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At least 433 records · Page 24Linked to original sources
PubMed2001

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

Adenoviridae↗
PubMed2002

[Assessment of the role of TLR-4 in shear-stress-induced IL-8 gene transcription activation in vascular endothelial cells by gene mutation and gene transfection technology].

This study is to evaluate the role of TLR-4 in lower laminar shear stress-induced interleukin-8 gene transcription activation in vascular endothelial cells by using gene mutation and transfection techniques. RT-PCR, Northern hybridization and immunocytochemical fluorescent staining showed that TLR-4 was expressed in human umbilical vein endothelial cells(HUVEC). When stimulated with 4.2 dyne/cm2 shear stress for 1 hour, an increase of TLR-4 mRNA expression was observed in HUVECs detected by RT-PCR and Northern hybridization. The intracellular domain deletion mutant TLR-4 cDNA (lacking the 155 COOH terminal amino acids of the wild type (TLR-4) and -102 -61 bp DNA sequence in 5'-flanking region of IL-8 gene (IL-8USCS) were isolated from endothelial cells by RT-PCR and PCR. These two DNA fragments were cloned into pcDNA3 and pEGFP1 respectively to construct TLR-4 dominant negative mutant pcDNA3-mTLR4 and IL-8 reporter gene pEGFP1-IL8USCS. ECV304 cells were transfected with the pEGFP1-IL8USCS or co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 by Dosper liposome transfectional reagent and selected by G418, then stimulated with 4.2 dyne/cm2 shear stress for 3 hours. Flow cytometric analysis showed that when exposed to 4.2 dyne/cm2 shear stress for 3 hours, there was a marked increase in the green fluorescent protein expression in pEGFP1-IL8USCS-transfected ECV304 cells. In contrast, there was almost no change in the green fluorescent protein expression in the cells co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 after the stimulation, suggesting the TLR-4 mutant depressed TLR-4 signaling. These experiments suggest that the inflammatory TLR-4/NF-kappa B signaling pathway would probably be involved in flow shear stress-induced IL-8 gene expression in human vascular endothelial cells.

Cells, Cultured↗
PubMed1990

The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the alpha-aminoadipyl-cysteinyl-valine synthetase and linkage to the pcbC and penDE genes.

Penicillium chrysogenum DNA fragments cloned in EMBL3 or cosmid vectors from the upstream region of the pcbC-penDE cluster carry a gene (pcbAB) that complemented the deficiency of alpha-aminoadipyl-cysteinyl-valine synthetase of mutants npe5 and npe10, and restored penicillin production to mutant npe5. A protein of about 250 kDa was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell-free extracts of complemented strains that was absent in the npe5 and npe10 mutants but exists in the parental strain from which the mutants were obtained. Transcriptional mapping studies showed the presence of one long transcript of about 11.5 kilobases that hybridized with several probes internal to the pcbAB gene, and two small transcripts of 1.15 kilobases that hybridized with the pcbC or the penDE gene, respectively. The transcription initiation and termination regions of the pcbAB gene were mapped by hybridization with several small probes. The region has been completely sequenced. It includes an open reading frame of 11,376 nucleotides that encodes a protein with a deduced Mr of 425,971. Three repeated dominia were found in the alpha-aminoadipyl-cysteinyl-valine synthetase which have high homology with the gramicidin synthetase I and tyrocidine synthetase I. The pcbAB is linked to the pcbC and penDE genes and is transcribed in the opposite orientation to them.

Amino Acid Sequence↗
PubMed1986

The rabbit kappa 1 b5 immunoglobulin gene: another J region gene cluster with only one functional J gene segment?

