Biosynthesis of vitamin B-12 Part I. Role of the ribosomal proteins in vitamin B-12 biosynthesis

1. 70 S ribosomes isolated from strains of Escherichia coli 113-3, K₁₂ and B take part in vitamin B-12 biosynthesis from AdoCbi-GDP, NAD and dimethylbenzimidazole in the presence of enzymes of the cytosol fraction. 2. 70 S ribosomes from E. coli 113-3 bind Ado[⁵⁸Co]Cbi-GDP. This reaction is independent of fusidic acid. 3. Proteins from 5 S RNA complex as well as L2 protein isolated from E. coli 113-3 ribosomes catalyze vitamin B-12 biosynthesis. The main catalytic function in this reaction is performed by protein L18.4. Vitamin B-12 biosynthesis proceeding in the presence of isolated ribosomal proteins is inhibited by fusidic acid, chloramphenicol and vernamycin but not by erythromycin. 5. Vitamin B-12 synthesized in the presence of isolated ribosomal proteins is biologically active.     

An FT-IR Study of DNA and RNA Conformational Transitions at Low Temperatures

Abstract

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO₂ and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant.

Marker infrared bands for the B conformer have been found to be the strong band at 825 cm^?1 (sugar conformer mode) and a band with medium intensity at 690 cm^?1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm^?1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm^?1 and at 665-600 cm^?1.     

Synthesis and characterization of amplified DNA in oocytes of the house cricket,Acheta domesticus (Orthoptera: Gryllidae)

At a time in the life cycle when a large proportion of the oocytes of Acheta incorporate ³H-thymidine into an extrachromosomal DNA body, synthesis of a satellite or minor band DNA, the density of which is greater than main band DNA, is readily detected. Synthesis of the satellite DNA is not detectable in tissues, the cells of which do not have a DNA body, or in ovaries in which synthesis of extrachromosomal DNA by the oocytes is completed. The DNA body contains the amplified genes which code for ribosomal RNA. However, less than 1 percent of the satellite DNA, all of which appears to be amplified in the oocyte, is complementary to ribosomal 18S and 28S RNA. In situ hybridization demonstrates that non-ribosomal elements, like the ribosomal elements of the satellite DNA, are localized in the DNA body.Abbreviations used rRNA ribosomal RNA, includes 18S and 28S RNA - rDNA gene sequences complementary to rRNA - cRNA complementary RNA synthesized in vitro     

Isolation and Characterization of Coal Solubilizing Aerobic Microorganisms from Salt Range Coal Mines,Pakistan

Microbial solubilization of coal has been considered as a promising technology to convert raw coal into valuable products. In the present study, initially a total of 50 different aerobic bacterial and fungal isolates have been isolated from soil, coal and water samples of Dulmial Coal Mines, Chakwal, Pakistan, but on the basis of solubilization potential, only four isolates were selected for further study. The intensity of biosolubilization was measured by determining the weight loss of the coal pieces, which was observed to be about 25.93% by Pseudomonas sp. AY2, 36.36% by Bacillus sp. AY3 and 50% by Trichoderma sp. AY6, while Phanerochaete sp. AY5 showed maximum coal solubilization potential i.e. 66.67% in 30 days. UV/Vis spectrum revealed an increase in the pattern of absorbance of all treated samples compared to control referring to solubilization. Fourier transform infrared spectroscopy indicated alterations in the structure of treated coal in comparison to control coal suggesting breakdown in the complex structure of coal. The major absorbance bands in infrared spectroscopy for solubilization product were attributed to carbonyl (1,600 cm^?1), hydroxyl (3,450 cm^?1), cyclane (2,925 cm^?1), ether linkage (1,000–1,300 cm^?1), carboxyl (3,300–2,500 cm^?1) and side chains of aromatic ring (1,000–500 cm^?1). The presence of microorganisms and surface erosion of coal residues compared to control samples were observed by scanning electron microscopy, which suggested that isolated microorganisms were able to survive in coal for a longer period of time. Therefore, the present study concluded that microorganisms isolated from coal mines have excellent potential for coal solubilization which is considered as a crucial step in coal methanogenesis allowing them to be used successfully for in situ methane production to meet future energy demands.     

Examination of the secondary structure of proteins by deconvolved FTIR spectra

Fourier transform ir (FTIR) spectra of 21 globular proteins have been obtained at 2 cm^?1 resolution from 1600 to 1700 cm^?1 in deuterium oxide solution. Fourier self-deconvolution was applied to all spectra, revealing that the amide I band of each protein except casein consists of six to nine components. The components are observed at 11 well-defined frequencies, although all proteins do not exhibit components at every characteristic frequency. The root mean square (RMS) deviation of 124 individual values from the 11 average characteristic frequencies is 1.9 cm^?1. The observed components are assigned to helical segments, extended beta-segments, unordered segments, and turns. Segments with similar structures do not necessarily exhibit band components with identical frequencies. For instance, the lower frequency beta-structure band can vary within a range of approximately 15 cm^?1. The relative areas of the individual components of the deconvolved spectra were determined by a Gauss–Newton, iterative curve-fitting procedure that assumed Gaussian band envelopes for the deconvolved components. The measured areas were used to estimate the percentage of helix and beta-structure for each of 21 globular proteins. The results are in good general agreement with values derived from x-ray data by Levitt and Greer. The RMS deviation between 22 values (alpha- and beta-content of 11 beta-rich proteins measured by both techniques) is 2.5 percentage points; the maximum absolute deviation is 4 percentage points.     

