RESUMO
We describe a novel repetitive DNA element isolated from three primate species belonging to the family Cercopithecidae. The unusually long 2.6-kb repeat unit of this DNA element is present in high copy number in the pericentromeric region of one pair of chromosomes in both baboon and macaque, forming chromosome-specific satellite-like DNA families. Besides these two very closely related species, the novel DNA element was also detected in the more distantly related African green monkey. However, the copy number of the repeat unit in this species is significantly lower than in macaque and baboon. Sequence analysis revealed that the repeat units of the new repetitive element show similarity to the human MER22 repeat and the Y chromosome-specific TTY2 element, which also exhibits retroelement-like features. Database searches indicate that tandemly arranged MER22-related DNA sequences can also be found in human, raising the possibility that these DNA elements may correspond to a novel primate-specific repetitive DNA group. Recent studies indicate that chromosome-specific pericentric repetitive elements, besides their potential involvement in centromere function, also facilitate homolog recognition during meiosis. In addition, rapid expansion of retroelements in the pericentric regions of chromosomes during interspecific hybridization has been described. In light of these data, we hypothesize that the novel repetitive element described here might have been involved in the speciation of the family Cercopithecidae.
Assuntos
Cercopithecidae/genética , DNA/genética , Sequências de Repetição em Tandem , Animais , Células COS , Linhagem Celular , Centrômero/genética , Chlorocebus aethiops/genética , Sequência Conservada , DNA Satélite/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Macaca fascicularis/genética , Masculino , Papio/genética , Especificidade da Espécie , Cromossomo Y/genéticaRESUMO
The use of baboons in experimental medicine, especially as organ and tissue donors, would be facilitated by the availability of ABO histo-blood group O animals, which are currently rare. To meet our need in breeding and identifying such animals, we have developed a single-stranded conformational polymorphism (SSCP)-based genotyping assay using fluorescence detection. Various buffers and labeling schemes were evaluated to optimize allele discrimination. Through the use of a nested PCR protocol, single-cell sensitivity was achieved, making the assay applicable to preimplantation diagnosis following in vitro fertilization. We discuss the comparative advantages of SSCP vs. alternative methodologies for genotyping.
Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Polimorfismo Conformacional de Fita Simples , Alelos , Animais , Soluções Tampão , Células Cultivadas , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Fluorescência , Genótipo , Papio , Reação em Cadeia da Polimerase/métodosRESUMO
Histo-blood group O has only rarely been observed in baboon. Recent discovery of such an animal has provided use the opportunity to investigate the molecular genetics of the ABO locus in baboons. The major baboon prototype O allele, observed in two homozygous and several heterozygous animals, is related to the A allele as is the case in humans. Additional apparent prototype O alleles have been observed in heterozygotes, one of which is related to the B allele. The nucleotide changes conferring the O phenotype in the two known human O alleles have not been observed in any baboon allele. This information will aid the identification of baboons useful for the development of xenotransplantation in humans.
Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Papio/genética , Polimorfismo Genético , Alelos , Animais , Sequência de Bases , Primers do DNA , Variação Genética , Heterozigoto , Homozigoto , Humanos , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
CD4 is the principal receptor for the human immunodeficiency virus (HIV). We have isolated and studied CD4-expressing tumor cell clones made by expressing CD4 in the T-cell tumor line HSB. Two clones, one designated HSBCD4, a clone expressing low levels of CD4, and the other, HSB10xCD4, a high-expresser CD4+ clone, were studied for their ability to bind and replicate HIV. In contrast to many other CD4+ cells that down-modulate CD4 following HIV infection, the HSB10xCD4 clones continued to express high levels of surface CD4 following infection with HIV. Unlike infection of HSBCD4 or many other human CD4+ cells, HIV infection of HSB10xCD4 clone was short lived: p24 antigen, provirus, or coculturable virus was present for less than 14 days following infection with several strains of HIV-1 or with HIV-2. When infection was initiated by transfection of proviral DNA, high and low CD4 expressers initially produced p24 antigen at approximately the same level. However, high CD4 expressers produced coculturable virus only during the first few days following transfection, whereas low CD4 expressers transfected with HIV continued to produce virus beyond 6 weeks. Monoclonal antibody-mediated down-modulation of CD4 surface expression on HSB10xCD4 clones permitted these formerly HIV-resistant cells to become persistently infected with HIV. Thus, high concentrations of CD4 on the surface of an HIV-infected cell prevent persistent HIV infection of CD4+ cells.
