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1.
Chromosome Res ; 23(4): 663-80, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25957863

RESUMO

The molecular mechanisms underlying spontaneous neoplastic transformation in cultured mammalian cells remain poorly understood, confounding recognition of parallels with the biology of naturally occurring cancer. The broad use of tumorigenic canine cell lines as research tools, coupled with the accumulation of cytogenomic data from naturally occurring canine cancers, makes the domestic dog an ideal system in which to investigate these relationships. We developed a canine kidney cell line, CKB1-3T7, which allows prospective examination of the onset of spontaneous immortalization and tumorigenicity. We documented the accumulation of cytogenomic aberrations in CKB1-3T7 over 24 months in continuous culture. The majority of aberrations emerged in parallel with key phenotypic changes in cell morphology, growth kinetics, and tumor incidence and latency. Focal deletion of CDKN2A/B emerged first, preceding the onset and progression of tumorigenic potential, and progressed to a homozygous deletion across the cell population during extended culture. Interestingly, CKB1-3T7 demonstrated a tumorigenic phenotype in vivo prior to exhibiting loss of contact inhibition in vitro. We also performed the first genome-wide characterization of the canine tumorigenic cell line MDCK, which also exhibited CDKN2A/B deletion. MDCK and CKB1-3T7 cells shared several additional aberrations that we have reported previously as being highly recurrent in spontaneous canine cancers, many of which, as with CDKN2A/B deletion, are evolutionarily conserved in their human counterparts. The conservation of these molecular events across multiple species, in vitro and in vivo, despite their contrasting karyotypic architecture, is a powerful indicator of a common mechanism underlying emerging neoplastic activity. Through integrated cytogenomic and phenotypic characterization of serial passages of CKB1-3T7 from initiation to development of a tumorigenic phenotype, we present a robust and readily accessible model (to be made available through the American Type Culture Collection) of spontaneous neoplastic transformation that overcomes many of the limitations of earlier studies.


Assuntos
Transformação Celular Neoplásica/genética , Aberrações Cromossômicas , Cariótipo , Neoplasias/genética , Animais , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Variações do Número de Cópias de DNA , Cães , Hibridização in Situ Fluorescente , Células Madin Darby de Rim Canino , Masculino , Neoplasias/patologia
2.
J Exp Med ; 185(11): 2015-23, 1997 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-9166430

RESUMO

The chemokine receptors CXCR4, CCR2B, CCR3, and CCR5 have recently been shown to serve along with CD4 as coreceptors for HIV-1. The tropisms of HIV-1 strains for subgroups of CD4(+) cells can be explained, at least partly, by the selective use of G protein-coupled receptors (GPCRs). We have identified a novel human gene, STRL33, located on chromosome 3 that encodes a GPCR with sequence similarity to chemokine receptors and to chemokine receptor-like orphan receptors. STRL33 is expressed in lymphoid tissues and activated T cells, and is induced in activated peripheral blood lymphocytes. When transfected into nonhuman NIH 3T3 cells expressing human CD4, the STRL33 cDNA rendered these cells competent to fuse with cells expressing HIV-1 envelope glycoproteins (Envs). Of greatest interest, STRL33, in contrast with CXCR4 or CCR5, was able to function as a cofactor for fusion mediated by Envs from both T cell line-tropic and macrophage-tropic HIV-1 strains. STRL33-transfected Jurkat cell lines also supported enhanced productive infection with HIV-1 compared with control Jurkat cells. Despite the sequence similarities between STRL33 and chemokine receptors, STRL33-transfected cell lines did not respond to any in a panel of chemokines. Based on the pattern of tissue expression of the STRL33 mRNA, and given the ability of STRL33 to function with Envs of differing tropisms, STRL33 may play a role in the establishment and/or progression of HIV-1 infection.


