Asunto(s)
Reflejo/fisiología , Torsión del Cordón Espermático/diagnóstico , Adolescente , Niño , Preescolar , Diagnóstico Diferencial , Humanos , Lactante , Recién Nacido , Masculino , Músculo Esquelético/inervación , Músculo Esquelético/fisiología , Piel/inervación , Torsión del Cordón Espermático/fisiopatología , Muslo/inervaciónRESUMEN
The cytotoxicity of a commonly used material to alleviate the symptoms of benign prostatic hyperplasia (BPH), Saw Palmetto Berry Extract (SPBE), was examined as neat oil using a set of prostatic cell lines; 267B-1, BRFF-41T and LNCaP. Proliferation of these prostatic derived cell lines is inhibited to different degrees when dosed for 3 days with SPBE. The amount of SPBE required to inhibit 50% growth (IC50) of these cell lines was 20-30 nl equivalents of SPBE per ml of medium for cell lines 267B-1 and BRFF-41T and approximately 10-fold more for the LNCaP cell line. The effect of SPBE dosing on these cell lines is not irreversible, since a 30 min treatment with SPBE at an IC50 concentration does not inhibit their growth. Normal prostate cells were inhibited by 20-25% when grown in the presence of 200 nl SPBE equivalent per ml media. Growth of other non-prostatic cancer cell lines, i.e. Jurkat and HT-29, was affected by approx. 50% and 40%, respectively. When LNCaP cells were grown in the presence of dihydrotestosterone and SPBE, the IC50 concentration decreased significantly compared to LNCaP cells grown in the presence of serum and SPBE. Reduced cellular growth after SPBE treatment of these cell lines may relate to decreased expression of Cox-2 and may be due to changes observed in the expression of Bcl-2. Expression of Cox-1 under similar conditions is not affected because of its constitutive expression. Since increased Cox-2 expression is associated with an increased incidence of prostate cancer, and decrease in its expression by SPBE would provide a basis for further investigation of its use against BPH and in prostatic cancer chemoprevention.
Asunto(s)
Antagonistas de Andrógenos/farmacología , Isoenzimas/biosíntesis , Extractos Vegetales/farmacología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Ciclooxigenasa 2 , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes bcl-2/fisiología , Inhibidores de Crecimiento/farmacología , Humanos , Isoenzimas/genética , Masculino , Proteínas de la Membrana , Prostaglandina-Endoperóxido Sintasas/genética , Serenoa , Células Tumorales CultivadasRESUMEN
In search for retinoic acid receptor (RAR) selective ligands, a series of 6-substituted 2-naphthoic acid retinoids were synthesized and evaluated in vitro in a transactivation assay and a competition binding assay for all RARs. These derivatives, in general, showed RAR beta,gamma selectivity. Among these naphthoic acids, oxime derivative 12 was identified as a potent RAR gamma-selective retinoid, while olefinic derivative 11 was found to be comparable to retinoic acid and slightly RAR beta,gamma selective. For the bioassays, a general correlation was observed between the binding affinity of the ligand to the receptors and the potency of the compounds in the transactivation assay. The structure-activity relationship of these naphthoic acids will be discussed.
Asunto(s)
Naftalenos/química , Receptores de Ácido Retinoico/efectos de los fármacos , Retinoides/farmacología , Unión Competitiva , Diseño de Fármacos , Genes Reporteros , Células HeLa/efectos de los fármacos , Humanos , Ligandos , Estructura Molecular , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Retinoides/síntesis química , Retinoides/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Activación Transcripcional/efectos de los fármacos , Tretinoina/metabolismo , Receptor de Ácido Retinoico gammaRESUMEN
Among retinoic acid receptors (RARs) alpha, beta, and gamma, the messenger RNA level of RAR-gamma is the most readily detectable by Northern blotting in human and mouse skin. This observation suggests that RAR-gamma may play a critical role in the modulation of the therapeutic benefits and side effects of retinoids in skin. To test this hypothesis, 11 RAR-gamma selective retinoids were synthesized based on three related structures. Each compound was found to prefer RAR-gamma when assessed by retinoid-induced transcriptional activity (RAR-gamma > RAR-beta > RAR-alpha). The apparent Kd for binding to recombinant receptor protein was found to follow a similar trend. To correlate this receptor selectivity with in vivo activity, the compounds were tested topically in the Rhino mouse utriculi reduction and rabbit irritation models, two assays widely used to screen retinoids for efficacy and side effects, respectively. The results indicated that for these compounds, both efficacy in the utriculi reduction assay and irritation potential in rabbits correlated positively with the RAR-gamma transactivation activity, with r2 of 0.9 and 0.8, respectively. These data suggest that RAR-gamma is an important regulator of retinoic acid efficacy in skin and further, that the irritation associated with the use of retinoids is most likely a receptor-mediated process.
