RESUMEN
We have used FISH with microdissected chromosome arm-specific DNA probes that allow direct visualization of chromosome 1 folding in human sperm nuclei. We described the overall topology and the mode of packaging of sperm chromosome 1 and thus established new elements of ordered genome architecture in these specialized cells.
Asunto(s)
Núcleo Celular/genética , Cromosomas Humanos Par 1/metabolismo , Espermatozoides/citología , Cromosomas Humanos Par 1/ultraestructura , Genoma , Humanos , Masculino , Microscopía FluorescenteRESUMEN
Mammalian telomeres contain long tandem (TTAGGG)n repeats, which are protected by a complex of different proteins. Telomeric repeat-binding factors TRF1 and TRF2 play the key role in protection of telomeres through the formation of terminal loops (called T-loop). A T-loop isolates the 3' strand telomeric end and with this mechanism protects telomeres from the influence of enzymes of DNA reparation and telomere fusions and also interferes with the interaction of telomerase with telomeres. Many vertebrate species also contain large blocks of (TTAGGG)n sequences in pericentric and interstitial chromosome bands. The Chinese hamster genome contains a total of 18 arrays of these non-telomeric internal (TTAGGG)n sequences (ITs). Chromosome bands containing these arrays are unstable and should be protected with the help of another mechanism, rather than that using telomeres. In this study we analysed association of Green Fluorescent Protein (GFP)-tagged TRF1 in Chinese hamster V79 cells with ITs. We found that in these cells GFP-TRF1 associates with ITs in the interphase nucleus. We detected a little overlap between IT-associated GFP-TRF1 and random DSB sites visualized after the treatment of V79 cells with ionizing radiation. We found that the treatment of V79 cells with WM significantly increases the frequency of spontaneous chromosome aberrations. These WM effects are possible due to inhibiting phosphorylation of TRF1 by ATM. TRF1 is known to be eliminated from telomeres by overexpression of TANK1, which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding TANK1 and found that the frequency of chromosome rearrangements increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 may be involved in the sequence-specific protection of internal non-telomeric (TTAGGG)n repeats.
Asunto(s)
Inestabilidad Cromosómica , Secuencias Repetidas en Tándem , Telómero/genética , Proteína 1 de Unión a Repeticiones Teloméricas/fisiología , Animales , Línea Celular/efectos de la radiación , Cricetinae , Cricetulus , Interfase , Tanquirasas/biosíntesis , Tanquirasas/genética , Telómero/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/fisiología , TransfecciónRESUMEN
By screening with labeled Alu DNA, a clone was isolated from cDNA expression library, which appeared identical in sequence to the well-known Ca-phospholipid-binding protein annexin II. To evidence the DNA-binding activity of recombinant annexin II and its presence in the cell nucleus, we have expressed full-length mouse annexin II cDNA in bacteria with pGEMEX vector. The expressed protein was studied with electrophoretic mobility shift assay and for its reaction with polyclonal antibody to chromatin-associated ribonucleoprotein (alpha-RNP), which is one of the major acid-dissolvent components of the nucleus. The obtained results confirm the DNA-binding activity of recombinant annexin II. Annexin II reacts with polyclonal antibody to rat alpha-RNP. So, annexin II is a major nuclear DNA-binding protein in mammalian cells.