Assuntos
Fusão Celular , Proteínas de Ligação ao GTP/fisiologia , HIV-1/fisiologia , Receptores de Citocinas/fisiologia , Receptores Acoplados a Proteínas G , Receptores de HIV/fisiologia , Receptores Virais , Sequência de Aminoácidos , Animais , Antígenos CD4/fisiologia , Linhagem Celular , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Cromossomos Humanos Par 3 , Clonagem Molecular , Humanos , Macrófagos/virologia , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptores CXCR6 , Receptores de Quimiocinas , Receptores de Citocinas/química , Receptores de Citocinas/genética , Alinhamento de Sequência , Linfócitos T/virologia , Transfecção , Proteínas do Envelope Viral/metabolismo
3.
J Cell Biol ; 105(5): 2315-25, 1987 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2824529

RESUMO

Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2-negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.


Assuntos
Antígenos Transformantes de Poliomavirus/genética , Transformação Celular Viral , Genes Virais , Genes , Metalotioneína/genética , Regiões Promotoras Genéticas , Células de Schwann/imunologia , Vírus 40 dos Símios/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/ultraestrutura , Microscopia Eletrônica , Ratos , Ratos Endogâmicos , Células de Schwann/citologia , Células de Schwann/ultraestrutura
4.
Science ; 209(4463): 1392-6, 1980 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-6251547

RESUMO

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.


Assuntos
Antígenos de Neoplasias/genética , DNA Viral/genética , Mutação , Vírus 40 dos Símios/genética , Proteínas Virais/genética , Animais , Antígenos Virais/genética , Transformação Celular Viral , Células Cultivadas , Deleção Cromossômica , DNA Recombinante , Escherichia coli , Replicação Viral
5.
Neuron ; 12(3): 627-37, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7512351

RESUMO

Expression of myelin proteins has been shown to be altered in transgenic mice that express papovaviral large tumor (T) antigens. This paper analyzes the effect on P0 gene expression in secondary Schwann cells transfected with the SV40 T antigen gene and in Schwann cells immortalized by T antigen. In secondary Schwann cells, both T antigen and c-Jun are required for significant inhibition of the P0 promoter; expression of only one of the proteins is insufficient for repression of the P0 gene. T antigen, c-Jun (p39), and c-Jun-related protein (p47) form an immunoprecipitable complex in SV40 immortalized Schwann cell lines, and T antigen and c-Jun bind independently and as a complex to the P0 promoter. Our data suggest that the probable molecular mechanism underlying the hypomyelination observed in transgenic animals expressing T antigen may be due to the repression of the P0 gene by T antigen and c-Jun.


Assuntos
Antígenos Transformantes de Poliomavirus/genética , Expressão Gênica , Proteínas da Mielina/genética , Proteínas Proto-Oncogênicas c-jun/fisiologia , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Antígenos Virais/imunologia , Antígenos Virais de Tumores/imunologia , Sequência de Bases , Doenças Desmielinizantes/imunologia , Regulação para Baixo , Dados de Sequência Molecular , Proteína P0 da Mielina , Proteínas da Mielina/antagonistas & inibidores , Proteínas da Mielina/metabolismo , Sondas de Oligonucleotídeos/genética , Papillomaviridae/imunologia , Polyomaviridae , Testes de Precipitina , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Células de Schwann/metabolismo , Transcrição Gênica
6.
Mol Cell Biol ; 3(2): 203-13, 1983 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-6300656

RESUMO

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.


Assuntos
Antígenos Virais/genética , Genes Virais , Vírus 40 dos Símios/genética , Sequência de Aminoácidos , Antígenos Virais/imunologia , Antígenos Virais de Tumores , Sequência de Bases , Transformação Celular Viral , Deleção Cromossômica , Replicação do DNA , Mutação , Replicação Viral
7.
Mol Cell Biol ; 6(4): 1117-28, 1986 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-3023870

RESUMO

To define the sequence elements involved in initiation of DNA synthesis at the simian virus 40 origin of replication, we determined the relative replication efficiencies in vitro and in vivo of templates containing a variety of mutations within the origin region. Replication of the mutants in vitro was assayed by the cell-free DNA replication system that we recently described (J.J. Li and T.J. Kelly, Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984; J.J. Li and T.J. Kelly, Mol. Cell. Biol. 5:1238-1246, 1985), and replication in vivo was assayed after transfection of the mutant templates into COS-1 cells. The minimal origin of replication defined by both assays included a 15-base-pair (bp) imperfect inverted repeat, a 27-bp perfect inverted repeat, and a 17-bp A/T-rich region. T-antigen binding site I was not required for DNA replication, but its presence increased replication efficiency severalfold both in vitro and in vivo. Although SP1 binding sites and enhancers had little or no effect on replication in vitro, the presence of either element markedly increased replication in vivo. Thus, the biological role of these elements is not restricted to stimulating transcription but may be more general.