Asunto(s)
Receptores de Ácido Retinoico/fisiología , Retinoides/farmacología , Piel/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Pelados , Conejos , Retinoides/química , Relación Estructura-Actividad , Receptor de Ácido Retinoico gammaRESUMEN
The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.
Asunto(s)
Azidas/metabolismo , Dihidropiridinas/metabolismo , Resistencia a Medicamentos/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Estructura Secundaria de Proteína , Células 3T3 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein. Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein. Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein. We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein. Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling. These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins. We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.
Asunto(s)
Azidas/metabolismo , Dihidropiridinas/metabolismo , Resistencia a Medicamentos/fisiología , Glicoproteínas de Membrana/metabolismo , Vinblastina/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Colchicina/farmacología , Bromuro de Cianógeno , Humanos , Células KB , Cinética , Peso Molecular , Fragmentos de Péptidos/aislamiento & purificación , Transfección , Tripsina , Verapamilo/farmacología , Vinblastina/farmacología , Vincristina/farmacologíaRESUMEN
P-Glycoprotein (Pgp) expression in cell lines derived from tumors arising from cells which normally express Pgp can be increased by sodium butyrate and other differentiating agents. Although the Pgp level increased 25-fold after sodium butyrate treatment in SW620 human colon carcinoma cells, the intracellular accumulation of vinblastine, adriamycin, and actinomycin D increased rather than decreased. In contrast, colchicine showed the expected decrease in accumulation, as a result of increased efflux. Likewise, treatment of a Pgp-expressing multidrug-resistant SW620 subline with sodium butyrate resulted in active interference with Pgp function. This effect was partially reversed by phorbol esters with a resulting decrease in the accumulation of vinblastine, adriamycin, and actinomycin D. Sodium butyrate, while increasing Pgp levels, inhibited the phosphorylation of Pgp. Time course studies revealed a tight relationship between decreased Pgp phosphorylation and increased vinblastine accumulation after sodium butyrate treatment. Either treatment with phorbol esters or withdrawal of sodium butyrate increased Pgp phosphorylation while decreasing vinblastine accumulation. These studies suggest that the specificity of Pgp transport can be modulated by phosphorylation and that vinblastine, adriamycin, or actinomycin D transport, but not that of colchicine, is diminished after dephosphorylation by sodium butyrate.
Asunto(s)
Butiratos/farmacología , Resistencia a Medicamentos , Glicoproteínas de Membrana/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Transporte Biológico/efectos de los fármacos , Ácido Butírico , Línea Celular , Colchicina/metabolismo , Dactinomicina/metabolismo , Doxorrubicina/metabolismo , Humanos , Técnicas In Vitro , Ésteres del Forbol/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Vinblastina/metabolismoRESUMEN
The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs. We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene. The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy. We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes. This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells. When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises. After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically. However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs. These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.
Asunto(s)
Daunorrubicina/metabolismo , Doxorrubicina/metabolismo , Resistencia a Medicamentos/fisiología , Rodaminas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Línea Celular , Daunorrubicina/análisis , Doxorrubicina/análisis , Resistencia a Medicamentos/genética , Humanos , Células KB , Rayos Láser , Glicoproteínas de Membrana/genética , Ratones , Microscopía Fluorescente/métodos , Rodamina 123 , Rodaminas/análisis , Transfección , Verapamilo/farmacologíaRESUMEN
We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Resistencia a Medicamentos , Glicoproteínas de Membrana/inmunología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Línea Celular , Epítopos/análisis , Humanos , Células KB , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Pruebas de PrecipitinaRESUMEN
Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm its capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pump, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.
Asunto(s)
Álcalis/metabolismo , Citosol/metabolismo , Portadores de Fármacos/metabolismo , Colorantes Fluorescentes , Animales , Benzopiranos , Línea Celular , Colchicina/farmacología , Dextranos , Resistencia a Medicamentos , Concentración de Iones de Hidrógeno , Microinyecciones , Verapamilo/farmacología , Vinblastina/farmacologíaRESUMEN
Multidrug-resistance of human cancer cells may result from expression of a 170,000 dalton multidrug efflux pump called P-glycoprotein. To identify this multidrug transporter, and to study its structure and function, we have generated polyclonal rabbit antibodies against the amino-terminal and carboxy-terminal halves of the molecule using recombinant protein fragments produced in Escherichia coli. Two recombinant P-glycoprotein fragments, representing amino acids 140-228 and 919-1280, were overproduced in Escherichia coli by an inducible T7 expression system, gel-purified and injected into rabbits. Both antisera specifically immunoprecipitate 3H-azidopine and 35S-methionine labeled P-glycoprotein from multidrug-resistant cells and detect P-glycoprotein on Western blots with high sensitivity. Because these antisera were raised against epitopes in the amino- and carboxy-terminal halves of P-glycoprotein, they should be useful as research tools to define the function of these two halves of the molecule.