Assuntos
Antígenos CD4/fisiologia , HIV/crescimento & desenvolvimento , Linfócitos T/microbiologia , Replicação Viral/fisiologia , Sequência de Bases , Regulação para Baixo , Vetores Genéticos , Células Gigantes/microbiologia , HIV/patogenicidade , Proteína do Núcleo p24 do HIV/biossíntese , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , HIV-2/crescimento & desenvolvimento , HIV-2/patogenicidade , Humanos , Dados de Sequência Molecular , Linfócitos T/imunologia , Fatores de Tempo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , VirulênciaRESUMO
CD4 is an integral cell surface glycoprotein that is able to enhance T cell specific antigen responses when it interacts with its physiological ligand, class II major histocompatibility (MHC) molecules. In addition, CD4 is a specific cell-surface receptor for the human immunodeficiency virus-1 (HIV-1). Infection by HIV-1 is initiated by the binding of the envelope glycoprotein, gp120, to the first domain of CD4. The binding of CD4 to class II MHC is inhibited by gp120, one possible mechanism for immunosuppression in AIDS patients. In addition, the CD4/gp120 interaction may directly inhibit T cell function. Recently we have synthesized small molecules (CPFs) that specifically inhibit this interaction. CPFs bind to gp120 and prevent the binding of gp120 to CD4, and also inhibit the infectivity of HIV-1.
Assuntos
Antígenos CD4/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , Animais , Antivirais/farmacologia , Sítios de Ligação , Infecções por HIV/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Mutação , Fosfatidilinositóis/análiseRESUMO
Two broad roles have been revealed for the CD4 molecule. It serves as a receptor for both class II major histocompatibility complex molecules and human immunodeficiency virus (HIV). Upon binding class II major histocompatibility molecules, CD4 functions to enhance T-cell activation. By binding to CD4, HIV gains entry into the cell. We have used a chimeric molecule of CD4 and lymphocyte function-associated antigen 3 (LFA-3), CD4PI, which lacks a membrane-spanning domain and is instead anchored in the membrane by linkage to glycosyl-phosphatidylinositol. To further define the structural attributes of viral receptors, and specifically those of CD4 required for HIV infection, we have expressed CD4PI and CD4 in a human T-cell line, HSB-2. We find that CD4PI is able to mediate infection of these cells by HIV with similar, if not greater efficiency, compared with wild-type CD4. Thus the membrane-spanning region of CD4 is not required for HIV infection, and a lipid-anchored protein can serve as a viral receptor.
Assuntos
Antígenos CD4 , Glicolipídeos/fisiologia , HIV-1/fisiologia , Fosfatidilinositóis/fisiologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos CD4/análise , Antígenos CD4/genética , Linhagem Celular , Imunofluorescência , Expressão Gênica , Glicosilfosfatidilinositóis , HIV-1/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Dados de Sequência Molecular , Retroviridae/genética , Linfócitos TRESUMO
Infection by human immunodeficiency virus type-1 (HIV-1) is initiated when its envelope protein, gp120, binds to its receptor, the cell surface glycoprotein CD4. Small molecules, termed N-carbomethoxycarbonyl-prolyl-phenylalanyl benzyl esters (CPFs), blocked this binding. CPFs interacted with gp120 and did not interfere with the binding of CD4 to class II major histocompatibility complex molecules. One CPF isomer, CPF(DD), preserved CD4-dependent T cell function while inhibiting HIV-1 infection of H9 tumor cells and human T cells. Although the production of viral proteins in infected T cells is unaltered by CPF(DD), this compound prevents the spread of infection in an in vitro model system.
