Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.     

Compact structure of ribosomal chromatin in Xenopus laevis.

Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X. laevis. It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin. Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion. A greater accessibility of the coding region in comparison to the non-coding spacer was found. In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight. Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion. The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation.     

Nucleosome structure.

Electron microscopic and biochemical results are presented supporting the following conclusions: (1) Two molecules of each histone H2A, H2B, H3 and H4 are necessary and sufficient to form a nucleosome with a diameter of 12.5 +/- 1 nm and containing about 200 base pairs of DNA. (2) H3 plus H4 alone can compact 129 +/- 8 DNA base pairs into a sub-nucleosomal particle with a diameter of 8 +/- 1 nm. In such a particle the DNA duplex is under a constraint equivalent to negative superhelicity. (3) Chromatin should be viewed as a dynamic structure, oscillating between a compact structure (the nucleosome) and more open structures, depending on the environmental conditions.     

Ophirasterol, a new C31 sterol from the marine sponge Topsentia ophiraphidites

Analysis of the sterol fraction obtained from the Colombian Caribbean sponge Topsentia ophiraphidites revealed that this sponge is a rich source of C30 and C31 sterols. Among them, a new C31 sterol, named ophirasterol, was isolated, and its structure was established as (22E,24R)-24-(1-buten-2-yl)cholesta-5,22-dien-3beta-ol (1) by spectral means and comparison with synthetic C-24 epimers with known configuration. Other isolated C30 and C31 sterols were the known 24-ethyl-24-methyl-22-dehydrocholesterol (2), 24-isopropyl-22-dehydrocholesterol (3), 24-isopropylcholesterol (4), 24-ethyl-24-methylcholesterol (5), 24-isopropenyl-25-methyl-22-dehydrocholesterol (6) and 24-isopropenyl-25-methylcholesterol (7), and 24-isopropenyl-22-dehydrocholesterol (8).     

Prospective study of BKV nephropathy in 117 renal transplant recipients

Nephropathy caused by poliomavirus (BKVAN) in transplant recipients is responsible for the loss of the transplanted organ. In this study we suggest a non-invasive diagnostic protocol for the early identification of BKVAN during follow-up treatments. In 117 kidney transplant recipients follow-up was performed every three months during a two year period after transplantation and a positive screening result was confirmed and assessed by quantitative assays (BKV DNA load in plasma and urine). The definitive diagnosis of BKV requires allograft biopsy. Of the 117 patients 4 had BKVAN (3.4%), and the consequential reduction of immunosuppression improved kidney function and plasma clearance of the virus was achieved.     

Inheritance and development of molecular markers linked to angular leaf spot resistance genes in the common bean accession G10909

Angular leaf spot (ALS) is one of the major diseases of the common bean (Phaseolus vulgaris L.). Different sources of resistance have been identified but few have been characterized. Studies were conducted to elucidate the inheritance of ALS resistance in the bean accession G10909 and to identify molecular markers linked to these genes. Evaluation of parental genotypes, F₁, F₂ and backcross to susceptible parent (Sprite) populations revealed that two dominant and complementary genes conditioned ALS resistance. Allelism tests showed that the ALS resistance genes in G10909 were different from those in the Mesoamerican cultivars Mexico 54, MAR 2, G10474 and Cornell 49-242. Three sequence-characterized amplified region (SCAR) markers, PF13₃₁₀, PF9₂₆₀ and OPE04₇₀₉, and a microsatellite, Pv-gaat001, segregated in coupling with the resistance genes in G10909. Pairwise segregation analysis revealed that markers PF13₃₁₀, PF9₂₆₀ and OPE04₇₀₉ were linked, while Pv-gaat001 segregated in a 9:3:3:1 ratio from all markers. Markers PF13₃₁₀, PF9₂₆₀ and OPE04₇₀₉ were mapped to linkage group B08, and segregated with resistance gene Phg _G10909B at 4.9, 7.4 and 9.9 cM, respectively. Pv-gaat001, previously mapped to linkage group B04, segregated with resistance gene Phg _G10909A at 13 cM. The potential utility of these markers to aid breeding for ALS resistance is discussed.     

ATM-dependent CHK2 activation induced by anticancer agent, irofulven

Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semisynthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor types. Mechanisms of action studies indicate that irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in ovarian cancer cells, CHK2 kinase is activated by irofulven while CHK1 kinase is not activated even when treated at higher concentrations of the drug. By using GM00847 human fibroblast expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd), it was demonstrated that ATR kinase does not play a major role in irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by irofulven is mediated by the upstream ATM kinase. Phosphorylation of ATM on Ser(1981), which is critical for kinase activation, was observed in ovarian cancer cell lines treated with irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing siRNA for ATM. Finally, experiments done with human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing kinase-dead CHK2 in an ovarian cancer cell line demonstrated that CHK2 activation contributes to irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on serine 20 is dependent on CHK2 after irofulven treatment. In summary, we found that the anticancer agent, irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by irofulven.     

A site of discontinuity in the interaction between DNA and histones in nucleosomes of sea urchin embryo chromatin.

Digestion of chromatin DNA in nuclei of sea urchin embryos with pancreatic nuclease and with micrococcal nuclease give additional details concerning the interaction between DNA and histones. A specific site of hydrolysis appears to be located on the nucleosome in such a position as to split the DNA unit length in two equivalent fragments of about 60–70 base pairs in length. The complete digestion of chromatin DNA appears to depend on the low stability of the nucleosome containing the split DNA fragments.