RESUMEN

The sequence encoding part of the 100 kDa refractile body protein (Ea1A) from Eimeria acervulina was cloned into the US10 locus of herpesvirus of turkeys (HVT) downstream of LTR promoter. Expression of the fusion protein was shown in vitro. Recombinant HVT showed a delayed and slightly reduced level of viremia compared to the parent strain in SPF chickens as well as in broilers. Effect on the performance of broilers vaccinated with recombinant or parent HVT was measured by challenge at day 24 with a high dose of E. acervulina and E. maxima oocysts. A significant improvement in weight of animals vaccinated with the recombinant HVT was detected at the end of the challenge period.

Asunto(s)

Antígenos de Protozoos/inmunología , Coccidiosis/veterinaria , Eimeria/inmunología , Gammaherpesvirinae/genética , Vacunas Antiprotozoos/inmunología , Vacunas Sintéticas/inmunología , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Southern Blotting , Western Blotting , Células Cultivadas , Embrión de Pollo , Pollos , Coccidiosis/parasitología , Coccidiosis/prevención & control , Técnica del Anticuerpo Fluorescente Indirecta , Gammaherpesvirinae/metabolismo , Masculino , Enfermedades de las Aves de Corral/parasitología , Enfermedades de las Aves de Corral/prevención & control , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Organismos Libres de Patógenos Específicos , Pavos/virología , Vacunación/veterinaria , Viremia/veterinaria

RESUMEN

A recombinant feline herpesvirus type 1 (FHV-1) was constructed expressing the envelope glycoprotein gene from feline leukaemia virus (FeLV). The expression cassette containing the long terminal repeat promoter from Rous sarcoma virus was stably integrated at the locus downstream of the gC homologue in FHV-1. Oronasal vaccination with recombinant FHV-1 engendered significant protection against challenge with the homologous FelV-A/Glasgow-1 isolate. Three of four vaccinated cats did not become viraemic for FeLV and developed serum neutralizing antibodies while five of six controls became persistently infected after challenge. However, latent FeLV was detected at 12 weeks after challenge in bone marrow cultures of all animals except one. The potential of this new vector to protect against FeLV was compared with previous reports using live recombinant vaccines.

Asunto(s)

Vectores Genéticos/inmunología , Herpesviridae/genética , Virus de la Leucemia Felina/inmunología , Leucemia Felina/prevención & control , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Administración Intranasal , Animales , Gatos , Regulación Viral de la Expresión Génica/inmunología , Genes env/inmunología , Vectores Genéticos/administración & dosificación , Vectores Genéticos/metabolismo , Herpesviridae/inmunología , Virus de la Leucemia Felina/genética , Leucemia Felina/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Virales/administración & dosificación , Vacunas Virales/genética

RESUMEN

Subunit vaccines prepared against feline immunodeficiency virus (FIV) infection were evaluated in two trials. First, cats were immunized with bacterial expression products of an envelope fragment that contained the V3 neutralization domain of the FIV surface protein fused to either galactokinase (K-SU3) or glutathione-S-transferase (G-SU3). Quantitative and qualitative differences in the humoral immune response were observed with three adjuvants of which Quil A was the best in terms of total and virus neutralizing antibody. Notwithstanding the responses induced, 19 of 20 immunized cats did not resist challenge and became infected. To determine whether priming with a live viral vector would confer protection, cats were inoculated oronasally and subcutaneously with a feline herpesvirus (FHV) mutant expressing the FIV env gene; two booster immunizations followed using the K-SU3 product in either Quil A or a mineral oill Al(OH)3 adjuvant. FIV-specific antibody responses were only weak, and the vaccinates did not withstand challenge with a low dose of homologous virus.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida del Felino/prevención & control , Virus de la Inmunodeficiencia Felina/inmunología , Vacunas Virales/uso terapéutico , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Gatos , Ensayo de Inmunoadsorción Enzimática , Estudios de Evaluación como Asunto , Síndrome de Inmunodeficiencia Adquirida del Felino/sangre , Productos del Gen env/biosíntesis , Productos del Gen env/genética , Productos del Gen env/inmunología , Virus de la Inmunodeficiencia Felina/genética , Vacunas Atenuadas/uso terapéutico , Proteínas Virales de Fusión/biosíntesis , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología

