Adult chicken alpha-globin gene expression in transfected QT6 quail cells: evidence for a negative regulatory element in the alpha D gene region.

The chicken adult alpha-globin genes, alpha A and alpha D, are closely linked in chromosomal DNA and are coordinately expressed in vivo in an approximate 3:1 ratio, respectively. When subcloned DNAs containing one or the other gene are stably transfected into QT6 quail fibroblasts, the alpha A-globin gene is expressed at measurable RNA levels, but the alpha D gene is not. The alpha A gene expression can be considerably increased by the presence of a linked Rous sarcoma virus long terminal repeat enhancer, but that of the alpha D gene remains undetectable. Transfection with subclones containing both genes, either in cis or in trans, leads to considerably reduced alpha A RNA levels and still no observable alpha D gene expression. Transfection with deleted subclones suggests that maximal expression levels in this system require the alpha A-globin gene promoter, as opposed to that of the alpha D gene, but that such expression is greatly reduced by one or more DNA sequences which lie approximately 2,000 base pairs upstream of the alpha A gene, within the body of the alpha D gene.     

Function of the upstream hypersensitive sites of the chicken beta-globin gene cluster in mice.

We have shown previously that the chicken beta A-globin gene, with its 3' enhancer, is expressed in a copy number-dependent manner in transgenic mice. The expression level was low but increased approximately 6-fold upon inclusion of 11 kb of upstream DNA containing four DNase I hypersensitive sites. To study the effect of the individual upstream hypersensitive sites on transgene expression, we produced lines of mice in which the individual upstream sites were linked to the beta A gene and enhancer. RNA levels were measured in blood from adult animals. With each of these four constructs, the level of transgene RNA per DNA copy varied over a > 20-fold range. These data suggest that addition of a hypersensitive site to the beta A-globin/enhancer region abrogates its position independent expression. The average beta A-globin expression per copy in the lines carrying an upstream site was comparable with that in lines without an upstream site. Thus, no single upstream hypersensitive site accounts for the higher level of beta A-globin expression seen in mice containing the complete upstream region. We had shown previously that control of the chicken beta-globin cluster is distributed between at least two regions, the beta A/epsilon enhancer and the upstream region. Our current results suggest that the control mediated by the upstream DNA is itself distributed and is not due to a single hypersensitive site.     

A biochemical analysis of topoisomerase II alpha and beta kinase activity found in HIV-1 infected cells and virus

Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (alpha) and topoisomerase II beta (beta). The alpha isoform is localized predominantly in the nucleus, while the beta isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II alpha, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II beta. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II alpha kinase activity after 8h p.i and topoisomerase beta kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II alpha and beta kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II alpha and beta, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II alpha. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II alpha and beta. The catalytic inhibitions by serine kinase inhibitors further suggest that the alpha and beta kinase activities associated with virus are distinctly different.     

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells.

We have studied the effect of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. We tested inhibitors of DNA polymerase alpha and delta (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topoisomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, we tested the effect of the potential topoisomerase I activator, beta-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; beta-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair.     

Disruption of a DNA topoisomerase I gene affects morphogenesis in Arabidopsis

The genesis of phyllotaxis, which often is associated with the Fibonacci series of numbers, is an old unsolved puzzle in plant morphogenesis. Here, we show that disruption of an Arabidopsis topoisomerase (topo) I gene named TOP1alpha affects phyllotaxis and plant architecture. The divergence angles and internode lengths between two successive flowers were more random in the top1alpha mutant than in the wild type. The top1alpha plants sporadically produced multiple flowers from one node, and the number of floral organ primordia often was different. The mutation also caused the twisting of inflorescences and individual flowers and the serration of leaf margins. These morphological abnormalities indicate that TOP1alpha may play a critical role in the maintenance of a regular pattern of organ initiation. The top1alpha mutant transformed with the RNA interference construct for TOP1beta, another topo I gene arrayed tandemly with TOP1alpha, was found to be lethal at young seedling stages, suggesting that topo I activity is essential in plants.     

Topoisomerase I is essential in Cryptococcus neoformans: role In pathobiology and as an antifungal target

Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, gamma-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.     

Characterisation of chicken erythroid nuclear proteins which bind to the nuclease hypersensitive regions upstream of the beta A- and beta H-globin genes.

Chicken erythrocyte sequence-specific nuclear DNA-binding proteins, which bind to the 5'-flanking DNAseI hypersensitive sites of the erythrocyte chromosomal beta A- and beta H-globin genes, have been fractionated by HPLC gel filtration. Three beta A-globin gene DNA binding activities (to sites A, B and B' (10-12)) were separated. The erythroid precursor cell line HD3 has beta A-globin gene sites B and B' binding activities, but binding to site A is detected only after the HD3 cells are induced to differentiate. The fractionated protein binds to a redefined site B', which contains at its center the globin CACCC consensus sequence. The chromosomal beta H-globin gene has two 5'-flanking DNAseI hypersensitive sites which bracket two sequences (H and H') bound by erythrocyte and HD3 nuclear protein in vitro. The beta H- and beta A-globin gene binding sites (H and B) contain variants of the sequences bound by Nuclear Factor 1 and the TGGCA-binding protein, and their protein binding activity(ies) co-purify after HPLC gel filtration.     

Androgenic regulation of enzymes involved in DNA synthesis in epididymis of young rats.

In the epididymis of young rats, activities of DNA polymerases alpha, beta and gamma and DNA topoisomerase I decreased after castration. DNA polymerase alpha and gamma increased with androgen administration and activity reached 81.3% and 78.0%, respectively, of the activity in the sham-operated group on day 21. Activity of DNA polymerase beta remained at the activity of day 7 during androgen administration and was almost the same as that in the sham-operated group on day 21. DNA topoisomerase I activity showed a slight increase with androgen administration and reached 50.3% of that in the sham-operated group. The activities of these enzymes were not fully restored to those in the sham-operated group. These results indicate that in young rats activities of epididymal DNA polymerase alpha and gamma and DNA topoisomerase I are partially, and that of DNA polymerase beta wholly, dependent on androgens and may provide a means of investigating the regulation of epididymal cell proliferation.     

Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development.

The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.     

A nuclear factor which interacts with an AT-cluster in the first intron of rat alpha 2-macroglobulin gene

We have identified a nuclear protein which binds specifically to the first intron of rat alpha 2-macroglobulin gene. The protein became insoluble at low salt concentration retaining the binding specificity. Its molecular weight was estimated to be 22 kilodalton by a protein blotting procedure. The binding site of the protein determined by DNase I footprinting was an AT-strech which shared 80% homology with the cleavage consensus of Drosophila topoisomerase II.     

Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24.

Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.