RESUMEN
Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.
Asunto(s)
Proteínas Portadoras , Colesterol/metabolismo , Proteínas de Drosophila , Glicoproteínas de Membrana , Enfermedades de Niemann-Pick/genética , Proteínas/genética , Secuencia de Aminoácidos , LDL-Colesterol/metabolismo , Mapeo Cromosómico , Cromosomas Humanos Par 18 , Clonación Molecular , Homeostasis , Humanos , Hidroximetilglutaril-CoA Reductasas/química , Proteínas de Insectos/química , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutación , Proteína Niemann-Pick C1 , Enfermedades de Niemann-Pick/metabolismo , Polimorfismo Conformacional Retorcido-Simple , Proteínas/química , Proteínas/fisiología , Receptores de Superficie Celular/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
We developed an assay to test for inhibition of myristoyl-CoA:protein N-myristoyltransferase (NMT) activity since this enzyme is important in viral replication and cellular biochemistry. Saccharomyces cerevisae NMT was harvested from Escherichia coli carrying a plasmid vector containing the yeast NMT cDNA. Following the enzyme-catalyzed reaction of [3H]myristoyl-CoA and an octapeptide substrate (GlyAsnAla4Arg2-NH2), the assay mixture was loaded on AG1-8X anion-exchange resin which bound negatively charged reactants and by-products and left a doubly positively charged and nonbinding [3H]myristoyl-peptide product in the supernatant. Optimum conditions for separating reactants and by-products from myristoyl-peptide in a 100-pmol reaction were 450 mg resin and 25% methanol at pH 5.8. Under these conditions 97% of myristic acid and 98% of myristoyl-CoA bound to the resin, whereas 99% of myristoyl peptide remained in the supernatant. The potent inhibitor S-(2-oxopentadecyl)-CoA was tested in our assay system. In addition, high-specific-activity [3H]myristoyl-CoA, synthesized using acyl-CoA synthetase, was purified on a 200-microCi scale (60 nmol) using a reverse-phase C-18 silica gel cartridge. Impurities, including free CoA, were washed from the column using 10% acetonitrile in 10 mM potassium phosphate buffer, pH 7.5, while purified (95% by radiochemical scan) myristoyl-CoA was eluted from the column using 1:1 acetonitrile:phosphate buffer.
Asunto(s)
Aciltransferasas/análisis , Péptidos/química , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Datos de Secuencia Molecular , TritioRESUMEN
Type 3 neuronopathic Gaucher's disease (GD3) is phenotypically heterogeneous. In many GD3 patients, progressive myoclonus and dementia dominate the illness, with death secondary to progressive CNS disease. We have designated this group as GD3a. We studied 14 children with Gaucher's disease, isolated horizontal supranuclear gaze palsy, and aggressive systemic disease, and designated this group as GD3b. In comparison with 13 children with type 1 non-neuronopathic Gaucher's disease, the GD3b children presented earlier, and were shorter, underweight, and more prone to cardiopulmonary, hepatic, and skeletal complications. One-half of the children died in childhood or adolescence of systemic complications. Patients with at least one copy of the mutation that causes substitution of asparagine for serine at amino acid 370 of glucocerebrosidase did not develop neurologic signs. Patients homoallelic for the mutation causing substitution of leucine for proline at position 444 had severe systemic disease; neurologic signs were frequently, but not invariably, present. Early diagnosis and timely enzyme replacement therapy promise to improve the prognosis in GD3b.
Asunto(s)
Enfermedad de Gaucher/diagnóstico , Enfermedad de Gaucher/fisiopatología , Parálisis Supranuclear Progresiva/etiología , Adolescente , Edad de Inicio , Niño , Preescolar , ADN/sangre , Estudios de Seguimiento , Enfermedad de Gaucher/genética , Genotipo , Humanos , Lactante , Parálisis Supranuclear Progresiva/fisiopatología , Factores de TiempoRESUMEN
Butyric acid has many strong effects on gene expression in mammalian and viral systems, as well as in increasing the expression of recombinant DNAs artificially introduced into cultured cells. We screened 14 analogues of butyric acid for their ability to upregulate expression from 3 different recombinant chloramphenicol acetyltransferase expression vectors stably integrated into NIH 3T3 cells that had been transformed by calcium phosphate transfection or electroporation. Butyric acid, 2-bromobutyric acid, 3-bromopropionic acid, 3-mercaptopropionic acid, vinylacetic acid, and butyraldehyde were found to upregulate human immunodeficiency viral long terminal repeat-, SV40 early gene promoter-, and glucocerebrosidase promoter-directed expression of heterologous genes in cultured cells. Three other analogues had lesser effects; and 6 additional analogues had very little, if any, effect.