Assuntos
Antivirais/farmacologia , Antígenos CD4/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/fisiologia , Animais , Compostos de Benzil/farmacologia , Linhagem Celular , Genes MHC da Classe II , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Cinética , Linfócitos T/imunologiaRESUMO
The CD4 molecule is functionally involved in the class II MHC-restricted T cell response to Ag. CD4 is also the receptor for HIV-1, the major etiologic agent of AIDS. We have assessed whether the interaction of the HIV-1 envelope protein with the CD4 molecule might interfere with the normal function of CD4, thereby contributing to the immunosuppression observed after HIV infection. Using a murine T cell hybridoma which expresses the human CD4 protein and exhibits a CD4-dependent response to Ag, we demonstrate that the HIV envelope protein gp120 can specifically inhibit this response.
Assuntos
Antígenos de Diferenciação de Linfócitos T , HIV/imunologia , Imunossupressores/fisiologia , Proteínas dos Retroviridae/fisiologia , Linfócitos T/metabolismo , Proteínas do Envelope Viral/fisiologia , Animais , Antígenos de Diferenciação de Linfócitos T/imunologia , Citoplasma/imunologia , Proteína gp120 do Envelope de HIV , Humanos , Interleucina-2/biossíntese , Camundongos , Fenótipo , Linfócitos T/classificaçãoRESUMO
T cell hybridomas with specificity for VSV (vesicular stomatitis virus)-infected cells were generated in an attempt to better define the la-restricted helper T cell response to VSV. The hybridomas were created by fusing BALB/c (H-2d) anti-VSV immune spleen cells to the murine thymoma BW 5147. These hybridomas produce IL-2 when stimulated with VSV-infected spleen cells. They were found to recognize viral antigens in association with I-Ad and, in addition, could also be stimulated by VSV-infected A20 cells (an Ia-positive B cell lymphoma of H-2d origin). The purified viral membrane glycoprotein, G protein, and Gs (secreted G protein that lacks the hydrophobic and intracytoplasmic domains) both stimulated IL-2 production when added to cultures of A20 and the hybridomas. These hybridomas therefore recognize a viral antigenic determinant on G protein. Since chemically-fixed antigen-presenting cells fail to stimulate the hybridomas after exogenous addition of purified G protein we can conclude that these T cell hybridomas recognize a processed form of the G protein. Stimulator cells created by expression in A20 of a transfected cDNA encoding G protein were also recognized. Recognition in this case was I-Ad-restricted, as anti-I-Ad monoclonal antibodies blocked stimulation, and an Ia-negative cell (P815) expressing a transfected G protein gene failed to stimulate the hybridomas. Even after paraformaldehyde fixation, G gene-transfected, Ia-positive cells could stimulate the hybridomas, suggesting that processing of this endogenously-synthesized antigen has occurred.
Assuntos
Genes MHC da Classe II , Linfócitos T/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Viroses/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular , Células Clonais , Genes Virais , Vetores Genéticos , Antígenos de Histocompatibilidade Classe II/imunologia , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mapeamento por Restrição , Linfócitos T Auxiliares-Indutores/imunologia , Vírus da Estomatite Vesicular Indiana/genética , Viroses/genéticaRESUMO
Recently Najarian et al. reported a complete cDNA sequence of the genomic RNA of hepatitis A virus (HAV) and the amino acid sequence inferred from it. As a picornavirus, HAV contains a single-stranded plus-sense RNA encoding a single 'polyprotein' which is post-translationally cleaved to yield the mature structural and non-structural proteins. In order to identify putative cleavage sites a combined function of predicted secondary structure and hydropathy was calculated by Najarian et al. for the polyproteins of HAV and poliovirus type 1 (Mahoney) (PV-1) and the two plots were aligned on the basis of a short homology in capsid protein VP3. Several of the proteins thereby predicted fail to conserve features found in all other picornaviruses that have been examined and, indeed, on the basis of these predictions HAV would hardly be a picornavirus. By an alternate analysis utilizing the computer programs FASTP and PRTALN we find that a putative protein processing map which does preserve these features can be constructed.