Our previous analysis of an immunoglobulin gene encoding the rabbit kappa chain of b4 allotype revealed that of the five J-like sequences in the J kappa cluster of this gene, only one, J2, appeared to be functional. This unusual ratio of J pseudogenes to functional J genes is unique among all J clusters of light and heavy chain genes of all species examined to date, including the cluster from the rabbit kappa 2 isotype, and must have consequences for diversity generation of b4 immunoglobulins. The fact that the only two known b5 J kappa sequences are different from the functional J2 of the b4 allotype prompted investigation of the b5 J kappa cluster to determine whether it resembled the b4 cluster, or the more typical mouse or human J kappa clusters. Our analysis of the b5 gene reveals a J kappa cluster strikingly similar to that of b4; apparent defects occur in all J sequences except J2. Although J2 is apparently functional, it differs from the J2 of the b4 locus by four nucleotide and three amino acid substitutions. The unusually high degree of sequence similarity previously observed between the b4 and b5 loci in the noncoding (vs the coding) regions extends through the newly sequenced DNA segment and remains an enigma.

Animals↗
PubMed1988

Studies on the mutator gene, mutT of Escherichia coli. Molecular cloning of the gene, purification of the gene product, and identification of a novel nucleoside triphosphatase.

The mutator gene, mutT, has been cloned into an expression vector and overproduced in Escherichia coli. The gene product has been purified to over 90% homogeneity as judged by gel electrophoresis and amino acid analysis. The amino acid composition of the protein and the sequence of the 20 amino acids of the N-terminal region agree well with the nucleotide sequence of the gene reported by Akiyama et al. (Akiyama, M., Horiuchi, T., and Sekiguchi, M. (1987) Mol. Gen. Genet. 206, 9-16) and indicate that the first of the potential initiation codons (position 164) of the open reading frame in the PvuII fragment carrying the mutT gene is the site of initiation of translation of the 15,000-Da polypeptide. A novel nucleoside triphosphatase activity which has a preference for dGTP is associated with the purified protein, and preliminary experiments are consistent with the notion that the mutT gene product is the enzyme responsible for this activity.

Amino Acid Sequence↗
PubMedDate not supplied

A defective herpes simplex virus vector system for gene delivery into the brain: comparison with alternative gene delivery systems and usefulness for gene therapy.

The delivery of exogenous genes into the brain is becoming an increasingly important strategy for answering questions about the molecular mechanisms of brain function. Answers to these questions may be applied to many of the disorders that affect the brain. For example, a detailed understanding of the mechanisms that modulate long-term changes in neurotransmitter release will almost certainly lead to new approaches to diseases such as the epilepsies, in which neurotransmitter release is altered. Knowledge of the molecular means by which neurotransmitters shape neuronal development and cause neurodegeneration, or how trophic factors regulate neuronal health, will lead to insights into how defects in these pathways cause specific diseases. To answer these questions, we have developed a defective herpes simplex virus (HSV) system for the delivery of exogenous genes into the brain. This system directs precise spatial and temporal expression of recombinant genes in the brain. We discuss its utility in comparison to other methods of gene transfer into the brain for answering basic questions about the molecular basis for neuronal physiology and for gene therapy.

Animals↗
PubMed1997

Gene structure, promoter activity, and chromosomal location of the DR-nm23 gene, a related member of the nm23 gene family.

DR-nm23 cDNA was cloned recently by differential screening of a cDNA library derived from chronic myelogenous leukemia-blast crisis primary cells. It is highly homologous to the putative metastasis suppressor nm23-H1 gene and the closely related nm23-H2 gene. When overexpressed in the myeloid precursor 32Dcl3 cell line, it inhibited granulocyte colony-stimulating factor-stimulated granulocytic differentiation and induced apoptosis. We have now found that the expression of DR-nm23 is not restricted to hematopoietic cells but is also detected in an array of solid tumor cell lines, including carcinoma of the breast, colon, and prostate, as well as the glioblastoma cell line T98G. We have also isolated both the gene and its 5'-flanking region and found that DR-nm23 localizes on chromosome 16q13. The gene consists of six exons and five introns. When fused in-frame to the nucleotide sequence for the green fluorescent protein and transfected in SAOS-2 cells, it generates a protein of the predicted size that localizes to the cytoplasm. The 5'-flanking region of DR-nm23 does not contain a canonical TATA box or a CAAT box, but it is G+C rich and contains two binding sites for the developmentally regulated transcription factor activator protein 2 (AP-2). Transient expression assays of DR-nm23 promoter-chloramphenicol acetyltransferase constructs demonstrated that the segment from nucleotides -1028 to +123 has the highest activity in hematopoietic K562 cells and in TK-ts13 hamster fibroblasts. Moreover, AP-2 induced a 3-fold transactivation of the DR-nm23 5'-flanking segment from nucleotides -1676 to +123 and interacted specifically with oligomers containing putative AP-2 binding sites (-936 to -909, and -548 to -519) as indicated by electrophoretic mobility shift assay. Furthermore, nuclear run-on assays from high and low DR-nm23-expressing cells (K562 and CCRF-CEM, respectively) revealed similar transcription rates. Therefore, the regulation of the DR-nm23 gene expression might involve other mechanisms occurring at posttranscriptional and/or translational levels.