Assuntos
Replicação do DNA , Vírus 40 dos Símios/genética , Antígenos Transformantes de Poliomavirus , Antígenos Virais de Tumores/genética , Sequência de Bases , DNA Viral/genética , Mutação , Proteínas Oncogênicas Virais/genética , Moldes Genéticos
8.
Mol Cell Biol ; 3(2): 220-8, 1983 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-6300658

RESUMO

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.


Assuntos
Antígenos Virais/genética , Vírus 40 dos Símios/genética , Adenosina Trifosfatases/genética , Antígenos Virais de Tumores , Deleção Cromossômica , DNA Viral/genética , Genes Virais , Peso Molecular , Replicação Viral
9.
Mol Cell Biol ; 3(2): 214-9, 1983 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-6300657

RESUMO

The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).


Assuntos
Antígenos Virais/genética , Regulação da Expressão Gênica , Genes Virais , Vírus 40 dos Símios/genética , Animais , Antígenos Virais de Tumores , Células Cultivadas , Deleção Cromossômica , Cricetinae , Replicação do DNA , Mutação , RNA Ribossômico/genética
10.
Dev Biol (Basel) ; 123: 45-53; discussion 55-73, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16566436

RESUMO

Vaccines and other biological products manufactured in cells contain contaminating residual DNA derived from that production cell substrate, with the amount and form of this DNA depending mainly on the type of vaccine. The potential risk of this cellular DNA has been debated for over 40 years without resolution. Opinions on residual DNA have varied from it being considered an inert contaminant, and thus its presence should not be deemed to be a risk to vaccine recipients, to it being considered an important risk factor, particularly for vaccines manufactured in certain cell substrates, such as cells derived from tumours or cells that are tumorigenic. We are not of the opinion that DNA can be considered biologically inert, but whether or what risk residual cell-substrate DNA poses remains to be determined. In this paper, we discuss our approaches to address this issue and describe some preliminary work.


Assuntos
Carcinógenos , Meios de Cultura/efeitos adversos , DNA/toxicidade , Vacinas , DNA de Neoplasias/toxicidade , DNA Viral/toxicidade , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Humanos , Neoplasias/prevenção & controle , Mapeamento por Restrição , Retroviridae/genética , Transfecção
11.
Oncogene ; 18(51): 7343-50, 1999 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-10602490

RESUMO

We have used two different, but complementary assays to characterize functions of SV40 T antigen that are necessary for its ability to immortalize rat embryo fibroblasts. In accordance with previous work, we found that several functions were required. These include activities that map to the p53 binding domain and the amino terminal 176 amino acids which contain the J domain as well as the CR1 and CR2 domain required for binding and sequestering the RB family of pocket proteins. Moreover, we found that even though activities dependent only upon the amino terminus were sufficient for immortalization they were unable to maintain it. This suggests that immortalization by these amino terminal functions requires either additional events or immortalization of a subset of cells within the heterogeneous rat embryo fibroblast population. We further found that an activity dependent upon amino acids 17 - 27 which remove a portion of the CR1 domain and the predicted alpha-1 helix of the J domain was not necessary to maintain growth but was required for direct immortalization suggesting that at least one of the functions required initially was not required to maintain the immortal state. This represents the first demonstration that some of the functions required for maintenance of the immortal state differ from those required for initiation of immortalization.