Asunto(s)
Butiratos/farmacología , ADN Recombinante/genética , Expresión Génica/efectos de los fármacos , Transfección , Células 3T3 , Animales , División Celular/efectos de los fármacos , Cloranfenicol O-Acetiltransferasa/genética , ADN Recombinante/efectos de los fármacos , Vectores Genéticos , Duplicado del Terminal Largo de VIH , Humanos , Ratones , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
We analyzed the involvement of chromosome 18 in Niemann-Pick disease type C (NPC), an autosomal recessive cholesterol-processing disorder. Within affected offspring, the chromosome 18 parental contributions were identified by using allele-specific microsatellite markers. Significant linkage of NPC to an 18p genomic marker, D18S40, was indicated by a two-point lod score of 3.84. Analysis of meiotic chromosomal breakpoint patterns among the affected individuals indicated that the NPC gene is pericentromerically localized on human chromosome 18.
Asunto(s)
Cromosomas Humanos Par 18 , Enfermedades de Niemann-Pick/genética , Ligamiento Genético , Marcadores Genéticos , Humanos , Linaje , Reacción en Cadena de la PolimerasaRESUMEN
Fluorescent microscopic examination of fibroblasts cultured with low density lipoprotein (LDL) and progesterone (10 micrograms/ml) for 24 h revealed extensive filipin-cholesterol staining of perinuclear lysosomes. Levels of unesterified cholesterol were 2-fold greater than in fibroblasts cultured with LDL alone. Progesterone strongly blocked cholesteryl ester synthesis. When cellular uptake of LDL was monitored in the presence of 58035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase, excess unesterified cholesterol was not stored in lysosomes. Discontinuation of LDL uptake in conjunction with progesterone washout markedly reversed the filipin-cholesterol staining of lysosomes. Reversal of the lysosomal cholesterol lipidosis was associated with a rapid burst of cholesteryl ester synthesis and a normalization of the cellular levels of free and esterified cholesterol. In contrast to normal cells, progesterone removal from Niemann-Pick C fibroblasts did not reverse the lysosomal cholesterol accumulation of these mutant cultures. The metabolic precursor of progesterone, pregnenolone, also induced extensive accumulation of cholesterol in lysosomes. Other steroids induced less vacuolar cholesterol accumulation in the following decreasing order: corticosterone and testosterone, promegestone, RU 486. The relative inhibition of cellular cholesterol esterification by the steroids paralleled their respective abilities to sequester cholesterol in lysosomes rather than their inhibition of acyl-CoA:cholesterol acyltransferase activity in cell-free extracts. The progesterone-related inhibition and restoration of lysosomal cholesterol trafficking is a useful experimental means of studying intracellular cholesterol transport. A particularly important feature of its utility is the facile reversibility of the steroid-induced block. The lysosomal cholesterol lipidosis established with a hydrophobic amine, U18666A, was not as readily reversed.
