SEARCH · PubMed Health

Results for “regulatory element transcripts”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources

PubMed2004

The role of apoptosis in the development of AGM hematopoietic stem cells revealed by Bcl-2 overexpression.

Apoptosis is an essential process in embryonic tissue remodeling and adult tissue homeostasis. Within the adult hematopoietic system, it allows for tight regulation of hematopoietic cell subsets. Previously, it was shown that B-cell leukemia 2 (Bcl-2) overexpression in the adult increases the viability and activity of hematopoietic cells under normal and/or stressful conditions. However, a role for apoptosis in the embryonic hematopoietic system has not yet been established. Since the first hematopoietic stem cells (HSCs) are generated within the aortagonad-mesonephros (AGM; an actively remodeling tissue) region beginning at embryonic day 10.5, we examined this tissue for expression of apoptosis-related genes and ongoing apoptosis. Here, we show expression of several proapoptotic and antiapoptotic genes in the AGM. We also generated transgenic mice overexpressing Bcl-2 under the control of the transcriptional regulatory elements of the HSC marker stem cell antigen-1 (Sca-1), to test for the role of cell survival in the regulation of AGM HSCs. We provide evidence for increased numbers and viability of Sca-1(+) cells in the AGM and subdissected midgestation aortas, the site where HSCs are localized. Most important, our in vivo transplantation data show that Bcl-2 overexpression increases AGM and fetal liver HSC activity, strongly suggesting that apoptosis plays a role in HSC development.

Age Factors↗

PubMed2005

NemaFootPrinter: a web based software for the identification of conserved non-coding genome sequence regions between C. elegans and C. briggsae.

BACKGROUND: NemaFootPrinter (Nematode Transcription Factor Scan Through Philogenetic Footprinting) is a web-based software for interactive identification of conserved, non-exonic DNA segments in the genomes of C. elegans and C. briggsae. It has been implemented according to the following project specifications:a) Automated identification of orthologous gene pairs. b) Interactive selection of the boundaries of the genes to be compared. c) Pairwise sequence comparison with a range of different methods. d) Identification of putative transcription factor binding sites on conserved, non-exonic DNA segments. RESULTS: Starting from a C. elegans or C. briggsae gene name or identifier, the software identifies the putative ortholog (if any), based on information derived from public nematode genome annotation databases. The investigator can then retrieve the genome DNA sequences of the two orthologous genes; visualize graphically the genes' intron/exon structure and the surrounding DNA regions; select, through an interactive graphical user interface, subsequences of the two gene regions. Using a bioinformatics toolbox (Blast2seq, Dotmatcher, Ssearch and connection to the rVista database) the investigator is able at the end of the procedure to identify and analyze significant sequences similarities, detecting the presence of transcription factor binding sites corresponding to the conserved segments. The software automatically masks exons. DISCUSSION: This software is intended as a practical and intuitive tool for the researchers interested in the identification of non-exonic conserved sequence segments between C. elegans and C. briggsae. These sequences may contain regulatory transcriptional elements since they are conserved between two related, but rapidly evolving genomes. This software also highlights the power of genome annotation databases when they are conceived as an open resource and the possibilities offered by seamless integration of different web services via the http protocol. AVAILABILITY: The program is freely available at http://bio.ifom-firc.it/NTFootPrinter.

Animals↗

PubMed1988

Stem cells in mammary gland differentiation and cancer.

