The evolutionarily conserved RNA Polymerase II Rpb4/7 sub-complex has been thoroughly studied in yeast and impacts gene expression at multiple levels including transcription, mRNA processing and decay. In addition Rpb4/7 exerts differential effects on gene expression in yeast and Rpb4 is not obligatory for yeast (S. cerevisiae) survival. Specialised roles for human (hs) Rpb4/7 have not been extensively described and we have probed this question by depleting hsRpb4/7 in established human cell lines using RNA interference. We find that Rpb4/7 protein levels are inter-dependent and accordingly, the functional effects of depleting either protein are co-incident. hsRpb4/7 exhibits gene-specific effects and cells initially remain viable upon hsRpb4/7 depletion. However prolonged hsRpb4/7 depletion is cytotoxic in the range of cell lines tested. Protracted cell death occurs by an unknown mechanism and in some cases is accompanied by a pronounced elongated cell morphology. In conclusion we provide evidence for a gene-specific role of hsRpb4/7 in human cell viability.
Cell Line ; Cell Nucleus ; metabolism ; Cell Survival ; drug effects ; Gene Expression Profiling ; HeLa Cells ; Humans ; RNA Interference ; RNA Polymerase II ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Small Interfering ; pharmacology

OBJECTIVETo study the imprinting status and expression level of insulin-like growth factor 2 (IGF2) gene in colorectal cancer and to provide a clue for the mechanism of carcinogenesis of colorectal cancer.

METHODSThe expression levels of IGF2 in the paired colorectal cancer and adjacent normal tissue were examined and compared by use of semi-quantitative reverse transcription-polymerase chain reaction. The imprinting status of IGF2 was detected by restriction fragment length polymorphism. The relationships between the expression level of IGF2, its imprinting status, and the carcinogenesis of colorectal cancer were analyzed.

RESULTSIGF2 was overexpressed in 82.4% (28/34) of colorectal cancer tissues which was significantly higher than those of the matched normal tissues (P<0.01, t=3.01). 87.5% (14/16) of colorectal cancer showed loss of imprinting(LOI), while 71.4%(10/14) of normal tissues also displayed LOI of IGF2.

CONCLUSIONOverexpression of IGF2 was found to play an important role in carcinogenesis of colorectal cancer. LOI of IGF2 may be a prophase manifestation of colorectal cancer.

Colorectal Neoplasms ; genetics ; pathology ; DNA, Neoplasm ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Genomic Imprinting ; Humans ; Insulin-Like Growth Factor II ; genetics ; Male ; Middle Aged ; RNA, Neoplasm ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area.

METHODSTwenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis.

RESULTSThe PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4.

CONCLUSIONA simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

DNA Restriction Enzymes ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Genotype ; Hepatitis E ; blood ; genetics ; immunology ; Hepatitis E virus ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

The largest subunit of eukaryotic RNA polymerase II contains a unique domain at its carboxy-terminus, which is referred to as the carboxy-terminal domain (CTD). The CTD is made up of an evolutionarily conserved heptapeptide repeat (YSPTSPS). Over the past decade, there has been increasing attention on the role of the CTD in transcription regulation in the view of mRNA processing and chromatin remodeling. This paper provides a brief overview of the recent progress in the dynamic changes in CTD phosphorylation and its role in integrating multiple nuclear events.
Amino Acid Motifs ; Amino Acid Sequence ; Animals ; Chromatin/*physiology ; Chromatin Assembly and Disassembly ; Conserved Sequence ; Histones/metabolism ; Humans ; Isomerism ; Phosphorylation ; Protein Structure, Tertiary ; RNA Polymerase II/chemistry/*physiology ; RNA, Messenger/metabolism ; Transcription, Genetic

Blood-borne angiotensin-II (Ang-II) has profound effects in the brain. We tested the hypothesis that Ang-II-dependent hypertension involves differential Ang-II type I (AT1) receptors expression in the subfornical organ (SFO) and the rostral ventrolateral medulla (RVLM). Male Wistar rats were implanted with 14-day osmotic minipump filled with Ang-II (150 ng/kg/min) or saline. AT1 receptor mRNA levels were detected in the SFO and RVLM by reverse transcription-polymerase chain reaction (RT-PCR). Ang-II caused hypertension (134 +/- 10 mmHg vs. 98 +/- 9 mmHg, n = 9, p < 0.05). RT-PCR revealed that Ang-II infusion induced increased AT1 receptor mRNA levels in RVLM and decreased in SFO. Our data suggest that Ang-II-induced hypertension involves differential expression of brain AT1 receptors.
Angiotensin II/*metabolism ; Animals ; Hypertension/chemically induced/*metabolism ; Male ; Medulla Oblongata/*metabolism ; RNA, Messenger/genetics ; Rats ; Rats, Wistar ; Receptor, Angiotensin, Type 1/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Subfornical Organ/*metabolism