Asunto(s)
Anticuerpos , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Marcadores de Afinidad , Animales , Anticuerpos Monoclonales , Reacciones Antígeno-Anticuerpo , Azidas , Secuencia de Bases , Western Blotting , Clonación Molecular/métodos , Dihidropiridinas , Resistencia a Medicamentos , Escherichia coli/genética , Vectores Genéticos , Humanos , Glicoproteínas de Membrana/genética , Metionina/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Plásmidos , Conejos/inmunología , Proteínas Recombinantes/inmunologíaRESUMEN
We studied transepithelial transport of 3H-labeled hydrophobic cationic drugs in epithelia formed by wild-type and by drug-resistant Madin-Darby canine kidney (MDCk) cells that had been infected with a retrovirus carrying the multidrug-resistance (MDR1) cDNA which encodes the P-glycoprotein. P-glycoprotein is an ATP consuming plasma membrane multidrug transporter responsible for the efflux of cytotoxic chemotherapeutic drugs from resistant cancer cells. Wild-type MDCK cells have small amounts of P-glycoprotein detected by immunoprecipitation. Net transepithelial transport across wild-type MDCK epithelia was demonstrated. Basal to apical flux of 100 nM vinblastine was about six times higher than apical to basal flux. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. Daunomycin, vincristine, and actinomycin D were also actively transported and at 20 microM these agents inhibited transport of vinblastine, suggesting that wild-type MDCK cells have a common transporter for all these drugs. Vinblastine transport was also inhibited by 20 microM verapamil, which inhibits the multidrug transporter and reverses multidrug-resistance in non-polarized cells. Net transepithelial transport of all these cytotoxic drugs and of verapamil was much higher in epithelia formed by MDCK cells infected with a human MDR1 virus (MDR-MDCK) which is expressed on the apical surface of MDR-MDCK monolayers. Because the transport of these cytotoxic drugs and verapamil is increased in MDR-MDCK epithelia compared to wild-type MDCK epithelia, transport in both these cell populations can be attributed to P-glycoprotein. These results are consistent with a role for P-glycoprotein in multidrug secretory transport across the epithelium of the proximal tubule since P-glycoprotein is normally expressed on the apical membrane of proximal tubule cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Riñón/metabolismo , Glicoproteínas de Membrana/metabolismo , Preparaciones Farmacéuticas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Dactinomicina/metabolismo , Dactinomicina/farmacología , Daunorrubicina/metabolismo , Daunorrubicina/farmacología , Perros , Resistencia a Medicamentos , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Riñón/efectos de los fármacos , Verapamilo/metabolismo , Verapamilo/farmacología , Vinblastina/metabolismo , Vincristina/metabolismo , Vincristina/farmacologíaRESUMEN
The multidrug transporter is a 170,000-dalton membrane glycoprotein which confers multidrug resistance through its activity as an ATP-dependent efflux pump for hydrophobic, cytotoxic drugs. To determine the essential structural components of this complex membrane transporter we have altered an MDR1 cDNA in an expression vector by deletion and insertion mutations. The structure of the transporter deduced from its amino acid sequence suggests that it consists of two homologous, perhaps functionally autonomous, halves each with six transmembrane segments and a cytoplasmic ATP-binding domain. However, several carboxyl-terminal deletions, one involving 53 amino acids, the second removing 253 amino acids, and an internal deletion within the carboxyl-terminal half of the molecule, totally eliminate the ability of the mutant transporter to confer drug resistance. An internal deletion of the amino-terminal half, which removed residues 140-229, is also nonfunctional. Small carboxylterminal deletions of up to 23 amino acids leave a functional transporter, although the removal of 23 COOH-terminal amino acids reduces its ability to confer colchicine resistance. Insertions of 4 amino acids in a transmembrane domain, and in one of the two ATP-binding regions, have no effect on activity. These studies define some of the limits of allowable deletions and insertions in the MDR1 gene, and demonstrate the requirement for two intact halves of the molecule for a functional multidrug transporter.