Asunto(s)
Colesterol/metabolismo , Lisosomas/efectos de los fármacos , Enfermedades de Niemann-Pick/metabolismo , Progesterona/farmacología , Androstenos/farmacología , Anticolesterolemiantes/farmacología , Células Cultivadas , Ésteres del Colesterol/metabolismo , Corticosterona/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Lipoproteínas LDL/metabolismo , Lisosomas/metabolismo , Microscopía Fluorescente , Mifepristona/farmacología , Ácido Oléico , Ácidos Oléicos/metabolismo , Valores de Referencia , Testosterona/farmacologíaRESUMEN
A genomic clone of glucocerebrosidase (D-glucosyl-N-acyl-sphingosine glucohydrolase; E.C. 3.2.1.45) purified from a genomic library derived from a Balb/c mouse was analyzed by restriction mapping and nucleotide sequencing of its promoter and protein coding regions. Promoter activity was functionally assessed by ligation of a 2 kb glucocerebrosidase fragment to the protein coding segment of a bacterial neomycin resistance gene. Smaller segments of the 5' flanking sequence were then analyzed for their ability to initiate transcription of the chloramphenicol acetyltransferase reporter gene. A 319 bp Eco RI-Bgl II fragment (containing 259 bp upstream of the cDNA 5' limit) ligated to the chloramphenicol acetyltransferase open reading frame produced considerable activity.
Asunto(s)
Genes , Glucosilceramidasa/genética , Regiones Promotoras Genéticas , Células 3T3 , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , Exones , Biblioteca Genómica , Glucosilceramidasa/metabolismo , Humanos , Kanamicina Quinasa , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Proteínas Recombinantes de Fusión , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
To study structure-function relationships and molecular evolution, we determined the nucleotide sequence and chromosomal location of the gene encoding murine glucocerebrosidase (glucosylceramidase; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45). In the protein coding region of the murine cDNA, the nucleotide sequence and the corresponding deduced amino acid sequences were 82% and 86% identical to the respective human sequences. All five amino acids presently known to be essential for normal enzymatic activity were conserved between mouse and man. The murine enzyme had a single deletion relative to the human enzyme at amino acid number 273. One ATG translation initiation signal was present in the mouse sequence in contrast to the human sequence, where two start codons have been reported. Nucleotide sequencing of a clone derived from murine genomic DNA revealed that the murine signal for translation initiation was located in exon 2. The locations of all 10 introns were conserved among mouse and man. We mapped the genetic locus for glucocerebrosidase to mouse chromosome 3, at a position 7.6 +/- 3.2 centimorgans from the locus for the beta subunit of nerve growth factor. Comparison of linkage relationships in the human and murine genome indicates that these closely linked mouse genes are also syntenic on human chromosome 1 but in positions that span the centromere.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 1 , Genes , Glucosidasas/genética , Glucosilceramidasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Ligamiento Genético , Humanos , Ratones , Datos de Secuencia Molecular , Mapeo Restrictivo , Especificidad de la Especie , Bazo/enzimologíaRESUMEN
Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.
Asunto(s)
Endorribonucleasas/genética , Virus de la Leucemia Murina de Moloney/genética , ADN Polimerasa Dirigida por ARN/genética , Replicación Viral , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Endorribonucleasas/metabolismo , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Ribonucleasa H , Relación Estructura-ActividadRESUMEN
In order to develop a sensitive method that can reliably assess the amount of previous genotoxic exposure, we examined whether relative loss of the supercoiled form of mitochondrial DNA in tissue culture cells could retrospectively reflect exposure to x-irradiation of 375,750, and 1500 roentgens. Five routines of a graphic analysis program named IMAGE developed for the Apple Macintosh II computer were used to densitometrically quantitate the relative amounts of supercoiled and nicked-circular forms of mitochondrial DNA blotted onto nitrocellulose. After x-irradiation, there were dose-dependent losses of supercoiled relative to nicked-circular DNA forms. Further developments in the methodology to evaluate changes in form of mitochondrial DNA should increase the sensitivity and reliability of the dosimetry. The IMAGE program is useful for a wide variety of studies in molecular biology, and is available at no charge.
