RESUMO
We describe conditions for optimal recovery of recombinant plasmids after blunt-end ligation. It was found that one of the most critical parameters of the blunt-end ligation reaction is total DNA concentration (vector plus incoming DNA). This concentration was optimal in the range of 1-5 micrograms/ml of reaction mixture. Concentrations larger than 10 micrograms/ml result in strong inhibition. The optimal molar relationship between incoming DNA and vector was found to be 1 or less. Under these conditions, using dephosphorylated vector, recombinants are generated at a frequency of 10(6) colonies per microgram of insert, provided that transforming efficiency is about 5 x 10(7) colonies per microgram of plasmid DNA.
Assuntos
Clonagem Molecular/métodos , DNA Ligases/metabolismo , DNA Recombinante/isolamento & purificação , Plasmídeos , Fenômenos Químicos , Físico-Química , DNA Recombinante/metabolismoRESUMO
Human papillomavirus type 6 subtype a (HPV-6a) was detected in a human invasive tonsillar carcinoma. Southern blot hybridization analysis showed the presence of additional bands when using non-cutting and single-cut restriction enzymes. Molecular cloning yielded two recombinant clones of 8.0 and 1.4 kb in size. The first represents the complete HPV-6a genome. Sequence analysis of the second clone showed a 0.6 kb DNA sequence corresponding to the L2 region of HPV-6a, whereas the rest belongs to cellular sequences. These data show the presence of a usually low risk HPV type in an invasive carcinoma, at an unusual infection site, with viral DNA integrated into the host genome. These findings add evidence in support of the hypothesis of a relationship between HPV infection and at least some ororespiratory cancers.
Assuntos
Carcinoma de Células Escamosas/microbiologia , Papillomaviridae/isolamento & purificação , Neoplasias Tonsilares/microbiologia , Infecções Tumorais por Vírus/microbiologia , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Viral , Feminino , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Papillomaviridae/genéticaRESUMO
A prospective study was carried out in 49 families of newborns in Avellaneda District, Argentina, recruited when the mother was in the last trimester of pregnancy and followed until the newborn was aged two years, to study the epidemiology of rotavirus infection under natural conditions. Weekly surveillance of all family members for diarrhea and rotavirus-related diarrhea was carried out from May 1983 to July 1986. Serum samples were also collected every six months to study the incidence of rotavirus infection in all family members. The peak of diarrhea incidence was observed in children aged less than two years (2.1 cases per person-year). Rotavirus infection was a common rather than a sporadic phenomenon (0.63 cases per person-year, on average) in all age groups, and at least 61% of rotavirus infections were reinfections. In contrast, the peak of rotavirus infection in seronegative persons (0.58 cases per person-year) and the peak of rotavirus-related diarrhea incidence (0.25 cases per person-year) were found during the first year of life. These results showed that infants aged less than one year are the most appropriate candidates for a rotavirus vaccine in the Avellaneda population. However, improvements in sanitary conditions and personal hygiene and education of the high-risk population should be considered the primary means of reducing infectious diarrhea in Argentina.
Assuntos
Gastroenterite/epidemiologia , Infecções por Rotavirus/epidemiologia , Adolescente , Adulto , Fatores Etários , Argentina , Pré-Escolar , Diarreia/epidemiologia , Diarreia/microbiologia , Diarreia Infantil/microbiologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Gastroenterite/sangue , Humanos , Higiene , Lactente , Masculino , Projetos Piloto , Gravidez , Terceiro Trimestre da Gravidez , Estudos Prospectivos , Infecções por Rotavirus/sangue , Infecções por Rotavirus/transmissão , Estações do Ano , Fatores Socioeconômicos , Saúde da População UrbanaRESUMO
Rotavirus gastroenteritis was studied by enzyme-linked immunosorbent assay (ELISA) and by PAGE from April 1983 to April 1985 at the Buenos Aires Ricardo Gutierrez Children's Hospital and at the San Justo Children's Hospital. Based on examination of 576 cases, rotavirus was identified in 109 (18.9%) cases by ELISA and in 99 (17.2%) cases by PAGE. As a diagnostic tool PAGE presented a sensitivity of 90.8% compared with ELISA. Compared with the control SA-11 genome, 84 samples (84.9%) presented a long electropherotype and 15 isolates (15.1%) presented a short electropherotype. We detected 35 different long electropherotypes and report a short electropherotype that did not show genome variation and was responsible for an outbreak in the San Justo Children's Hospital population from September 1983 to July 1984.
Assuntos
Infecções por Rotavirus/epidemiologia , Rotavirus/classificação , Argentina , Eletroforese , Humanos , RNA Viral/genética , Rotavirus/genética , Infecções por Rotavirus/genética , Infecções por Rotavirus/microbiologiaRESUMO
Detection of human rotaviruses by enzyme-linked immunosorbent assay (ELISA), provided by the World Health Organization, in 100 stool specimens was compared with the sensitivity of detection by counterimmunoelectrophoresis (CIED) and polyacrylamide gel electrophoresis (PAGE). Table 1 shows that 34 samples were found to contain rotavirus antigens by ELISA and in other 4 specimens false negative results were observed in the ELISA confirmatory test because of high antigen concentration. The ELISA confirmatory test was repeated with the 4 samples diluted 1/40 and they became positives. It was found that, of the 38 ELISA positive samples, 35 were positive by PAGE (92%) and 24 by CIED (63%). Detection of human rotaviruses in stools by PAGE were almost as sensitive as ELISA, but the direct visualization of viral RNA fragments, provided a distinct advantage since no confirmatory test was necessary. Besides, the surveillance of rotavirus electrophoretypes constitutes a very useful tool in epidemiological studies, in order to the future possible use of a rotavirus vaccine in our country. By the other hand, it was found CIED less sensitive than ELISA and PAGE. This disadvantage might be tolerated in a diagnostic setting, only if no other test was available for rotavirus diagnosis.
Assuntos
Contraimunoeletroforese , Diarreia/diagnóstico , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Imunoeletroforese , Infecções por Rotavirus/diagnóstico , Diarreia/etiologia , Fezes/microbiologia , Humanos , Valor Preditivo dos Testes , Rotavirus/imunologiaRESUMO
Detection of human rotaviruses by enzyme-linked immunosorbent assay (ELISA), provided by the World Health Organization, in 100 stool specimens was compared with the sensitivity of detection by counterimmunoelectrophoresis (CIED) and polyacrylamide gel electrophoresis (PAGE). Table 1 shows that 34 samples were found to contain rotavirus antigens by ELISA and in other 4 specimens false negative results were observed in the ELISA confirmatory test because of high antigen concentration. The ELISA confirmatory test was repeated with the 4 samples diluted 1/40 and they became positives. It was found that, of the 38 ELISA positive samples, 35 were positive by PAGE (92
) and 24 by CIED (63
). Detection of human rotaviruses in stools by PAGE were almost as sensitive as ELISA, but the direct visualization of viral RNA fragments, provided a distinct advantage since no confirmatory test was necessary. Besides, the surveillance of rotavirus electrophoretypes constitutes a very useful tool in epidemiological studies, in order to the future possible use of a rotavirus vaccine in our country. By the other hand, it was found CIED less sensitive than ELISA and PAGE. This disadvantage might be tolerated in a diagnostic setting, only if no other test was available for rotavirus diagnosis.