The core promoter is an important yet often overlooked component in the regulation of transcription by RNA polymerase II. In fact, the core promoter is the ultimate target of action of all of the factors and coregulators that control the transcriptional activity of every gene. In this review, I describe our current knowledge of a downstream core promoter element termed the DPE, which is a TFIID recognition site that is conserved from Drosophila to humans. The DPE is located from +28 to +32 relative to the +1 transcription start site, and is mainly present in core promoters that lack a TATA box motif. Moreover, in Drosophila, the DPE appears to be about as common as the TATA box. There are distinct mechanisms of basal transcription from DPE- versus TATA-dependent core promoters. For instance, NC2/Dr1-Drap1 is a repressor of TATA-dependent transcription and an activator of DPE-dependent transcription. In addition, DPE-specific and TATA-specific transcriptional enhancers have been identified. These findings further indicate that the core promoter is an active participant in the regulation of eukaryotic gene expression.
Animals ; Base Sequence ; DNA-Binding Proteins/metabolism ; Enhancer Elements (Genetics) ; Feedback, Biochemical ; Gene Expression Regulation ; Human ; Models, Genetic ; *Promoter Regions (Genetics) ; RNA Polymerase II/*metabolism ; Transcription Factors/chemistry/genetics/*metabolism ; *Transcription, Genetic

The core promoter is an important yet often overlooked component in the regulation of transcription by RNA polymerase II. In fact, the core promoter is the ultimate target of action of all of the factors and coregulators that control the transcriptional activity of every gene. In this review, I describe our current knowledge of a downstream core promoter element termed the DPE, which is a TFIID recognition site that is conserved from Drosophila to humans. The DPE is located from +28 to +32 relative to the +1 transcription start site, and is mainly present in core promoters that lack a TATA box motif. Moreover, in Drosophila, the DPE appears to be about as common as the TATA box. There are distinct mechanisms of basal transcription from DPE- versus TATA-dependent core promoters. For instance, NC2/Dr1-Drap1 is a repressor of TATA-dependent transcription and an activator of DPE-dependent transcription. In addition, DPE-specific and TATA-specific transcriptional enhancers have been identified. These findings further indicate that the core promoter is an active participant in the regulation of eukaryotic gene expression.
Animals ; Base Sequence ; DNA-Binding Proteins/metabolism ; Enhancer Elements (Genetics) ; Feedback, Biochemical ; Gene Expression Regulation ; Human ; Models, Genetic ; *Promoter Regions (Genetics) ; RNA Polymerase II/*metabolism ; Transcription Factors/chemistry/genetics/*metabolism ; *Transcription, Genetic

OBJECTIVETo explore the influence of lanthanum chloride (LaCl3) on inducible nitric oxide synthase (iNOS) expression in RAW264.7 macrophages with lipopolysaccharide (LPS) induction, and to investigate its possible mechanisms.

METHODSThe RAW264.7 macrophages were randomly divided into four groups: i. e, control group (without treatment), LaCl3 group (with treatment of 2.5 micromol/L of LaCl3 for 24 hrs), LaCl3 + LPS group (with treatment of 2.5 micromol/L LaCl3 for 24h), and LPS group (with treatment of 1 mg/L LPS for 24 hrs). The iNOS protein expression was measured by immunofluorescence and Western blot. iNOS gene expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). NO production in culture supernatant was assayed by nitrate reductase method.

RESULTSImmunofluorescence analysis showed that iNOS was located mainly in the cytoplasm. RAW264.7 cells with overexpression of iNOS accounted for 44.4%, which was obviously higher than that in LaCl3 + LPS group (11.8%, P < 0.05). There was a faint signal of FITC-labeled green tint in control group or LaCl3 group. The iNOS mRNA and protein expression, and the NO content in LPS group were significantly higher than those in control, LaCl3, and LaCl3 + LPS groups (P < 0.05).

CONCLUSIONLaCl3 can suppress LPS-induced iNOS overexpression at mRNA and protein level and reduce NO production, indicating that LaCl3 can antagonize the excessive activation of iNOS induced by LPS.

