RESUMEN
The mannose-specific snowdrop lectin [Galanthus nivalis agglutinin (GNA)] displays toxicity to the rice brown planthopper Nilaparvata lugens. A 26kDa GNA-binding polypeptide from N. lugens midgut was identified by lectin blotting and affinity chromatography, and characterized by N-terminal sequencing. This polypeptide is the most abundant binding protein for GNA in the N. lugens midgut. A cDNA (fersub2) encoding this protein was isolated from an N. lugens cDNA library. The deduced amino acid sequence shows significant homology to ferritin subunits from Manduca sexta and other arthropods, plants and vertebrates, and contains a putative N-glycosylation site. Native ferritin was purified from whole insects as a protein of more than 400kDa in size and characterized biochemically. Three subunits of 20, 26 and 27kDa were released from the native complex. The 26kDa subunit binds GNA, and its N-terminal sequence was identical to that of fersub2. A second cDNA (fersub1), exhibiting strong homology with dipteran ferritin, was identified as an abundant cDNA in an N. lugens midgut-specific cDNA library, and could encode the larger ferritin subunit. The fersub1 cDNA carries a stem-loop structure (iron-responsive element) upstream from the start codon, similar to structures that have been shown to play a role in the control of ferritin synthesis in other insects.
Asunto(s)
Ferritinas/metabolismo , Proteínas de Insectos/metabolismo , Insectos/metabolismo , Lectinas/metabolismo , Lectinas de Unión a Manosa , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Sistema Digestivo/metabolismo , Ferritinas/genética , Ferritinas/aislamiento & purificación , Galanthus , Proteínas de Insectos/genética , Proteínas de Insectos/aislamiento & purificación , Datos de Secuencia Molecular , Lectinas de Plantas , Homología de Secuencia de AminoácidoRESUMEN
The increase in thyroid carcinoma post-Chernobyl has been largely confined to a specific subtype of papillary carcinoma (solid/follicular). This subtype is observed predominantly in children under 10 in unirradiated populations, but maintains a high frequency in those aged 10-15 from those areas exposed to fallout from the Chernobyl accident. The aim of this study was to link morphology with molecular biology. We examined 106 papillary carcinomas from children under the age of 15 at operation. All were examined for rearrangements of the RET oncogene by reverse transcription polymerase chain reaction (RT-PCR); a subset of these cases were also examined for mutations of the three ras oncogenes, exon 10 of the thyroid stimulating hormone receptor, associated more usually with a follicular rather than papillary morphology, and exons 5, 6, 7 and 8 of the p53 gene, commonly involved in undifferentiated thyroid carcinoma. Rearrangements of the REToncogene were found in 44% of papillary carcinomas in which we studied fresh material; none of the tumours examined showed mutation in any of the other genes. The two rearrangements resulting from inversion of part of chromosome 10 (PTC1 and PTC3) accounted for the majority of RET rearrangements identified, with PTC1 being associated with papillary carcinomas of the classic and diffuse sclerosing variants and PTC3 with the solid/follicular variant.
Asunto(s)
Carcinoma Papilar/genética , Desastres , Neoplasias Inducidas por Radiación/genética , Proto-Oncogenes/genética , Liberación de Radiactividad Peligrosa , Neoplasias de la Tiroides/genética , Adolescente , Carcinoma Papilar/etiología , Niño , Preescolar , Cromosomas Humanos Par 10/genética , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Centrales Eléctricas , Neoplasias de la Tiroides/etiología , UcraniaRESUMEN
Missense germline mutations of the RET proto-oncogene have recently been identified in the hereditary cancer syndromes MEN2A, MEN2B, and FMTC, all characterized by medullary carcinoma, but also including phaeochromocytoma in MEN2A and MEN2B and parathyroid disease in MEN2A. In addition, somatic RET proto-oncogene mutations have been identified in a subset of sporadic medullary carcinomas and phaeochromocytomas. This study investigated the possibility that RET plays a role in sporadic parathyroid neoplasia. Firstly, normal and neoplastic parathyroid tissues were screened for expression of the RET proto-oncogene, using an RT-PCR approach on autopsy material. Secondly, 20 archival parathyroid adenomas were screened for somatic mutations in the transmembrane region of RET, the region associated with germline mutations in MEN2A and hence parathyroid disease, using a PCR-solid phase direct sequencing approach. RET expression was identified in all the parathyroid tissues analysed. However, no mutations were identified in any of the 20 adenomas, suggesting either that other mechanisms of RET activation occur, such as translocation, or that RET plays a more minor role in the growth control of the parathyroid cells than in C cells or phaeochromocytes.