Evidence based on ultrastructure and immunocytochemical staining suggests that morphological gradations between epithelial and myoepithelial cells, and possibly between epithelial and alveolar-like cells, can occur in terminal ductal structures of rat and human mammary glands. In neoplastic disease the benign, carcinogen-induced rat and benign, human mammary tumours can contain epithelial, myoepithelial-like and alveolar-like cells, whereas their malignant counterparts mainly contain only epithelial-like cells. Clonal epithelial cell lines from normal rat mammary glands, from benign tumours and from SV40-transformed human mammary cultures can differentiate to either myoepithelial-like or alveolar-like cells. In those of the rat, the differentiation processes occur in steps, intermediate cells along the myoepithelial-like pathway resemble the morphological intermediates in the terminal ductal structures in vivo. Changes in specific polypeptides characterize each of the intermediate cells in vitro. One of the earliest increases observed in the myoepithelial-like pathway in vitro is that due to a novel protein p9Ka, whereas the major increases in Thy-1 antigen and the basement membrane proteins laminin and type IV collagen occur at later steps. The nucleotide sequence of the gene for p9Ka is related to that of the small, regulatory calcium-binding proteins, and antibodies raised to synthetic fragments of its predicted amino acid sequence react with only myoepithelial cells within the rat mammary parenchyma. Increases in the production of p9Ka and Thy-1 are largely due to increases in their messenger RNAs, possibly arising at the level of transcription of the DNA, whereas the increases in production of laminin and type IV collagen occur at a post-transcriptional level. The normal transcriptional promoter sequences of TATA or CAAT are not found adjacent to the genes for p9Ka or Thy-1. Cells and cell lines from malignant rat mammary tumours of increasing metastatic potential and from malignant areas of human ductal carcinomas largely fail to yield fully differentiated myoepithelial-like or alveolar-like cells in culture; however, weakly metastasizing rat cells yield variants which may retain a vestige of the myoepithelial phenotype. It is suggested that novel regulatory transcriptional element(s) may control the production of some of the proteins along the normal myoepithelial-like pathway, and that these elements may be relatively unique in their capacity to become inoperative in the malignant breast cancer cell.

Adolescent↗

PubMed2004

Generation of eight adjacent mutations in a single step using a site-directed mutagenesis kit.

Studies that characterize transcriptional regulatory elements involve the site-directed mutagenesis methodology to generate deletions, insertions and point mutations. Commercial PCR-based system kits are widely used to introduce up to four base changes in a target sequence. Sequential mutagenesis experiments allow more than four bases to be altered. In this work, we show that the QuikChange site-directed mutagenesis kit developed by Stratagene, with an optimum primer design, can change eight adjacent bases. This approach allows us to study the effect of DNA sequence changes on functionality of specific sequences from gene target regions, including promoters, exons and introns. As a result of this methodology, a faster and cheaper way of introducing this number of mutations is achieved in a single step with only one pair of primers, thus reducing the possibility of potential random mutation in the rest of the target sequence.

DNA↗

PubMed2003

Angelman syndrome: etiology, clinical features, diagnosis, and management of symptoms.

It is estimated that Angelman syndrome (AS) accounts for up to 6% of all children presenting with severe mental retardation and epilepsy. The main clinical features of AS may not be apparent early in life. Clinical findings present in all patients include developmental delay, which becomes apparent by 6-12 months of age, severely impaired expressive language, ataxic gait, tremulousness of limbs, and a typical behavioral profile, including a happy demeanor, hypermotoric behavior, and low attention span. Seizures, abnormal electroencephalography, microcephaly, and scoliosis are observed in >80% of patients. Approximately 70% of patients show a deletion involving the maternally inherited chromosome 15q11-q13, encompassing a cluster of gamma-aminobutyric acid receptor subunit genes, 3% show chromosome 15 paternal uniparental disomy (UPD), 1% harbor a mutation in the imprinting center (a transcriptional regulatory element), and 6% harbor intragenic mutations of the ubiquitin-protein ligase E3A (UBE3A) gene. Twenty percent of patients have no detectable genetic abnormality. Rare cases of familial recurrence of AS show either imprinting center (IC) or UBE3A mutations. Approximately 75% of cases are detected through the methylation test, which allows the detection of AS due to deletions, UPD and IC mutations. Mutation analysis of the UBE3A gene should be performed when the methylation test is negative. Individuals with chromosome 15q11-q13 deletions have a more severe clinical picture and are more prone to develop severe epilepsy. Epilepsy has typical features, including absence and myoclonic seizures, and insidious episodes of nonconvulsive or subtle myoclonic status which are easily overlooked as children appear apathetic or in a state of neurologic regression. Tremulousness, present in all patients even when seizures are well controlled or absent, is related to distal cortical myoclonus. Valproic acid (sodium valproate), benzodiazepines, and ethosuximide, in various combinations, are quite effective in treating the typical seizure types. Piracetam may help in reducing distal myoclonus. Carbamazepine and vigabatrin may seriously aggravate absence and myoclonic seizures and should be avoided. Cognitive, language, and orthopedic problems must be addressed with vigorous rehabilitation programs, including early physical therapy, which may help to develop communicative skills and prevent severe scoliosis and subsequent immobility. Where these treatment strategies are applied, individuals with AS may reach an appreciable level of integration, self care, and have a normal life span.