Assuntos
Antígenos Virais de Tumores , Transformação Celular Viral , Fibroblastos/patologia , Vírus 40 dos Símios , Animais , Linhagem Celular , Fibroblastos/virologia , Ratos , Proteína Supressora de Tumor p53
12.
Gene ; 22(2-3): 277-80, 1983.
Artigo em Inglês | MEDLINE | ID: mdl-6307828

RESUMO

A revised sequence of the tetracycline-resistance gene of pBR322 is reported. The change, the presence of an additional CG base pair at position 526, adjusts the published sequence to allow an open reading frame from nucleotides 86-1273 (new number) and increases the size of the plasmid to 4363 bp. The predicted polypeptide encoded by this region would contain 396 amino acid residues and have a calculated Mr of 41518. A polypeptide of the predicted size has been reported previously when pBR322 is used as template in the maxicell system.


Assuntos
Genes Bacterianos/efeitos dos fármacos , Genes/efeitos dos fármacos , Plasmídeos/efeitos dos fármacos , Tetraciclina/farmacologia , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Enzimas de Restrição do DNA , Resistência Microbiana a Medicamentos , Escherichia coli/genética
13.
Artigo em Inglês | MEDLINE | ID: mdl-1740758

RESUMO

Plasmid proviral molecular clones of the LAI isolate of HIV-1 and the ROD isolate of HIV-2 were originally prepared in moderate copy number plasmids derived from pBR322, which contains the colE1 origin of replication. In these plasmid vectors, the HIV sequences are stable to continuous passage in bacteria. However, when the colE1 origin was replaced by the mutant origin from pUC18, pGEM, or pBluescript plasmids, which replicates to much higher copy numbers in bacteria, then deletions of HIV sequences occurred even in RecA defective strains. Deletions occurred in two different media, at room temperature and 37 degrees C, and with or without plasmid amplification in the presence of chloramphenicol. These results raise a cautionary note when cloning immunodeficiency viral sequences into plasmid vectors containing a high copy number origin of replication.


Assuntos
Replicação do DNA , DNA Viral/biossíntese , HIV-1/genética , HIV-2/genética , Plasmídeos , Sequência de Bases , Amplificação de Genes , Vetores Genéticos , Provírus/genética
14.
Biotechniques ; 7(4): 356-9, 1989 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2698200

RESUMO

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.


Assuntos
Clonagem Molecular/métodos , Oligodesoxirribonucleotídeos/genética , Biotecnologia , Escherichia coli/genética , Vetores Genéticos , Mutação , Plasmídeos
15.
AIDS Res Hum Retroviruses ; 14(16): 1397-406, 1998 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-9824317

RESUMO

The effect of macrophage (M)-tropic and T cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) infection on antigen-specific CD4 cytotoxic T lymphocytes (CTLs) has been studied using a CD4 CTL line specific for a peptide from influenza B virus hemagglutinin. In the absence of antigen presentation, the production of CC chemokines was low. Both the M-tropic HIV-1 strain (HIV-1AD) and the T-tropic HIV-1 strain (HIV-1LAI) established productive infections in the CD4 CTLs, decreasing antigen-specific cytotoxicity. Peptide presented to the CD4 CTLs increased their secretion of RANTES and MIP-1beta, suppressed M-tropic HIV-1 replication, downmodulated CCR5 expression, and preserved CTL recognition. The suppression of M-tropic HIV-1 replication and downmodulation of the CCR5 receptor likely resulted from CC chemokine secretion since antibodies to CC chemokines restored M-tropic HIV-1 replication. Antigen presentation did not protect CD4 CTLs from T-tropic HIV-1 infection or preserve their CTL recognition. Thus, these CD4 CTLs do not make suppressor factors that inhibit the T-tropic HIV-1LAI isolate. The results indicate that these CD4 CTLs can either harbor or suppress M-tropic HIV-1 infection, depending on whether antigen is present. CD4 CTLs might therefore provide some protection in the early stages of HIV-1 infection when M-tropic isolates are present.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Linfócitos T Citotóxicos/virologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Quimiocina CCL4 , Quimiocina CCL5/metabolismo , Regulação para Baixo , HIV-1/imunologia , Humanos , Proteínas Inflamatórias de Macrófagos/metabolismo , Receptores CCR5/biossíntese , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
16.
Mol Cell Endocrinol ; 64(2): 205-12, 1989 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2792562