RESUMEN

The major cause of upper respiratory tract disease in cats in the feline herpesvirus type 1 (FHV-1). FHV-1 replicates predominantly in the mucosal epithelium of the oral and nasal cavities, local immunity should therefore be the key target for vaccine development. Recombinant DNA technology enables accurate manipulation of the genetic content of the FHV-1 genome, hopefully resulting in a next generation of safe vaccine strains that can be used intranasally in cats. Integration of a reporter gene into the glycoprotein I (gI) homologous gene of FHV-1, resulted in strain C4-1-4-1 which displayed reduced replication not only in cell culture but also in the respiratory tract of infected cats. Oronasal application of strain C4-1-4-1 caused less severe clinical signs than local administration of the parent virus. In addition, oronasally vaccinated cats were better protected against the clinical signs of a challenge infection than cats vaccinated subcutaneously.

Asunto(s)

Alphaherpesvirinae/genética , Elementos Transponibles de ADN/inmunología , Genes Virales/inmunología , Infecciones por Herpesviridae/veterinaria , Operón Lac/inmunología , Mutagénesis Insercional , Infecciones del Sistema Respiratorio/veterinaria , Proteínas del Envoltorio Viral/genética , Vacunas Virales/administración & dosificación , Animales , Gatos , ADN Viral/inmunología , Infecciones por Herpesviridae/prevención & control , Infecciones del Sistema Respiratorio/prevención & control , Vacunas Atenuadas/administración & dosificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología

RESUMEN

Transcription mapping was performed in the short region of the feline herpesvirus type 1 (FHV-1) genome as a first approach to the functional analysis of open reading frames encoding the homologs of the herpes simplex virus type 1 (HSV-1) gD, gl, gE, US9, and probably also US8.5. All reading frames appeared to be transcribed. Transcripts were grouped into two nested RNA sets; namely, the coterminal transcripts of gD and gl and the coterminal transcripts of gE, US8.5, and US9. This situation was similar to that found in the equivalent region of HSV-1. The FHV-1 ORFs US8.5 and US9 have not been described previously. Sequence analysis and comparison of the putative polypeptide encoded by US8.5 revealed that this ORF was unique to FHV-1. However, US8.5 of FHV-1 might be functionally related to its positional homologs in HSV-1 and equine herpesvirus type 1. In all three viruses, US8.5 does not seem to be critical for virus propagation in cell culture. This was shown for FHV-1 by isolating a mutant containing an insertion in US8.5 and comparing its growth properties in cell culture to those of the parent virus G2620. With regard to US9, conscientious amino acid sequence alignment of FHV-1 US9 and homologs in related herpesviruses suggests that this particular protein could contribute to the virus infectivity in vivo. This speculation was based on the highly conserved C-terminus of US9, starting with a characteristic YYSES motif and followed by a nuclear target sequence and a transmembrane region.

Asunto(s)

Alphaherpesvirinae/genética , Genoma Viral , Mutagénesis Insercional , Sistemas de Lectura Abierta/genética , Transcripción Genética , Alphaherpesvirinae/crecimiento & desarrollo , Alphaherpesvirinae/patogenicidad , Secuencia de Aminoácidos , Animales , Gatos , Línea Celular , Clonación Molecular , Secuencia Conservada/genética , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Viral/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Simplexvirus/genética

RESUMEN

Feline herpesvirus type 1 (FHV-1) mutants were constructed, carrying a beta-galactosidase marker gene integrated into the region downstream of the gene encoding the homologue of glycoprotein C (gC) of herpes simplex virus type 1. In cell culture, no differences in replication were observed between mutants and the parent FHV-1 strain. However, in experimentally infected cats, mutants caused fewer clinical signs after oronasal administration although they replicated to the same extent as the parental strain. Sequence analysis in the region of the UL segment surrounding the insertion site revealed an open reading frame (ORF 2) encoding a putative polypeptide of 21K. RNA analysis indicated a corresponding transcript of 0.8 kb that was detected late after infection of cells in culture. This particular UL locus downstream of the gC gene has not been thoroughly investigated in any of the herpesviruses. The putative gene product showed only limited evolutionary conservation since similarity could be found only with the assumed homologue of equine herpesvirus type 1. Further characterization of this newly identified FHV-1 gene involved in virulence may provide insight into the development of disease owing to herpesvirus infection.

