RESUMO
Primate genomes show a great karyological variability while the DNA content variation is scarce. The biggest genome size occurs in Cercophitecus cephus (Catarrhini, Cercophitecidae) with 5.26 pg whereas the smallest one is described for Callicebus torquatus (Platyrrhini, Callithricidae) with 2.26 pg. Over the last 20 years different authors have been studying the Platyrrhini genomes on a chromosomal level. Among them, Cebus (Cebidae) being considered the most ancestral and conserved karyotype in relation to human karyotype has been extensively studied. Cebus genome sizes range from 3.40 to 3.98 pg. The species that inhabit Argentina, where they reach the most southern natural distribution, Cebus paraguayanus (CPA) and Cebus nigritus (CNI), have been extensively studied with classical cytogenetic comparisons focusing on banding pattern behavior. In the present study we performed comparative genomic hybridization (CGH) between these two closely related species with the aim of going a step further in the dissection of Cebus genomes. CGH evidenced that the DNA imbalances between them involved different genome regions, i.e. preferentially repetitive DNA in CPA and coding or very disperse DNA in CNI. Particularly, CNI showed species-specific DNA in more than 9 chromosomal pairs with a red/green (r/g) ratio ranging from 1.7 to 4, meaning that CNI presents at least twice as much DNA than CPA in those chromosomal segments. CPA showed species-specific DNA in the telomeric region of at least 3 chromosomal pairs with an r/g ratio of 0.5. They also showed a DNA gain in the chromosomal pairs with extracentromeric heterochromatin. Our findings modify the widespread idea of considering the heterochromatin proportion as the only difference between CPA and CNI. In Cebus then, the diversification process could be mediated by little changes in DNA content accompanied by a euchromatin-heterochromatin interaction although maintaining a minimum proportion like the one observed in CNI.
Assuntos
Cebus/genética , Cercopithecus/genética , Animais , Argentina , Hibridização Genômica Comparativa/métodos , DNA/genética , DNA/isolamento & purificação , Feminino , Genoma , Humanos , Cariotipagem , Masculino , Primatas/genética , Especificidade da EspécieRESUMO
Genetic data are very important for conservation programs in wild population as well as in captive conditions. Primates in zoos or breeding centers are often maintained in groups without geographic origin or genetic heritage information. These lead to the incorrect assignment of species and introduce an artificial reproductive barrier, which in turn constitutes inadequate management of the colonies. A karyological analysis of specimens from a Primate Reproduction Center, considered as Cebus apella (Platyrrhini), was performed. Cell cultures were conducted from peripheral blood samples following standard cytogenetic methods. A fluorescence in situ hybridization (FISH) procedure was applied in mitotic metaphases using two probes: A specific probe of the extracentromeric heterochromatin (He+) of Cebus, and a human chromosome 21 probe. The latter was chosen due to the known homeology with the euchromatic region limiting with 11qHe+ of Cebus. The species status was determined for at least half of the animals and identified a hybrid specimen using this combined FISH protocol. This procedure is an accurate diagnostic methodology for taxonomic determinations and, therefore can be used for management of reproduction in colonies.
Assuntos
Cebus/classificação , Cebus/genética , Hibridização Genética/genética , Cariotipagem/veterinária , Animais , Animais de Zoológico , Feminino , Masculino , FenótipoRESUMO
We studied chromosomal abnormalities in arrested embryos produced by assisted reproductive technology with fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in order to determine the best technique for evaluating chromosomal aneusomies to be implemented in different situations. We examined individual blastomeres from arrested embryos by FISH and arrested whole embryos by CGH. All of the 10 FISH-analyzed embryos gave results, while only 7 of the 30 embryos analyzed by CGH were usable. Fifteen of the 17 embryos were chromosomally abnormal. CGH provided more accurate data for arrested embryos; however, FISH is the technique of choice for screening in preimplantation genetic diagnosis, because the results can be obtained within a day, while the embryos are still in culture.
