L-type calcium channel alpha 1-subunit is increased by cyclic adenosine monophosphate: messenger RNA and protein expression in intact bone.

L-type calcium channels have been identified previously in both osteoblast-like osteosarcoma cell lines and primary cultures of osteoblasts using numerous techniques such as patch clamp recording, drug inhibited 45Ca2+ uptake, and Fura-2 measurements, but intact bone has not been investigated. Using reverse-transcription polymerase chain reaction (RT-PCR) we found that the three major isoforms of the alpha 1-subunit of L-type calcium channels, (alpha 1C, alpha 1D, and alpha 1S) are present in RNA extracted from ROS 17/2.8 osteosarcoma cells, rat femur, and rat skull. Sequencing of most of the alpha 1C-subunit from rat femur and ROS cells revealed that the splice variants in osteosarcoma cells and intact bone differ, but there are no unique sequence variations compared with those found in other tissues. Northern blot analysis of ROS cell RNA indicated that cyclic adenosine monophosphate (cAMP), but not 1 alpha, 25-dihydroxyvitamin D3, increased the messenger RNA (mRNA) of the alpha 1C-subunit. Western blot of ROS cell lysates revealed a band of more then 220 kDa, the amount of which increased in cells treated with cAMP. Using confocal microscopy combined with immunohistochemistry in ROS cells, intact bone, and cartilage, we found that the alpha 1C-subunit of this channel is expressed in osteoblasts and chondrocytes suggesting this channel may be a pathway for signal transduction in intact tissue, because it is in osteosarcoma cell lines and primary osteoblasts grown in tissue culture.

Animals↗
PubMedDate not supplied

Characterization of the genome of avian encephalomyelitis virus with cloned cDNA fragments.

cDNA fragments were generated from RNA extracted from preparations of avian encephalomyelitis virus (AEV) by a reverse transcription-polymerase chain reaction (RT-PCR) strategy, which exploited the probability that AEV is a picornavirus. Rapid amplification of the 3' cDNA ends, which utilized an oligo d(T)-based primer that hybrizes to the putative Poly (A) tract at the 3' terminus of picornavirus RNA, produced a 3.8-kbp fragment (3.8-kbp 3' RACE fragment), from which a 2.5-kbp cDNA fragment specific to the extreme 3' terminal region of the AEV genome was cloned. Positive hybridization reactions between RNA from gradient-purified virus and radiolabeled probes confirmed that the cloned 2.5-kbp fragment was AEV specific. The success of the RT-PCR amplification strategy adopted and the results of northern blotting hybridization experiments indicated that the AEV genome is a polyadenylated, single-stranded RNA, approximately 7.5 kb in size. Sequence analysis of a 869-base region at the 3' terminal of the genome indicated that this region encoded a protein with close homologies to picornaviral RNA polymerase proteins. On the basis that the highest levels of protein homologies were observed with hepatitis A virus, it is likely that AEV will be reassigned to a genus other than the enterovirus genus within the virus family Picornaviridae. The AEV-specific cloned DNA fragments and nucleotide sequence information resulting from this investigation may facilitate the development of in situ hybridization and RT-PCR methods that will be useful in AEV diagnosis.

Amino Acid Sequence↗
PubMed1995

Quantification of hepatitis C virus RNA by competitive amplification of RNA from denatured serum and hybridization on microtiter plates.

The direct detection of hepatitis C virus (HCV) RNA by PCR is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. We report a technique of competitive amplification allowing the estimation of HCV RNA copy number in biological samples. We constructed a standard competitive RNA template containing only two point mutations compared with its wild-type counterpart. The competitor was added in titrated amounts to the target RNA, and the mixture was then reverse transcribed and amplified in the same reaction tube. The relative amounts of target and competitor were determined by differential hybridization on microtiter plates with nonradioactive probes. The evaluation of HCV RNA titer required a single coamplification with the competitor and could be read from a standard curve. Furthermore, this method proved suitable for amplification of HCV RNA directly from serum, thus avoiding the intrinsic variability of the RNA extraction step.

Base Sequence↗
PubMed1984

Gene amplification as a mechanism of reversion at the HPRT locus in V79 Chinese hamster cells.

Spontaneous phenotypic revertants of hypoxanthine phosphoribosyl-transferase (HPRT) temperature-sensitive V79 Chinese hamster cells were selected by plating a temperature-sensitive mutant in HAT medium at 39 degrees C. The incidence of such revertants was approximately 2 X 10(-4) per cell. The majority of the revertants examined had increases of between three- and tenfold in their specific activity of the enzyme, and they were able to grow continuously in the presence of HAT medium at 39 degrees C. When the revertants were cultivated in the absence of HAT, they recovered their HAT-sensitive phenotype and their lowered level of HPRT. Three of the revertants were examined for their temperature inactivation profiles, and all were found to have profiles identical to the ts parent, and quite different from the V79 wild type. The kinetic properties of the cell lines were studied: the Km for both PRPP and hypoxanthine was significantly different in the temperature-sensitive cells but was not significantly altered in the revertants with respect to the ts mutants. A specific antibody to Chinese hamster brain HPRT was employed in immunoprecipitation experiments. By measuring the point at which the immunoprecipitation of the antibody to HPRT was overcome by increasing concentrations of cell supernatant, it was possible to estimate the relative amount of enzyme molecules in the cell lines. From these data, it could be concluded that the revertants overproduced an enzyme with the same immunological properties as the ts line. Southern blots of the Hind III restricted DNA from the ts mutant and two revertant cell lines were examined with an HPRT cDNA probe. This established that the HPRT gene was amplified twofold in one of the revertants, and threefold in the other. However, if the revertants were reintroduced into nonselective medium, the gene copy number declined to one. Finally, northern blots of RNA extracted from the various cell lines demonstrated that the HPRT mRNA was augmented 1.5-fold in one revertant and 1.4-fold in the other. Reintroduction into non-selective medium resulted in a decline in mRNA level for the second mutant, whereas the first mutant appeared to be stabilized. We conclude that gene amplification and concomitant amplification of messenger RNA and enzyme levels are mechanisms of phenotypic reversion at the HPRT locus in Chinese hamster cells.