Angelman Syndrome↗

PubMed2006

Role of c-Myc in intestinal tumorigenesis of the ApcMin/+ mouse.

The c-MYC oncogene plays an important role in tumorigenesis and is commonly highly expressed in gastrointestinal cancers. In colon cells, c-MYC is regulated by the adenomatous polyposis coli (Apc) tumor suppressor gene. Multiple intestinal neoplasia (ApcMin/+ or Min) mice are heterozygous for a truncating Apc mutation and serve as a model of familial adenomatous polyposis (FAP) disease. To study the role of c-Myc in the mutant Apc-mediated colon tumorigenesis, we have developed a transgenic mouse with the conditional deletion of the floxed c-Myc alleles in the intestinal crypts of ApcMin/+ mice (ApcMin/+; c-Mycfl/fl). The floxed c-Myc deletion was initiated via a Cre recombinase controlled by the intestine-specific transcriptional regulatory elements of the liver fatty acid-binding protein gene (Fabpl4xat-132). Fabpl4xat-132-mediated Cre expression and recombination resulted in a two-fold decrease in c-MYC protein expression with no effect on intestinal tract morphology. Small intestinal tumorigenesis was significantly suppressed throughout the small intestinal tract of ApcMin/+; c-Mycfl/fl mice compared to c-Myc wild type littermates. In ApcMin/+; c-Mycfl/fl mice, the intestinal apoptosis was higher in the areas of the small intestine with the decreased c-Myc protein expression (P=0.0016, compared to their littermates with the wild type c-Myc). Thus, conditional inactivation of c-Myc, mediated by Fabpl4xat-132-driven Cre-recombinase, suppresses Apc-dependent intestinal tumorigenesis in adult ApcMin/+ mice, without apparent effect on normal intestinal mucosa.

Animals↗

PubMed2006

Epigenetic inactivation implies a tumor suppressor function in hematologic malignancies for Polo-like kinase 2 but not Polo-like kinase 3.

The Polo-Like kinases (Plk) are a family of highly conserved cell cycle kinases, of which there are four members in humans. Whilst many studies support an oncogenic role for Plk1 in neoplasia, there is little definitive evidence at present to support involvement of the other family members in human cancer. Both Plk2 and Plk3 function in pathways of DNA damage response. Plk2 is a target gene for p53 and imposes a G2 checkpoint. More recent evidence reveals a novel function for Plk2 in mediating apoptosis in high grade B lymphomas. Epigenetic inactivation of Plk2 via aberrant CpG methylation in the transcriptional regulatory elements of the gene is a common event in B cell neoplasia, whereas epigenetic inactivation of Plk3 is exceedingly rare in lymphomas. Further, in every case lacking Plk2 expression, there is concomitant overexpression of Plk3, consistent with functional degeneracy between the two proteins. These results imply that Plk2 may function as a tumor suppressor in hematologic neoplasia and have pharmaco-epigenomic implications.

Animals↗

PubMed2002

Detection of bcl-2/J(H) translocation by polymerase chain reaction: a summary of the experience of the Molecular Oncology Survey of the College of American Pathologist.

CONTEXT: The t(14;18)(q32;;t6q21) translocation, found in about 85% of follicular lymphomas, brings the bcl-2 gene on 18q21 under control of the immunoglobulin heavy-chain gene transcriptional regulatory elements on 14q32. Detection of this translocation in a clinical sample suspected of containing lymphoma can assist the pathologist in diagnosis and classification of lymphoma. Polymerase chain reaction is a technology that is frequently used to detect the t(14;18)(q32;q21) translocation (bcl-2/J(H)). This article reviews the utility of polymerase chain reaction testing for bcl-2/J(H) detection and summarizes the experience of participants in the Molecular Oncology Proficiency Survey of the College of American Pathologists from 1997 through 2000. OBJECTIVE: To describe current practice and encourage improvement of bcl-2/J(H) testing in clinical laboratories. DESIGN: Retrospective analysis of Molecular Oncology Proficiency Survey data. PARTICIPANTS: Laboratory participants in the College of American Pathology Molecular Oncology Proficiency Survey. RESULTS: Twenty-four well-characterized specimens were sent to participants, of which 6 contained bcl-2/J(H) major breakpoint region translocations. Eight hundred nineteen major breakpoint region and 323 minor cluster region determinations were performed, with an overall correct response rate of 91% and 94%, respectively. No significant difference in correct response could be found for frozen versus paraffin-embedded tissues. Many laboratories did not know their assay sensitivity. CONCLUSION: Overall performance was good; however, there was great variability in the methods reported and lack of knowledge of the limits of detection was common. Continued participation in external quality control programs, such as the Molecular Oncology Survey; dissemination of information that impacts on test performance; and technical recommendations from the molecular diagnostics community are critical for improved testing for bcl-2/J(H).

