Reverse Transcriptase PCR Method Research Articles

Combination of 5-Fluorouracil and N1,N11-Diethylnorspermine Markedly Activates Spermidine/Spermine N1-Acetyltransferase Expression, Depletes Polyamines, and Synergistically Induces Apoptosis in Colon Carcinoma Cells

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) is used widely for chemotherapy of colorectal carcinoma. Recent studies showed that 5-FU affects polyamine metabolism in colon carcinoma cells. We therefore examined whether combinations of 5-FU with drugs that specifically target polyamine metabolism, i.e. N1,N11-diethylnorspermine (DENSPM) or alpha-difluoromethylornithine (DFMO), have synergistic effects in killing HCT116 colon carcinoma cells with wild-type or absent p53. Our results showed that simultaneous 5-FU and DENSPM, a spermine analogue, synergistically increased transcript levels of the polyamine catabolism enzyme spermidine/spermine N1-acetyltransferase, depleted spermine and spermidine, increased acetylated spermidine, and produced synergistic tumor cell apoptosis in both p53 wild-type and p53-null variants. By contrast, simultaneous combination of 5-FU with DFMO, an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, depleted putrescine but did not produce synergistic cell killing. Some pre-treatment and post-treatment regimens of DENSPM and DFMO were antagonistic to 5-FU depending on cellular p53 status. Protein and transcriptome expression analysis showed that combined 5-FU and DENSPM treatment activated caspase 9, but not caspase 3, and significantly suppressed NADH dehydrogenases and cytochrome c oxidases, consistent with the observed increase in hydrogen peroxide, loss of mitochondrial membrane potential, and release of cytochrome c. Our findings demonstrate the importance of the polyamine pathway in 5-FU effects and suggest that the combination of 5-FU with DENSPM has potential for development as therapy for colorectal carcinoma.

Method for detection of respiratory viruses in the sputa of patients with cystic fibrosis

Since the role of respiratory viruses in lung exacerbations of patients with cystic fibrosis has been hampered by the difficulty of detecting viruses in viscous sputum specimens, a multiplex reverse transcriptase PCR (RT-PCR) assay combined with colorimetric amplicon detection was tested for the identification of seven common respiratory viruses in the sputa of cystic fibrosis patients. Of 52 sputa from 38 patients, 12 (23%) samples from 12 patients were positive for a respiratory virus (4 for influenza B, 3 for parainfluenza 1, 3 for influenza A and 2 for respiratory syncytial virus). These results suggest that the RT-PCR method carried out on sputum may provide a convenient means of investigating the role of virus infection in lung exacerbations of cystic fibrosis patients.

ATP‐binding cassette superfamily transporter gene expression in human soft tissue sarcomas

The phenomenon of multidrug resistance (MDR) in various malignant neoplasms has been reported as being caused by one or multiple expressions of ATP-binding cassette (ABC) superfamily protein, including P-glycoprotein/multidrug resistance (MDR) 1 and the MDR protein (MRP) family. However, their expression levels and distribution within soft tissue sarcomas remain controversial. In 86 cases of surgically resected soft tissue sarcoma, intrinsic mRNA levels of MDR1, MRP1, MRP2 and MRP3 were assessed using a quantitative reverse transcriptase-PCR (RT-PCR) method. Moreover, immunohistochemical protein expressions of P-glycoprotein (P-gp), MRP1, MRP2, MRP3 and p53 protein were evaluated in concordant paraffin-embedded material. The mRNA expression and immunohistochemical expression of ABC superfamily transporters were compared to clinicopathologic parameters and proliferative activities as evaluated by the MIB-1-labeling index (LI). Among the various histologic types, malignant peripheral nerve sheath tumor (MPNST) showed significantly high levels of MDR1 (p=0.017) and MRP3 (p=0.0384) mRNA expression, compared to the other tumor types. When the immunohistochemical method was compared to the RT-PCR technique to assess ABC transported expression at the protein and mRNA levels, a significantly close relationship was found between the 2 methods (p<0.05). P-gp expression was significantly correlated with large tumor size (> or =5 cm, p=0.041) and high AJCC stage (stages III and IV) (p=0.0365). Furthermore, cases with nuclear expression of p53 revealed significantly higher levels of MDR1 mRNA expression, compared to those with negative immunoreaction for p53 (p=0.0328). Our results suggest that MDR1/P-gp expression may have an important role to play in tumor progression in the cases of soft tissue sarcoma, and p53 may be one of the active regulators of the MDR1 transcript. In addition, the high levels of both MDR1 and MRP3 mRNA expression in MPNST may help to explain the poor response of this tumor to anticancer-drugs.