Assuntos
Hepatovirus/genética , Mapeamento Cromossômico , Genes Virais , Proteínas Virais/genéticaRESUMO
Sequence analysis of the genomic RNA of interstrain guanidine-resistant and antibody-resistant variant recombinants of poliovirus type 1 mapped the resistance of mutants capable of growth in 2.0 mM guanidine hydrochloride to a region located 3' of nucleotide 4444. This region of the viral genome specifies the nonstructural protein 2C. The sequence of genomic RNA encoding 2C from six independently isolated mutants resistant to 2.0 mM guanidine was determined. All six isolates contained a mutation in 2C at the same position in all cases, resulting in two types of amino acid changes. Dependent mutants were examined and found to contain two amino acid changes each within 2C. Mutants resistant to 0.53 mM guanidine were isolated and found to lack the mutations seen in variants resistant to 2.0 mM guanidine. A comparison of the amino acid sequences of the 2C proteins of poliovirus, foot-and-mouth disease virus, rhinovirus types 2 and 14, and encephalomyocarditis virus revealed a strong homology over regions totaling 115 residues. All of the mutations observed in guanidine-selected mutants were contained within this region. The amino acid region containing the mutations observed in poliovirus mutants resistant to 2.0 mM guanidine was compared with the homologous region in the other picornaviruses; a strong correlation was found between the amino acid present at this position and the sensitivity of the virus to 2.0 mM guanidine.
Assuntos
Resistência Microbiana a Medicamentos , Guanidinas/farmacologia , Poliovirus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Relação Dose-Resposta a Droga , Vírus da Encefalomiocardite/genética , Guanidina , Poliovirus/efeitos dos fármacos , RNA Viral/genética , Seleção Genética , Homologia de Sequência do Ácido Nucleico , Replicação Viral/efeitos dos fármacosRESUMO
Mutations have been identified in variants of poliovirus, type 1 (Mahoney) on the basis of their resistance to neutralization by individual monoclonal antibodies. The phenotypes of these variants were defined in terms of antibody binding; the pattern of epitopes expressed or able to be exploited for neutralization were complex. Single amino acid changes can have distant (in terms of linear sequence) and generalized effects on the antigenic structure of poliovirus and similarly constituted virions.
Assuntos
Epitopos/análise , Poliovirus/imunologia , Sequência de Aminoácidos , Aminoácidos/análise , Anticorpos Monoclonais , Imunidade Inata , Mutação , Fenótipo , Poliovirus/genética , Vírion/imunologiaRESUMO
A neutralizing monoclonal antibody-resistant variant of the Sabin vaccine strain of poliovirus type 1 and a guanidine-resistant variant of the virulent parent Mahoney strain were derived. The two variants were used, in a coinfection, to generate recombinant virus containing both resistance markers. Recombinants appeared on the order of 1.0 PFU for every 10(4) total PFU. Two independently derived recombinant viruses were isolated. Each isolate contained the P1 (structural protein) gene region of the Sabin strain virus and the P3 (nonstructural replicase protein) gene region of the Mahoney strain virus. The recombinant virus phenotypes were compared with certain characteristic in vitro phenotypes of both parents. It was found that the slow growth in cell culture, temperature sensitivity, and virion surface charge characteristics of the Sabin virus mapped entirely to the structural protein gene region whereas the phenotype of the actinomycin D sensitivity of the Sabin virus mapped to the gene region specifying the nonstructural replication proteins. Sequence analysis of the recombinant RNA revealed that the crossover occurred 3' of nucleotide 3919. This result showed that the resistance of poliovirus mutants to growth in 2 mM guanidine hydrochloride maps in the 3'-terminal region of the viral genome specifying the nonstructural proteins.
Assuntos
Guanidinas/farmacologia , Poliovirus/genética , Recombinação Genética , Proteínas Virais/genética , Antígenos Virais/análise , Sequência de Bases , Enzimas de Restrição do DNA , Resistência Microbiana a Medicamentos , Guanidina , Células HeLa/fisiologia , Humanos , Fenótipo , Poliovirus/efeitos dos fármacos , RNA Viral/isolamento & purificação , Especificidade da EspécieRESUMO
This study shows that, when homogeneous classes of pictures are used in a picture-recognition task, developmental trends do emerge whereas when a heterogeneous class of pictures is used such developmental trends are not found. These results are consistent with the view that as children get older (3-4 yr., 6-7 yr., 9-10 yr., adolescents, adults) they get better at organizing material that is more difficult to differentiate or remember.