Isolation and characterization of a gene encoding DNA topoisomerase I in Drosophila melanogaster.

We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.     

Incomplete reversion of double stranded DNA cleavage mediated by Drosophila topoisomerase II: formation of single stranded DNA cleavage complex in the presence of an anti-tumor drug VM26.

Anti-tumor drug VM26 greatly stimulates topoisomerase II mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase II remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase II covalently linked to the nicked DNA. Chelation of Mg++ by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase II remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCl density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3, ..... topoisomerase II molecules bound per DNA molecule. Analysis of topoisomerase II/DNA complexes isolated from the CsCl gradient indicates that there are single stranded DNA breaks associated with the CsCl stable complexes. Therefore, topoisomerase II/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase II reactions.     

Confirmation of the human cathepsin B gene (CTSB) assignment to chromosome 8

Summary Human cathepsin B gene (CTSB) has been mapped to two locations: 8p22 and 13q14. Here we confirm the chromosome 8 assignment by three independent methods: (1) analysis of human-hamster somatic cell hybrid DNA by polymerase chain reaction; (2) comparison of hybridization signals to cathepsin B in interphase nuclei of normal fibroblasts and fibroblasts with a chromosome 8 deletion; and (3) fluorescence in situ hybridization to metaphase spreads using cathepsin B cosmid clones. Our results indicate that human CTSB is located at 8p22-p23.1.     

Shear-induced platelet-derived growth factor gene expression in human endothelial cells is mediated by protein kinase C.

Our previous studies have shown that steady shear stress causes a transient increase of platelet-derived growth factor (PDGF) A and B chain mRNA levels in human umbilical vein endothelial cells (HUVEC). In the present study, we elucidated the signaling pathway of shear stress in HUVEC by examining the roles of protein kineses, intracellular calcium, cyclooxygenase, and guanine nucleotide-binding proteins (G proteins) in the PDGF gene induction by shear. The protein kinase C inhibitors, H7 and staurosporine, strongly inhibited the shear-induced PDGF gene expression in HUVEC. In contrast, HA1004, a cAMP- and cGMP-dependent protein kinases inhibitor, was only slightly inhibitory. BAPTA/AM, an intracellular calcium chelator, partially (50%) inhibited the shear-induced PDGF gene expression. The cyclooxygenase inhibitors, ibuprofen and indomethacin, were slightly inhibitory. A 35-50% inhibition of shear-induced PDGF gene expression was found with GDP-beta-S, an inhibitor of G proteins. These results suggest that shear-induced PDGF gene expression in HUVEC is mainly mediated by protein kinase C activation and requires intracellular calcium. Furthermore, G proteins seem to be involved in this process, whereas prostaglandin synthesis via cyclooxygenase pathway is not. We propose a mechanism of shear-induced PDGF gene expression in HUVEC: Shear stress, either directly or indirectly (G protein-mediated), enhances the membrane phosphoinositide turnover via phospholipase C, producing diacylglycerol, an activator of protein kinase C. The activated protein kinase C then triggers the subsequent PDGF gene expression.     

Production of extracellular and cell-associated biopolymers byPseudomonas atlantica

Summary Pseudomonas atlantica is shown to produce both cell-associated and free forms of polymer in a partially growth associated manner. The gross characteristics of the polymer were independent of the physiological state of the culture.     

Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor

Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acysyst-P^® (Endotronic) with a total fiber surface area of 7.2 m² (6×1.2 m²) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics-and serum-free IMDM medium, supplemented with 50g/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20–40 g protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.     

Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts of Aspergillus parasiticus.

A cell-free extract, prepared from Aspergillus parasiticus ATCC 15517 grown in synthetic medium, was active in converting [14C]sterigmatocystin into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate. The activity was demonstrated by the time course of conversion and the linear dependence of the yield of product on enzyme concentrations. Optimum activity was obtained at pH 7.5 to 7.8 at 27 C. The results confirm sterigmatocystin as a biogenetic precursor of aflatoxin B1. Techniques were developed for enzymatic studies on aflatoxin biosynthesis.     

Studies on the female-sterile mutant rudimentary of Drosophila melanogaster,: I. An analysis of the rudimentary wing phenotype

The rudimentary wing phenotype was examined in detail, using six different alleles of rudimentary, and a number of points about the genesis of the r phenotype were made. (1) All of the r alleles in which the wings are defective produce wings in which the area of individual hair cells is reduced. The more severely affected the allele, the greater is the reduction in wing cell area. This reduction in area is probably uniform throughout the wing rather than localized to specific wing regions. (2) The total number of cells per wing is also greatly reduced in phenotypically r wings. As with cell area, the more severely affected the allele, the greater the reduction in cell number. However, the reduction in cell number is not uniform throughout the wing. In the less severely affected alleles, the cell number reduction is much greater in those regions of the wing which are drastically altered in shape (truncated), while those wing regions which show only slight size reductions but no overall shape changes have near normal numbers of cells. In the most deformed wings, there is a reduction in cell number throughout the wing, but again those regions with are severely truncated are the most drastically reduced in cell number. Measurements of the amount of chitin per wing indicated that the three most severely affected alleles had as much or more chitin than the wild type. It is suggested that overproduction of chitin in these alleles prevents normal expansion of the wing cells, thus increasing the severity of the wing defect. Finally, the validity and limitations of a quantitative measure of the r phenotype were defined. This measure was utilized to demonstrate a clear-cut effect of nutrition on the expression of the r phenotype.