Effect of R Factors on Rifampicm Resistance in <Emphasis Type="Italic">E. coli</Emphasis>

THE bactericidal effect of rifampicin, a semi-synthetic rifamycin, is due to its action on DNA-dependent RNA polymerase¹ and all rifampicin-resistant mutants of Escherichia coli contain an altered RNA polymerase with an increased resistance to rifampicin in vitro^2–4. While studying a possible curing effect of rifampicin on E. coli R factors, we observed that R⁺ recombinants of some rif-r mutants are more sensitive to rifampicin (Table 1). Of the cells harbouring certain R factors, less than 1% are able to form colonies on rifampicin-supplemented agar, while with certain others there is no detectable effect.     

Stereochemistry of Intercalation: Interaction of Daunomycin with DNA

DAUNOMYCIN^1–3, a glycosidic anthracycline antibiotic from Streptomyces peucetius⁴, is being used in the treatment of acute leukaemia and solid tumours in man^5,6. The biological activity seems to be due to complex formation with the DNA of deoxyribonucleoprotein⁴. In vivo, daunomycin inhibits both RNA and DNA synthesis^7,8 and, in vitro, DNA-dependent RNA polymerase and DNA polymerase^7–9.     

Mutant tRNA<Superscript>His</Superscript> Ineffective in Repression and Lacking Two Pseudouridine Modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons^1–4. Histidyl-tRNA^His has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium^5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNA^His as the wild type⁴ and in the one example sequenced, contains tRNA^HIS with a structure identical to that of the wild type⁸. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNA^HISin vitro². The amounts of charged and uncharged tRNA^His present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined⁹. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNA^Hisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNA^His. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNA^His) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNA^His which these mutants contain.     

RNA Polymerase III of <Emphasis Type="Italic">Blastocladiella emersonii</Emphasis> is Mitochondrial

MULTIPLE RNA polymerases have been shown to exist in a wide variety of eukaryotic organisms^1–5. Two nuclear polymerases have been found in all the cells studied, each with a specific location and a specific function: the DEAE fraction I enzyme is located in the nucleolus and may be involved in the synthesis of ribosomal RNA^1,2,5,6; the DEAE fraction II enzyme is located in the non-nucleolar nucleoplasm and functions in the synthesis of DNA-like RNA^2–5,7. The DEAE fraction III enzyme was reported to exist in sea urchin¹, the aquatic fungus B. emersonii⁵ and to be present sometimes in rat liver preparations^1,8. Although there have been some reports that polymerase III is nuclear, Horgen and Griffin⁵ showed that the enzyme was sensitive to the prokaryotic RNA polymerase inhibitor rifampicin. They suggested that the fraction III enzyme may be mitochondrial, formed as the result of organelle contamination in their crude nuclear preparations. The results of this study show that the DEAE fraction III enzyme in B. emersonii is a mitochondrial enzyme, most likely functioning in the synthesis of mitochondrial RNA. The rifampicin sensitivity of the enzyme is further evidence of a prokaryotic origin of mitochondria^9,10.     

Mutant Ribosomal Protein with Defective RNA Binding Site

THE 30S ribosomal subunits of Escherichia coli contain twenty-one different proteins^1–4, which together with 16S RNA can reassemble in vitro to form functional 30S particles⁵. Five proteins can individually bind to specific sites on the 16S RNA^6–8 and these are S4, S7, S8, S15 and S20 (in the nomenclature recently adopted by several laboratories to report results with the E. coli system⁹). We report here the first identification of a mutation that affects a ribosomal protein-nucleic acid interaction.     

On the Mechanism of Action of <Emphasis Type="Italic">lac</Emphasis> Repressor

Chen et al. have proved conclusively that lac repressor and RNA polymerase bind independently to wild type lac DNA in vitro. To explain the lacp ^s mutation, which causes competitive binding between repressor and polymerase, they suggest that a new promoter site has been created near the lac operator.     

Initial Nucleotide Frequencies of Bacterial RNA synthesized during Amino-acid Starvation or Changes of Carbon Source

THERE is growing evidence to indicate that RNA synthesis in bacteria is regulated through adjustment of the frequency of initiation of new RNA molecules. In this framework an understanding of the process of initiation of RNA synthesis takes on a special importance and for this reason we have investigated in varying conditions the composition of the 5′ terminal or first-inserted nucleotide. It has been previously shown that such initiations do not occur randomly, either in vivo¹ or in the proper conditions in vitro^2,3, but that RNA chains are exclusively initiated with the purines, adenosine and guanosine. Additionally, there was a recent suggestion based on in vitro studies that the nucleotide guanosine-3′-diphosphate-5′-diphosphate (MS1), proposed to be a regulatory agent in RNA synthesis, functioned by specifically depressing the frequency of initiation of a large fraction of RNA molecules beginning with guanosine⁴. Here we report, however, that in vivo, in conditions in which regulation of RNA synthesis is manifest, the ratio of molecules initiated with adenosine and guanosine is not changed.     

