Structure of DNA in metaphase chromosomes of mouse fibroblasts.

We show that N-1 in adenine of chromosomal DNA is methylated by treatment of metaphase chromosomes with dimethylsulphate while this is not the case in chromatin. The data on methylation are consistent with those obtained from the experiments with S1-nuclease treatment of chromatin and chromosomes. This suggests a disarrangement of DNA secondary structure in the metaphase chromosomes.

Localization of proteins forming the outer surface of isolated metaphase chromosomes.

The outer surface of isolated metaphase chromosomes has been investigated by a method of thermally activated tritium labelling. We show that both chromosomal proteins and DNA are tritium-labelled. Fractionation of the chromosomal proteins reveals that scaffold proteins are the most labelled in condensed and EDTA-decondensed chromosomes. Exposition of some scaffold proteins on the outer surface of metaphase chromosomes is suggested.

Juvenile hormone signaling during oogenesis in Drosophila melanogaster.

Juvenile hormone (JH) participates both in the control of insect development and the establishment of reproductive maturity. In cultured Drosophila cells and in ovarian nurse cells, JH and its synthetic analog, methoprene, induce the expression of two related genes. These genes encode highly similar amino acid transport proteins that are homologous to transporters found in a variety of eukaryotes. JhI-21 is a novel Drosophila gene, and minidiscs (mnd) is a gene that was identified earlier. Two JH-inducible genes are regulated by different molecular mechanisms; JhI-21 behaves as a secondary JH-responsive gene, while mnd behaves as a primary responsive gene. Both JhI-21 and mnd transcripts show developmental profiles, which are consistent with JH regulation. Following eclosion, transcripts from JhI-21 and mnd are synthesized in ovarian nurse cells and subsequently sequestered in the mature egg. Their ectopic accumulation in ovaries can be induced by topical methoprene application. In apterous (ap4) mutant adults defective in JH secretion, mnd and JhI-21 RNA levels are severely reduced, but normal abundance is rescued to a high degree by topical methoprene treatment. Based on the evidence, we propose that during sexual maturation of Drosophila, JH provides a signal to the ovary that leads to the production of several maternally inherited mRNAs.

Selective binding of Drosophila BR-C isoforms to a distal regulatory element in the hsp23 promoter.

The Broad-Complex (BR-C) gene plays a key role in the ecdysone regulatory hierarchy. Together with other early ecdysone-inducible genes BR-C transmits the hormonal signal to a set of secondary response genes in a tissue-specific manner. Among its targets is the hsp23 gene. Previously we showed that expression of the hsp23 gene in late third instar is BR-C-dependent, and accompanied by the appearance of a BR-C-dependent DNase I hypersensitive site at position -1400 (DHS-1400). BR-C encodes a family of transcription factors, and we show here that at least three BR-C protein isoforms--Z1, Z2, and Z3--bind to the sequences around DHS-1400 in vitro. A DNase I footprinting assay reveals five protected regions, designated site 1 to site 5, each of which specifically associates with one or several BR-C protein isoforms. We also show that a 100 bp region overlapping site 5, which binds all three isoforms in vitro, is required for hsp23 activity in vivo. The deletion of binding site 5 in a reporter gene construct reproduced the effect of the npr class mutations, that is, hsp23 is no longer expressed in any tissue tested except brain. Thus, BR-C regulates hsp23 expression via direct interaction of the predominant isoform with the distal regulatory element.

Effects of Cr(VI) on the expression of the oxidative stress genes in human lung cells.

Intracellular metabolism of chromium(VI) [Cr(VI)] may lead to oxidative stress and this may account for the ability of Cr(VI) to act as a complete carcinogen. Therefore, we examined the effects of Cr(VI) treatment on the expression of oxidative stress genes in normal human lung LL 24 cells and human lung adenocarcinoma A549 cells. RT-PCR and northern blot analyses were used to determine the steady-state mRNA levels of catalase, glutathione S-transferase, glutathione reductase, Cu/Zn- and Mn-superoxide dismutases, glutathione peroxidase, NAD(P)H:quinone oxidoreductase, heme oxygenase and interleukin 8 in control cells and cells treated with 5-200 microM of Cr(VI). We found that only expression of the heme oxygenase gene is strongly elevated under the treatment with Cr(VI), and only in normal human lung LL 24 cells. Our data showed that even in the absence of Cr(VI) treatment, the level of heme oxygenase gene expression is much higher in A549 cells than in LL 24 cells. As glutathione is believed to play a protective role in cells against different forms of oxidative stress, we studied the correlation between intracellular glutathione levels and the inducibility of the heme oxygenase gene after treatment of cells with Cr(VI). Our results demonstrate that glutathione levels are increased by 35 % of control values in LL 24 cells treated with Cr(VI). The data obtained indicate that heme oxygenase, known to be a stress-inducible gene, may be involved in cellular pathways critical to the carcinogenic activity of Cr(VI) in normal human lung cells. Intracellular glutathione levels and reactive oxygen species do not appear to be primarily responsible for the stress response, induced by Cr(VI) in the studied human cells.

The isolation of two juvenile hormone-inducible genes in Drosophila melanogaster.

Juvenile hormone (JH) is an important regulator of both insect development and reproductive maturation. Although the molecular mechanism of JH action is not yet known, there is growing circumstantial evidence that JH directly regulates gene expression. In the absence of a JH target gene, however, this suggestion has remained speculative. Cultured Drosophila S2 cells have been used to identify genes whose expression is regulated by JH. Employing differential display we identified several genes whose transcripts accumulate in cells treated with the JH agonist methoprene. Two of the genes-JhI-1 and JhI-26-were cloned and characterized in detail. For both genes, transcripts showed rapid and specific induction in the presence of either methoprene or JHIII, but not in the presence of other biologically inactive compounds of similar chemical structure. Accumulation of JhI-1 and JhI-26 RNAs requires continuous hormone presence. The developmental expression of the two JH-inducible genes corresponds to the abundance profile of JH in vivo. Furthermore, topical methoprene application to pupae leads to the ectopic accumulation of JhI-1 and JhI-26 transcripts.

Membrane-mediated changes in the structure of chromosomes.

In the present paper the interaction of metaphase chromosomes and chromatin with model and natural lipid membranes was studied. It was shown that chromatin and chromosomes are able to form complexes with membranes in the presence of divalent cations. In such complexes, the typical structure of chromosomes is altered. The character of this alteration in chromosomal structure was investigated with the use of electron microscopy and chemical modification with dimethylsulphate (DMS). The latter is possible because, according to the presented data, the condensation of chromatin into chromosomes is associated with a decrease in accessibility of N-3 in adenine (the protection of the minor groove of DNA) to modifications, and with an increased methylation of N-1 in adenine (the disarrangement of the secondary structure of DNA). It was shown that the interaction of chromosomes with liposomes provides various levels of unfolding up to the appearance of chromatin-like structures. The secondary DNA structure of decondensed chromosomes coincides with the secondary structure of chromosomal but not chromatin DNA, whereas the extent of shielding of the minor groove of DNA in such decondensed structures typical for chromatin DNA. It is possible to suggest that the chromosomal decondensation in telophase of mitosis is initiated by the action of a membrane component of the developing nuclear envelope.