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PubMed1994

Storage phosphor imaging technique improves the accuracy of RNA quantitation using 32P-labeled cDNA probes.

The use of the storage phosphor imaging technique to quantitate radioactivity on blots generated by hybridization with 32P-cDNA probes was evaluated and compared with screen-enhanced x-ray film autoradiography. Quantitation of RNA dot blots hybridized with a 28S ribosomal RNA-specific cDNA probe showed that storage phosphor imaging was more sensitive than screen-enhanced x-ray film autoradiography in identifying low amounts of total RNA (1-10 micrograms). Evaluation of Northern blots containing 30 micrograms of total RNA from human skin biopsies hybridized with a cDNA probe for the human acidic ribosomal phosphoprotein, PO, showed that both techniques detected random biological variability of this housekeeping gene in a similar manner. The two techniques exhibited a strong linear correlation in their ability to quantitate mRNA levels of a retinoic acid-inducible gene (RIS-1). This correlation was stronger at levels corresponding to 1-fold to 30-fold increases of signal and decreased beyond this range because of the insensitivity of the x-ray film. In conclusion, storage phosphor imaging is more accurate than screen-enhanced x-ray film autoradiography in identifying different RNA amounts (higher sensitivity) and in detecting increasing RNA signals at high levels of radioactivity (higher dynamic range).

Animals↗
PubMed1998

Human gap junction protein connexin31: molecular cloning and expression analysis.

We have isolated and characterized a human genomic clone containing the complete coding region of connexin31 (Cx31). Similar to rodent Cx31, the coding region of human Cx31 is completely contained within the second exon and consists of 810 nucleotides. The deduced human Cx31 polypeptide consists of 270 amino acids with a predicted molecular mass of 30.818 kDa. Its sequence is most similar to mouse Cx31 (82.6% identical amino acids) and rat (83.0% identical amino acids), but shows considerably fewer potential sites of phosphorylation. After Northern blot hybridization, two Cx31 transcripts of 2.2 and 1.8 kb were detected in total RNA of the human keratinocyte cell line HaCaT and two transcripts of 2.2 and 1.9 kb in total RNA of E6/E7 transfected human keratinocytes (HEK cells). Using affinity-purified rabbit antibodies to mouse Cx31, immunofluorescence analysis demonstrated relatively weak expression of human Cx31 in HaCaT and HEK cells. The Cx31 gene exists as a single copy gene in the human genome and was mapped to the chromosomal region 1p34-p36 by analyzing human-mouse somatic cell hybrids.

Amino Acid Sequence↗
PubMed2005

Differential expression of genes within the cochlea as defined by a custom mouse inner ear microarray.

Microarray analyses have contributed greatly to the rapid understanding of functional genomics through the identification of gene networks as well as gene discovery. To facilitate functional genomics of the inner ear, we have developed a mouse inner-ear-pertinent custom microarray chip (CMA-IE1). Nonredundant cDNA clones were obtained from two cDNA library resources: the RIKEN subtracted inner ear set and the NIH organ of Corti library. At least 2000 cDNAs unique to the inner ear were present on the chip. Comparisons were performed to examine the relative expression levels of these unique cDNAs within the organ of Corti, lateral wall, and spiral ganglion. Total RNA samples were obtained from the three cochlear-dissected fractions from adult CF-1 mice. The total RNA was linearly amplified, and a dendrimer-based system was utilized to enhance the hybridization signal. Differentially expressed genes were verified by comparison to known gene expression patterns in the cochlea or by correlation with genes and gene families deduced to be present in the three tissue types. Approximately 22-25% of the genes on the array had significant levels of expression. A number of differentially expressed genes were detected in each tissue fraction. These included genes with known functional roles, hypothetical genes, and various unknown or uncharacterized genes. Four of the differentially expressed genes found in the organ of Corti are linked to deafness loci. None of these are hypothetical or unknown genes.

Animals↗
PubMed1991

Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR).

We have identified a rat smooth muscle cell mRNA, 2B7, which distinguishes smooth muscle cell phenotypes in vitro. Sequence and tissue distribution data strongly suggest this mRNA to be identical to osteopontin (secreted phosphoprotein I, 2aR). In vivo, 2B7 mRNA is expressed in normal rat aorta and carotid arteries at levels 50-60-fold greater than heart, and about 3-4 times the levels found in adult rat kidney on a per micrograms of total RNA basis. Of the other smooth muscle sources surveyed, significant levels of 2B7 mRNA were detectable in total RNA prepared from rat uterus and stomach, but not intestine. 2B7 mRNA levels in both carotid and aortic artery increase with age, and are elevated approximately 5-fold in the carotid artery 48 h after balloon angioplasty. The presence of osteopontin (secreted phosphoprotein I, 2aR) in the normal artery wall and its increased expression after injury suggests a previously unappreciated role for this molecule in the vascular system.