Lack of Discrimination between Early and Late T4 mRNA by Host Initiation Factors

DURING development of T4 phage in E. coli, control at the translational level may play an important part in switching the reading of early to late T4 messenger RNAs. In vitro experiments have shown that protein factors isolated from ribosomes of T4 infected cells can restrict translation of either host mRNA or R17 phage RNA, whilst permitting normal translation of late T4 mRNA^1–4. This alteration of specificity has been attributed to an initiation factor F3^5,6. It is unlikely that this switch is related to the shut-off of host protein synthesis which occurs immediately after infection^7,8, because they occur at distinctly different times in vivo^{1, 3}.     

Macrophage Functional Heterogeneity in the <Emphasis Type="Italic">in vitro</Emphasis> Induced Immune Response

RNA from antigenically stimulated peritoneal macrophages is immunogenic in vitro^1–4. The studies of Fishman and Adler^5–6 suggest that the peritoneal macrophage population consists of at least three functionally distinct subpopulations. Although most peritoneal macrophages act as scavenger cells⁷, a second population—possibly less than one cell per 1,000—consists of cells that produce but do not necessarily secrete antibody^8,9 and respond to antigen by synthesizing informational RNA. On transfer to normal lymphoid cells, this RNA elicits IgM antibody with the allotypic specificity of the macrophage donor¹⁰. A third type of macrophage gives rise to the RNA-antigen complex responsible for the in vitro synthesis of IgG antibody with the allotypic specificity of the lymphocyte donor¹⁰.     

Transfer of Immunity to Tumour Isografts by the Systemic Administration of Xenogeneic “Immune” RNA

SPECIFIC immunoreactivity can be conferred on lymphoid cells by incubation with RNA-rich extracts prepared from lymphoid tissues exposed to specific antigens in vivo¹ and in vitro^2,3. We have shown transfer of immunity to tumour specific antigens in vivo⁴ and in vitro⁵ by incubation of syngeneic spleen cells in vitro with RNA extracted from the lymphoid tissues of xenogeneic or syngeneic animals immunized with the tumour to be treated. Administration of these spleen cells to normal animals decreased the development and growth of isografts of the same tumour.     

Comprehensive dataset of mangrove tree weights in Southeast Asia

We assembled a dataset tabulating the weights of Thai and Indonesian mangrove trees that we measured between 1982 and 2001. We selected four Thai study sites in Phang Nga, Ranong, Satun, and Trat Provinces and one site in eastern Indonesia on Halmahera Island in Maluku Province. The stands in Ranong Province and on Halmahera Island were in primary forests with data collected in the 1980s and the remaining stands were in secondary forests with data collected later. We collected 124 tree samples from ten species (Avicennia alba, Bruguiera cylindrica, B. gymnorrhiza, Ceriops tagal, Rhizophora apiculata, R. mucronata, Sonneratia alba, S. caseolaris, Xylocarpus granatum, and X. moluccensis) and measured the root weights of 32 individuals of nine species (A. alba, B. cylindrica, B. gymnorrhiza, C. tagal, R. apiculata, R. mucronata, S. alba, S. caseolaris, and X. granatum). All sampled trees were subjected to a standardized protocol to obtain aboveground weights. The trunks were divided into horizontal segments from which the leaves and branches were collected separately. Roots were collected by winching them out of the ground, by trench digging, or by complete excavation. Thus, we were able to compile the weights of the trunk, branches, leaves, and roots of each tree sampled. Aerial roots were included in root weight measurements, although they were collected above ground. We compiled separate lists of trunk diameters, trunk heights, heights of the lowest living branches, and the heights of aerial roots on the trunks of trees in different size categories. Our dataset includes a wide range of tree sizes (maximum trunk diameter 48.9 cm), geographical locations (1°10′N–12°24′N, 98°32′E–123°49′E) and organ weights (trunks, branches, leaves, and roots), and therefore should prove useful in future biomass studies of mangrove forests.     

Molecular characteristics of the ompA gene of serotype B Chlamydia trachomatis in Qinghai Tibetan primary school students