Asunto(s)
ADN Mitocondrial/química , Procesamiento de Imagen Asistido por Computador , Animales , Southern Blotting , Células Cultivadas , ADN Circular/química , ADN Circular/efectos de la radiación , ADN Mitocondrial/efectos de la radiación , ADN Superhelicoidal/química , ADN Superhelicoidal/efectos de la radiación , Ratones , Ratones Endogámicos , Conformación de Ácido Nucleico , Sensibilidad y Especificidad , Rayos XAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Enfermedades del Sistema Nervioso Central/complicaciones , Barrera Hematoencefálica , Enfermedades del Sistema Nervioso Central/terapia , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Humanos , Inmunoterapia , Biología Molecular/métodos , Retroviridae/genética , Timidina/análogos & derivados , Timidina/uso terapéutico , Zalcitabina , ZidovudinaRESUMEN
We analyzed wild mouse DNAs for the number and type of proviral genes related to the env sequences of various murine leukemia viruses (MuLVs). Only Mus species closely related to laboratory mice carried these retroviral sequences, and the different subclasses of viral env genes tended to be restricted to specific taxonomic groups. Only Mus musculus molossinus carried proviral genes which cross-reacted with the inbred mouse ecotropic MuLV env gene. The ecotropic viral env sequence associated with the Fv-4 resistance gene was found in the Asian mice M. musculus molossinus and Mus musculus castaneus and in California mice from Lake Casitas (LC). Both M. musculus castaneus and LC mice carried many additional Fv-4 env-related proviruses, two of which are common to both mouse populations, which suggests that these mice share a recent common ancestry. Xenotropic and mink cell focus-forming (MCF) virus env sequences were more widely dispersed in wild mice than the ecotropic viral env genes, which suggests that nonecotropic MuLVs were integrated into the Mus germ line at an earlier date. Xenotropic MuLVs represented the major component of MuLV env-reactive genes in Asian and eastern European mice classified as M. musculus molossinus, M. musculus castaneus, and Mus musculus musculus, whereas Mus musculus domesticus from western Europe, the Mediterranean, and North America contained almost exclusively MCF virus env copies. M. musculus musculus mice from central Europe trapped near the M. musculus domesticus/M. musculus musculus hybrid zone carried multiple copies of both types of env genes. LC mice also carried both xenotropic and MCF viral env genes, which is consistent with the above conclusion that they represent natural hybrids of M. musculus domesticus and M. musculus castaneus.
Asunto(s)
ADN Viral/análisis , Genes Virales , Virus de la Leucemia Murina/genética , Virus Inductores de Focos en Células del Visón/genética , Muridae/genética , Animales , Enzimas de Restricción del ADN , Ratones/clasificación , Ratones/genética , Ratones/microbiología , Muridae/clasificación , Muridae/microbiología , Hibridación de Ácido NucleicoRESUMEN
A murine sarcoma virus (MSV) was recovered from an (NFS X NS.C58v-1) F1 mouse which developed splenic sarcoma and erythroleukemia 6 months after inoculation with a mink cell focus-inducing murine leukemia virus (MuLV) isolated from an NFS mouse infected with a wild mouse ecotropic MuLV. The MSV, designated NS.C58 MSV-1, induced foci of transformation in mouse and rat fibroblasts, and inoculation of mice of various strains 2 weeks of age or younger resulted in erythroleukemia and sarcomatous lesions in spleen, lymph node, and brain. The MSV provirus was molecularly cloned from a genomic library prepared from transformed non-producer rat cells. The 8.8-kilobase proviral DNA contained a 1.0-kilobase p21 ras coding segment which replaced most of the gp70-encoding portion of an MuLV, most likely the endogenous C58v-1 ecotropic virus. The ras oncogene is closely related to v-Ha-ras by hybridization, expression of p21 protein, and nucleotide sequence. It is nearly identical in sequence to v-bas, the only previously described transduced, activated mouse c-ras. At position 12 in the p21 coding region, arginine is substituted for the naturally occurring glycine present in c-ras. A second MSV isolate is described which is similar to NS.C58 MSV-1 except for a 100- to 200-base-pair deletion in the noncoding region of the ras-containing insert.
