DNase I and II present in avian oocytes: a possible involvement in sperm degradation at polyspermic fertilisation

During polyspermic fertilisation in birds numerous spermatozoa enter the eggs, in contrast to the situation in mammals where fertilisation is monospermic. However, in birds only one of the spermatozoa which have entered an egg participates in zygote nucleus formation, while the supernumerary spermatozoa degenerate at early embryogenesis. Our previous work has demonstrated the presence in preovulatory quail oocytes of DNase I and II activities able to digest naked lambdaDNA/HindIII substrate in vitro. In the present studies, the activities of both DNases in quail oocytes at different stages of oogenesis and in ovulated mouse oocytes were assayed in vitro using the same substrate. Degradation of quail spermatozoa by quail oocyte extracts was also checked. Digestion of the DNA substrate was evaluated by electrophoresis on agarose gels. The activities of DNase I and II in quail oocytes increased during oogenesis and were the highest in mature oocytes. The activities were present not only in germinal discs but also in a thin layer of cytoplasm adhering to the perivitelline layer surrounding the yolk. At all stages of oogenesis the activity of DNase II was much higher than that of DNase I. DNA contained in spermatozoa was also degraded by the quail oocyte extracts under conditions optimal for both DNases. In contrast to what is observed in quail oocytes, no DNase activities were detected in ovulated mouse eggs; this is logical as they would be useless or even harmful in monospermic fertilisation. The possible role of DNase activities in avian oocytes, in degradation of accessory spermatozoa during polyspermic fertilisation, is discussed.     

Calcium protects DNase I from proteinase K: a new method for the removal of contaminating RNase from DNase I

DNase I and proteinase K are two enzymes commonly used in the purification of highly polymerized RNA. In the presence of EDTA DNase I is rapidly inactivated by proteinase K while in 10 mm Ca²⁺ DNase is totally immune to proteinase K inactivation even at protease concentrations of up to 1 mg/ml. RNase A, a common contaminant of “RNase-free” DNase was inactivated by proteinase K in the presence or absence of Ca²⁺. Treatment of DNase I with proteinase K in the presence of Ca²⁺ selectively removed RNase A activity as judged by rRNA and poly(A⁺ RNA ribosomal RNA degradation monitored by sucrose gradient centrifugation. These results suggest that (i) DNase A and proteinase K can be used together in the presence of Ca²⁺ to obtain better digestion of nucleoprotein complexes, and (ii) proteinase K treatment of Ca²⁺ DNase can be used to selectively remove contaminating RNase.     

Coexistence ofTetranychus urticae with different diapause capacities: a mathematical model

JapaneseTetranychus urticae is highly variable in diapause traits both among populations from different localities and host plants, as well as within populations. Many southern populations have almost lost their diapause capacity, and those from central Japan (34–37°N) are a mixture of diapausing (DP) and non-diapausing (ND) individuals. A simple mathematical model was constructed for analyzing the conditions under which the ND is more favourable than the DP in a system consisting of two different microhabitats: L, in which winter is lethal for the ND, and O, in which some of the ND can overwinter successfully. The model suggests that if winter mortality is not very high for the ND, and the annual reproductive rate of the ND is higher than that of the DP in microhabitat L, then a higher dispersal rate of the mites from L to O, and an equal number of patches of the two microhabitats will favour the ND more than the DP.This study was supported in part by a Grant-in Aid (Bio Cosmos Program BCP 92-I-B-3) from the Ministry of Agriculture, Forestry and Fisheries, Japan.     

The distribution of cofilin and DNase I in vivo

Actin is the principal component of the cytoskeleton, a structure that can be disassembled and reassembled in a matter of seconds in vivo. The state of assembly of actin in vivo is primarily regulated by one or more actin binding proteins (ABPs). Typically, the actions of ABPs have been studied one by one, however, we propose that multiple ABPs, acting cooperatively, may be involved in the control of actin filament length. Cofilin and DNase I are two ABPs that have previously been demonstrated to form a ternary complex with actin in vitro. This is the first report to demonstrate their co-localisation in vivo, and differences in their distributions. Our observations strongly suggest a physiological role for higher order complexes of actin in regulation of cytoskeletal assembly during processes such as cell division.     

The distribution of cofilin and DNase I in vivo

Actin is the principal component of the cytoskeleton,a structure that can be disassembled and reassem-bled in a matter of seconds in vivo.The state of assembly of actin vivo is primarily regulated by one or more actin binding proteins(ABPs).Typically,the actions of ABPs have been studied one by one,however,we propose that multiple ABPs ,acting cooperatively,may be involved in the control of actin filament length.Cofilin and Dnase Iare two ABPs that have previously been demonstrated to form a ternary complex with actin in vitro.This is the first report to demonstrate their co-localisation in vivo,and differences in their distributions.Our observations strongly suggest a physiological role for higher order complexes of actin in regulation of cytoskeletal assembly during processes such as cell division.     