Asunto(s)

Genes Virales , Herpesviridae/genética , Herpesviridae/patogenicidad , Proteínas del Envoltorio Viral/biosíntesis , Algoritmos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Gatos , Línea Celular , Clonación Molecular , Elementos Transponibles de ADN , Genoma Viral , Herpesviridae/metabolismo , Herpesvirus Équido 1/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Mapeo Restrictivo , Transcripción Genética , Proteínas del Envoltorio Viral/genética , Virulencia/genética , Replicación Viral

RESUMEN

The onset of protection against Newcastle disease and the effect of maternal antibodies to Newcastle disease virus (NDV) and Marek's disease virus (MDV) on vaccine efficacy were determined following vaccination of chickens with a recombinant herpesvirus of turkeys (HVT) vaccine expressing the fusion (F) glycoprotein gene of NDV. Onset of protection following intra-abdominal administration of the recombinant HVT/F vaccine at 1 day of age and subsequent ocular challenge with the neurotropic velogenic Texas GB strain of NDV was determined to occur between days 14 and 21 post-vaccination (PV). Vaccination with the Hitchner B1 strain of NDV resulted in protection by day 6 PV, and vaccination with an inactivated NDV oil-emulsion vaccine induced protection by day 14 PV. One-day-old broiler-type chickens with maternal antibodies to both NDV and MDV and 1-day-old specific-pathogen-free (SPF) white leghorn chickens lacking maternal antibodies were vaccinated with the recombinant HVT/F vaccine or with control vaccines, challenged intra-abdominally with the very virulent RB1B strain of MDV on day 8 PV, and challenged with the Texas GB strain of NDV on day 29 PV. The HVT/F and NDV strain Hitchner B1 vaccines provided 73% and 80% protection, respectively, against NDV in broilers, whereas both vaccines resulted in 100% protection in SPF leghorns.

Asunto(s)

Anticuerpos Antivirales/inmunología , Herpesvirus Gallináceo 2/inmunología , Enfermedad de Newcastle/prevención & control , Virus de la Enfermedad de Newcastle/inmunología , Vacunas Sintéticas/uso terapéutico , Animales , Pollos , Ensayo de Inmunoadsorción Enzimática , Femenino , Genes Virales , Herpesvirus Gallináceo 2/genética , Virus de la Enfermedad de Newcastle/genética , Pavos , Vacunas Sintéticas/administración & dosificación

RESUMEN

Control of Marek's disease in the poultry industry has been successfully achieved for several decades by large-scale vaccination of day-old chickens with live herpesvirus of turkeys (HVT) strains. Several features of this virus including lack of pathogenicity and long-term immune protection due to a persistent viraemic infection made us decide to use HVT as a live viral vector for the expression of foreign antigens. Potential sites for the integration of foreign DNA in the unique short region of the HVT genome were identified by the insertion of a beta-galactosidase expression cassette. Vaccination trials with recombinant virus strains indicated that the marker gene was expressed and stably maintained during animal passage. Based on an insertion site mapping in one of the open reading frames of the unique short region, a general recombination vector was designed for the integration of foreign genes into HVT. Recombinant virus-directed expression of individual antigens from Newcastle disease virus was driven by a strong promoter element derived from the lung terminal repeat sequence of Rous sarcoma virus.

Asunto(s)

Antígenos Virales/genética , Herpesviridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , ADN Viral/genética , Expresión Génica , Genes Virales , Vectores Genéticos , Herpesviridae/inmunología , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/inmunología , Enfermedad de Marek/prevención & control , Datos de Secuencia Molecular , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Pavos

RESUMEN

Recombinant strains of herpesvirus of turkeys (HVT) were constructed that contain either the fusion protein gene or the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) inserted into a nonessential gene of HVT. Expression of the NDV antigens was regulated from a strong promoter element derived from the Rous sarcoma virus long terminal repeat. Recombinant HVT strains were stable and fully infectious in cell culture and in chickens. Chickens receiving a single intra-abdominal inoculation at 1 day of age with recombinant HVT expressing the NDV fusion protein had an immunological response and were protected (> 90%) against lethal intramuscular challenge at 28 days of age with the neurotropic velogenic NDV strain Texas GB. Recombinant HVT expressing the NDV hemagglutinin-neuraminidase provided partial protection (47%) against the same challenge. Chickens vaccinated with recombinant HVT vaccines had low levels of protection against NDV replication in the trachea when challenged ocularly. Recombinant HVT vaccines and the parent HVT strain provided similar levels of protection to chickens challenged with the very virulent RB1B strain of Marek's disease virus, indicating that insertion of foreign sequences into the HVT genome did not compromise the ability of HVT to protect against Marek's disease.