Association of HLA Class I and Class II genes with bcr-abl transcripts in leukemia patients with t(9;22) (q34;q11).

BackgroundBased on the site of breakpoint in t(9;22) (q34;q11), bcr-abl fusion in leukemia patients is associated with different types of transcript proteins. In this study we have seen the association of HLA genes with different types of bcr-abl transcripts. The association could predict the bcr-abl peptide presentation by particular HLA molecules.MethodsThe study included a total of 189 patients of mixed ethnicity with chronic myelogenous leukemia and acute lymphocytic leukemia who were being considered for bone marrow transplantation. Typing of bcr-abl transcripts was done by reverse transcriptase PCR method. HLA typing was performed by molecular methods. The bcr-abl and HLA association was studied by calculating the relative risks and chi-square test.ResultsSignificant negative associations (p < 0.05) were observed with HLA-A*02 (b2a2, e1a2), -A*68 (b2a2, b3a2, e1a2), -B*14 (b2a2, b3a2, e1a2), -B*15 (b2a2, b3a2), -B*40 (b2a2), -DQB1*0303 (b2a2, b3a2), -DQB1*0603 (b2a2), -DRB1*0401 (e1a2), -DRB1*0701 (b3a2), and -DRB1*1101 (b2a2).ConclusionsThe negative associations of a particular bcr-abl transcript with specific HLA alleles suggests that these alleles play a critical role in presenting peptides derived from the chimeric proteins and eliciting a successful T-cell cytotoxic response. Knowledge of differential associations between HLA phenotypes and bcr-abl fusion transcript types would help in developing better strategies for immunization with the bcr-abl peptides against t(9;22) (q34;q11)-positive leukemia.

No evidence of a correlation between BCL10 expression and API2-MALT1 gene rearrangement in ocular adnexal MALT lymphoma.

In the present study, 62 cases of ocular adnexal lymphoproliferative disorders were reviewed clinicopathologically. Of them, 51 were extranodal marginal zone B-cell lymphoma (MALT lymphoma), five were diffuse large B-cell lymphoma (DLBCL), one was peripheral T-cell lymphoma, one was NK/T cell lymphoma, nasal type, and four were reactive lymphoid hyperplasia. These lymphoma cases showed a favorable clinical course and localized disease, except for the case of NK/T cell lymphoma, although 19 cases (32.8%) had a recurrence of disease. To clarify the correlation between BCL10 protein expression and API2-MALT1 gene rearrangement, the 51 cases of MALT lymphoma and 5 cases of DLBCL were analyzed by immunohistochemical and RT-PCR methods. Nuclear BCL10 expression was identified in 58% of MALT lymphoma cases, but not in any DLBCL cases. There was no evidence of a correlation between aberrant nuclear BCL10 expression and the clinical parameters examined in the present study. API2-MALT1 transcription was not demonstrated in either the MALT lymphoma cases or the DLBCL cases studied using a multiplex one-tube reverse transcriptase-PCR method. These findings indicate that the nuclear expression of BCL10 is unlikely to correlate with the API2-MALT1 fusion gene in ocular adnexal MALT lymphoma.

Anisomycin superinduces annexin V mRNA expression through the ERK1/2 but not the p38 MAP kinase pathway

Annexin V is a Ca 2+-dependent phospholipid-binding protein belonging to the annexin family whose regulation is currently not well understood. In this study, we utilized anisomycin, a protein synthesis inhibitor that activates MAP kinases (MAPKs), to examine the role of MAPKs in annexin V expression in the MCAS ovarian carcinoma cell line. A one-step real-time TaqMan-based reverse transcriptase-PCR method was developed to quantify annexin V mRNA expression. We found that annexin V was induced 13.3-fold by anisomycin and that this superinduction was attenuated by pretreatment with the MEK inhibitors, U0126 and PD98059, but not with the p38 MAPK inhibitor, SB203580. In addition, immunoblotting showed that anisomycin stimulated the phosphorylation of ERK1/2 as well as p38 MAPK and that the phosphorylations were blocked by the three kinase inhibitors. Taken together, these results suggest that anisomycin superinduces annexin V mRNA expression through the ERK1/2 MAPK pathway, but not through the p38 MAPK pathway.