Amino Acid Sequence↗
PubMed2003

Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats.

BACKGROUND/PURPOSE: Newborns with congenital diaphragmatic hernia (CDH) still have a high mortality rate, which has been attributed to pulmonary hypoplasia and pulmonary hypertension. Fibroblast growth factors (FGFs) are essential components of the gene network that regulates lung development. Recent studies suggest that the new member of FGF family, FGF-10, plays a fundamental role in branching morphogenesis and is essential for lung formation. FGF-10-deficient mice exhibit complete absence of lungs. FGF-7 promotes epithelial proliferation and expansion leading to the formation of cystlike structures. The aim of this study was to determine the gene level expression of FGF-10 and FGF-7 in the lung of nitrofen-induced CDH. METHODS: Congenital diaphragmatic hernia (CDH) was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: normal controls (n = 16), nitrofen induced without CDH (n = 16), and nitrofen-induced CDH (n = 16). Total RNA and DNA were extracted from the lung in each group and measured. mRNA was extracted from total RNA. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expressions of FGF-10 and FGF-7. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin, a house-keeping gene. RESULTS: FGF-10 mRNA expression was decreased significantly in CDH lung (2.914 +/- 0.320) compared with controls (4.062 +/- 0.307; P <.05) and nitrofen induced without CDH lung (3.923 +/- 0.250; P <.01). FGF-7 mRNA expression was decreased significantly in CDH lung (0.777 +/- 0.097) compared with controls (1.028 +/- 0.093; P <.01). CONCLUSIONS: Decreased gene expression of FGF-10 and FGF-7 in the hypoplastic lung suggests that pulmonary hypoplasia in nitrofen-induced CDH rat may be caused by reduced synthesis of FGF-10 and FGF-7 during lung morphogenesis.

Animals↗
PubMed2004

Identification of pancreatic exocrinopathy in non-obese diabetic mice by gene subtraction analysis.

Type 1 diabetes is an immune-mediated disease with pancreatic infiltration and subsequent beta cell destruction. In this study pancreatic exocrinopathy in non-obese diabetic mice (NOD) was identified using gene subtraction methods (SSH) and macroarray analysis. Female NOD mice were treated with cyclophosphamide for acceleration and synchronization of the disease process at 70 d of age and analysed 10 d later, before the onset of overt diabetes. Extraction of total RNA of pancreas was followed by subtraction using the SSH technique. Pools of cDNA were generated using total RNA from treated and untreated NOD mice. Subtraction of cDNA pools of cyclophosphamide treated mice from cDNA pools of untreated mice resulted in a cDNA library, from which 480 clones were randomly selected. The clones were hybridized against labelled cDNA-probes generated from cyclophosphamide-treated and control NOD mice. Fifty-three clones (11 %) revealed at least twofold differential gene expression after cyclophosphamide treatment. Three of the downregulated genes (amylase, carboxypeptidase and preprotrypsin) were selected for evaluation of macroarray data by quantitative real-time PCR. Analysis of real-time PCR data confirmed suppression of gene expression with highest fold change for amylase (4.68-fold) followed by carboxypeptidase (2.79-fold) and preprotrypsin (2.14-fold). These results lead to the conclusion that inflammation in this animal model of type 1 diabetes is not restricted to pancreatic islets and that subtraction followed by macroarray analysis is capable of identifying genes responsible associated with disease progression.

Amylases↗
PubMed1982

Message-specific sequestration of maternal histone mRNA in the sea urchin egg.

