RESUMEN
The effect of magnesium and phosphate limitation on the molecular weight distribution of poly-beta-hydroxybutyrate (PHB) in Alcaligens europhus in cotinuons culture has been stuied. Conditions of nitrogen limitation both with glucose excess (above ca. 20 g/L) and without excess were investigated Under N-limitation and glucose excess, M(w) decreases when the magnesium content is decreased below 50% (19.7 mg/L) of the basal medium content; this also results in a broadenng of molecular weight distribution (M(w)/M(n)) from 2 to 5 and a decrease in M(w) fron 2 x 10(6) to 0.9 x 10(6). Below 20% of the basal content of magnesium (7.9 mg/L) these two trends were reversed. This behaviour was not observed in the absence of glucose excess, phshate had virtually no effect on PHB M(w) or its distribution, whereas wih no (or little) glucose excess M(w) of the PHB decreased with phosphate concentrations below 50% of the basal level (0.705 g/L). Hence, in continuous or fed-batch cultures, in addition to nitrogen limitation to alklow for PHB accumulation, it is necesary to control both the addition of glucose (no excess) and also to maintain magnesium limitation (ca. 25% of basal medium level, 9.9 mg/L) and phosphate above 50% of he basal level (0.705 g/L). Thus, when broadening of molecular weight destribution (increase in M(w)/M(n)) is observed at the end of fed-batch culture it is probably caused by phosphate limitation and/or glucose excess. (c) 1995 John Wiley & Sons, Inc.
RESUMEN
Diagnosis of paratuberculosis using the IDEXX DNA probe test and 3 methods for cultivation of Mycobacterium paratuberculosis from fecal specimens were compared. Twenty-one of 170 fecal specimens were DNA probe test positive, whereas 35 specimens were positive by 1 or more of the cultivation methods evaluated. Four specimens were DNA probe test positive but were negative by fecal culture. The probe test detected M. paratuberculosis DNA in 62.9% of the specimens positive by a sedimentation culture method, in 56.6% of those positive by a centrifugation culture method, and in 65.4% of the specimens positive by the Cornell culture method. Specificity of the DNA probe test was approximately 97% relative to all culture methods. Generally, the probe test detected M. paratuberculosis DNA in fecal specimens from animals shedding at least 10(4) M. paratuberculosis colony forming units per gram of feces. Although the probe test did not detect all of the cattle shedding M. paratuberculosis, it was possible to identify cattle shedding the greatest number of organisms in 3 days compared with a minimum of 6 weeks required for positive culture results. The centrifugation method resulted in the most isolations of M. paratuberculosis after 12 weeks of incubation. However, contamination also was greatest when the centrifugation method was used. Contamination was best controlled using the Cornell method. The sedimentation method was the least time consuming and yielded results similar to those of the other 2 methods.
Asunto(s)
Enfermedades de los Bovinos/diagnóstico , ADN Bacteriano/análisis , Heces/microbiología , Mycobacterium tuberculosis/aislamiento & purificación , Paratuberculosis/diagnóstico , Animales , Bovinos , Enfermedades de los Bovinos/microbiología , Centrifugación , Sondas de ADN , Mycobacterium tuberculosis/genética , Paratuberculosis/microbiología , Reacción en Cadena de la Polimerasa , Juego de Reactivos para Diagnóstico/veterinaria , Sensibilidad y EspecificidadRESUMEN
DNA probes that hybridize to a mycobacterial insertion sequence, IS900, present in multiple copies in the genome of Mycobacterium paratuberculosis were found to be highly specific for M. paratuberculosis. DNA sequences derived from IS900 were used to prepare DNA primers for detection and identification of M. paratuberculosis by the polymerase chain reaction. Highly specific direct detection of M. paratuberculosis DNA in feces from cattle with Johne's disease was obtained. The polymerase chain reaction test had a sensitivity equal to or greater than that obtained by standard culture techniques and was much more rapid, taking only hours compared with 6 to 12 weeks for culture.
Asunto(s)
Sondas de ADN , Amplificación de Genes , Mycobacterium/genética , Paratuberculosis/diagnóstico , Reacción en Cadena de la Polimerasa , Animales , Secuencia de Bases , Bovinos , ADN Bacteriano/genética , Datos de Secuencia Molecular , Mycobacterium/aislamiento & purificación , Paratuberculosis/microbiologíaRESUMEN
The feline immunodeficiency virus (FIV) is a recently identified feline lentivirus that has been found at significant levels in domestic cat populations worldwide. A microdilution plate format, monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of the FIV group-associated antigen (gag) designated p24. Assays of serially diluted samples containing disrupted virus showed that the assay had a sensitivity limit of approximately 0.2 ng/ml for FIV p24. The assay was approximately eightfold more sensitive than the assay for viral reverse transcriptase activity when it was tested with diluted tissue culture samples. A qualitative confirmation assay by standard antibody inhibition techniques was coupled to the screening test methodology. The test was used to detect and confirm the presence of virus in cultured feline lymphocytes from infected animals.
