RESUMO
Homologous regions of five amino acids each, were identified in the NH2-terminal domain of human class II beta chains and the COOH terminus of HIV I envelope protein. The homologous regions are highly conserved among different DR and DQ alleles and also among different isolates of HIV. Septamers containing these sequences were synthesized and used for the generation of murine mAbs. The mAbs selected for this study were raised against the HIV I-derived peptide and reacted strongly not only with the immunizing peptide, but also with the homologous class II-derived peptide. These mAbs also reacted with native MHC class II antigens expressed on human B cell lines and on murine fibroblast L cell lines transfected with the genes coding for the alpha and beta chains of human class II antigens. Furthermore, sera from 36% of AIDS patients tested contained antibodies that reacted with the class II-derived peptide, as well as with intact class II molecule-rich cell extracts. Such antibodies in HIV I-infected individuals may recognize self class II antigens, triggering autoimmune mechanisms that could contribute to the development of immunodeficiency in AIDS patients.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Doenças Autoimunes/imunologia , Genes MHC da Classe II , Genes Virais , HIV/genética , Antígenos HLA-D/genética , Antígenos HLA-DR/genética , Proteínas dos Retroviridae/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Autoanticorpos/imunologia , Reações Cruzadas , Genes , HIV/imunologia , Anticorpos Anti-HIV , Proteína gp41 do Envelope de HIV , Humanos , Dados de Sequência Molecular , Proteínas dos Retroviridae/imunologia , Homologia de Sequência do Ácido Nucleico , Proteínas do Envelope Viral/imunologiaAssuntos
Desoxirribonucleases/isolamento & purificação , Endodesoxirribonucleases , Endonucleases/isolamento & purificação , Escherichia coli/enzimologia , Centrifugação com Gradiente de Concentração/métodos , Cromatografia por Troca Iônica/métodos , Desoxirribonucleases/metabolismo , Endonucleases/metabolismo , Cinética , Peso Molecular , Especificidade por SubstratoAssuntos
Sequência de Aminoácidos , Proteínas , Aminoácidos/análise , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Meios de Cultura , Camundongos , Neoplasias Experimentais/metabolismo , Fragmentos de Peptídeos/análise , Biossíntese de Proteínas , Técnica de Diluição de Radioisótopos , Radioisótopos de Enxofre , TrítioAssuntos
beta-Globulinas , Antígenos H-2 , Microglobulina beta-2 , Animais , Fenômenos Químicos , Química , Antígenos H-2/imunologia , Antígenos H-2/isolamento & purificação , Humanos , Camundongos , Camundongos Endogâmicos , Microglobulina beta-2/imunologia , Microglobulina beta-2/isolamento & purificaçãoRESUMO
A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.
Assuntos
Desoxirribonucleases , Endonucleases , Escherichia coli/enzimologia , Cátions Bivalentes , Desoxirribonucleases/isolamento & purificação , Desoxirribonucleases/metabolismo , Endonucleases/isolamento & purificação , Endonucleases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática , Concentração de Íons de Hidrogênio , Ferro/farmacologia , Cinética , Magnésio/farmacologia , Mutação , Especificidade da EspécieRESUMO
An endonuclease which is active upon DNA exposed to ultraviolet light at a photoproduct other than thymine dimers has been extensively purified from Escherichia coli. The small (2.7 S) enzyme is active in the presence of EDTA, has a neutral pH optimum, and is inhibited by tRNA and 1 M NaCl. It has no detectable exonuclease, DNA-N-glycosidase, or ribonuclease activities. The enzyme also nicks duplex DNA exposed to OsO4, x-rays, or acid, but it does not act upon undamaged DNA or irradiated single-stranded DNA. The majority of sites of action in DNA exposed to ultraviolet light or OsO4 appear to be alkali-stable, but those in DNA exposed to x-rays or acid are not. The incisions created by the endonuclease contain 5'-phosphate termini. The enzyme is possibly the same as E. coli endonuclease III described by Radman (Radman, M. (1976) J. Biol. Chem. 251, 1438-1445), but it is distinguishable from the other endodeoxyribonucleases described from that organism.