Nucleate and anucleate fragments of sea urchin eggs were prepared by centrifugation on sucrose step gradients. The amount of total RNA, poly(A)+ RNA, histone mRNA, actin mRNA, alpha-tubulin mRNA, and mitochondrial rRNA was determined for each fragment. Total RNA, poly(A)+ RNA, actin mRNA, and alpha-tubulin mRNA all distributed in the same ratio as the volume of the fragments. In contrast, the mitochondrial rRNA was found preferentially distributed in the anucleate fragments, coinciding with the distribution of the mitochondria. Histone mRNAs did not follow the fragment volume ratios, but rather were always found associated with the fragment containing the nucleus. To distinguish between nuclear association and possible artifacts associated with centrifugation, eggs were manually cut into nucleate and anucleate fragments and the amount of histone mRNA was determined for each set. Again only the fragments containing the nucleus had detectable amounts of histone mRNA. Although histone mRNAs were always associated with the nucleate fragment, very little histone mRNA was found associated with isolated egg nuclei prepared under gentle isotonic isolation conditions. Furthermore, embryos that have had first nuclear breakdown blocked with 6-dimethylaminopurine still initiated the recruitment of histone mRNAs into polysomes at the same time as control embryos, thus indicating that nuclear breakdown is not necessary for normal histone message utilization. These results demonstrate a message-specific sequestration of maternal histone mRNA which is physically different from that of other maternal mRNAs and which may govern the timing of maternal histone synthesis in sea urchin embryos.

Actins↗
PubMed2003

Expression of Ku80 in cervical cancer correlates with response to radiotherapy and survival.

To reveal the genes relevant for prediction of cervical cancer after radiotherapy, we previously carried out cDNA microarray experiments on primary cervical cancer comparing patients with a complete response (CR) and those with no change (NC). Some of these genes had already been associated with the radiation response, such as x-ray repair cross-complementing 5 (XRCC5), which was found more in radioresistant tumors than in radiosensitive ones. The aim of this study was to confirm the possible roles of XRCC5 mRNA levels by a real-time polymerase chain reaction method in 20 cervical cancers, and Ku80 protein, which is the gene product of XRCC5, using a histopathologic method of formalin-fixed sections of tumor biopsies in determining tumor response to radiotherapy and survival in 89 patients with cervical cancer. The levels of XRCC5 mRNA were 10(4.82) +/- 10(0.2) copies/microg total RNA in tumor tissues in the CR group (mean +/- standard deviation) and 10(4.95) +/- 10(0.32) copies/microg total RNA in those in the NC group. The levels of XRCC5 mRNA were not significantly different between the CR and NC groups. Histopathologic methods revealed 29.2% (26 of 89) of the patients to be Ku80-negative, with Ku80-positive findings in 70.8% (63 of 89). Of the Ku80-negative patients, 19 had CR, 3 had a partial response (PR), and 4 had NC. Of the Ku80-positive patients, 25 had CR, 22 had PR, and 16 had NC. Ku80-negative tumors showed significantly better responses than Ku80-positive ones, comparing CR and PR/NC responses (p = 0.01). In addition, overall survival was significantly better in the Ku80-negative patients as compared with those who were Ku80-positive (p = 0.04). The results of this study suggest that a low expression of Ku80 protein leads to radiosensitivity in cervical cancer and that Ku80 might play a role in treatment outcome.

Aged↗
PubMed1983

Ribonucleic Acid and Protein Metabolism in Pea Epicotyls : III. Response to Auxin in Aged Tissue.

Applications of auxin to the tips of intact aged pea Pisum sativum L. var Alaska epicotyls resulted in an increase in the content of polyribosomes and poly(A) and in the capacity of isolated polysomes to support protein synthesis in vitro. Few changes were seen in the two-dimensional gel patterns of silver-stained proteins accumulated (or degraded) in vivo even after 15 hours of auxin treatment. In contrast, substantial changes were evident in the two-dimensional gel fluorographs of polypeptides generated in vitro by total RNA and by polysomal RNA from tissue treated with auxin for only 6 hours. Of the 200 spots resolved by fluorography, total RNA from auxin-treated tissue generated 33 spots with increased intensity and 10 with decreased intensity; polysomal RNA yielded 33 spots which increased and only three that decreased. In general, the polypeptides that increased in intensity were higher molecular weight and those that decreased were lower molecular weight. These changes occurred prior to growth and might be prerequisite for the auxin-induced slow growth response seen in this aged tissue.Comparisons were made between the changes in RNA and protein metabolism occuring during aging and after wounding and auxin treatment of aged tissue. Aging causes a decline in poly(A), polysomes, and protein synthesizing capacity, whereas wounding and auxin treatment cause increases. Wounding appears to act primarily at the level of translation, whereas auxin has a greater effect on transcription. It is argued that the use of excised tissue to study auxin effects on RNA and protein metabolism should be avoided.

Journal Article↗
PubMed2000

Regulated expression and potential roles of p53 and Wilms' tumor suppressor gene (WT1) during follicular development in the human ovary.