Animals↗
PubMed1985

Regulation of the expression of iso 2-cytochrome c gene in S. cerevisiae: cloning of the positive regulatory gene CYP1 and identification of the region of its target sequence on the structural gene CYP3.

CYP1 is a trans acting regulatory locus modulating both iso 1- and iso 2-cytochrome c synthesis. Genetical analysis of various mutated alleles has allowed us to identify the gene product as a positive regulatory element. The region of the target sequence of the CYP1 product on the iso 2-cytochrome c structural gene was located by molecular and genetic analysis of two cis acting mutations located at the CYP3 locus: CYP3-36 and CYP3-4, which have been shown to arise from the integration of TY1 elements near the promoter site. Determination of the amount of iso 2-cytochrome c synthesized by strains bearing various genetic constructions, in which the cis acting mutations were associated with different alleles of the CYP1 trans acting locus, showed that TY1 inserted into CYP3-36 extinguishes the activation function due to a mutated overproducer allele CYP1-18, while CYP3-4 amplifies this function. This result identifies at least a part of the target sequence of the CYP1 product within the region separating the two TY1 insertions. To clone the CYP1 gene, we took advantage of the iso 2-cytochrome c overproducer phenotype of the mutated allele CYP1-18, which confers a Lactate+ phenotype on an iso 1-cytochrome c-deficient strain. Such a phenotype allowed the isolation of a recombinant plasmid YEpJFM1 carrying the mutated allele, able to complement on lactate medium a lactate- recipient strain. The identity of the YEpJFM1 sequence with the chromosomal gene was confirmed by homologous recombination at the CYP1 locus.

Alleles↗
PubMed1990

Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes.

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the Sc1 gene [Dons et al., EMBO J. 3 (1984) 2101-2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43-62% (63-69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.

Amino Acid Sequence↗
PubMed2004

Gene therapy progress and prospects: cancer gene therapy using tumour suppressor genes.

Targeting tumour suppressor gene pathways is an attractive therapeutic strategy in cancer. Since the first clinical trial took place in 1996, at least 20 other trials have investigated the possibility of restoring p53 function, either alone or in combination with chemotherapy, but with limited success. Other recent clinical trials have sought to harness abnormalities in the p53 pathway to permit tumour-selective replication of adenoviral vectors such as dl1520 (Onyx-015). Other tumour suppressor genes, such as retinoblastoma (Rb) and PTEN (phosphatase, tensin homologue, deleted on chromosome 10), are the targets for imminent clinical trials, while microarray technologies are revealing multiple new genes that are potential targets for future gene therapy.

Gene Targeting↗
PubMed1993

Mechanism of expression of DNA repair gene vsr, an Escherichia coli gene that overlaps the DNA cytosine methylase gene, dcm.