Rapid RT-PCR amplification of full-length poliovirus genomes allows rapid discrimination between wild-type and recombinant vaccine-derived polioviruses

Poliomyelitis outbreaks in areas that were free for a long time of wild-type polioviruses have been reported. Characterization at nucleotide level of the causative agents showed that the isolated viruses were recombinant oral polio vaccine (OPV)-derived polioviruses. To allow rapid identification and detailed analysis of such recombinant polioviruses, a robust full-length reverse transcriptase-PCR (RT-PCR) was developed using SuperScript II (RT) and expand (PCR). Without extensive purification, it was possible to amplify and characterize the full-length genomes of all selected vaccine, wild-type, and recombinant vaccine-derived polioviruses within a week. Endonuclease nuclease analysis ( SpeI) of the full-length amplicons allowed easy discrimination between recombinant and non-recombinant polioviruses. Furthermore, sequence analysis of cloned full-length amplicons of a recombinant vaccine-derived poliovirus strain showed that the quasi-species nature of a viral stock is preserved during the RT-PCR procedure. This robust and rapid RT-PCR method will allow rapid characterization of (recombinant) poliovirus strains in case of a local poliomyelitis outbreak, and will help to assess the risk of the appearance of such strains after wild-type poliovirus has been eradicated globally.

A sensitive, real-time, RNA-specific PCR method for the detection of recombinant AAV-CFTR vector expression.

Following adeno-associated virus (AAV)-mediated transduction, cellular RNA preparations can be contaminated with AAV single-stranded DNA. The single-stranded DNA genome of recombinant AAV vectors can serve as an efficient, but undesirable, template for traditional reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Consequently, recombinant AAV gene therapy presents a unique challenge to the design of sensitive and reliable methods to detect vector-derived mRNA. Several methods have been proposed to reduce the presence of single- and double-stranded vector DNA without compromising RNA specificity. For example, DNase I, although widely used, can be ineffective at completely removing the AAV single-stranded DNA genome. We have developed a sensitive real-time RNA-Specific reverse transcriptase PCR (RS-PCR) method that is independent of DNase I treatment. The RS-PCR method relies on the generation of a first-strand cDNA template using a primer with a linker sequence, X, at the 5'- end such that synthesis of second-strand cDNA incorporates the X-linker sequence into the cDNA template. The RS-PCR then utilizes forward and reverse primers targeting AAV vector sequence and the X-primer site, respectively, while a vector-specific Taqman probe makes sensitive real-time detection possible. We present data to validate the sensitivity and RNA specificity of the RS-PCR method and propose two unique endogenous control strategies by monitoring expression of both beta-glucuronidase and endogenous cystic fibrosis transmembrane conductance regulator (CFTR). Finally, we demonstrate the utility of this new RS-PCR method in detecting recombinant AAV-CFTR expression, including, an in vitro transduction assay and methods to support both preclinical and clinical trials.

Nuclear Factor-κB and Mitogen-activated Protein Kinases Mediate Nitric Oxide-enhanced Transcriptional Expression of Interferon-β

Mitogen-activated protein (MAP) kinase and nuclear factor-kappaB (NF-kappaB) activation are critical for initiating the transcriptional expression of cytokines, cell adhesion molecules, and other factors in the macrophage immune response. Nitric oxide (NO), an endogenous free radical, is a product of macrophages that mediates inflammatory and cytotoxic processes in the immune system. Here we report the effects of NO on MAP kinase signaling and NF-kappaB activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and correlate these effects to the induction target genes, including interferon-beta (IFN-beta) and IkappaB-alpha. LPS alone induced a rapid phosphorylation of the stress-activated MAP kinases: c-Jun N-terminal kinase (JNK) and p38. Simultaneous treatment with LPS and the NO donor, diethylamine NONOate (DEA/NO), enhanced and prolonged JNK and p38 phosphorylation. Similarly, DEA/NO prolonged the LPS-induced degradation of the NF-kappaB inhibitory subunit, IkappaB-alpha, despite an increase in IkappaB-alpha mRNA levels. Whereas DEA/NO alone was sufficient to induce JNK and p38 phosphorylation, it was not sufficient to cause IkappaB-alpha degradation. The enhancement of IkappaB-alpha degradation by DEA/NO correlated with an increase in the nuclear levels of the p50 and p65 subunits and DNA-binding activity determined by electrophoretic mobility shift assay. DEA/NO and an additional NO donor, MAHMA/NO, are further demonstrated to enhance the transcriptional expression of the IFN-beta gene. The results suggest a role for NO in enhancing and propagating inflammatory conditions and the immune response.