Asunto(s)
Genes Virales , Virus Helper/aislamiento & purificación , Hemangiosarcoma/microbiología , Virus de la Leucemia Murina/aislamiento & purificación , Oncogenes , Virus del Sarcoma Murino/aislamiento & purificación , Neoplasias del Bazo/microbiología , Animales , Neoplasias Encefálicas/microbiología , Transformación Celular Viral , Virus Helper/genética , Virus de la Leucemia Murina/genética , Leucemia Eritroblástica Aguda/microbiología , Linfoma no Hodgkin/microbiología , Ratones , Ratones Endogámicos/microbiología , Virus Inductores de Focos en Células del Visón/aislamiento & purificación , Proteína Oncogénica p21(ras) , Proteínas Oncogénicas Virales/genética , Virus del Sarcoma Murino/genética , Homología de Secuencia de Ácido Nucleico , Transducción GenéticaRESUMEN
We derived an amphotropic murine leukemia virus (MuLV) type-specific probe for use in Southern blot hybridizations with cloned and genomic DNAs. A 133-base-pair RsaI-RsaI fragment from the 5' env region of the amphotropic viral isolate 4070A was subcloned into M13mp18 and radiolabeled in vitro. The probe detected the proviral DNAs in mink cells infected with seven different amphotropic MuLV isolates. The probe did not cross hybridize with the DNAs of molecular clones of ecotropic, mink cell focus-forming, or xenotropic MuLVs; nor did it anneal to the proviral DNAs of four xenotropic or six mink cell focus-forming viral isolates grown in mink cells. DNAs of 12 inbred laboratory mouse strains and more than 15 different wild mouse species and subspecies were examined for the presence of endogenous amphotropic env-related fragments. Amphotropic env-related sequences were found only in the DNAs of wild mice trapped in southern California in an area previously shown to harbor mice producing infectious amphotropic virus. Restriction enzyme analyses of DNAs from these mice showed that amphotropic sequences were not present as germ line copies but were the result of congenital or horizontal infection or both in this population. The DNAs of 11 various mammalian and avian species, including both natural predators of mice and squabs from the farms with virus-positive mice, lacked amphotropic envelope-related sequences.
Asunto(s)
Animales Salvajes/microbiología , Virus de la Leucemia Murina/aislamiento & purificación , Ratones/microbiología , Infecciones por Retroviridae/veterinaria , Proteínas de los Retroviridae/genética , Enfermedades de los Roedores/transmisión , Proteínas del Envoltorio Viral/genética , Animales , Animales Salvajes/genética , Secuencia de Bases , Gatos/genética , Gatos/microbiología , Columbidae/genética , Columbidae/microbiología , ADN Recombinante , ADN Viral/análisis , ADN Viral/genética , Virus de la Leucemia Murina/genética , Ratones/genética , Ratones Endogámicos/genética , Ratones Endogámicos/microbiología , Virus Inductores de Focos en Células del Visón/genética , Virus Inductores de Focos en Células del Visón/aislamiento & purificación , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/transmisión , Enfermedades de los Roedores/genética , Especificidad de la EspecieRESUMEN
We used hybridization probes that react specifically with xenotropic and mink cell focus-forming virus envelope sequences to characterize the nonecotropic proviruses of BALB/c and NFS/N mice. Analysis of somatic cell hybrids with different BALB/c chromosomes showed that the 9 xenotropic and more than 20 MCF virus-related proviral sequences in this mouse were present on more than nine BALB/c chromosomes. Multiple copies were found on chromosomes 1, 4, 7, 12, and probably 11, and the copies found on a single chromosome were not identical by restriction enzyme mapping. We also identified and characterized the proviral sequences that give rise to infectious xenotropic virus in both BALB/c and NFS/N mice. BALB/c contains the major locus for induction of infectious virus in inbred mice, Bxv-1, which is on chromosome 1. We showed that this locus contains a single xenotropic provirus on an 18-kilobase HindIII fragment. Restriction enzyme analysis of a hybrid cell DNA that contains only the Bxv-1 xenotropic provirus showed that the Bxv-1 provirus contains restriction enzyme sites characteristic of the infectious virus induced from BALB/c fibroblasts. The Bxv-1 provirus and its flanking sequences also contain the same restriction sites as the provirus thought to contribute U3 long terminal repeat sequences to leukemogenic (class I) AKR MCF viruses. Analysis of cell hybrids made with the nonvirus-inducible strain NFS/N showed that the single xenotropic virus env gene of NFS mice, here termed Nfxv-1, is not on chromosome 1. Unlike that of Bxv-1, the restriction map of Nfxv-1 does not resemble that of any known infectious xenotropic virus including xenotropic viruses isolated from NFS mice. These data suggest that Bxv-1, but not Nfxv-1, is a full-length xenotropic provirus that can be transcribed directly to produce infectious virus.