Asunto(s)
Antígenos Virales/análisis , Ensayo de Inmunoadsorción Enzimática , Retroviridae/inmunología , Proteínas del Núcleo Viral/inmunología , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Gatos , Estudios de Evaluación como Asunto , Infecciones por Retroviridae/diagnósticoRESUMEN
Feline immunodeficiency virus (FIV) structural proteins were identified using sera obtained from experimentally inoculated cats. Proteins analysed by both radioimmunoprecipitation and Western blotting were specific for FIV infection and failed to cross-react with either antisera to feline leukaemia virus of feline syncytium-forming virus. Western blot analysis of purified virus revealed immunoreactive proteins with apparent Mr of 65K, 50K, 40K, 32K, 24K, 15K and 10K. The major core structural proteins of the virus were isolated by reverse phase HPLC and the aminoterminal sequences of p10 and p24 were determined. Monoclonal antibodies specific for p24 suggested the presence of a precursor protein that could be detected in 35[S]methionine/cysteine-labelled, virus-infected cell extracts. This putative precursor protein possessed an apparent Mr of 50K (Pr50gag). Further analysis revealed the presence of two additional proteins of 130K and 40K. Experiments utilizing tunicamycin, endoglycosidase H and glycopeptidase F revealed that p130 and p40 exhibited properties characteristic of glycoproteins. Our studies also indicated that FIV is immunologically related to other lentiviruses.
Asunto(s)
Proteínas Estructurales Virales/análisis , Virus Visna-Maedi/análisis , Secuencia de Aminoácidos , Animales , Western Blotting , Gatos , Línea Celular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Sueros Inmunes , Metionina/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Radioisótopos de Azufre , Proteínas Estructurales Virales/biosíntesis , Proteínas Estructurales Virales/inmunología , Virus Visna-Maedi/genéticaRESUMEN
The feline T-cell lymphotropic lentivirus (feline immunodeficiency virus) is a recently described feline-specific retrovirus that can produce chronic immunodeficiency-like disorders in cats. A microdilution plate format enzyme-linked immunosorbent assay has been developed to detect the presence of antibody to the virus in feline serum or plasma. Temporal studies performed with experimentally infected animals show that seroconversion can be demonstrated 3 to 4 weeks after exposure to the virus. Results of a serosurvey (n = 1,556 samples) indicate that infection is fairly common in both clinic (5.2%) and sick cat (15.2%) populations. Western blot (immunoblot) and sodium dodecyl sulfate radioimmunoprecipitation assays were developed to confirm microdilution plate test results and to identify peptides specific for the feline immunodeficiency virus. All microdilution plate test positive results and selected negative results were confirmed by one or both of these procedures. These data demonstrate that this microassay plate enzyme-linked immunosorbent assay is a very sensitive and specific test for detection of antibody to the feline immunodeficiency virus.
Asunto(s)
Anticuerpos Antivirales/análisis , Enfermedades de los Gatos/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Infecciones por Retroviridae/veterinaria , Retroviridae/inmunología , Animales , Western Blotting , Gatos , Electroforesis en Gel de Poliacrilamida , Pruebas de Precipitina , Valor Predictivo de las Pruebas , Infecciones por Retroviridae/diagnósticoRESUMEN
Feline immunodeficiency virus (FIV; formerly, feline T-lymphotropic lentivirus) is a typical lentivirus resembling human and simian immunodeficiency viruses in morphologic features, protein structure, and reverse transcriptase enzyme. It is antigenically dissimilar, however. The virus is tropic for primary and permanent feline T-lymphoblastoid cells and Crandell feline kidney cells. The virus did not grow in other permanent feline non-lymphoblastoid cells that were tested, or in lymphoid and non-lymphoid cells from man, dogs, mice, and sheep. During short-term inoculation studies in cats, the feline immunodeficiency-like syndrome found in nature was not experimentally induced, but a distinct primary phase of infection was observed. Fever and neutropenia were observed 4 to 5 weeks after inoculation; fever lasted several days, and neutropenia persisted from 1 to 9 weeks. Generalized lymphadenopathy that persisted for 2 to 9 months appeared at the same time. Antibodies to FIV appeared 2 weeks after inoculation and then plateaued. Virus was reisolated from the blood of all infected cats within 4 to 5 weeks after inoculation and persisted indefinitely in the face of humoral antibody response. Virus was recovered from blood, plasma, CSF and saliva, but not from colostrum or milk. Contact transmission was achieved slowly in one colony of naturally infected cats, but not between experimentally infected and susceptible specific-pathogen-free cats kept together for periods as long as 4 to 14 months. The infection was transmitted readily, however, by parenteral inoculation with blood, plasma, or infective cell culture fluids. In utero and lactogenic transmission were not observed in kittens born to naturally or experimentally infected queens. Lymphadenopathy observed during the initial stage of FIV infection was ascribed to lymphoid hyperplasia and follicular dysplasia. A myeloproliferative disorder was observed in 1 cat with experimentally induced infection.