Asunto(s)

Pollos/microbiología , Enfermedad de Marek/prevención & control , Enfermedad de Newcastle/prevención & control , Enfermedades de las Aves de Corral/prevención & control , Proteínas Virales de Fusión/genética , Vacunas Virales , Animales , Pollos/inmunología , Herpesviridae , Enfermedad de Marek/inmunología , Enfermedad de Newcastle/inmunología , Enfermedades de las Aves de Corral/inmunología , Pavos/microbiología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales de Fusión/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunología , Viremia/prevención & control , Viremia/veterinaria

RESUMEN

The identification of unique Marek's disease (MD) virus (MDV) antigens expressed not only in lytically infected cells but also in latently infected MD lymphoblastoid tumor cell lines is important in understanding the molecular mechanisms of latency and transformation by MDV, an oncogenic lymphotropic herpesvirus of chickens. Through cDNA and nucleotide sequence analysis, an open reading frame (designated the pp38 ORF) which encodes a predicted polypeptide of 290 amino acids was identified in BamHI-H. Demonstration that the pp38 ORF spans the junction of the MDV long unique and long internal repeat regions (MDV has an alphaherpesvirus genome structure) precludes the presence of the gene encoding the B-antigen complex (gp100, gp60, and gp49) in the same region of BamHI-H, where it was originally thought to exist. Duplication of the complete pp38 ORF was not observed in BamHI-D, but part of it (encoding 45 amino acids) was found in the long terminal repeat region of the fragment. By use of trpE-pp38 fusion proteins, antisera against pp38 were prepared. By immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a predominant virus-specific 38,000-dalton polypeptide (designated pp38) and a minor 24,000-dalton polypeptide (designated p24) were found. No precursor-product relationship was found between pp38 and p24 by pulse-chase analysis, and only pp38 was detected by Western blot (immunoblot) analysis with antiserum to pp38. pp38 was found to be phosphorylated and present in oncogenic serotype 1-but not nononcogenic serotype 3-infected cells. Expression of the gene encoding pp38 was relatively insensitive to phosphonoacetic acid inhibition, suggesting that pp38 may belong to one of the early classes of herpesvirus proteins. pp38 was also detected in the latently infected MSB-1 lymphoblastoid tumor cell line. The detection of antibody against pp38 in immune chicken sera indicates that pp38 is an immunogen in birds with MD. Most of the properties described here for a protein detected by methods based on finding the ORF first are identical to those of a 38-kDa phosphoprotein reported by others, suggesting that they are the same. Collectively, the data reported here provide (i) more definitive information on the complete ORF of another MDV gene and the protein that it encodes, (ii) clarification of the gene content within a specific region of the MDV genome, and (iii) the molecular means to conduct further studies to determine whether pp38 plays a role in MDV latency and transformation.

Asunto(s)

Antígenos Virales/genética , Genes Virales , Herpesvirus Gallináceo 2/genética , Fosfoproteínas/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Secuencia de Bases , Northern Blotting , Células Cultivadas , ADN Viral/análisis , ADN Viral/metabolismo , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Herpesvirus Gallináceo 2/fisiología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Transcripción Genética , Células Tumorales Cultivadas , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Replicación Viral

RESUMEN

The replicative form (RF) DNA of chicken anaemia agent (CAA) was isolated and cloned into bacterial plasmids. After religation of the cloned CAA DNA and transfection into MDCC-MSB1 cells, the DNA could induce c.p.e. characteristic of that caused by CAA, and an antigen was produced which gave positive immunofluorescence when detected with an anti-CAA serum. Sanger sequencing of the 2298 bp genome revealed several open reading frames (ORFs); the major ORF encoded a polypeptide of 51.8K. In SDS-PAGE of CAA viral particles a 50K protein has been reported as the only detectable viral protein. The genomic region downstream of the major ORF had several predicted GC-rich inverted repeats, a poly(A) signal and four copies of an 18 bp repeat element. Database searches did not reveal any sequence with homology to the viral genomic DNA, nor to the amino acid sequence of any of the ORFs, apart from the N-terminal 40 amino acids of the major ORF which showed a limited similarity to the structure of protamines.

