Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex.

Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-kDa subunit of RPA. Rpa4 associates with the 70- and 13-kDa subunits to form a trimeric complex capable of binding to single-stranded DNA. Rpa4 is preferentially expressed in placental and colon mucosa tissues. In the placenta, Rpa4 is more abundant than the 70-kDa Rpa1 subunit and is not associated with either Rpa1 or with any other single-stranded DNA-binding protein. In proliferating cells in culture, Rpa4 is considerably less abundant than Rpa1 and Rpa2. Northern (RNA) blot analysis suggest that there are alternatively processed forms of the RPA4 mRNA, and Southern blot analysis indicates that beside RPA4 there may be other members of the RPA2 gene family.     

Assembly of nucleosomal DNA in a cell-free extract from wild-type and top1− strains ofUstilago maydis

An in vitro nucleosome assembly system has been established from cell-free extracts of the fungusUstilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as ATP and MgCl₂ and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared fromSaccharomyces cerevisiae andCandida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain ofU. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1^? extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation.     

Synthesis properties of the naturally occurring N-[(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl]-L-threonine (mt-6A) and other related synthetic analogs.

The naturally occurring modified nucleoside, N-[(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl]-L-threonine (mt6A), and the corresponding glycine analog mg6A were synthesized from N6-methyl-2',3',5'-tri-O-acetyladenosine and the appropriately blocked isocyanates derived from threonine and glycine. The natural mt6A isolated from Escherichia coli tRNA (F. Kimura-Harada et al. (1972), Biochemistry 11, 3910), from wheat embryo tRNA (R. Cunningham and M. W. Gray (1974), Biochemistry 13, 543), and from rat liver tRNA (Rogg et al. (1975), Eur. J. Biochem. 53, 115) was found to be identical with the synthetic mt6A in paper and thin-layer chromatography and electrophoresis. Several analogs of the parent 6-ureidopurine ribonucleoside, N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]-L-thronine (t6A), were also prepared. Starting from 2',3',5'-tri-O-acetylguanosine and 2',3',5'-tri-O-acetylcytidine and the above isocyanates, the t6A analogs, N-[(9-beta-D-ribofuranosyl-6-oxo-1H-purin-2-yl)carbamoyl]-L-threonine (t2G) and N-[(1-beta-D-ribofuranosyl-2-oxypyrimidin-4-yl)carbamoyl]-L-threonine (t4C), were prepared. Also synthesized were the corresponding glycine analogs, g2G and g4C, from guanosine and cytidine, respectively. The 2'-deoxyribosyl analog, N-[(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)carbamoyl]-L-threonine (2'-deoxy-t6A), and the arabinosyl derivative, N-[(9-beta-D-arabinofuranosylpurin-6-yl)carbamoyl]-L-threonine (t6AraA), were synthesized from the appropriate urethane and the requisite amino acid. The ureido group in mt6A could not be hydrolyzed by the enzymes urease, peptidase, and protease. Various chemical and biological properties of the naturally occurring mt6A and the related analogs are discussed.     

Serum alkaline phosphatase and lactic dehydrogenase activity in cows with retained fetal membranes

Serum alkaline phosphatase (AKP) and lactic dehydrogenase (LDH) activities were determined for 15 cows with retained fetal membranes (RFM) and 15 cows without retained fetal membranes (WRFM). The results revealed that the levels of both AKP and LDH were significantly higher in cows with RFM during late gestation and continued to be higher until 5th day postpartum. The usefulness of these findings for the prediction of RFM before parturition and for evaluating the therapeutic response of RFM is discussed.     