Asunto(s)
Adenoma/genética , Proteínas de Drosophila , Neoplasias de las Paratiroides/genética , Proto-Oncogenes/genética , Carcinoma Medular/genética , Humanos , Neoplasia Endocrina Múltiple Tipo 2a/genética , Mutación , Reacción en Cadena de la Polimerasa , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras , Transcripción GenéticaRESUMEN
Fifteen human herpesvirus 6 (HHV-6) strain variants were analysed by PCR amplifications, restriction enzyme site polymorphism and sequence analyses. Three DNA regions were chosen for study: a fragment of a variable glycoprotein gene (210 bp), the conserved glycoprotein H (gH) gene complete with intergenic sequences (2381 bp) and the 5' intergenic region with the N-terminal coding sequence of gH up to a polymorphic BamHI site (427 bp). Infected cell DNA from five laboratory reference strains including GS, U1102, AJ, Z29 and KF were examined together with DNA from peripheral blood lymphocytes infected with HHV-6 reactivated from blood and/or marrow from five bone marrow transplant (BMT) patients. Separate blood and marrow isolates were obtained from four BMT patients. In addition, HHV-6 sequences were examined directly from one of six Hodgkin's lymphomas and six B cell proliferations which contained HHV-6 DNA as detected by PCR amplification. The results show two groups of very closely related but heterogeneous strains which correlate with previous groupings by antigenic and restriction site differences. These are variant A strains (including laboratory strains GS, U1102 and AJ) and variant B strains (including laboratory strains Z29 and KF, the Hodgkin's lymphoma strain, and the nine BMT patient isolates). Variations between the groups were 4 to 6% in nucleotide sequence and 5 to 8.5% in amino acid sequence. Within each group maximum heterogeneity was observed in different genes. Variant A strains differed by 2.0% in the variable glycoprotein gene sequence whereas variant B strains were identical in this region; conversely, variant B strains differed by 2 to 3% in the gH N-terminal and intergenic sequences whereas variant A strains differed there by less than 0.2%. There was evidence for sequence drift independent of selection: relationships between the groups were shown by analyses of amino acid sequence, coding nucleotide sequence as well as intergenic sequence, and the B variant-specific BamHI site in the gH gene was due to a non-coding nucleotide substitution. There was little evidence for in vivo or in vitro variation: the gH nucleotide sequence from the uncultured lymphoma strain (first variant B gH gene identified) was almost identical to the gH sequence from four BMT isolates, and matched BMT isolates from blood and marrow were identical or with a single nucleotide substitution.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Herpesvirus Humano 6/clasificación , Secuencia de Aminoácidos , Secuencia de Bases , Trasplante de Médula Ósea , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/aislamiento & purificación , Enfermedad de Hodgkin/microbiología , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Filogenia , Reacción en Cadena de la Polimerasa , Alineación de SecuenciaRESUMEN
The nucleotide sequence was determined for a 9.3 kb BamHI DNA fragment derived from a cosmid clone (Lorist 6) library of the 160 kb human herpesvirus-6 (HHV-6) strain U1102 genome. Analysis of the sequence showed two different sources for the DNA; 8.0 kb was derived from HHV-6, while 1.3 kb was derived from the right repeat of transposon Tn10, the insertion sequence (IS) element IS10R. The IS element sequence is shown to be derived from the host bacteria of the plasmid. The HHV-6 sequence represents a highly conserved part of the genome encoding 15% of the genes conserved among the other human herpesviruses in only 5% of the genome. Six genes were identified, five encoding products with amino acid sequence similarity to homologues in herpes simplex virus (HSV), Varicella Zoster Virus (VZV), human cytomegalovirus (CMV) and Epstein-Barr Virus (EBV). All had closest amino acid similarity to CMV proteins. Three clustered structural genes, included glycoprotein H, a major conserved determinant of infectivity, were jointed to a putative dUTPase homologue in an arrangement distinct to CMV and HHV-6. In the other herpes viruses these genes are separated by over 50 kb. The gene at this point of genetic rearrangement had no sequence similarity to proteins of other herpesviruses. However, there is a protein at this locus in CMV with similar composition and character. Both appear to be highly glycosylated, secreted glycoproteins with repetitive elements similar to those of human mucins. Comparison of sequence available in the HHV-6 GS strain also shows this to be a variable region (5% nucleotide differences) in an overall conserved DNA sequence (0.5%).
Asunto(s)
Genes Virales , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/patogenicidad , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Elementos Transponibles de ADN , ADN Viral , Desoxirribonucleasa BamHI/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas del Envoltorio Viral/genética , Virulencia/genéticaRESUMEN
Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells ("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared with those of wild-type and syncytial laboratory strains SC16, strain 17, and HFEM. The sequences were highly conserved in contrast to the diversity observed between gH sequences from herpesviruses of different subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites distributed over one-half of the gH external domain, Glu-86, Asp-168, or Arg-329, while the 52S-resistant mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168 with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope could be formed in the absence of other virus products, virus gene expression was required for stable presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol. 63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those produced by the wild-type virus, with the exception of one variant which produced lower yields.(ABSTRACT TRUNCATED AT 400 WORDS)