The distribution of cofilin and DNase I in vivo

Actin is the principal component of the cytoskeleton, a structure that can be disassembled and reassembled in a matter of seconds in vivo. The state of assembly of actin in vivo is primarily regulated by one or more actin binding proteins (ABPs). Typically, the actions of ABPs have been studied one by one, however, we propose that multiple ABPs, acting cooperatively, may be involved in the control of actin filament length. Cofilin and DNase I are two ABPs that have previously been demonstrated to form a ternary complex with actin in vitro. This is the first report to demonstrate their co-localisation in vivo, and differences in their distributions. Our observations strongly suggest a physiological role for higher order complexes of actin in regulation of cytoskeletal assembly during processes such as cell division.     

DNase I footprinting of the nucleosome in whole nuclei

DNase I was used to footprint the 147 bp DNA fragment of the nucleosome in whole chicken erythrocyte nuclei. It was found that the higher-order structure imposes an additional protection on nucleosomes at sites close to the entry and exit points of the linker DNA, around the dyad axis (site S 0). The observed protection is extended up to 20 bp on either side of S 0. It is partial (∼50%) and most probably reflects a full protection of different regions in alternatively oriented nucleosomes. These are the same regions which interact with linker histones. The results strongly support the findings by simulation of DNase I digests of unlabelled oligonucleosome fragments in the 30 nm fibre that in all nucleosomes sites S −5 to S −3 and S +3 to S +5 ara on the outside of the fibre exposed to DNase I.     

DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

Mapping DNase I hypersensitive sites is an accurate method of identifying the location of gene regulatory elements, including promoters, enhancers, silencers and locus control regions. Although Southern blots are the traditional method of identifying DNase I hypersensitive sites, the conventional manual method is not readily scalable to studying large chromosomal regions, much less the entire genome. Here we describe DNase-chip, an approach that can rapidly identify DNase I hypersensitive sites for any region of interest, or potentially for the entire genome, by using tiled microarrays. We used DNase-chip to identify DNase I hypersensitive sites accurately from a representative 1% of the human genome in both primary and immortalized cell types. We found that although most DNase I hypersensitive sites were present in both cell types studied, some of them were cell-type specific. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome.     

24-Hour dopamine-beta-hydroxylase pattern: a possible biological index of manic-depression

Intravenous administration of the neurotoxic agent, 6-hydroxydopa, to mice lowered both epinephrine and norepinephrine concentrations in the brainstem. Pretreatment with the tricyclic antidepressant drugs, imipramine, iprindole, and protriptyline, differentially blocked these depletions. Imipramine and protriptyline blocked both the effects on epinephrine and norepinephrine, although higher doses were required to protect epinephrine. Iprindole selectively blocked epinephrine depletions.     

C-value paradox in angiosperm plant species. I. Sensitivity to DNase I in species with different 2C DNA content.

The experimental conditions for DNAase I digestion in situ for plant nuclei have been presented. Cytophotometric measurements of DNA loss performed on Feulgen-stained nuclei of three species differing in 2C DNA, heterochromatin and condensed euchromatin contents have shown that the lower 2C DNA amount the higher is DNase I sensitivity. Heterochromatin and some fractions of euchromatin are DNase I resistant. Microdensitometric measurements along M chromosome in Vicia faba have demonstrated the sites hypersensitive to DNase I.     

A single amino acid substitution can shift the optimum pH of DNase I for enzyme activity: biochemical and molecular analysis of the piscine DNase I family

We purified four piscine deoxyribonucleases I (DNases I) from Anguilla japonica, Pagrus major, Cryprus carpio and Oreochromis mossambica. The purified enzymes had an optimum pH for activity of approximately 8.0, significantly higher than those of mammalian enzymes. cDNAs encoding the first three of these piscine DNases I were cloned, and the sequence of the Takifugu rubripes enzyme was obtained from a database search. Nucleotide sequence analyses revealed relatively greater structural variations among the piscine DNase I family than among the other vertebrate DNase I families. From comparison of their catalytic properties, the vertebrate DNases I could be classified into two groups: a low-pH group, such as the mammalian enzymes, with a pH optimum of 6.5-7.0, and a high-pH group, such as the reptile, amphibian and piscine enzymes, with a pH optimum of approximately 8.0. The His residue at position 44 of the former group is replaced by Asp in the latter. Replacement of Asp44 of piscine and amphibian DNases I by His decreased their optimum pH to a value similar to that of the low-pH group. Therefore, Asp44His might be involved in an evolutionarily critical change in the optimum pH for the activity of vertebrate DNases I.     

Site of attachment of mercuribenzoate in crystals of an actin:DNase I complex.

Crystals of a complex of chicken gizzard G-actin and DNase I were soaked in a solution of radioactive 4-hydroxymercuribenzoate (MB). The soaked crystals, which contained 0.93 mol of MB per mol of G-actin, were dissolved in "G-buffer" and digested with trypsin, and the resulting peptides were fractionated by thin-layer chromatography. The MB is exchangeable between peptides that contain cysteine residues, but the data obtained here suggested that MB attached to the cysteine residue at the 373rd position of the G-actin molecule.