Expression of excretory and secretory protein genes of Trichinella at muscle stage differs before and after cyst formation

By adapting a semi-quantitative reverse transcriptase-PCR (RT-PCR) method, we investigated kinetics of gene expression at different developmental stages of Trichinella spiralis and T. pseudospiralis. The analyzed genes included four kinds of excretory and secretory (ES) proteins, a heat shock protein (HSP) and a DNA binding protein and showed that T. spiralis and T. pseudospiralis expressed ES proteins in a stage-specific manner. The gene encoding a 43 kDa ES protein was expressed by muscle larvae, either pre-cyst or post-cyst larvae. The genes encoding: the 53 kDa ES protein of T. spiralis; 53 kDa ES protein of T. pseudospiralis; and 19.6 kDa ES protein of T. spiralis were expressed by post-cyst larvae and adult worms, but not expressed by pre-cyst larvae or newborn larvae. The results showed that pre-cyst larvae and post-cyst larvae are similar but different in the expression of 53 and 19.6 kDa ES proteins. On the other hand, genes of housekeeping proteins, such as HSP and the DNA binding protein, were expressed at all stages although there were some differences in the expression level.

Quantitative Analysis of Presenilin‐1 mRNA Expression in Sporadic Alzheimer's Disease Brain

Background: The majority of familial Alzheimer's disease (FAD) is caused by mutations in the presenilin‐1 (PS‐1) or presenilin‐2 (PS‐2) genes. The neuropathological changes in both FAD and sporadic late‐onset Alzheimer's disease (AD) are similar, suggesting that they share a common etiological pathway. Despite many attempts to investigate the function of PS in sporadic AD, no consensus has been reached as to the levels of expression in sporadic AD. Methods: PS‐1 mRNA content was measured in the frontal cortex of post‐mortem human brains from four sporadic AD cases and five controls. Real‐time quantitative polymerase chain reaction (PCR) was performed using a LightCycler rapid thermal cycler (Roche) with SYBR Green I fluorescence. Results: The results showed that the content of PS‐1 mRNA in the frontal cortex of sporadic AD cases was higher than those in controls (p&lt;0.05). Conclusion: We quantified PS‐1 mRNA in the frontal cortex from sporadic AD and controls with a quantitative real‐time reverse transcriptase‐PCR (RT‐PCR) method. We found that the PS‐1 mRNA content in sporadic AD was higher than in controls suggesting that up‐regulated PS‐1 expression may be associated with some of the pathological changes found in sporadic AD.

Calcitonin gene-related peptide-enhanced nitric oxide release and inducible NOS activity and mRNA expression in LPS-stimulated mouse peritoneal macrophages.

Previously we have shown that calcitonin gene-related peptide (CGRP), a neuropeptide increases lipopolysaccharide- (LPS) induced nitric oxide (NO) production in mouse peritoneal macrophages by using the Griess method. In this study we further examined whether CGRP could modulate inducible NO synthase (iNOS) protein and mRNA expression from mouse peritoneal macrophages. Macrophages were obtained from the peritoneal exude of male Balb/c mouse. The cells were plated on culture dishes at a density of 5 x 10(5) cells per well and were allowed to adhere for 2 h. After incubation for 24 h, the macrophages were cultured with 0.01 to 1 microg/mL LPS with or without CGRP (1-1,000 nM) for 24 h. The results showed that CGRP markedly enhanced 0.5 microg/mL LPS-induced NO release as compared with that of lower doses of LPS, such as 0.01 and 0.1 microg/mL LPS. NO was increased from 19.8 +/- 2.6 to the highest level of 31.5 +/- 4.2 microM in 5 x 10(5) cells by 10 nM CGRP in 0.5 microg/mL LPS-stimulated macrophages. The cGMP level in macrophages was augmented when CGRP was added with LPS. However, when using higher dose (1.0 microg/mL) of LPS to stimulate the macrophages, CGRP had no effect at all on NO release. CGRP had no direct effect on NO and cGMP production. CGRP increased the expression of inducible NOS protein in LPS-stimulated macrophages shown by immunocytochemistry method. The activity of iNOS was also enhanced by CGRP as compared with LPS-stimulation alone by detecting the 3H-L-citruline formation from 3H-L-arginine. We found that CGRP also increased the LPS-induced iNOS mRNA levels by using reverse transcriptase-PCR method. These data suggest that CGRP enhances LPS-induced NO release, iNOS activity, and iNOS mRNA in mouse peritoneal macrophages.