Asunto(s)
Enfermedades de los Gatos/etiología , Infecciones por Retroviridae/veterinaria , Retroviridae/aislamiento & purificación , Animales , Enfermedades de los Gatos/transmisión , Gatos , Femenino , Enfermedades Linfáticas/etiología , Enfermedades Linfáticas/veterinaria , Masculino , Microscopía Electrónica , ADN Polimerasa Dirigida por ARN/metabolismo , Retroviridae/fisiología , Retroviridae/ultraestructura , Infecciones por Retroviridae/etiología , Infecciones por Retroviridae/transmisiónRESUMEN
In an effort to evaluate data on genomic relatedness among the various human immunodeficiency viruses (HIVs), we have molecularly cloned a virus isolate designated HIV (CDC-451). Preliminary characterization of the HIV (CDC-451) clone indicated that the restriction enzyme map was distinct from those of other known HIV isolates. Analysis of the primary nucleotide sequence of the regions encoding the structural proteins and comparison with sequences known for other HIV isolates indicated substantial differences for HIV (CDC-451). The sequences encoding the group-specific antigen gene, although they showed some variation, were conserved to a greater extent than were those encoding envelope proteins. In the envelope gene sequences, most of the changes (up to 24.5% divergence) were located in the amino-terminal region encoding a glycoprotein with a Mr of 120,000. The carboxyl-terminal region, encoding a protein of Mr 41,000, was more highly conserved. The variation in the sequences encoding envelope proteins may have important implications for the antigenic properties and/or pathogenicity of the disease and for its detection and ultimate eradication.
Asunto(s)
Clonación Molecular , Genes Virales , VIH/genética , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Productos del Gen gag , Genes Reguladores , Glicoproteínas/análisis , Humanos , Masculino , Proteínas de los Retroviridae/genética , Proteínas del Envoltorio Viral/análisis , Proteínas del Envoltorio Viral/genéticaRESUMEN
The major internal structural protein of human T-cell lymphotropic virus type III (HTLV-III), a virus etiologically implicated in acquired immunodeficiency syndrome (AIDS), was purified to homogeneity. This 24,000-molecular-weight protein (p24) was shown to lack immunologic cross-reacting antigenic determinants shared by other known retroviruses, including HTLV-I and HTLV-II, with the exception of equine infectious anemia virus (EIAV). A broadly reactive competition immunoassay was developed in which antiserum to EIAV was used to precipitate 125I-labeled HTLV-III p24. Although the major structural proteins of HTLV-III and EIAV competed in this assay, other type B, C, and D retroviral proteins lacked detectable reactivity. Thus, HTLV-III is more related to EIAV than to any other retroviruses. That the HTLV-III isolate is very distinct from HTLV-I and HTLV-II was further confirmed by the amino acid compositions of the major internal antigens of all three isolates. Moreover, comparison of the amino-terminal amino acid sequence of HTLV-III p24 with analogous sequences for HTLV-I and HTLV-II p24 showed that these proteins do not share significant sequence homology. In an attempt to evaluate immune response in individuals exposed to HTLV-III, sera from AIDS and lymphadenopathy syndrome patients as well as from clinically normal blood donor controls were tested for antibodies to HTLV-III p24. The results showed that sera from 93% of lymphadenopathy syndrome patients and 73% of AIDS patients exhibited high-titered antibodies to HTLV-III p24. In contrast, none of the normal control sera showed detectable reactivity to HTLV-III p24.