RESUMO

Expressing foreign proteins in heterologous eukaryotic cells has been a powerful tool for analyzing protein structure and function. The inducible mouse metallothionein-I promoter has been particularly useful for expression studies. However, the levels of expression achieved with this promoter in heterologous eukaryotic expression systems have not equaled those observed in vivo for the metallothionein-I gene. We have constructed expression plasmids placing either the gene for chloramphenicol acetyltransferase (CAT) or the cDNA for human neuropeptide Y (NPY) under control of the mouse metallothionein-I promoter. These two expression vectors were used to transfect mouse anterior pituitary tumor cells, from which stable transformants were isolated. The resulting cell lines, Mt.NPY1a and Mt.CAT, were used to maximize functional product expression from the metallothionein-I promoter. In both cell lines, a 35-fold induction of mRNA accumulation, peptide synthesis, or CAT activity was observed.


Assuntos
Regulação da Expressão Gênica , Metalotioneína/genética , Regiões Promotoras Genéticas , Animais , Cádmio/farmacologia , Cloranfenicol O-Acetiltransferase/genética , Dexametasona/farmacologia , Camundongos , Neuropeptídeo Y/biossíntese , Neuropeptídeo Y/genética , Adeno-Hipófise/metabolismo , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Zinco/farmacologia
17.
J Clin Virol ; 11(1): 19-28, 1998 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-9784140

RESUMO

BACKGROUND: A recent publication reporting the presence of low levels of reverse transcriptase (RT) activity in certain vaccines for human use necessitated that regulatory agencies address the issue of whether this RT activity presented a risk to humans. Detection of low levels of RT activity corresponding to fewer than ten virions became possible with the development of highly-sensitive polymerase chain reaction (PCR)-based RT (PBRT) assays. Variations of the PBRT assay were developed in three laboratories. These assays were reported as being at least one million-fold more sensitive than conventional RT assays. OBJECTIVE: To ascertain the sensitivity and reliability of PBRT assays in different laboratories and to determine which vaccine samples possessed RT activity. STUDY DESIGN: Coded panels of licensed vaccines together with positive and negative controls was assembled at the Center for Biologics Evaluation and Research (CBER) of the Food and Drug Administration (FDA) and distributed to five cooperating laboratories as well as to our laboratory at CBER. Each laboratory carried out their version of the PBRT assay and submitted the results to the coordinator at CBER. RESULTS: Results of the PBRT analyses carried out in the six laboratories are presented. Five of the six laboratories reported results that were highly consistent. RT activity was detected in live attenuated vaccines that were prepared in chick embryo cells (mumps, measles and yellow fever), but very low or undetectable RT activity was found in vaccines produced in mammalian cells (rabies and rubella). Influenza vaccines from several manufacturers included in the panel displayed the most variability, with different products of this inactivated vaccine having differing amounts of RT activity. CONCLUSIONS: Only vaccines produced in chick embryo cells had significant RT activity. Because RT activity was present in the allantoic fluid of uninfected chick embryos and culture medium from chick embryo fibroblasts, the RT activity arises from the cell substrate used for vaccine production. The PBRT assays were reliably able to detect the low levels of RT activity in chicken-derived vaccines.


Assuntos
Contaminação de Medicamentos , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas Virais/normas , Animais , Southern Blotting , Células Cultivadas , Embrião de Galinha , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estados Unidos , United States Food and Drug Administration , Vacinas Atenuadas/normas , Vacinas Atenuadas/virologia
18.
J Clin Virol ; 11(1): 7-18, 1998 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-9784139