Analysis of the P1 gene sequences and the 3'-terminal sequences and secondary structures of the single-stranded RNA genome of Potato virus V.

The immunocapture reverse transcriptase PCR method (IC-RT-PCR) was used to selectively amplify specific genome sequences of an isolate of Potato virus V (PVV, genus Potyvirus) from a potato plant infected by multiple viruses in the field in Finland. The sequences of the 5'- and 3'-non-translated regions (NTR) and the P1-and coat protein (CP)-encoding sequences were determined because they are the most variable genomic regions in potyviruses. The sequences of the new Finnish PVV isolate obtained in this study were compared to the sequences of eight PVV isolates characterized from other European countries. The results indicated little genetic variability among the European PVV isolates (nt identity values > 96% for all regions examined). Most PVV isolates were grouped according to their geographical origin using P1-sequences for a phylogenetic analysis. The nucleotide substitution patterns for P1 and the CP genes revealed that variability is due to a random genetic drift. Comparison of the secondary structure predictions for the unusually long 3'-non-translated region (3'-NTR) of PVV to the 3'-NTR of Tobacco etch virus (TEV) and other potyviruses revealed that some of the structures defining regions crucial for viral genome replication in TEV are present in PVV and other potyviruses.

Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-microm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the alpha- and gamma-subdivisions of the division Proteobacteria (alpha- and gamma-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, alpha-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

Protective effect of thioredoxin upon NO-mediated cell injury in THP1 monocytic human cells

Although NO has been postulated to play important roles in host defences, it is potentially damaging for exposed cells, including for the macrophages producing the NO. Thus a network of radical acceptors and enzymes is thought to play an important redox-buffering role to protect cells against NO-mediated injury. We examined the properties of the redox systems superoxide dismutase (SOD)/catalase, glutathione (GSH) and thioredoxin (Trx), in regulating the viability of two human monocytic cell lines (THP1 and U937) exposed to the NO-generating compound diethylene triamine-nitric oxide (DETA-NO). We observed that NO-induced cytotoxic effects were time- and dose-dependent towards the two cell lines. After vitamin-induced differentiation in vitro with retinoic acid (RA) and 1,25-dihydroxy vitamin D3 (VD), termed RA/VD, we observed that THP1 RA/VD cells became more resistant to NO-mediated cytotoxicity whereas the susceptibility of U937 cells was not modified. Using Western blotting and reverse-transcriptase PCR methods, we observed that gene transcription and protein expression of Trx and thioredoxin reductase were significantly increased upon RA/VD treatment and differentiation in THP1 cells. By contrast, SOD/catalase and GSH redox state remained unmodified. Finally, a stable transfectant THP1 line overexpressing Trx was found to be more resistant than THP1 control cells that were untransfected or transfected with an empty plasmid, when exposed to DETA-NO in vitro. In conclusion, we observed an inverse correlation between cell susceptibility to NO damaging effects and Trx expression, suggesting that the Trx system may have important preventative capacities towards NO-mediated cellular injury in monocytic macrophage cells.

Protective effect of thioredoxin upon NO-mediated cell injury in THP1 monocytic human cells

Although NO has been postulated to play important roles in host defences, it is potentially damaging for exposed cells, including for the macrophages producing the NO. Thus a network of radical acceptors and enzymes is thought to play an important redox-buffering role to protect cells against NO-mediated injury. We examined the properties of the redox systems superoxide dismutase (SOD)/catalase, glutathione (GSH) and thioredoxin (Trx), in regulating the viability of two human monocytic cell lines (THP1 and U937) exposed to the NO-generating compound diethylene triamine-nitric oxide (DETA-NO). We observed that NO-induced cytotoxic effects were time- and dose-dependent towards the two cell lines. After vitamin-induced differentiation in vitro with retinoic acid (RA) and 1,25-dihydroxy vitamin D(3) (VD), termed RA/VD, we observed that THP1 RA/VD cells became more resistant to NO-mediated cytotoxicity whereas the susceptibility of U937 cells was not modified. Using Western blotting and reverse-transcriptase PCR methods, we observed that gene transcription and protein expression of Trx and thioredoxin reductase were significantly increased upon RA/VD treatment and differentiation in THP1 cells. By contrast, SOD/catalase and GSH redox state remained unmodified. Finally, a stable transfectant THP1 line overexpressing Trx was found to be more resistant than THP1 control cells that were untransfected or transfected with an empty plasmid, when exposed to DETA-NO in vitro. In conclusion, we observed an inverse correlation between cell susceptibility to NO damaging effects and Trx expression, suggesting that the Trx system may have important preventative capacities towards NO-mediated cellular injury in monocytic macrophage cells.