The DNA cytosine methylase gene of Escherichia coli, dcm, overlaps an open reading frame (ORF) that continues in +1 register past the end of dcm. This ORF codes for a gene, vsr, that is required for a T:G to C:G base mismatch correction process. In this study, mutants that affect the level of expression of the two genes were constructed and characterized. Further, a previously isolated mutant, dcm-6, was cloned and mutations within it were identified. Northern blots were used to identify dcm-specific RNA species in wild type and dcm-6 cells. Based on these studies we conclude that there is a six-codon overlap between vsr and dcm. The two proteins appear to be made from a single RNA transcript and translation of dcm is required for the efficient synthesis of Vsr. Further, Vsr is active by itself and may not be produced as a fusion with Dcm. This is the first example of chromosomal genes that overlap in their coding regions and produce proteins with distinct functions.

Alleles↗
PubMed1990

Gene amplification in human lung cancer. The myc family genes and other proto-oncogenes and growth factor genes.

The development of human lung cancer may require multiple genetic deletions affecting a number of chromosomes, e.g., 1, 3, 11, 13, and 17. These genetic aberrations may induce the activation of proto-oncogenes (c-jun, ras, c-raf1) and the loss of tumor suppressor genes (p53). Some of the activated proto-oncogenes and tumor suppressor genes are more selectively expressed or absent in small-cell lung cancer (L-myc, c-myb, c-scr, Rb gene) or non-small-cell lung cancer (c-erbB-2, c-sis, c-fes). These genes may thus be of importance for selection of differentiation pathway. The c-myc oncogene is frequently amplified in small-cell lung cancer cell lines in a much higher frequency than in vivo. This indicates that c-myc seems to be related to tumor progression and a relatively late event in the lung cancer development. The uncontrolled production of multiple growth factors has been identified in human lung cancer cell lines. These factors can promote and inhibit the proliferation via paracrine and autocrine loops via specific receptors. The products from some of the activated proto-oncogenes (c-sis, c-erbB-2) are sequences homologous to a certain growth factor (PDGF) and a receptor (EGF) identified in lung cancer. The production and action of these growth factors may be of major importance for further activation of proto-oncogenes via intracellular signal transduction and specific oncogenic activation leading to further tumor progression.

Carcinoma, Non-Small-Cell Lung↗
PubMed1996

[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. II. Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes].

The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum. The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively. These constructions were used for transformation of agrobacteria followed by transfer into plants. In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants. The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.

Cloning, Molecular↗
PubMed1998

Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia. Mutations in brief no. 135. Online.

We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.

DNA Mutational Analysis↗
PubMed1998

Two novel mutations in exon 11 of the PAH gene (V1163del TG and P362T) associated with classic phenylketonuira and mild phenylketonuria. Mutations in brief no. 143. Online.

PKU is one of the commonest genetic disease in man, affecting 1/10,000 individuals. It presents a wide phenotypical spectrum, from classic PKU to moderate Hyperpheylalaninemia depending on the residual enzymatic activity. Two novel mutations 1163/1164delTG and P362T in exon 11 have been detected during the mutational screening of the PAH gene in 84 families. 1163/1164delTC can be confused with V388M if the mutational screening is performed with BsaAI restriction enzyme, this mutation in heterozigosis presents a moderate phenotype. P362T mutation in heterozigosis with V388M shows a classical PKU phenotype. We report here two new mutations in exon 11 of the PAH gene (GenBank U49897), V1163delTG and P362T (using cDNA sequence), detected during the analysis of 57 PKU and 36 HPA patients belonging to 84 unrelated families detected under a neonatal screening program performed in Catalonia.

DNA Mutational Analysis↗
PubMed1998

Evolutionarily conserved, "acatalytic" carbonic anhydrase-related protein XI contains a sequence motif present in the neuropeptide sauvagine: the human CA-RP XI gene (CA11) is embedded between the secretor gene cluster and the DBP gene at 19q13.3.