RESUMO

BACKGROUND: Reverse transcriptase (RT) activity has previously been reported in concentrated medium of primary chicken embryo cell cultures using the traditional RT assay. Recently, using the newly-developed and highly-sensitive product-enhanced reverse transcriptase (PERT) assay, RT activity has been detected in live, attenuated vaccines grown in chicken cell substrates. Furthermore, this activity has been associated with particles that contain RNA related to an ancient, endogenous avian retrovirus family designated as EAV-0. OBJECTIVE: To investigate whether the RT activity present in vaccines produced in specific pathogen-free chicken cell substrates is associated with an infectious retrovirus that can replicate in human cells. STUDY DESIGN: The kinetics of RT activity produced by 10-day-old chicken embryo fibroblast (CEF) cultures was determined by analyzing cell-free medium in a PCR-based RT (PBRT) assay. Material containing the peak PBRT activity was used as the inoculum to infect various human cell lines and peripheral blood mononuclear cells. Filtered supernatants from control and test cultures were analyzed for the presence of replication-competent retroviruses by the PBRT assay. The cells were monitored for other adventitious agents by routine observation for cytopathic effect (CPE) and by transmission electron microscopy (TEM) at culture termination. RESULTS: The PBRT activity did not increase above the background level in the human target cells through at least five cell passages, thus indicating the absence of a replicating retrovirus. No other adventitious agents were detected based upon TEM analysis and the absence of CPE. CONCLUSION: The RT activity produced by chicken primary cell cultures is not associated with a retrovirus that can replicate in human cells.


Assuntos
DNA Polimerase Dirigida por RNA/metabolismo , Retroviridae/fisiologia , Replicação Viral , Animais , Células Cultivadas , Embrião de Galinha , Técnicas de Cocultura , Meios de Cultura , Efeito Citopatogênico Viral , Humanos , Cinética , Microscopia Eletrônica , Retroviridae/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Células Tumorais Cultivadas
19.
Ann N Y Acad Sci ; 605: 286-93, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2176444

RESUMO

The process of myelination in the central and peripheral nervous systems has been well characterized morphologically by a variety of techniques. It is evident from these studies that, in the peripheral nervous system myelin formation is a multistep process. Clearly, a 1:1 relationship must be established with the axon, which is followed by formation of the basal lamina and eventually myelin. Because immortalized Schwann cell lines obtained using SV40 T antigen under the control of an inducible promoter have many properties of untransfected Schwann cells in culture, including their ability to form myelin in vitro, these cells will enable us to dissect more easily the process of myelination. Having successfully immortalized rat Schwann cells without affecting their ability to differentiate fully, we are applying this approach to generate analogous cell lines from the peripheral nerves of other species such as mouse and human. Unlike rat Schwann cells, there are no known mitogens for human and mouse Schwann cells, making it impossible to expand these cell populations. The ability to produce large numbers of human Schwann cells from nerve biopsy and to analyze their biochemical properties would be of enormous value in identifying the cellular abnormalities that result in demyelinating disease. Likewise, there are several mutant mouse strains with defects in myelin formation, and cell lines from these animals would facilitate our understanding of the process leading to dysmyelination.


Assuntos
Antígenos Virais de Tumores/genética , Bainha de Mielina/ultraestrutura , Oncogenes , Células de Schwann/citologia , Vírus 40 dos Símios/genética , Animais , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas , Técnicas de Cultura/métodos , Metalotioneína/genética , Neurônios/citologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Plasmídeos , Regiões Promotoras Genéticas , Ratos , Células de Schwann/ultraestrutura , Nervo Isquiático/citologia , Transfecção
20.
J Virol Methods ; 66(2): 247-61, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9255736

RESUMO

Three highly sensitive reverse transcriptase (RT) assays were recently published that are at least one million times more sensitive than conventional RT assays. These assays derive their high sensitivities through the ability to amplify the complementary DNA (cDNA) product of the RT reaction by the polymerase chain reaction (PCR). We describe a modified PCR-based RT (PBRT) assay that retains the high sensitivities of the original assays while reducing their inherent background signals. The background signal of the PBRT assay was found to be due to an intrinsic RNA-dependent DNA polymerase activity of the Taq DNA polymerase, the enzyme used for the PCR. It could be eliminated by inserting a ribonuclease digestion step prior to amplifying the cDNA product of the RT reaction by PCR and by using a thermostable DNA polymerase identified as having reduced RNA-dependent DNA polymerase activity. Comparable results were obtained using three RNA templates with two purified RT enzymes. This modified assay is capable of detecting reliably between 10 and 100 molecules of RT, which is equivalent to between 1 and 10 retrovirus particles.


Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , DNA Polimerase Dirigida por RNA , DNA Polimerase Dirigida por DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Hormônios Placentários/farmacologia , Ribonucleases/antagonistas & inibidores , Sensibilidade e Especificidade , Taq Polimerase
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