Assuntos
Brugia Malayi/genética , Cromossomos Artificiais Bacterianos/genética , DNA de Helmintos/genética , Biblioteca Gênica , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Animais , Biotinilação , Eletroforese em Gel de Poliacrilamida , Etiquetas de Sequências Expressas , Genes de Helmintos , Membranas Artificiais , Reação em Cadeia da Polimerase , PolivinilRESUMO
A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.
Assuntos
Brugia Malayi/genética , DNA Complementar/análise , DNA/análise , Biblioteca Gênica , Medições Luminescentes , Hibridização de Ácido Nucleico , Animais , Biotinilação , Brugia Malayi/patogenicidade , Células Clonais , Cosmídeos/genética , Sondas de DNA/genética , Filariose/genética , Filtração/métodos , Corantes Fluorescentes , Humanos , Análise de Sequência de DNA , Sitios de Sequências RotuladasRESUMO
The genes encoding the ApaLI (5'-GTGCAC-3'), NspI (5'-RCATGY-3'), NspHI (5'-RCATGY-3'), SacI (5'-GAGCTC-3'), SapI (5'-GCTCTTCN1-3', 5'-N4GAAGAGC-3') and ScaI (5'-AGTACT-3') restriction-modification systems have been cloned in E. coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5'-GCTCTTC-3' blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.
Assuntos
Proteínas de Bactérias , Clonagem Molecular/métodos , DNA (Citosina-5-)-Metiltransferases/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Escherichia coli/genética , Sequência de Aminoácidos , Sequência de Bases , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência MolecularRESUMO
Amniotic membrane was obtained from 36 mothers seronegative for hepatitis B surface antigen and syphilis, undergoing caesarean section. Membrane was separated from placenta and was washed first with saline and then saline solution containing penicillin. The processed membrane was found to be sterile and useable for up to one week. Of 36 placentas obtained, 33 were utilized in 22 patients, with no history of penicillin allergy, as biological dressing in acute burns. Each patient received three applications of membrane one every other day, over a period of six days. This method of obtaining amniotic membrane was simple and more practical for maintaining the biological effectiveness of membrane, as shown by quantitative reduction of bacterial counts in burn wounds.