Asunto(s)
Proteínas Portadoras/genética , Deleción Cromosómica , Elementos Transponibles de ADN , Glicoproteínas de Membrana/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Animales , Línea Celular , Colchicina/farmacología , Resistencia a Medicamentos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Vectores Genéticos , Humanos , Ratones , Mutación , FenotipoRESUMEN
Double-label fluorescence microscopy was used to demonstrate the efflux activity of the multidrug transporter in single cultured cells. NIH3T3 cells expressing a transfected MDR1 gene (NIH3T3-MDR) were treated with vinblastine or daunomycin. The accumulation of vinblastine was monitored by examining the morphology of tubulin in cells, using immunofluorescence. Overnight treatment of drug-sensitive cells caused disassembly of microtubules and formation of paracrystals; the absence of vinblastine effects was evident by the presence of intact microtubules. Daunomycin accumulation was detected in nuclei using the inherent fluorescence of the drug with rhodamine epifluorescence microscopy. Drug efflux in multidrug-resistant cells was inhibited with verapamil. When multidrug-resistant cells were treated overnight in vinblastine, an effect of 0.5 microM vinblastine on microtubules was seen only in the presence of verapamil. Similarly, when cells were treated with daunomycin, this drug accumulated in nuclei only when verapamil was present. When cells incubated with vinblastine and verapamil were washed free of drugs, they did not accumulate daunomycin in a subsequent incubation, indicating that the multidrug transporter was still active; this occurred even though the morphologic effects of vinblastine persisted. Cells incubated with vinblastine alone showed an immediate inhibition of efflux activity when verapamil was subsequently added with daunomycin. These results show that the efflux activity of the multidrug transporter can be rapidly manipulated by agents such as verapamil, despite a prior history of drug treatment, and that the effects of inhibition of the transporter are rapidly reversible.
Asunto(s)
Daunorrubicina/farmacología , Resistencia a Medicamentos , Glicoproteínas de Membrana/metabolismo , Vinblastina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Transporte Biológico/efectos de los fármacos , Proteínas Sanguíneas , Línea Celular , Núcleo Celular/metabolismo , Cristalización , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Citoplasma/ultraestructura , Daunorrubicina/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Glicoproteínas de Membrana/genética , Microscopía Fluorescente , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Transfección , Tubulina (Proteína)/metabolismo , Verapamilo/farmacología , Vinblastina/metabolismoRESUMEN
Overlapping cosmid clones of rabbit germ-line DNA containing VH, DH and JH gene segments were isolated. The map of this cluster of cosmid clones indicated that the rabbit VH and JH regions were separated by 63 kb. Hybridization of Southern blots of these cosmid clones with two different DH segment probes identified a total of six DH segments within the region between the VH and JH regions. The nucleotide sequences of the JH region and one of the DH segments have been determined. The DH segment has conserved heptamer and nonamer sequences separated by 12 and 11 bp at the 3' and 5' sides, respectively, of the coding region and hence, appears to be a functional gene. The nucleotide sequence of the JH region revealed four functional JH gene segments and one JH pseudogene. Inasmuch as the JH region had previously been linked by contiguous overlapping clones with C mu, C gamma, C epsilon, and one C alpha gene, this VH-DH-JH cluster and the clones containing the Ig H chain C region genes represent 190 kb of contiguous germ-line DNA of the Ig H chain locus.
Asunto(s)
Cósmidos , ADN/aislamiento & purificación , Genes de Inmunoglobulinas , Genes Sobrepuestos , Genes , Ligamiento Genético , Células Germinativas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Reordenamiento Génico , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Región de Unión de la Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Datos de Secuencia Molecular , Familia de Multigenes , Conejos , Homología de Secuencia de Ácido NucleicoRESUMEN
Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.
Asunto(s)
Mapeo Cromosómico , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Conejos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular/métodos , ADN/aislamiento & purificación , Células Germinativas/análisis , Datos de Secuencia Molecular , Familia de Multigenes , Conejos/genéticaRESUMEN
We analyzed the genes which encode the heavy chain constant region of rabbit IgG molecules. Five DNA clones derived from the chromosomal region which spans the C gamma coding sequences were isolated from a recombinant phage library of rabbit liver DNA. Four of these clones can be grouped together by overlaps; together they represent 37 kb of genomic DNA, and contain one C gamma gene and one tentatively identified C epsilon gene. A second C gamma gene was identified which did not overlap with the group of four clones because of restriction site differences found in the flanking regions 5' to the C gamma genes. Nucleotide sequences of the two C gamma genes were identical. Comparisons of the restriction maps of the two cloned C gamma genomic regions suggest that they may represent allelic regions of chromosomes of two different heavy chain haplotypes with polymorphisms in the regions flanking the C gamma genes.