RESUMO
The histone-like proteins of the monogenetic parasite Crithidia fasciculata were extracted with 0.2 M sulfuric acid either from purified nuclei, or from purified chromatin, in both cases in the presence of 1 mM tosyl lysylchloromethylketone and 2 mM phenyl methyl sulfonyl fluoride as proteinase inhibitors. The presence of histones in the flagellate, nonidentical with those from calf thymus used as controls, was shown by their electrophoretic patterns in three different polyacrylamide gel systems; their staining with Alkaline fast green, specific for basic proteins; their global amino acid composition and absorption spectrum and their molecular weights. The protein showing the slower mobility in SDS gels and the fastest mobility in the urea-acetic acid-Triton gels, seems to be an H1 histone, because of its metachromatic staining with Coomassie brilliant blue, solubility characteristics, differential destaining properties and amino acid composition. Band 5 in Triton-urea-acetic acid gels is probably an HMG protein. We conclude that C. fasciculata has a complete set of histones and that the lack of chromosome condensation during mitosis is not due to lack of histone H1.
Assuntos
Crithidia/análise , Histonas/isolamento & purificação , Aminoácidos/análise , Animais , Bovinos , Fracionamento Celular , Núcleo Celular/análise , Núcleo Celular/ultraestrutura , Cromatina/análise , Cromatina/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Indicadores e Reagentes , Peso Molecular , Especificidade da Espécie , Timo/análiseRESUMO
The binding of toluidine blue molecules under Mg2+ competitive staining conditions was investigated in chromocentres and the euchromatin of single- and multi-chromocentred nuclei of Triatoma infestans Malpighian tubule cells. It was demonstrated that the chromocentre of single-chromocentred nuclei exhibited the largest critical electrolyte concentration (CEC) value (0.4 M), followed by the chromocentres of multi-chromocentred nuclei (0.3 M) and the euchromatin (0.2 M). The differences in CEC values were assumed to be due to differences in availability of free DNA phosphates and in packing states of the DNA-protein complexes of these chromatin types. Differences in chromatin supra-organization were evident for the chromocentral heterochromatin of single vs multi-chromocentred nuclei. This was also valid for the chromocentral heterochromatin in some multi-chromocentred nuclei, when one of the heterochromatic bodies was especially larger than the others.
Assuntos
Cromatina/análise , Eletrólitos/análise , Heterocromatina/análise , Triatoma/genética , Triatominae/genética , Animais , Eucromatina , Cloreto de Magnésio , Túbulos de Malpighi/análise , Espectrofotometria , Coloração e Rotulagem , Cloreto de TolônioRESUMO
After fertilization the cone shaped sperm nucleus is transformed into a spheroidal male pronucleus which fuses with the female pronucleus reestablishing the diploid genome. These morphological changes are correlated with biochemical transitions of sperm-specific chromosomal proteins. To obtain information on the rearrangements of chromosomal proteins after fertilization, we have followed sperm-specific nonhistones (Sp NHCP) and the sperm-specific histones (SpH) in zygotes harvested at different times post-insemination (p.i.). The acquisition of proteins synthesized de novo was determined during the time of male pronucleus formation, estimated to occur until 30 min p.i. The results obtained may be summarized as follows: 1. The fate of Sp NHCP, followed by Western blot analysis with polyclonal antibodies obtained in rabbits against the whole complement of Sp NHCP, indicates that the majority of Sp NHCP are lost shortly p.i., except two proteins of 58 Kd and 61 Kd that were not distinguishable from eggs NHCP because a crossreaction was obtained. Acidic proteins synthesized de novo were bound to chromatin between 3 and 30 min p.i., and the majority of these proteins comigrated with egg NHCP in SDS/PAGE. 2. The histones from sperm differ from their counterparts found in unfertilized eggs, both in their amino acid composition and their microheterogeneity in bidimensional gels. Sperm contain the five major SpH whereas eggs have seven major histone variants, distinct from each other, with an electrophoretic behaviour consistent with histones but a different amino acid composition. The total complement of histone variants found in zygotes collected at amphimixis is identical with that found in unfertilized eggs, suggesting that SpH should be lost before that time.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cromatina/metabolismo , Ouriços-do-Mar/fisiologia , Espermatozoides/fisiologia , Zigoto/fisiologia , Aminoácidos/análise , Animais , Cromatina/análise , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Histonas/análise , Masculino , Espermatozoides/análise , Zigoto/análiseRESUMO
A close relationship between histone synthesis and DNA replication has been suggested for many biological systems. The presence of histonic proteins has been demonstrated in practically all kinds of eukaryotic cells. During the clevage period of sea urchins it has been postulated that histones are not present in nuclei, and that only at blastula stage they do associate with DNA. Our results suggest that the failure to isolate histonic proteins from nuclei derived from cleavage cells might be caused by the presence in those cells of proteases activated by NaHSO3. This compound has being widely used to inhibit proteolytic action in other biological systems. By changing the method for chromatin isolation, we have been able to isolate basic proteins from nuclei of gametes, zygotes and 2-4 blastomeres. These proteins behave like calf thymus histones in urea-acetic acid polyacrylamide gels, and they do not show great differences from proteins isolated from nuclei of blastula, gastrula, prism, and pluteus. The electrophoretic patterns of basic proteins obtained from eggs and zygotes are practically identical, except for one protein moving like lysine-rich calf thymus histones. This protein appears in zygote nuclei at the beginning of the first replication wave.