Assuntos
DNA Viral/metabolismo , Desoxirribonucleases/metabolismo , Endonucleases/metabolismo , Escherichia coli/enzimologia , Bacteriófagos , Sítios de Ligação , DNA Viral/efeitos da radiação , Desoxirribonucleases/isolamento & purificação , Endonucleases/isolamento & purificação , Concentração de Íons de Hidrogênio , Desnaturação de Ácido Nucleico , Osmio , Especificidade da Espécie , Relação Estrutura-Atividade , Raios Ultravioleta , Raios XRESUMO
This manuscript provides suggestions for monitoring the safety, purity, and potency of new drugs and biologics produced by recombinant DNA technology. We discussed: the Expression System, the Master Cell Bank, Production Procedure, Purification Procedure, and Characterization of the Product. These points represent our current concensus of opinion relating to the safe use of recombinant DNA products and should not be regarded as fixed or all-inclusive.
Assuntos
Produtos Biológicos/normas , Contenção de Riscos Biológicos , DNA Recombinante , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Produtos Biológicos/toxicidade , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas/análise , Controle de QualidadeRESUMO
Primary structural analyses of beta 3-microglobulin isolated from the tumor cell lines EL4.BU (derived from a C57BL/6 mouse) and C14 (derived from a BALB/c mouse) have revealed the presence of an amino acid difference at position 85 of this molecule. beta 2-Microglobulin isolated from histocompatibility antigens of EL4.BU has alanine at this position, whereas that from C14 has aspartic acid. Determination of the sequence of these molecules has employed radiochemical methodology that was developed in studies of murine histocompatibility antigens. The sequence obtained in this study is: Ile - Gln - Lys - Thr - Pro - Gln - Ile - Gln - Val - Tyr - Ser - Arg - His - Pro - Pro - Glu - Asn - Gly - Lys - Pro - Asn - Ile - Leu - Asn - Cys - Tyr - Val - Thr - Gln - Phe - His - Pro - Pro - His - Ile - Glu - Ile - Gln - Met - Leu - Lys - Asn - Gly - Lys - Lys - Ile Pro - Lys - Val - Glu - Met - Ser - Asp - Met - Ser - Phe - Ser - Lys - Asp - Trp - Ser - Phe - Tyr - Ile - Leu - Ala - His - Thr - Glu - Phe - Thr - Pro - Thr - Glu - Thr - Asp - Thr - Tyr - Ala - Cys - Arg - Val - Lys - His - Ala/Asp - Ser - Met - Ala - Glu - Pro - Lys - Thr - Val - Tyr - Trp - Asp - Arg - Asp - Met. Comparison of the sequence of murine beta 2-microglobulin to the sequences reported for the homologues from man, rabbit, and guinea pig indicate identities of 68%, 66%, and 61%, respectively.
Assuntos
beta-Globulinas , Camundongos/genética , Polimorfismo Genético , Microglobulina beta-2 , Sequência de Aminoácidos , Animais , beta-Globulinas/genética , Cobaias , Humanos , Coelhos , Especificidade da Espécie , Microglobulina beta-2/genética , Microglobulina beta-2/isolamento & purificaçãoRESUMO
A messenger RNA fraction from the C 3 H mouse myeloma 5563 was used to direct the synthesis of heavy (gamma 2a) and light (chi) chain precursors. The synthetic products were radiolabeled by inclusion of [3H] or [35S] amino acids in wheat-germ cell-free translation system. Precursor peptides for both H and L chains were indicated by comparison by polyacrylamide gel analysis of apparent molecular weights of chains synthesized in vitro vs. in vivo. The nonglycosylated H chain synthesized in vivo in the presence of tunicamycin was used for comparison. This approach should be generally applicable for the demonstration of precursor forms of N-glycosylated polypeptides. Following immunoprecipitation and preparative polyacrylamide gel electrophoresis, the isolated chains were subjected to automated Edman degradation. The amino acid sequence data obtained for these precursors were significantly different from those reported for BALB/c immunoglobulins. The 5563 H chain precursor bears little homology to the previously reported H chain precursor from MOPC 315 (IgA) (Jilka, J. A. and Pestka, S., Proc. Nat. Acad. Sci. USA 1977. 74:5692). The 5563 H chain can be assigned to a heretofore undescribed H chain variable region subgroup on the basis of partial sequence data. The amino terminal sequence of the 5563 L chain precursor has maximum homology (25-50%) to the ssequence o the precursor peptide of MOPC 41 chi chain.