Chromosomes, Human, Pair 14↗

PubMed2000

The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.

BACKGROUND AND OBJECTIVE: As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. DESIGN AND METHODS: To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). RESULTS: An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. INTERPRETATION AND CONCLUSIONS: A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.

3T3 Cells↗

PubMed2001

A hepatocellular carcinoma-specific adenovirus variant, CV890, eliminates distant human liver tumors in combination with doxorubicin.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world. Tumor resection remains the only curative treatment but is often not possible because of advanced stage and frequently unsuccessful because of intrahepatic or distant tumor recurrence. alpha-Fetoprotein (AFP), a tumor marker currently used for the diagnosis and management of HCC, is an oncofetal protein expressed in a majority of HCCs but rarely in normal hepatocytes. Because AFP gene expression is tightly regulated at the level of transcription, AFP transcriptional regulatory elements (TRE) are excellent candidates for generating HCC-specific oncolytic adenoviruses. We devised a new strategy for the AFP TRE to control an artificial E1A-IRES-E1B bicistronic cassette in an adenovirus 5 vector (Ad5) and constructed an HCC-specific oncolytic virus, CV890. In vitro, CV890 expression of the E1A and E1B genes, virus replication, and cytopathic effects were examined by Northern blot, Western blot, virus yield assay, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in AFP-producing cell lines (HepG2, Huh7, Hep3B, PLC/PRF/5, and SNU449), non-AFP-producing cell lines (Sk-Hep-1, Chang liver cell, LNCaP, HBL-100, PA-1, UM-UC-3, SW 780, Colo 201, and U118 MG), and non-AFP-producing human primary cells (lung fibroblast, bladder smooth muscle, and mammary epithelial). CV890 efficiently replicates in and destroys AFP-producing HCC cells as well as wild-type Ad5, but replication is highly attenuated in non-AFP-producing HCC cells or non-HCC cells. CV890 produced 5,000-100,000-fold less virus than wild-type Ad5 in non-AFP-producing cells. CV890 was attenuated 100-fold more than CV732, a virus containing the AFP TRE driving the E1A gene alone, in non-AFP-producing cells. These studies demonstrated that expression of both E1A and E1B genes under the control of a bicistronic AFP-E1A-IRES-E1B cassette yielded improvements in virus specificity equivalent to driving the E1A and E1B genes with two independent TREs yet requires only one TRE thereby conserving genomic space within the virus. Significantly, CV890 produced nearly the same yield of virus in cells that produced AFP over a 75-fold range, from a low of 60 ng AFP/10(6) cells/10 days to as high as 4585 ng AFP/10(6) cells/10 days. In vivo, antitumor efficacy of CV890 was examined in BALB/c-nu/nu mice containing large s.c. HepG2 or Hep3B tumor xenografts. Tumor volume of distant xenografts dropped below baseline 4 weeks after a single i.v. injection. Combination of CV890 with doxorubicin demonstrated synergistic antitumor efficacy, yielding complete elimination of distant Hep3B tumors 4 weeks after a single i.v. administration of both compounds. Our results support the clinical development of CV890 as an antineoplastic agent for the treatment of localized or metastatic HCC.

Adenoviridae↗

PubMed1999

[Immunogenicity and immunoprotection of a leptospiral DNA vaccine].