It has been previously demonstrated that the gonadotropin-mediated inhibition ofapoptosis in rat ovarian granulosa cells is associated with changes in the expression of several cell death-regulatory genes, including p53. In addition, it has been shown that the actions of p53 may be amplified through a cooperative interaction with the Wilms' tumor suppressor gene product (WT1). Based on these findings, the present studies were conducted to determine whether p53 and WT1 are expressed and gonadotropin regulated in the human ovary and to study the relationship between tumor suppressor gene expression and apoptosis in human granulosa/lutein cells (GCs). Analysis of total RNA prepared from human GCs using the RT-PCR demonstrated the presence of p53 messenger RNA (mRNA) and four WT1 mRNA splice variants. These observations were supported by Northern blot analysis of total RNA prepared from human GCs, which revealed the presence of a single (approximately 2.8 kb) p53 mRNA transcript and two primary (approximately 1.8 and approximately 3.5 kb) WT1 mRNA transcripts. Western blot analysis of nuclear protein extracts from human GCs yielded one immunoreactive protein of the expected size (approximately 53 kDa) recognized by a p53 antibody and one immunoreactive protein of the expected size (approximately 52-54 kDa) recognized by the WT1 antibody. Immunohistochemical staining showed that both molecules were localized to nuclei of human GCs and were coordinately regulated during follicular development. Immunofluorescence analysis showed that p53 protein was localized exclusively to nuclei of GCs undergoing apoptosis during in vitro culture and was similarly localized to nuclei and cytoplasm of apoptotic granulosa cells in atretic follicles in vivo. To further evaluate whether human GC apoptosis is linked to increased expression of tumor suppressor genes, we analyzed levels of p53 and WT1 mRNA and protein in GCs induced to undergo apoptosis in vitro. Healthy (nonapoptotic) GCs snap-frozen immediately after isolation from patients undergoing in vitro fertilization-embryo transfer possessed relatively low, but detectable, levels of p53 and WT1 mRNA and protein. However, following serum-free culture to induce apoptosis, p53 mRNA and protein levels increased significantly after 24 h, paralleling the increase in the number of apoptotic GCs. The induction of both p53 mRNA and protein in GCs was inhibited by the addition of human CG to the culture medium. In contrast, WT1 mRNA and protein levels remained constitutive in GCs incubated for 24 h compared with GCs snap-frozen immediately after isolation. We conclude that the p53 and WT1 genes are expressed at the mRNA and protein levels in human GCs and that expression of p53 is regulated during follicular maturation. Nuclear accumulation of p53 protein occurs in human GCs during apoptosis in vitro and in vivo, and p53 mRNA and protein are up-regulated in GCs starved of hormonal support but down-regulated by the presence of human CG. We propose that the products of these two principal tumor suppressor genes serve as important regulators of human follicular development and corpus luteum function.

Adult↗
PubMed1987

Effects of light deprivation on RNA synthesis, accumulation of guanosine 3'(2')-diphosphate 5'-diphosphate, and protein synthesis in heat-shocked Synechococcus sp. strain PCC 6301, a cyanobacterium.

The rate of total RNA synthesis, the extent of guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) accumulation, and the pattern of protein synthesis were studied in light-deprived and heat-shocked Synechococcus sp. strain PCC 6301 cells. There was an inverse correlation between the rate of total RNA synthesis and the pool of ppGpp, except immediately after a temperature shift up, when a parallel increase in the rate of RNA synthesis and accumulation of ppGpp was observed. The inverse correlation between RNA synthesis and ppGpp accumulation was more pronounced when cells were grown in the dark. Heat shock treatment (47 degrees C) had an unexpected effect on ppGpp accumulation; there was a fairly stable level of ppGpp under heat shock conditions, which coincided with a stable steady-state rate of RNA synthesis even in the dark. We found that the pattern of dark-specific proteins was altered in response to heat shock. The transient synthesis of several dark-specific proteins was abolished by an elevated temperature (47 degrees C) in the dark; moreover, the main heat shock proteins were synthesized even in the dark. This phenomenon might be of aid in the study of cyanobacterial gene expression.

Cyanobacteria↗
PubMed1987

Repression of rRNA synthesis during slug reconstruction in Dictyostelium discoideum.

When cells dissociated from "slugs" are allowed to reaggregate, they reconstruct slugs. During this process, the cells showed a marked decrease in the ratio of labeled RNA lacking poly(A) to labeled total RNA as compared with that of cells in normal slugs. Irrespective of the change in total labeled RNA, the ratio remained low even after 6 h of reconstruction. Sucrose density gradient analysis of RNA showed that the synthesis of high molecular weight rRNA (26S and 17S) was considerably repressed in reconstructed slugs as compared with normal slugs. Polyacrylamide gel electrophoresis of low molecular weight rRNA revealed that the synthesis of 5S rRNA, but not 4S tRNA, was repressed.