Assuntos
Sequência de Aminoácidos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Animais , Linhagem Celular , Camundongos , Camundongos Endogâmicos C3H , Mieloma Múltiplo/imunologia , Neoplasias Experimentais , RNA Neoplásico/imunologiaRESUMO
Standard cell-fusion techniques have been used to generate hybrid cells from rabbit spleen cells and mouse myeloma cell lines. The hybrids were selected for secretion of rabbit immunoglobulin. Detailed allotype analyses were carried out for 189 cell lines found to be immunoglobulin positive: 1 produced an intact immunoglobulin molecule with antibody activity, 143 produced rabbit light (L) chains, 36 produced rabbit heavy (H) chains, and 9 cell lines gave negative results in tests for group a and b allotypes. Fusions with a nonproducing murine myeloma cell line (SP-2) yielded only L chain-secreting hybrids, whereas 27% of hybrids resulting from fusion with an L chain-producing line (PU) secreted rabbit H chains paired with mouse L chains. Several of the hybridomas have maintained the ability to produce rabbit immunoglobulin chains in culture for almost 1 year and can be propagated as ascites tumors in athymic (nude) mice. Cytogenetic analyses have detected no rabbit chromosomes in the stable hybrid cell lines.
Assuntos
Células Híbridas/imunologia , Imunoglobulinas/biossíntese , Coelhos/imunologia , Animais , Ascite , Linhagem Celular , Alótipos de Imunoglobulina/biossíntese , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Cariotipagem , Camundongos , Mieloma Múltiplo/imunologia , Proteínas do Mieloma/biossíntese , Fatores de TempoRESUMO
Serologic and primary structural analyses of Ig chains secreted by several rabbit-mouse hybridomas have shown that these hybrid cells produce heavy (H) or light (L) chains identical to those isolated from rabbit sera. Two of the cell lines (7D2, 7D6) secreted rabbit H chains with a m.w. of 55,000 each of which expressed a full complement of variable and constant region allotypes (a3, d11, e15). These apparently normal rabbit H chains were secreted in a complex with a m.w. about 130,000, and serologic studies indicated that this complex contained a covalently linked mouse kappa L chain. Two other cell lines (4C1, 12F2) produced allotype b4 L chains with m.w. of 23,000 and 25,000, and a third (1D4P5) produced an allotype b5 L chain with a m.w. of 23,000. Serologic analyses indicated that the allotypes on these chains are equivalent to those expressed by normal rabbit Ig molecules. Partial amino acid sequence data obtained for the L chain products showed them to be typical of rabbit L chains, and to be significantly different from mouse L chains.
Assuntos
Células Híbridas/imunologia , Imunoglobulinas/biossíntese , Sequência de Aminoácidos , Animais , Ascite/imunologia , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Alótipos de Imunoglobulina , Cadeias Leves de Imunoglobulina , Cadeias kappa de Imunoglobulina , Focalização Isoelétrica , Camundongos , Camundongos Nus , Peso Molecular , CoelhosRESUMO
Improved procedures have expedited the formation and propagation of stable rabbit-mouse hybridomas (RMH) that secrete rabbit immunoglobulin (Ig) chains and that serve as sources for allotype-defined mRNA, specifying both constant and variable regions of rabbit Ig. The Ig-secreting hybridomas were stabilized by multiple recloning steps and eventually attained a 90% frequency of cells secreting rabbit Ig chains. Stabilized RMH were propagated in vivo in nude (athymic) and in some instances in conventional BALB/c mice. Rabbit Ig products secreted by these RMH cell lines were isolated and were shown to have amino acid sequence characteristics of rabbit Ig chains and that are distinct from those of the mouse. Preparative amounts of polyadenylated RNA-(poly(A) RNA) encoding allotype-defined rabbit Ig chains were isolated from RMH grown as solid tumors. Poly(A) RNA encoding a b4 L chain from one RMH (12F2) and an a3 H chain from another (7D2) were purified 10-fold by sucrose density gradient centrifugation and were characterized in an in vitro translation system. The mRNA encoding the b4 allotype had an S value of 12 and directed the synthesis of a rabbit precursor L chain with m.w. 27,500. Radiochemical amino acid sequence analysis of the L chain precursor has shown it to include a 22 residue leader sequence with a leucine profile similar to that of murine L chain precursors. Analysis of the mRNA encoding the a3 H chain revealed it has an S value of 16 and directed the synthesis of an Ig chain with a m.w. identical to that of the rabbit H chain secreted by the RMH.