RESUMO
Nick translation of the DNA of conventionally prepared human metaphase chromosomes using DNase I and biotin dUTP combined with streptavidin-phosphatase-detection assay produced a banding-like appearance. This pattern seems to be due to differences in DNase I sensitivity along the chromosomes. The Y chromosome could be clearly distinguished from the other chromosomes because of its intensely dark labelled heterochromatic region. In addition to DNase I concentration, hypotonic treatment seems to be an important methodological factor influencing band resolution. Together with recently published similar methods these results indicate that in situ nick translation using biotinylated nucleotides may develop into a useful technique to overcome several problems of human cytogenetics.
Assuntos
Fosfatase Alcalina , Biotina/análogos & derivados , Cromossomos Humanos/análise , Nucleotídeos de Desoxiuracil , Biossíntese de Proteínas , Líquido Amniótico/citologia , Proteínas de Bactérias , Bandeamento Cromossômico , Desoxirribonucleases/metabolismo , Feminino , Humanos , Masculino , Metáfase , EstreptavidinaRESUMO
In situ nick translation of mammalian chromosomes by restriction endonuclease treatment to nick the chromosomal DNA, and 'translation' in the presence of DNA polymerase I and biotinylated dUTP, results in a distinct banding pattern. Further experiments have elucidated the mechanisms producing these bands. The hypothesis is presented that differences in the local conformation of the DNA-protein complex, rather than the DNA sequence itself, lead to the nick translation bands. The different DNase I sensitivity along the chromosomes suggests that the bands, which were clearly evident, reflect morphological units closely related to biological functions.
Assuntos
Cromossomos/fisiologia , Enzimas de Restrição do DNA , Biossíntese de Proteínas , Animais , Linhagem Celular , Células Cultivadas , Desoxirribonuclease I , Humanos , Cariotipagem , Linfócitos/citologiaRESUMO
Here we present first results of an attempt to replace the nicking activity of DNAase I by ECO RI for in situ nick translation of human chromosomes. For the detection of nick translated sites we used biotinylated dUTP. The procedure resulted in a distinct banding pattern of the chromosomes which does not seem to correspond to that reached by any known banding technique.