Asunto(s)

Anemia/veterinaria , Virus ADN/genética , Genes Virales , Virus/genética , Secuencia de Aminoácidos , Anemia/microbiología , Animales , Secuencia de Bases , Pollos , Clonación Molecular , Replicación del ADN , ADN Viral/biosíntesis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Alineación de Secuencia , Proteínas Virales/genética

RESUMEN

cDNA, copied from nuclear RNA isolated from heat shocked Drosophila hydei cells, has been cloned. From this collection of clones a clone, N09-15, with a 450 bp insert has been isolated that hybridizes in situ to the heat shock locus-2-48B of Drosophila hydei. The N09-15 sequence is present in two different genomic arrangements, as shown by restriction mapping, in our wild type D. hydei population. These genomic arrangements are allelic. Both alleles contain multiple copies of the N09-15 sequence but differ in their lengths and in the distribution of Msp I and Taq I sites.

Asunto(s)

Alelos , Drosophila/genética , Proteínas/genética , Animales , Secuencia de Bases , Cromosomas/fisiología , ADN/metabolismo , Enzimas de Restricción del ADN , Embrión no Mamífero/fisiología , Proteínas de Choque Térmico , Calor , Hibridación de Ácido Nucleico , Plásmidos

RESUMEN

Fluorochrome-labeled RNA allows the rapid detection of in situ hybrids without the need for long exposure times as in the autoradiographical hybridisation methods. Resolution is high because of the high resolving power of fluorescence microscopy. The application of a previously reported method for the hybrido-cytochemical detection of DNA sequences to polytene chromosomes of Drosophilia is described. The specificity and sensitivity of the method are demonstrated by the hybridisation with polytene chromosomes of 1) rhodamine-labeled 5S RNA, to the 5S rRNA sites of D. melanogaster (56F) and D. hydei (23B), 2) rhodamine-labeled RNA complementary to a plasmid containing histone genes, to the 39DE region of D. melanogaster, 3) rhodamine-labeled D. melanogaster tRNA species (Gly-3 and Arg-2), to their respective loci in D. melanogaster, 4) rhodamine-labeled RNA complementary to the insert of plasmid 232.1 containing part of a D. melanogaster heat shock gene from locus 87C, to D. hydei heat shock locus 2-32A. In the latter instance it was possible to demonstrate the labeling of a double band which escaped unambiguous detection by autoradiography in the radioactive cytochemical hybridisation procedure because of the low topological resolution of autoradiograms. The sensitivity of the fluorochrome-labeled RNA method is compared with the radioactive methods which use 3H- or 125 I-labeled RNAs. The factors governing the sensitivity and the number of bound fluorochrome molecules to be expected are discussed.

Asunto(s)

Cromosomas/ultraestructura , ADN/genética , Drosophila/genética , Histocitoquímica/métodos , Hibridación de Ácido Nucleico , Animales , Drosophila melanogaster/genética , Colorantes Fluorescentes , ARN/genética

RESUMEN

Four cell lines have been isolated from Drosophila hydei embryos. Three lines have a normal XY karyotype, the fourth has an XO karyotype with an additional small heterochromatic fragment. The cells contain presumable cytoplasmic virus like particles.

Asunto(s)

Línea Celular , Drosophila/genética , Animales , Aberraciones Cromosómicas , Cromosomas/ultraestructura , Citoplasma/microbiología , Embrión no Mamífero , Cuerpos de Inclusión Viral/ultraestructura , Cariotipificación , Microscopía Electrónica , Fenotipo , Cromosomas Sexuales/ultraestructura

RESUMEN

In situ hybridization of cRNA transcribed from cloned D. melanogaster heat shock sequences to D. hydei chromosomes has shown that the D. hydei locus 2--32 A corresponds to the D. melanogaster locus 87 A/C and the D. hydei locus 2--36 A to the D. melanogaster locus 95 D, while the D. hydei locus 4--81 B corresponds to the D. melanogaster locus 63 BC. No hybridization to D. hydei chromosomes was found with cRNA transcribed from a clone containing the alpha beta sequences encoded by the D. melanogaster locus 87 C. Neither D. melanogaster heat shock RNA nor D virilis heat shock RNA hybridized significantly to the D. hydei heat shock locus 2--48 B. Furthermore, D. hydei heat shock RNA did not hybridize to the cytological homologs of locus 2--48 B found in D. repleta or in D. virilis. D. hydei heat shock. RNA did hybridize to the cytological homologs of locus 2--48 B in D. neohydei and D. eohydei, both of which belong to the hydei subgroup.