Studies on some fungi isolated from the rhizosphere of tomato plants and the consequent prospect for the control of Verticillium wilt

Summary Biological control of Verticillium wilt disease with antagonistic micro-organisms was studied. Antagonism of some fungi, isolated from tomato rhizosphere, toVerticillium albo-atrum R & B. was observedin vitro. A clearly defined zone, in which the growth of the pathogen was inhibited, was observed withPenicillium spp. (includingPenicillium chrysogenum Thom) andFusarium culmorum (S.G. Sm) Sacc., whileTrichoderma viride pers. ex Fries,Gliocladium spp. andPenicillium vermiculatum Dangeard, suppressed the growth ofV. albo-atrum by penetrating, and overgrowing it. OnlyT. viride andP. vermiculatum culture filtrate added to the Dox's agar, reduced the radial growth ofV. alboatrum. Root-dip application of culture filtrates ofT. viride andP. chrysogenum was found to be most effective in controlling the disease, followed by other species ofPenicillium andGliocladium spp. WhileFusarium culmorum provided no control. Improvement of plant height and vigour with a better yield due to culture filtrate treatment occurred. Root-dip application of antagonistic fungal propagules (T. viride, P. chrysogenum) to tomato seedlings was also very effective in controlling wilt in tomato plants grown inV. albo-atrum infested soil. Dedicated to the memory of the late Prof. Ivor Isaac with whom I had the pleasure of working     

Partial characterization of transfer RNA genes isolated from Neurospora crassa

Summary Purified DNA sequences that code for tRNA in Neurospora crassa were isolated and partially characterized. The tRNA cistrons comprise about 0.3 percent of the N. crassa genome. The tRNA:tDNA hybrids showed a buoyant density in cesium sulfate of 1.48 g cm^-3 and sedimented in an intermediate position between native DNA and tRNA of N. crassa as expected. Te 50 of hybridized tRNA:tDNA molecules, reassociated tDNA: native DNA and homoduplexes of native DNA were 83.0°C, 88.5°C and 89.5°C respectively from thermal stability studies using hydroxyapatite chromatography. The isolated tRNA cistrons react almost completely with unlabeled DNA and tRNA of N. crassa or tRNA of the slime mutant of N. crassa; but react poorly with either ribosomal RNA or ribosomal RNA cistrons of N. crassa and tRNA of Escherichia coli. It appears that tRNA genes of N. crassa are repeated.This research was supported from grants from U.S. Atomic Energy Commission No. At(40-1)-4182 and the Anna Fuller Fund, New Haven, Connecticut to S.K.D. we are grateful to Dr. J. White, Director of H.U. Cancer Res. Center for help.     

Flavonoid constituents of Eupatorium odoratum

Isosakuranetin and a new chalcone, odoratin, have been isolated from the leaves of Eupatorium odoratum. The structure of odoratin has been shown to be 2′-hydroxy-4,4′,5′,6′-tetramethoxy chalcone.     

Enhanced uptake and metabolism of riboflavin in erythrocytes infected with Plasmodium falciparum

Riboflavin deficiency inhibits the growth of malaria parasites both in vitro and in vivo in infected animals and humans. Although the precise mechanisms underlying this inhibition are unknown, they may involve enhanced requirements for riboflavin by parasites. To investigate this possibility, the rate of uptake of [14C]riboflavin and the biosynthesis of FMN and FAD from riboflavin were studied in infected (5-8% parasitemia) and uninfected human erythrocytes. All cells were incubated for 0-3 h at 37 degrees C in phosphate buffered saline containing MgCl2, glucose, and [14C]riboflavin (2.5-7.5 microM). At hourly intervals, samples were removed, centrifuged, washed twice with cold buffer, and lysed before counting the radioactivity. The rate of in vitro biosynthesis of FMN and FAD from riboflavin in erythrocytes was measured by ion exchange chromatography and reverse isotope dilution techniques. Results showed that the rate of riboflavin uptake and the biosynthesis of FMN and FAD were enhanced in erythrocytes with parasitemia as compared with results in unparasitized erythrocytes. Riboflavin uptake in erythrocytes was proportional to the extent of parasitemia and especially to percent of schizonts present in erythrocytes. These studies indicate that the requirement for riboflavin may be greater in the parasite than in the host erythrocyte. This increased riboflavin requirement may be due to rapid multiplication, higher metabolic rate, and extreme vulnerability to oxidative stress of malaria parasites compared with that of host erythrocytes. The differential requirement of riboflavin by the host and the malaria parasite may hold important potential for developing new strategies for malaria chemotherapy.