Asunto(s)
Antígenos Virales/inmunología , Deltaretrovirus/inmunología , Proteínas Virales/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Secuencia de Aminoácidos , Aminoácidos/análisis , Anticuerpos Antivirales/análisis , Donantes de Sangre , Reacciones Cruzadas , Deltaretrovirus/análisis , Epítopos/inmunología , Humanos , Virus de la Anemia Infecciosa Equina/inmunología , Enfermedades Linfáticas/inmunología , Peso Molecular , Retroviridae/inmunología , Síndrome , Proteínas Virales/análisis , Proteínas Virales/aislamiento & purificación , Proteínas Estructurales ViralesRESUMEN
We determined the nucleotide sequence of the v-H-ras-related oncogene of BALB/c murine sarcoma virus. This oncogene contains an open reading frame of 189 amino acids that initiates and terminates entirely within the mouse cell-derived ras sequence. The protein encoded by this open reading frame matches the sequence predicted for the T24 human bladder carcinoma oncogene product, p21, in all but two positions. The presence of a lysine residue in position 12 of BALB/c murine sarcoma virus p21 likely accounts for its oncogenic properties.
Asunto(s)
Oncogenes , Virus del Sarcoma Murino/genética , Animales , Secuencia de Bases , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
The closed circular form of the endogenous squirrel monkey type D retrovirus (SMRV) was molecularly cloned in a bacteriophage vector. The restriction map of the biologically active clone was determined and found to be identical to that of the parental SMRV linear DNA except for the deletion of one long terminal repeat. Restriction enzyme analysis and Southern blotting indicated that the SMRV long terminal repeat was approximately 300 base pairs long. The SMRV restriction map was oriented to the viral RNA by using a gene-specific probe from baboon endogenous virus. Restriction enzyme digests of a variety of vertebrate DNAs were analyzed for DNA sequence homology with SMRV by using the cloned SMRV genome as a probe. Consistent with earlier studies, multiple copies of SMRV were detected in squirrel monkey DNA. Related fragments were also detected in the DNAs from other primate species, including humans.
Asunto(s)
Clonación Molecular , ADN Viral/análisis , Genes Virales , Primates/genética , Retroviridae/genética , Animales , Secuencia de Bases , ADN/análisis , Enzimas de Restricción del ADN , Humanos , Hibridación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , SaimiriRESUMEN
The sequence of the transforming region of simian sarcoma virus (SSV) has been determined by using molecularly cloned viral DNA. This region encompassed the 1.0-kilobase pair woolly monkey cell-derived insertion sequence, v-sis, and flanking simian sarcoma-associated viral (SSAV) sequences. A 675-nucleotide-long open reading frame commenced 19 nucleotides within the SSAV sequences to the left of the v-sis helper viral junction and terminated within v-sis itself. Possible promoter and acceptor splice signals were detected in helper viral sequences upstream from this open reading frame, and potential polyadenylylation sites were identified downstream both within v-sis and in helper viral sequences beyond v-sis. The recombinational event that led to the generation of SSV occurred in the middle of two functional codons, indicating that SSAV provided the regulatory elements for transcription as well as the initiation codon for translation of SSV cell-derived transforming sequences.
Asunto(s)
Transformación Celular Viral , Genes Virales , Retroviridae/genética , Virus del Sarcoma del Mono Lanudo/genética , ADN Viral/genética , Regulación de la Expresión Génica , Genes , Genes Reguladores , Virus Helper/genética , ARN Viral , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
Polyadenylated RNAs of certain human tumour cell lines are shown to contain transcripts related to the cell-derived transforming onc genes of molecularly cloned primate, murine or avian transforming retrovirus genomes. Thus, analogues of retroviral transforming genes are both present and frequently expressed in human neoplastic cells.
Asunto(s)
Genes Virales , Neoplasias/genética , Poli A/genética , ARN/genética , Retroviridae/genética , Transcripción Genética , Secuencia de Bases , Línea Celular , Transformación Celular Viral , Femenino , Glioblastoma , Humanos , Melanoma , Biosíntesis de Proteínas , ARN Mensajero , Sarcoma , TeratomaRESUMEN
BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.