Dictyostelium↗
PubMed2002

Effect of creatine supplementation on cardiac muscle of exercise-stressed rats.

The role of creatine supplementation in altering the physiological parameters regulating cardiac muscle's functional capacity through the initiation of cardiac hypertrophy and altered contractile protein expression has not been determined. The purpose of this study was to determine the effect of creatine supplementation, with and without exercise stress, on physiological parameters regulating functional capacity through alterations in rat cardiac mass and contractile-protein expression. Thirty male Sprague-Dawley rats were subjected to 30 min of exercise stress 5 days/week for 3 weeks with 2% of total body mass attached to the tail. Animals were randomly assigned to one of four treatment groups: group 1 (Con) received (1 ml/day) sucrose water by intubation tube (n=8); group 2 (Cr) received (1 ml/day) sucrose/creatine solution (n=6); group 3 (EX) received 1 ml/day sucrose water and the exercise stimulus (n=8), and group 4 (Cr/EX) received (1 ml/day) sucrose/creatine solution and the exercise stimulus (n=8). At the conclusion of the 21-day exercise-training period, the heart was collected and weighed for determination of wet weight, total protein, total RNA, and myosin heavy chain protein expression. RNA concentration decreased significantly (13%) in the EX group, but not in the CR/EX group, indicating an interactive effect of creatine and exercise. Total RNA significantly decreased (15%) in the EX group. Protein concentration significantly increased (9%) in the exercising treatments, while total protein did not change. Cardiac myosin heavy chain expression significantly shifted towards a predominant expression of the beta-isoform in the Cr/EX group [54.53% (3.42) beta]. These results indicate an interaction of creatine supplementation and swimming exercise stress that potentially alters cardiac protein synthesis and demonstrates a possible mechanism through which the combination of creatine supplementation and swimming stress stimuli act to alter the physiological parameters regulating cardiac functional capacity.

Administration, Oral↗
PubMed1996

A rapid and highly specific technique to detect hepatitis C RNA in frozen sections of liver.

AIMS: To develop a polymerase chain reaction (PCR) based technique that would permit the rapid and highly specific detection of hepatitis C virus (HCV) RNA extracted from frozen liver tissue. METHODS: Samples of liver tissue from 18 patients undergoing orthotopic liver transplantation were studied. Nine patients were HCV positive. Total RNA was extracted from between one and 10 sections, 10 microns thick, from each tissue sample. HCV RNA was amplified by (1) conventional, multistep reverse transcription PCR (RT-PCR) and by (2) combined, single step RT-PCR using coupled oligonucleotide primers based on the sequence of the 5' untranslated region of the viral genome. Positive results were confirmed by dot blot analysis using a digoxigenin labelled oligoprobe (Alx 89). RESULTS: HCV RNA was detected in the nine HCV positive patients by both conventional and combined RT-PCR. HCV RNA was not detected in the HCV negative patients. As little as 500 ng total RNA was needed as the template to yield detectable amounts of amplified cDNA. The digoxigenin labelled oligoprobe hybridised with HCV RNA positive specimens only. CONCLUSIONS: The combined, single step RT-PCR is a rapid and sensitive technique for detecting HCV RNA in frozen liver tissue.

Base Sequence↗
PubMed2001

A quantitative analysis of purinoceptor expression in human fetal and adult bladders.