Assuntos
Humanos , Feminino , Gravidez , Hibridização in Situ Fluorescente , Cariotipagem/métodos , Diagnóstico Pré-Implantação/métodos , Genômica , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/embriologia , Transtornos Cromossômicos/genética , Técnicas de Reprodução AssistidaRESUMO
Neotropical Primate karyotypes are highly variable, particularly in the heterochromatic regions, not only regarding the amount of heterochromatin, but also the composition. G and C banding and FISH techniques provide useful information to characterize interspecific relationships. We used chromosome microdissection to develop a FISH probe of the chromosome 11 heterochromatic block (11qHe+) of Cebus apella paraguayanus (CAPp). Fragments of the 11qHe+ microdissected from fibroblast cell culture were collected in a PCR tube, amplified by degenerate oligonucleotide primer-PCR and subsequently labeled. The specificity of the FISH probe was confirmed in metaphases of some Ceboidea species. Signals were located in the He+ of chromosomes 4, 11, 12, 13, and 19 of CAPp and in the He+ of chromosomes 4, 12 and 13 of C. a. nigritus (CAPn); no signals were observed when other Ceboidea species were analyzed. We propose that the heterochromatin observed in CAPp and CAPn is specific for these species. We consider this C. apella heterochromatin identity as a possible key for the interpretation of chromosomal evolution in these Ceboidea.
Assuntos
Animais , Masculino , Feminino , Hibridização in Situ Fluorescente , Bandeamento Cromossômico/métodos , Cebus/genética , Evolução Molecular , Heterocromatina/genética , Microdissecção/métodos , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
The radiosensitive mutant cell line IRS-20, its wild type counterpart CHO and a derivative of IRS-20 with a transfected YAC clone (YAC-IRS) that restores radioresistance were tested for DNAse I sensitivity. The three cell lines were cultured under the same conditions and had a mitotic index of 2-5%. One drop of fixed cells from the three lines was always spread on the same microscopic slide. After one day of ageing, slides were exposed to DNAse I and stained with DAPI. Images from every field were captured and the intensity of blue fluorescence was measured with appropriate software. For untreated cells, the fluorescence intensity was similar for all of the cell lines. After DNAse I treatment, CHO and YAC-IRS had an intensity of 85% but IRS-20 had an intensity of 60%, when compared with the controls. DNAse I sensitivity differences between the cell lines indicate that overall conformation of chromatin might contribute to radiation sensitivity of the IRS-20 cells.
Assuntos
Células CHO/efeitos da radiação , Proteínas de Ligação a DNA , DNA/efeitos da radiação , Desoxirribonuclease I/farmacologia , Conformação de Ácido Nucleico , Tolerância a Radiação/genética , Animais , Células CHO/metabolismo , Cromossomos Artificiais de Levedura/genética , Cromossomos Humanos Par 8/genética , Cricetinae , Cricetulus , DNA/efeitos dos fármacos , Dano ao DNA , Reparo do DNA , Proteína Quinase Ativada por DNA , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Teste de Complementação Genética , Humanos , Índice Mitótico , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , TransfecçãoRESUMO
As the pioneer among molecular cytogenetics techniques, fluorescence in situ hybridization (FISH) allows identification of specific sequences in a structurally preserved cell, in metaphase or interphase. This technique, based on the complementary double-stranded nature of DNA, hybridizes labeled specific DNA (probe). The probe, bound to the target, will be developed into a fluorescent signal. The fact that the signal can be detected clearly, even when fixed in interphase, improves the accuracy of the results, since in some cases it is extremely difficult to obtain mitotic samples. FISH is still used mostly in research, but there are diagnostic applications. New nomenclature is being developed in order to define many of the aberrations that were not distinguished before FISH. Prenatal diagnosis of aneuploidies and malignancies are promptly detected with FISH, which is very useful in critical cases. In some tumors, where chromosomal abnormalities are too complicated to classify manually, the technique of comparative genomic hybridization (CGH), a competitive FISH, allows examiners to determine complete or partial gain or loss of chromosomes. CGH results allow the classification of many tumor cell lines and along with other complementary techniques, like microdissection-FISH, PRINS, etc., increase the possibility of choosing an appropriate treatment for cancer patients