Animals ; Cell Line ; Lanthanum ; pharmacology ; Lipopolysaccharides ; toxicity ; Macrophages ; drug effects ; metabolism ; Mice ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of fetal rats and to explore the relationship between TCDD-induced abnormal development in rats and the expression and the methylation of insulin-like growth factor 2 gene (Igf2).

METHODSA single dose of 10 microg/kg TCDD was given to gestation day (GD) 10 pregnant rats by gavage. On GD20, the fetuses were taken out and examined. The crown-rump length, the body weight and the placental weight were measured. The expression of Igf2 in liver was detected by real-time quantitative reverse transcription (RT-PCR) and Western blot. The methylation of Igf2 differentially methylated regions (DMRs) in liver was analyzed by a methylation-sensitive restriction enzyme Hpa II PCR assay and a bisulfite-modified DNA sequencing procedure.

RESULTSIn the treatment group, 12.2% of the fetuses were either dead or absorbed, and 11.6% of them were malformed. For the live fetuses, their crown-rump length, body weight and placental weight were significantly lower than those of the control group. The relative amount of Igf2 mRNA in the treated livers and the control livers was 0.77 +/- 0.11 and 0.27+/- 0.15, respectively. The number was significantly higher in the treatment group than in the control group (P < 0.01). Western blot also showed a remarkable up regulation of Igf2 protein in liver after treatment. The two groups showed no difference in the methylation status of Igf2 DMR1 in liver. The DMR2 Igf2 was significantly hypomethylated in the treated livers than in the control livers.

CONCLUSIONExposure to TCDD in pregnancy can lead fetal rats to death, absorption, malformation and intrauterine growth retardation (IUGR). The TCDD led abnormal development in rats may be associated with the hypomethylated DMR2 of Igf2 and the up regulation of Igf2 in liver.

Animals ; Blotting, Western ; DNA Methylation ; Female ; Fetal Growth Retardation ; chemically induced ; genetics ; metabolism ; Gene Expression ; drug effects ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Polychlorinated Dibenzodioxins ; toxicity ; Pregnancy ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDLittle is known about the role of dual angiotensin II forming pathways during heart failure. In the present study, the changes of chymase and angiotensin converting enzyme (ACE) expressions in the failing hearts of hamsters were analysed.

METHODSHeart failure was induced by ligation of left anterior descending branch of the coronary artery. Chymase, ACE and angiotensin II type 1 receptor (AT1R) mRNA levels were analysed by reverse transcription polymerase chain reaction (RT-PCR). The activities of chymase and ACE were determined by radioimmunoassay (RIA). Myocardial collagen fibre analysis was performed under optical microscope.

RESULTSLeft ventricular systolic pressure (LVSP) and maximum left ventricular developed pressure increase rate (dp/dtmax, mmHg/s) gradually moved lower at 2, 3, 4 and 8 weeks after operation. On the other hand, left ventricular end-diastolic pressure (LVEDP) increased gradually after operation. Compared with the control group (3.55 +/- 0.06, 4.79 +/- 0.70), the heart weight/body weight ratio in operation group had increased significantly at 4 weeks and 8 weeks (4.28 +/- 0.43, 6.17 +/- 0.73) (P < 0.01). Collagen staining showed that the quantity of myocardial collagen fibre increased significantly in the operation group. RT-PCR showed that the chymase mRNA level in the operation group was consistently greater than that in the control group. AT1R mRNA level was also increased significantly at 3 weeks and 4 weeks, both being 1.3 times that of the control group (P < 0.01), whereas ACE mRNA level was not changed. Higher activity of chymase was detected in operation group, being 4, 8, 13 and 19 times that of the control group at 2, 3, 4 and 8 weeks (P < 0.01), respectively. ACE activity was also significantly higher at the same time, being 7, 10, 10 and 3.5 times that of the control (P < 0.01). Angiotensin II (Ang II) level in operation group increased significantly, being 2.5, 2.7, 3.5 and 2 times that of the control group at 2, 3, 4 and 8 weeks, respectively (P < 0.01).

CONCLUSIONSA dual Ang II forming pathway from both ACE and chymase in the hamster hearts plays an important role during the development of heart failure. At the decompensatory stage, the reduction of AngII level may be associated with the decrease of ACE activity.

Angiotensin II ; analysis ; Animals ; Body Weight ; Chymases ; Cricetinae ; Heart Failure ; metabolism ; Male ; Myocardium ; metabolism ; Peptidyl-Dipeptidase A ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptor, Angiotensin, Type 1 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Serine Endopeptidases ; genetics ; physiology ; Ventricular Function, Left