Leptospirosis is one of the most widespread zoonosis. Immunization of NZW rabbit with plasmid DNA constructs encoding leptospiral endoflagellin can induce specific antibody response in the animal. The vector, VR1012 consists of a pUC18 backbone with the cytomegalovirus (CMV) IE1 enhancer, promoter, and intron A transcription regulatory elements and the BGH polyadenylation sequences driving the expression of leptospiral endoflagellar gene, flaB2. In this study, a pair of oligonucleotide primers were designed to amplify the endoflagellar of L. interrogans serovar lai. An approximately 840 bp fragment was generated with PCR and inserted into expressed plasmid vector, VR1012, after the fragment of VR1012 were digested respectively with EcoRV and Sall. A recombinant plasmid (designated VR1012 + flaB2) was obtained. Plasmid encoding leptospiral endoflagellin was injected into quadriceps of NZW rabbits. This resulted in the generation of specific leptospiral antibody with very high ELISA titer. Immunoprotection was performed in guinea pigs. The group "VR1012 + flaB2" had higher survival rate, compared with the group "VR1012 only" (control) and the group "Normal Saline only" (control). The result suggests that the technique of DNA injection should have an advantage over certain previous vaccine technologies.

Animals↗

PubMed2000

Protection against leptospirosis by immunization with plasmid DNA encoding 33 kDa endoflagellin of L. interrogans serovar lai.

OBJECTIVE: To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira. METHODS: A pair of oligonucleotide primers were designed to amplify the endoflagellar gene of L. interrogans sensu stricto serovar lai. An approximately 840bp fragment was generated with PCR and inserted into VR1012, a plasmid DNA expression vector, after the fragment and VR1012 were digested respectively with EcoRV and Sal I. A recombinant plasmid designated as VR1012+flaB2 was obtained. The vector, VR1012 consits of a pUC18 backbone with the cytomegalovirus (CMV) IE1 enhancer, promoter, and intron A, transcription regulatory elements and the BGH polyadenylation sequences driving the expressing of leptospiral endoflagellar gene of L. interrogans sensu stricto serovar lui. Plasmid encoding leptospiral endoflagellin gene was injected into quadriceps of NZW rabbits. RESULTS: This resulted in the generation of specific leptospiral antibody with high ELISA titer (1:32768) in the rabbits. Immuno/protection was performed in guinea pigs without adjuvant. The group "VR1012 + flaB2" showed higher survival rate (90%, 9/10 animals), compared with the group "VR1012 lack flaB2" and the group "normal saline". CONCLUSION: The technique of DNA vaccine has potential advantages over certain other vaccine preparation technologies. However whether DNA vaccine will be useful for vaccine development remains to be tested.

Animals↗

PubMed1990

Comparison of human lymphotoxin gene expression in CHO cells directed by genomic DNA or cDNA sequences.

Four recombinant plasmids coding for human lymphotoxin (LT) were constructed with genomic DNA (gDNA) or cDNA sequences. The simian virus 40 (SV40) early region, which contains the early promoter, an intron of the small-t-antigen-encoding gene, and polyadenylation signal sequences, was used for transcriptional and post-transcriptional regulatory elements in the construction of these plasmids. Two of them contained gDNA and the other two contained cDNA. One of the gDNA plasmids and one of the cDNA plasmids carry the SV40 intron between the structural gene and polyadenylation signal. Transient and stable gene expression levels of these plasmids in Chinese hamster ovary (CHO) cells were measured by assaying the secreted LT. The plasmid carrying gDNA without the SV40 intron was expressed more efficiently than the other three plasmids in both transient and stable gene expression assays.

Animals↗

PubMed2003

Statistical analysis and experimental design for mapping genes of complex traits in domestic animals.

Gene mapping for complex traits has been an active and challenging research area in humans, agricultural and laboratory species. In domestic animals and poultry, the goal of gene mapping is to find genes associated with production, reproduction and health traits. In humans, gene mapping for complex diseases has been a significant challenge. Although gene mapping results are accumulating rapidly, confirmed results are scarce. In domestic animals, gene mapping results are also accumulating rapidly and several large scale mapping projects in chickens, swine, and dairy cattle are currently in progress. Although new and more reliable results can be expected in the near future, the precise understanding of genes underlying complex traits is still some distance away. Recently, the approach of mapping genes of complex traits has been applied to gene expression data to map transcription regulatory elements, adding a new dimension to gene mapping. Statistical analysis of gene mapping data and experimental design are critical components of a gene mapping research. The purpose of this review article is to discuss current status as well as future directions and challenges in statistical analysis and experimental design for mapping genes of complex traits in domestic animals and poultry.