Discoordinate expression of pancreatic lipase and two related proteins in the human fetal pancreas.

The lipase gene family contains a large number of members. Among the most closely related are pancreatic triglyceride lipase (PTL) and two pancreatic lipase-related proteins (PLRP1 and PLRP2). Previous studies in rodents demonstrated divergent temporal expression of the genes encoding these proteins. PLRP1 and PLRP2 were expressed in fetal pancreas, whereas PTL was not expressed until pups were several weeks old. To determine whether the human pancreas has a similar expression pattern for these genes, we determined the levels of each mRNA in fetal pancreas at various ages. A reverse transcriptase-PCR method was developed and used to quantify the mRNA levels for the three species normalized to the mRNA encoding cyclophillin. The mRNA encoding PLRP1 and PLRP2 was present by 16 wk in the fetal pancreas. In contrast, the mRNA encoding PTL was not present in the fetal pancreas. This pattern of expression suggests that the genes encoding theses proteins have different regulatory elements controlling temporal expression and provides another example of nonparallel expression of genes encoding pancreatic exocrine proteins.

Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA.

AIM:To investigate the characteristics of newly established four hepatocellular carcinoma cell lines (SNU-739, SNU-761, SNU-878 and SNU-886) from Korean hepatocellular cancer patients.METHODS:Morphologic and genetic studies were done.RESULTS:All four lines grew as a monolayer with an adherent pattern, and their doubling times ranged from 20 to 29 hours. The viability rate was relatively high (88%-94%).Neither mycoplasmal nor bacterial contamination was present.The lines showed different patterns in fingerprinting analysis. The hepatitis B virus (HBV) DNA was integrated in the genomes of all four lines, and in all of them HBx,HBc and HBs transcripts were detected by reverse transcriptase-PCR methods. Among the three cell lines used as control (Hep 3B, SK Hep1 and Hep G2),only Hep 3B showed HBx expression, and this line was used as a HBV integrated control.The RNA of albumin was detected in three lines (SNU-761, SNU-878 and SNU-886), that of transferrin in two lines (SNU-878, SNU-886), and that of IGF-II was detected in none of the cell lines.CONCLUSION:These well characterized cell lines may be very useful for studying the biology of hepatocellular carcinoma in association with the hepatitis Bvirus.

The expression pattern of the Arabidopsis ACC synthase gene 1 during rosette leaf development

In Arabidopsis thaliana (L.) Heynh,, 1-aminocyclopropane-1-carboxylate (ACC) synthase, an enzyme of the ethylene biosynthesis pathway, is encoded by a gene family that contains at least five members, The AT-ACS1 gene is regulated in a tissue-specific manner and is induced during early development. Expression of AT-ACS1 during rosette leaf development was analysed in further detail by using a competitive reverse transcriptase PCR method to monitor transcript levels in individual leaves at different stages of rosette development. AT-ACS1 expression was the highest at leaf emergence and decreased sharply in rapidly expanding leaves, to increase again after the leaf expansion phase. Leaf emergence was also accompanied by an increase in ethylene production. AT-ACS1 transcript levels in rosette leaves were not influenced by the transition from the vegetative to the reproductive phase.

BRCA1 expression during prenatal development of the human mammary gland.

Germ-line alterations of BRCA1 are associated with elevated risk of breast cancer. Evidence for the involvement of Brca1 in cellular differentiation and morphogenesis has been obtained in mouse models during embryogenesis. Although the presence of well-conserved functional domains might suggest a similar function for both human and mouse genes, very few data on BRCA1 expression in human fetal tissues are available. We have, therefore, investigated the expression of BRCA1 in the mammary gland from human female fetuses aged between 15 and 33 weeks. Quantification of BRCA1 transcripts, using a competitive reverse transcriptase PCR method, indicates a progressive decrease in BRCA1 expression with increasing fetal age between the 15th and 30th week of gestation. Subsequently, the amount of BRCA1 transcripts becomes similar to that found in adult mammary gland. Analysis of BRCA1 protein revealed, in fetal samples, a 220 kDa band corresponding to the 220 kDa BRCA1 protein described in human cell lines. These later experiments confirm that the relative level of the 220 kDa BRCA1 protein is highest in the early stages of mammary gland development. The temporal patterns of BRCA1 expression in human fetuses suggest a role for BRCA1 in the morphogenesis and differentiation of the human mammary gland.