Asunto(s)
Clonación Molecular , Genes Virales , Recombinación Genética , Virus del Sarcoma Murino/genética , Animales , Secuencia de Bases , Línea Celular , Transformación Celular Neoplásica , Transformación Celular Viral , Enzimas de Restricción del ADN , ADN Recombinante , Virus Helper/genética , Ratones , Ratones Endogámicos BALB C , Hibridación de Ácido Nucleico , TransfecciónRESUMEN
The nature of the cell-derived (bas) sequences of BALB-MuSV, a spontaneous mouse sarcoma virus isolate, was determined. Molecularly cloned bas sequences demonstrated no detectable homology with the onc genes of other mouse transforming viruses, but exhibited a high degree of sequence homology with the ras gene of the rat-derived Harvey murine sarcoma virus (Ha-MuSV) genome. The Ha-MuSV cell-derived sequence (ras) shared a colinear 750 bp region of homology with bas. Moreover, BALB-MuSV transformation was associated with the expression of high levels of a 21,000 dalton protein, immunologically related to the ras gene products, p21. Thus bas and ras represent retroviral transforming gene homologs that were independently transduced by mouse type C viruses from the genomes of different species.
Asunto(s)
Transformación Celular Viral , Genes Virales , Virus del Sarcoma Murino/genética , Virus de la Leucemia Murina de Abelson/genética , Animales , Secuencia de Bases , Ratones , Ratones Endogámicos BALB C/microbiología , Peso Molecular , Virus de la Leucemia Murina de Moloney/genética , Hibridación de Ácido Nucleico , Retroviridae/genética , Transducción Genética , Proteínas Virales/genéticaAsunto(s)
Transformación Celular Viral , Genes Virales , Virus de la Leucemia Murina de Moloney/genética , Virus del Sarcoma Murino/genética , Animales , Células Cultivadas , ADN Viral/genética , Virus Defectuosos/genética , Virus Helper/genética , Ratones , Transformación Genética , Replicación ViralRESUMEN
The unintegrated circular DNA form of Moloney murine sarcoma virus (MSV) has been cloned in bacteriophage lambda. Discrete deletions in the viral genome were shown to occur during propagation of recombinant phage in Escherichia coli. Heteroduplex and restriction enzyme analyses indicated the deletion of tandemly repeated sequences within certain of the cloned MSV DNA inserts. Cloned MSV DNA was used to prepare a probe composed of its acquired cellular (src) sequences, shown previously to be necessary for MSV transformation. Analysis of EcoRI digests of normal mouse cellular DNA revealed the presence of a single 14-kilobase-pair fragment containing these sequences which lacked contiguity with endogenous type C helper viral information of the same cells. Thus, the sarcoma virus-specific sequences of MSV are represented within the normal mouse genome in a manner analogous to that of a cellular gene.
Asunto(s)
Clonación Molecular , ADN Viral/genética , ADN/genética , Genes Virales , Ratones/genética , Virus de la Leucemia Murina de Moloney/genética , Animales , Deleción Cromosómica , ADN Recombinante , Escherichia coli/genética , Amplificación de Genes , Virus Helper/genética , Recombinación Genética , Sarcoma Experimental/genéticaRESUMEN
A retrovirus previously isolated from a tumored Russell's viper is shown by molecular hybridization to be an endogenous virus of this reptilian species. Radio-immunologic techniques revealed that the viper retrovirus is immunologically and, hence, evolutionarily related to endogenous type D retorviruses of Old World primates. These findings extend the number of vertebrate classes possessing endogenous retroviruses and suggest that type D retroviruses may even be more widely distributed in nature than type C retroviruses.
Asunto(s)
Evolución Biológica , Genes Virales , Primates/microbiología , Retroviridae/genética , Serpientes/microbiología , Animales , Antígenos Virales/análisis , Secuencia de Bases , Hibridación de Ácido Nucleico , Retroviridae/inmunología , Serpientes/genética , Proteínas Virales/inmunologíaRESUMEN
(1) Two viper cells lines were investigated, one which harbors IMV in the mitochondria (VSW cells) and one without detectable IMV (VH3 cells). (2) The size of closed circular mtDNA molecules from both VSW and VH3 cells was found to be significantly greater (5.4 to 5.6 micron) than the contour lengths of typical mammalian cells (4.8 to 5.2 micron). (3) A small percentage of mini-circles ranging in size from 0.1 to 0.6 micron was observed to band with closed circular mtDNA from both cell lines. Minicircles were especially abundant in VH3 cells. (4) MtDNA from VSW cells contained 34.1% dimers plus oligomers (10.2% oligomers), whereas VH3 cells had only 14.8% dimeric and oligomeric forms (5.4% oligomers). (5) Treatment of VSW cells with 1 microng/ml ethidium bromide for 48 hours resulted in an increased incidence of IMV (IMV in 15% of mitochondrial sections) as compared with untreated VSW cells (IMV in 3% of mitochondrial sections).