Animals↗

PubMed1991

Expression of the diphtheria toxin A-chain coding sequence under the control of promoters and enhancers from immunoglobulin genes as a means of directing toxicity to B-lymphoid cells.

Previous results have shown that cells can be killed by the expression of an introduced gene encoding diphtheria toxin A-fragment (DT-A) and that killing can be targeted using tissue-specific transcriptional regulatory elements. Here, we describe expression plasmids containing the DT-A gene linked with promoters and enhancers from immunoglobulin heavy chain or kappa-light chain genes. When these plasmids were transfected into cultured cells, DT-A was expressed in B-lymphoid cells but not detectably in HeLa cells or fibroblasts. A high specificity for B-cells was confirmed by assaying for luciferase reporter gene expression from a plasmid containing an analogous combination of immunoglobulin heavy chain regulatory elements. A plasmid containing an immunoglobulin-kappa promoter and enhancer was substantially less active in expressing DT-A in a pre-B-cell line than in B-lymphoma cells, suggesting the possibility of targeting DT-A expression to mature, malignant B-cells while sparing normal B-cell progenitors. By means of viral delivery vehicles, the constructs described might be applied in gene therapy for B-cell leukemias or lymphomas.

Animals↗

PubMed1991

Regulation of HLA class I gene expression in human colorectal carcinoma.

The under-representation of HLA antigens in human tumors is usually associated with a poor prognosis. In this report, the expression of HLA class I genes in human colorectal carcinomas was studied using HLA-A and HLA-B locus-specific probes. Over 50% of the colorectal carcinomas studied showed a reduction in the amount of steady state HLA class I mRNA. For some carcinomas, non-coordinated regulation of the HLA-A and HLA-B genes was observed. The mechanism of HLA suppression was investigated and is most likely due to the presence of transcriptional regulatory elements.

Blotting, Northern↗

PubMed1986

Structure and expression of the gene locus encoding the phosphatidylglycerophosphate synthase of Escherichia coli.

This paper presents definitive results which establishes a direct gene-protein product relationship between the pgsA gene and the phosphatidylglycerophosphate synthase of Escherichia coli. The predicted protein sequence derived from the determined DNA sequence of pgsA is in close agreement with the amino acid composition and partially determined amino acid sequence of the purified enzyme. The purified synthase has the same apparent molecular mass as the gene product made by a plasmid-directed transcription-translation system. The plasmid-borne copy of the pgsA gene is also capable of expressing enzymatically active synthase in vitro. The DNA sequence analysis has established the exact linear relationship between the uvrC, pgsA, and glyW loci and revealed that these three genes are transcribed in the same direction. The terminal coding regions of these three genes also share common sequences with transcriptional regulatory elements for the adjacent genes.

Amino Acid Sequence↗

PubMed1995

Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alpha-fetoprotein gene promoter.

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but is reactivated in hepatocellular carcinoma. It has been shown that the positive and negative transcriptionally regulatory elements of the human AFP gene, which play an important role in its developmental regulation, exist over the quite extended region (4 kb). We constructed a hybrid gene consisting of herpes simplex virus thymidine kinase (HSV-tk) gene under the control of the 0.3-kb human AFP gene promoter and inserted it into a retroviral vector. When AFP-producing hepatoma cells were infected with this recombinant retrovirus (LNAF0.3TK virus), the cells expressed HSV-tk gene and exhibited increased sensitivity to ganciclovir parallel with the ability of AFP production. On the other hand, the retroviral infection had little effect on ganciclovir-mediated cytotoxicity in AFP-nonproducing hepatoma or non-hepatoma cells. Moreover, the addition of dexamethasone increased the cytotoxicity of aciclovir to the virus-infected, AFP-producing cells through a glucocorticoid-responsive element in the AFP promoter, although aciclovir, by itself, had little cytotoxicity. These results demonstrate that the AFP promoter sequence alone can provide enough tumor-specific activity for therapeutic gene expression and induce selective growth inhibition by ganciclovir in the virus-infected, AFP-producing human hepatoma cells. In addition, it is possible that expression of the therapeutic gene is modulated by administration of dexamethasone or other agents that alter AFP promoter activity after gene transduction.

3T3 Cells↗