Asunto(s)

ADN/análisis , Drosophila melanogaster/genética , Drosophila/genética , Genes , Calor , Animales , Secuencia de Bases , ADN/genética , Hibridación de Ácido Nucleico , ARN/genética , Especificidad de la Especie

RESUMEN

Complementary DNA was made to poly A+ nuclear or polysomal RNA isolated from heat shock tissue culture cells of Drosophila hydei. A number of loci other than the four major heat shock loci are labelled after in situ hybridization of these cDNA preparations, while solution hybridization indicated that only about 10% of the cDNA was specific for heat shocked cells. Removal of the fraction of cDNA which could react with 25 degrees C RNA and subsequent in situ hybridization of heat shock specific cDNA indicated that locus 4-81 B, a major salivary gland heat shock locus, is also active at 25 degrees C in tissue culture cells, while locus 4-85 B is specifically activated by heat shock in tissue culture cells. This latter locus is not seen to be clearly puffed in salivary glands, but was shown to be active in that tissue both by direct autoradiography of salivary gland chromosomes after 3H-uridine labeling and by hybridization of cDNA to chromosomal RNA.

Asunto(s)

Drosophila/genética , Genes , Calor , Animales , Células Cultivadas , ADN/genética , Hibridación de Ácido Nucleico , ARN Ribosómico/genética , Glándulas Salivales/ultraestructura

Asunto(s)

Drosophila/metabolismo , Biosíntesis de Péptidos , Glándulas Salivales/metabolismo , Animales , Calor , Plantas/metabolismo , Poli A/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Triticum/metabolismo

RESUMEN

Antibody was raised against total Drosophila hydei embryonic cellular protein with a molecular weight between 65,000 and 70,000 dalton. This antiserum reacted with the 70,000 MW "heat-shock" peptide found in "S labelled cell extracts of "heat-shocked" D. hydei tissue culture cells or salivary glands.--The antibody was coupled to Sepharose 4B and this material was used to absorb polysomes obtained from tissue culture cells incubated at 37 degrees C in the presence of tritiated RNA precursors. The relative concentrations of various RNA species complementary to the "heat-shock" loci 2-32A, 2-36A, and 2-48C in either bound, non-bound, or total polysomal material was then determined by in situ hybridization. The RNA species complementary to locus 2-36A was found to be enriched in the bound polysomal material.

Asunto(s)

Drosophila , ARN/biosíntesis , Animales , Calor , Peso Molecular , Hibridación de Ácido Nucleico , Péptidos , Polirribosomas

RESUMEN

The maximum grain density over the "heat-shock" locus 2-48BC of Drosophila hydei polytene chromosomes obtained after in situ hybridization of nuclear RNA extracted from tissue culture cells labelled during incubation at 37 degrees C is five times higher than that obtainable by using polysomal RNA isolated from the same cells. Furthermore, the addition of a large excess of unlabelled polysomal RNA reduced the amount of in situ hybridization of nuclear RNA by only 20% showing that nuclear 2-48BC RNA contains sequences not present in polysomal 2-48BC RNA.--The polysomal 2-48BC RNA is polyadenylated, as are the RNA sequences present in the polysomes complementary to the other two major "heat shock" loci 2-32A and 2-36A. Polyadenylated RNA, with an apparent size of 15S, complementary to locus 2-48BC is also found in the cytoplasm of D. hydei salivary glands.

Asunto(s)

Hibridación de Ácido Nucleico , ARN , Animales , Núcleo Celular , Técnicas de Cultivo , Citoplasma , Drosophila , Polirribosomas , ARN Ribosómico

RESUMEN

The defective phages of Bacillus subtilis cannot be counted by plating as they do not form plaques. In addition, counting under the electron microscope with latex spheres as an internal standard is not possible. The reliability of a method using Escherichia coli phage T4 as a substitute for the latex spheres has been tested and the results compared with those of other methods. Using this method, we determined the burst sizes of the defective phages PBS X, PBS Y and PBS Z under various conditions.

Asunto(s)

Bacillus subtilis , Bacteriófagos , Virus Defectuosos , Técnicas Bacteriológicas , Colifagos