Asunto(s)
Virus de la Leucemia Murina/crecimiento & desarrollo , Ratones Endogámicos/microbiología , Animales , Mapeo Cromosómico , Enzimas de Restricción del ADN , ADN Viral/genética , Regulación de la Expresión Génica , Genes Virales , Virus de la Leucemia Murina/genética , Ratones , Ratones Endogámicos/genética , Proteínas del Envoltorio Viral/genética , Replicación ViralRESUMEN
We have derived hybridization probes from analogous 100-base-pair segments located within the N-terminal region of gp70 coding sequences which differentiate xenotropic from mink cell focus-forming (MCF)-related murine leukemia virus (MuLV) DNAs. The MCF probe annealed to the integrated proviruses of all six MCF MuLV isolates tested; the xenotropic probe hybridized to the DNAs of all four xenotropic proviral isolates examined. No cross-hybridization was observed, and neither probe reacted with the env segments of amphotropic or ecotropic MuLV DNAs. Southern blot analysis of HindIII- or EcoRI-digested genomic DNAs from a variety of inbred laboratory mice demonstrated the presence of more MCF- than xenotropic MuLV-related segments in every strain tested.
Asunto(s)
ADN Viral/análisis , Genes Virales , Virus de la Leucemia Murina/genética , Ratones Endogámicos/microbiología , Virus Inductores de Focos en Células del Visón/genética , Recombinación Genética , Animales , Línea Celular , Clonación Molecular , ADN/análisis , ADN/genética , Ratones , Ratones Endogámicos/genética , Visón , Hibridación de Ácido Nucleico , Proteínas del Envoltorio Viral/genéticaRESUMEN
The nucleotide sequence of a full-length (8.8-kilobase) endogenous C-type human retroviral DNA (clone 4-1) is presented and compared with that of Moloney murine leukemia virus (MoMuLV) DNA. Colinearity of deduced amino acids of clone 4-1 with MoMuLV in the gag and pol regions was clearly evident, and overall amino acid homology in these regions was about 40%. Identification of the putative N terminus of gag and p30, the gag-pol junction, and the C terminus of pol could be established on the basis of sequence homology with MoMuLV. Unique characteristics of the endogenous human retroviral DNA included a tRNA Glu primer binding site separated from the 5' long terminal repeat by a pentanucleotide and a putative env sequence which does not appear to overlap the C terminus of pol and has virtually no homology with the env gene of known infectious retroviruses. Clone 4-1 represents a defective prototype of a human C-type retrovirus which integrated into the germ line some time in the distant past.
Asunto(s)
ADN Viral/análisis , Retroviridae/genética , Secuencia de Bases , Codón/análisis , Humanos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
An infectious NZB xenotropic murine leukemia virus (MuLV) provirus (NZB was molecularly cloned from the Hirt supernatant of NZB-IU-6-infected mink cells, and the nucleotide sequence of its env gene and long terminal repeat (LTR) was determined. The partial nucleotide sequence previously reported for the env gene of NFS-Th-1 xenotropic proviral DNA (Repaske, et al., J. Virol. 46:204-211, 1983) is identical to that of the infectious NZB xenotropic MuLV DNA reported here. Alignment of nucleotide or deduced amino acid sequences, or both, of xenotropic, mink cell focus-forming, and ecotropic MuLV proviral DNAs in the env region identified sequence differences among the three host range classes of C-type MuLVs. Major differences were confined to the 5' half of env; a high degree of homology was found among the three classes of MuLVs in the 3' half of env. Alignment of the nucleotide sequence of the LTR of NZB xenotropic MuLV with those of the LTRs of NFS-Th-1 xenotropic, mink cell focus-forming, and ecotropic MuLVs revealed extensive homology between the LTRs of xenotropic and MCF247 MuLVs. An inserted 6-base-pair repeat 5' to the TATA box was a unique feature of both NZB and NFS-Th-1 xenotropic LTRs.