Conserved amino acid motifs are found in numerous expressed genes. Proteins and peptides with functional relationships may be identified using probes designed to hybridize with these motifs. An oligonucleotide probe was prepared to match the sequence of the expected active region of a frog corticotropin-releasing factor-like peptide sauvagine and used to screen a sheep brain cDNA library. A novel 1331-bp cDNA encoding a putative 328-residue protein with a theoretical mass of 36 kDa was identified. The presence of a strong signal sequence indicates that it is a secreted protein. The amino- and carboxy-terminal regions are characterized by several potential phosphorylation sites and binding motifs, suggesting a role in intracellular signal transduction. Although the protein possesses a 7-residue sequence identical to that found in sauvagine, its overall primary structure most closely resembles those of the alpha-carbonic anhydrases (alpha-CAs). Moreover, the detection of the human and mouse orthologues in the EST databases, together with an evolutionary analysis, indicates that the protein represents a new member of the alpha-CA gene family, which we designate carbonic anhydrase-related protein XI (CA-RP XI), encoded by CA11 (human) and Car11 (mouse, rat). The human CA11 gene appears to be located between the secretor type alpha(1,2)-fucosyltransferase gene cluster (FUT1-FUT2-FUT2P) and the D-site binding protein gene (DBP) on chromosome 19q13.3. Despite potentially inactivating changes in the active-site residues, CA-RP XI is evolving very slowly in mammals, a property indicative of an important function, which has also been observed in the two other "acatalytic" CA isoforms, CA-RP VIII and CA-RP X, whose functions are unknown.

Amino Acid Sequence↗
PubMed1987

Structure and expression of HLA-DQ alpha and -DX alpha genes: interallelic alternate splicing of the HLA-DQ alpha gene and functional splicing of the HLA-DQ alpha gene using a retroviral vector.

The nucleotide sequences of the two closely related HLA-DQ alpha and HLA-DX alpha genes have been determined. Exons coding for the signal peptide, alpha 2 and transmembrane domains are 94-99% homologous, whereas the alpha 1 exon and the promoter region have diverged as much as or more than introns and the 3' untranslated region. The promoter regions of both genes contain two short sequences thought to be important for regulation of transcription by gamma-interferon. Transfection studies established that the DQ alpha and DQ beta genes encode the HLA-DQ antigen. Transcripts of varying length are produced from different alleles as the result of the use of alternate splice and polyadenylation signals at the 3' end of the DQ alpha gene. Thus typing at the DQ alpha locus can be achieved by Northern blot analysis. No transcript of DX alpha was detected in B lymphocytes. The DX alpha gene was accurately spliced when introduced into a retroviral vector, suggesting that the lack of expression of DX alpha is not due to aberrant splice signals.

Alleles↗
PubMed1998

The mouse gene encoding the testis-specific isoform of Poly(A) binding protein (Pabp2) is an expressed retroposon: intimations that gene expression in spermatogenic cells facilitates the creation of new genes.

The gene encoding the testis-specific isoform of mouse poly(A) binding protein (Pabp2) has been isolated and sequenced. Unexpectedly, comparison of the sequence of genomic and cDNAs demonstrated that the Pabp2 gene lacks introns, whereas all other functional Pabp genes in plants, amphibians, and mammals contain introns. Thus, the mouse Pabp2 gene is a retroposon, created by synthesizing a reverse transcriptase copy of a processed mRNA and inserting the copy into the genome. The Pabp2 retroposon is unusual because it is functional: previous work demonstrates that its promoter drives the accumulation of Pabp2 mRNA in meiotic and early haploid spermatogenic cells, and the Pabp2 mRNA encodes a protein whose size and RNA-binding specificities are characteristic of PABP in plants, yeast, and mammals (Kleene et al. 1994). Two novel factors can be implicated in the retention of function of the Pabp2 retroposon. First, the promoter of the Pabp2 gene is not derived from its intron-containing progenitor, Pabp1. Second, mRNAs encoding somatic PABP isoform, PABP1, are present at high levels in meiotic and haploid spermatogenic cells. Both features contrast with the phosphoglycerate kinase 2 retroposon, which is believed to compensate for the depletion of the somatic isoform due to X-chromosome inactivation in meiotic spermatogenic cells. We also document that more functional retroposons are expressed in meiotic and haploid spermatogenic cells than in any other tissue and speculate that transcriptional derepression in spermatogenic cells favors the creation of expressed retroposons.

Amino Acid Sequence↗