Animals↗
PubMed2004

Quantitation of MLV-based retroviral vectors using real-time RT-PCR.

Murine leukaemia virus-based vectors quantitation is a time consuming process that can take up to five days. In order to reduce this time a real-time RT-PCR was developed. This method quantifies vectors without an RNA extraction step, using AMV reverse transcriptase and LightCycler technology. Besides a low quantitation time, this method has the advantages of using a plasmid DNA standard curve with good reproducibility, and of having a high sensitivity (3 x 10(2) particles/microl) as well as an excellent intra- and inter-assay reproducibility. Although the method described quantifies vector particles with RNA whether these particles are infectious or not, it is possible to use it to determine infectious particles concentration after the establishment of a correlation between particles with RNA and infectious particles, for a given set of conditions. This method can also be used to study vector stability by comparison of infectious particles, total particles and particles with RNA.

Genetic Vectors↗
PubMed2006

Use of dried high-phenolic laden host leaves for virus and viroid preservation and detection by PCR methods.

The efficiency of RNA extraction for Apricot latent virus (ApLV), Plum bark necrosis stem pitting associated virus (PBNSPaV), Prunus necrotic ring spot virus (PNRSV), Potato virus Y (PVY), and Apple scar skin viroid (ASSVd) from infected hosts is of great importance for molecular diagnosis by the polymerase chain reaction (PCR). A method is described for drying tissue to overcome phenolic inhibitors of viral RNA. This study showed that the infected host leaves, dried at 65 degrees C for 2 days and conserved at 4 degrees C in air proof conditions, serve as good sources for detection of viral and viroid pathogens by PCR methods. Preliminary results suggest that ApLV, PNRSV, PVY, and ASSVd were detected easily by reverse transcriptase-polymerase chain reaction (RT-PCR) and PBNSPaV by nested-RT-PCR with high amplification yields. No significant difference was observed between ethidium bromide-stained band profiles of dried compared to fresh leaves of infected samples. The RNA derived from dry leaf samples was suitable for detection studies. This simple and inexpensive method has proved very effective for long term conservation of virus and viroid isolates.

Desiccation↗
PubMed2003

The use of genomics and proteomics to understand oocyte and early embryo functions in farm animals.

Oocyte maturation, a simple and visible phenomenon, is about to be transformed into a complex and not so visible molecular cascade leading to the marking of the following generation. The study of oocyte maturation in mammals is progressively changing towards a more molecular approach. This review addresses the main challenges in the study of RNA extraction and quantification in oocytes and embryos as well as the importance of the mRNA maturation. The identification of specific genes in oocytes and embryos is now possible with the use of powerful tools, such as library analysis or subtractions, DNA array, comparative analysis of databanks from other mammals or animals and two-dimensional gel electrophoresis analysis. Finally, RNA interference is a useful tool for studying gene function by knocking out the activity of specific genes and will be used in oocytes and embryos.

Animals↗
PubMedDate not supplied

[Nucleic acid and proteins content in Trypanosoma cruzi epimastigotes].

The nucleic acids and protein content in epimastigote forms of Trypanosoma cruzi during its exponential growth in vitro were studied. The parasites were cultivated in a diphasic blood-agar medium at 28 degrees C. The RNA extraction and hydrolisis were performed by the method of Fleck and Begg and the acid hydrolisis of DNA precipitate was done according to Webb and Lindstrom. Schneider's colorimetric method was used for ribose and desoxyribose measurement, employing as reagents orcein and diphenylamine respectively. Protein content was determined by the method of Lowry et al. Calf thymus DNA, yeast RNA and bovine plasma albumin were used as standards. It was established that 1.00 g of wet parasites contained 1.05 (+/-0.17) X 10(10) epimastigote forms. The contents of DNA, RNA and protein were 6.1, 12.3 and 105.4 mg/g of wet epimastigote, or 5.8, 11.7 and 100.1 X 10(-7) microgram per epimastigote, respectively. The method employed for nucleic acid determinations did not show great differences when compared with the Schmidt Thamhauser-Schneider technique, and it has the advantage of being more rapid.

Animals↗
PubMed1994

A rapid method for detection and identification of flaviviruses by polymerase chain reaction and nucleic acid hybridization.

A polymerase chain reaction (PCR) technique was developed and evaluated for the detection of flaviviruses. A set of sense and antisense oligomeric DNA primers were constructed from nucleotide sequences of the conserved region of the genome of several different flaviviruses. Virus specific complementary DNA (cDNA) was prepared by reverse transcription of total RNA extracted from infected cell cultures. Amplified cDNA was identified by nucleic acid hybridization with specific oligomeric internal probes. Various conditions, such as number of cycles and annealing temperature were examined to optimize the detection of viral RNAs from infected cell cultures. Slot blot hybridization with a radioactive probe was used to evaluate the sensitivity of PCR amplification. The PCR amplified RNA sequences of dengue 2 (DEN-2), West Nile (WN), St. Louis encephalitis (SLE) and Kunjin (KUN) virus and detected 0.1 to 1 pg of viral RNA. Japanese encephalitis (JE), Yellow Fever virus (YF), DEN-1, 3, and 4 viruses were not amplified. The more frequent occurrence of mismatches in the 3' primer binding site may explain the failure to amplify cDNA of these viruses.

Aedes↗