To study the molecular characteristics of Chlamydia trachomatis, the major outer membrane protein gene(omp A) of C. trachomatis from primary school students with trachoma residing in the Qinghai Tibetan area was sequenced and compared with the same serotype in Gen Bank. In Jianshetang Primary School and Galeng Central Primary School in the Galeng Tibetan Township of Qinghai Haidong Sala Autonomous County, scraped samples were collected from the upper tarsal conjunctiva and lower conjunctival sac of both eyes of 45 students with trachoma, stored at 4°C, and transported to Beijing Tongren Hospital by air within 24 h. The samples were screened for C. trachomatis by real-time PCR. The omp A gene from the C. trachomatis-positive samples was amplified by nested PCR. The serotype was confirmed by National Center for Biotechnology Information(NCBI) BLAST search and homology analysis. The entire omp A gene sequence was compared with the corresponding gene sequences of serotype B strains available in Gen Bank. Of the 45 students aged 6–13 years with trachoma, 26 C. trachomatis-positive students were identified by the initial real-time PCR screening(average age,(9.09±1.63) years; sex ratio, 1.0), accounting for 57.78%(26/45). The cycle threshold values for real-time PCR were 16.79–37.77. Half(13/26) of C. trachomatis-positive students had a bacterial copy number of 105. The compliance rate of the omp A gene sequences with the C. trachomatis serotype B strains in Gen Bank was up to 99%. Two novel genetic mutations were found when the omp A gene was compared with those of the 11 serotype B strains in Gen Bank. The two non-synonymous mutations were located at(i) position 271 in the second constant domain, an adenine(A) to guanine(G) substitution(ACT?GCT), changing the amino acid at position 91 from threonine to alanine(Thr?Ala) in all 26 strains; and(ii) position 887 in the fourth variable domain, a cytosine(C) to thymine(T) substitution(GCA?GTA), changing the amino acid at residue 296 from alanine to valine(Ala?Val) in four of the 26 strains. Six mutations were identified relative to ATCC VR-573. The strains could be divided into two gene clusters according to the mutation at nucleotide position 887: CQZ-1(China Qinghai Tibetan-1) and CQZ-2(China Qinghai Tibetan-2). We thus detected two novel serotype B mutant strains of C. trachomatis among study subjects with trachoma.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.     

Chromosome Replication in Toluenized <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cells

BIOCHEMICAL studies of chromosome replication have been hampered by the unavailability of an adequate in vitro system with the basic features of in vivo DNA replication. The criteria for such a system are: (1) semiconservative replication; (2) normal biological activity of newly synthesized DNA; (3) normal advancement of the original replication fork; (4) rate of DNA replication equivalent to in vivo; and (5) expected phenotypic behaviour of temperature-sensitive dna mutants. Systems in Escherichia coli, a membrane-DNA fraction¹, an agar-embedded cell lysate² and toluene-treated cells³ have met two or three of the requirements. Several laboratories have also reported the expected behaviour of ts-dna E. coli mutants in toluenized cells^3–5.     

Acceleration of Senescence induced in Wheat by Light

CELLS from patients with G-trisomy or E-trisomy and XXY cells from patients with XY/XXY mosaic Klinefelter's syndrome are more susceptible to transformation in vitro by SV40 than are cells from normal individuals^1–3. We have used triploid (69XXY) human cells to determine whether the presence of extra chromosomes per se increases susceptibility to transformation.     

Poly U Tracts absent from Viral RNA

Polyadenylic acid (poly A) is covalently attached to the RNA molecules in which it occurs^1–4, but its exact location is not definitely established. It was at first thought to exist only at the 3′OH terminus of RNA molecules^5–6 but recently Ryskov et al. claimed to have found it at the 5′ terminus of light nuclear RNA7 and it is possible that it also exists internally.     

Imide Products from Photo-oxidation of Bilirubin and Mesobilirubin

IN 1958 Cremer et al.¹ observed that the concentration of bilirubin (la) in the plasma could be reduced by exposing newborn infants to fluorescent light. Since that time phototherapy has come into wide use to lower elevated bilirubin levels associated with neonatal jaundice (hyperbilirubin-aemia)^{2, 3}. This condition in the newborn has been associated with retarded motor development, irreversible brain damage or even death. Phototherapy lowers bilirubin levels (by conversion of this lipid-soluble pigment to water-soluble products)⁴ and therefore presumably helps to prevent brain damage. But there are two possible dangers in this treatment: light may have other deleterious effects on the newborn and the photo-products of bilirubin may themselves be toxic. At present neither the structures of the bilirubin photo-products nor their toxicities have been established. Although the photo-destruction of bilirubin has been studied in vivo and in vitro by Ostrow^5–7, Schmid^{4, 7} and others^{8, 9}, these authors investigated principally the visible-ultraviolet spectral changes during the course of bilirubin photo-oxidation and recorded paper chromatographic separations of the photo-products for comparison with the bile or urine of the congenitally jaundiced Gunn rat. More recently McDonagh has shown that singlet oxygen is involved in the self-sensitized photo-oxidation of bilirubin¹⁰. In view of the paucity of structural.information on the bilirubin photo-products, we wish to report preliminary findings on the in vitro photo-oxidation products from bilirubin IXa (1a), mesobilirubin IXa (1b) and 5′-oxo-3′,4,4′-triethyl-3,5-dimethyl-l′,5′-dihydro-(2.2′)-dipyrrylmethene (2). We synthesized and carried out our initial studies on 2, which serves as a simplified model for rings I and II of 1a and 1b because it lacks the vinyl group of 1a and the propionic acid β-substituent of 1a and 1b. The visible-ultraviolet spectrum of 2 is quite similar to that of either 1a or 1b^11–13.