PURPOSE: In adults there is evidence that adenosine triphosphate acting at P2X receptors functions as a co-transmitter at vesical smooth muscle. The contractile mechanisms of human fetal bladder have been studied to a limited extent and it remains undetermined whether P2X receptors contribute. We compared the expression of the 7 known P2X receptors in fetal and adult human bladders using a quantitative polymerase chain reaction (PCR) based method. MATERIALS AND METHODS: Real-time quantitative reverse transcriptase-PCR provides a system for the detection and analysis of RNA. Four complete cadaver fetal bladders were obtained at 16 weeks to full-term gestation and divided into a total of 12 segments. Adult bladder samples were obtained from 4 patients requiring bladder biopsy. Total RNA was extracted from each sample and 10 ng. were used for individual PCR reactions. An ABI 7700 machine (PE Applied Biosystems, California) determined expression levels of the 7 P2X genes in total RNA. RESULTS: In adult bladders P2X1 was by far the predominant purinergic receptor at the messenger RNA level. The remaining purinergic receptors were consistently present in the order P2X1 >> P2X4 > P2X7 >> P2X5 > P2X2 >> P2X3 = P2X6 = 0. In fetal bladders the expression of P2X1 transcripts was much lower than in adult bladders, and P2X4 and P2X7 were also present. The rank order of the P2X transcript level was P2X1 = P2X4 > P2X7 >> P2X5 >> P2X2 >> P2X3 = P2X6 = 0. With increasing gestation the P2X receptor transcript level (expression) shifted from the dome to the body of the bladder. CONCLUSIONS: P2X1 is the predominant purinoceptor subtype in adult human bladders, consistent with pharmacological evidence. The fetal expression of all P2X receptor transcripts is much lower than in adults, suggesting that purinergic transmission is of less importance. However, there are also several marked developmental changes in purinoceptor expression in the bladder, in that P2X4 is expressed in developing bladders at relatively high levels. There is also a marked developmental change in the regional distribution of purinoceptors. These changes are likely to reflect the changing role of purinergic transmission in the control of bladder motility during fetal maturation.

Adult↗
PubMedDate not supplied

Direct reverse transcription--polymerase chain reaction from whole blood without RNA extraction.

A simple, fast, efficient method of direct reverse transcription-polymerase chain reaction (RT-PCR) from whole blood without RNA extraction is described. After the erythrocytes in a few microliters of fresh blood were destroyed and removed, the total RNA was released from the leukocytes by means of water treated with diethyl pyrocarbonate. The solution was used as RNA template and directly introduced in the RT-PCR. Following this method, which is called direct RT-PCR, three different cDNA fragments were successfully amplified with three different pairs of primers, respectively. Meanwhile, the direct RT-PCR was compared with the RT-PCR from the extracted total RNA template. The results showed no obvious difference.

DNA, Single-Stranded↗
PubMed1992

Unique nucleotide differences in the conserved 3' termini of brome mosaic virus RNAs are maintained through their optimization of genome replication.

To explore the functionality and conservation of specific base differences in the 3' 200 nucleotides of brome mosaic virus (BMV) RNA-1 (1t) and RNA-2 (2t) with respect to the 3' end of RNA-3 (3t), all possible permutations were used to exchange these regions among the genomic RNAs. When all RNAs bore the 1t promoter, total RNA accumulation was only 15% of wild type; when the 2t or 3t promoter was present on all three RNAs total RNA accumulation was reduced to 30 or 35% of wild type. Two major processes were found to be involved in these dramatic differences. The first reflects the distinct and competitive strengths of the (-)-strand promoters in these sequences, which were shown to have a 3t greater than 1t greater than 2t hierarchy. The second is the importance of the context of upstream sequences in which the 3' promoter is placed. Important contributions of the 3t promoter in preferential amplification of RNA-3 were apparent from changed RNA 1 + 2: 3 ratios and reduced progeny accumulation from transfections using the RNA-3/1t chimera. These interactions contribute to temporal modulation as well as overall optimization of viral RNA functions, leading to selection and maintenance of the specific base differences present in the otherwise highly conserved 3' 200 nucleotides of each genomic RNA component of BMV.

Base Composition↗
PubMed2004

[Cloning of differentially expressed genes of eosinophils from asthmatic patients by suppression subtractive hybridization].

OBJECTIVE: To explore the molecular mechanism of eosinophils for its role in bronchial asthma. METHODS: The total RNA extracted from the eosinophils of patients during the onset of asthma was used as the tester and the total RNA obtained after treatment served as the driver. cDNA suppression subtractive hybridization (SSH) was performed using the protocols described in the Clontech SMART PCR cDNA Syn thesis Kit and PCR-Select cDNA Subtraction Kit. The subtracted cDNA was then inserted into T vector to generate subtracted cDNA library. Clones of the subtracted cDNA library were screened by hybridization and the insert sequence of the positive clones was compared with the sequence in the GenBank. RESULTS: A total of 400 clones selected from the subtracted cDNA library were amplified by PCR and about 85% of these clones contained inserts. Six differential cDNA fragments were acquired after two differential screening. These genes were involved in the regulation of proinflammatory response, signal transduction, energy metabolism and cell apoptosis. CONCLUSION: Differentially expressed genes of the eosinophils during the onset and the remission stage of bronchial asthma can be effectively cloned by SSH, which provides a solid foundation for clarifying the molecular mechanism of eosinophils in asthma and a theoretical base for clinical treatment and prevention of asthma.

Asthma↗