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.     

An Examination of the Differential Sensitivity to Ketolide Antibiotics in <Emphasis Type="Italic">ermB</Emphasis> Strains of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC₅₀ values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC₅₀ values for inhibition of translation and subunit formation in S. pneumoniae (ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC₅₀ values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.     

Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast

A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized.It was found that the enzyme utilizes GTP and ATP as phosphoryl donors. Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 × 10^-6M and 5.5 × 10^-5M, respectively.Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the [-³²P] nucleotides used. It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro. The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E. coli ribosomes. The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.     

Ribosomes of Brachionus plicatilis (Rotifera) – distribution in homogenate fractions and general properties

(1) The content of DNA, RNA and of proteins of Brachionus plicatilis was estimated and the distribution of RNA and of proteins of different homogenate fractions characterised. (2) Ribosomes were isolated from Brachionus plicatilis homogenates and were characterised by gradient centrifugation. (3) Unlike the RNA content, the yield of ribosomes from different homogenate fractions is strongly dependent on the concentration of Mg²⁺-ions in the buffers. Likewise resuspension of ribosomes is more effective in Mg²⁺- (or Ca²⁺-) free buffers. (4) Dissociation of ribosomes was brought about by centrifugation of ribosomes in gradients containing less than 4 mM Mg²⁺. In this case, beside the peaks of subunits, a peak in the region of 80 S remained which vanished only under conditions destroying ribosomal material altogether. (5) Proteins were isolated from ribosomal subunits and from undissociated ribosomes and were characterised by two-dimensional gel electrophoresis techniques. Patterns of 51 spots were regularly obtained from large subunits and patterns of 41 spots from small subunits. The undissociated ribosomes showed 83 spots, most of which could be attributed to the large or the small subunit. The ribosomal proteins have molecular masses of between 11000 and 56000 Da, while the molecular mass of the total protein content of Brachionus ribosomes was estimated to be 1.8 ±0.5) ×10⁶ Da.     

Affinity of PIP-aquaporins to sterol-enriched domains in plasma membrane of the cells of etiolated pea seedlings

The hypothesis that sterol-enriched domains represent sites of preferred localization of PIP-aquaporins was tested in experiments on plasma membranes isolated from cells of etiolated pea (Pisum sativum L.) seedlings. Plasma membranes were isolated from microsomes by the partition in the aqueous two-phase polymer system and separated into vesicle fractions of different buoyant density by flotation in discontinuous OptiPrep gradient. Two types of plasma membrane preparations were used: one was treated with cold 1% Triton X-100 and the other was not. In untreated preparations, three populations of plasma membrane vesicles were obtained, while in the case of treated preparations, fractions of detergent-resistant membranes (DRM) and solubilized membrane proteins were obtained. In all membrane fractions collected after OptiPrep flotation, the amounts of proteins, sterols, and PIP-aquaporins were determined. The highest sterol content was detected in the membrane fraction with buoyant density 1.098 g/cm³ and in the DRM fraction (1.146 g/cm³). These fractions contained much more PIP-aquaporins than the other ones. Phase state of the lipid bilayer was determined by measuring generalized polarization excitation of fluorescence (GPEX) of laurdan incorporated into the membranes of different fractions. It was revealed that the lipid bilayer of the membranes with density of 1.098 g/cm³ had a higher extent of ordering than that of the fractions with density of ∼1.146 g/cm³. The results indicated that uppermost local concentrations of PIP-aquaporins were associated with tightly packed sterol-enriched domains. Moreover, upon solubilization of plasma membrane with Triton X-100, PIP-aquaporins mainly resided in DRM, thus exhibiting a high affinity to sterols.     

Heterogeneity of ribosomal populations in Escherichia coli cells grown in different media

Summary In order to establish whether ribosomes exhibit heterogeneity with respect to their protein pattern in vivo, E. coli cells were grown in rich or minimal medium and labeled with ¹⁴C and ³H amino acid mixture, respectively. After harvesting, the cells from the different media were mixed, the differently labeled ribosomes isolated and the ribosomal proteins separated. For each protein the ratio of ¹⁴C to ³H was determined and used as an indication of whether differences exist in ribosomal populations synthesized under different growth conditions.With respect to their ratio the ribosomal proteins can be classified as follows: Many of the proteins have a ratio of 1, i. e. they are present in the same amount in both preparations. The ratios for about 30% of the proteins differ only slightly from 1 whereas three proteins namely S6, S21 and L12 have ratios of 2.5 and 3.1 respectively. This means that ribosomal populations isolated from cells grown in rich medium contain these three proteins in two to three fold greater amounts compared to those synthesized in minimal medium.The relevance of these results with respect to the occurrence of heterogeneous ribosomal populations in vivo is discussed.Paper Nr. 36 on Ribosomal Proteins. Preceding paper is by H. J. Weber, Mol. Gen. Genetics 119, 233–248 (1972).     

Plastid changes during the conversion of chloroplasts to chromoplasts in ripening tomatoes

Methods were developed for the isolation of plastids from mature green and ripening tomatoes (Lycopersicon esculentum Mill.) and purification by sucrose or Percoll density-gradient centrifugation. Assessment of the purity of preparations involved phase-contrast and electron microscopy, assays for marker enzymes and RNA extraction and analysis. Proteins were extracted from isolated plastids at different ripening stages and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The profiles obtained from chloroplasts and chromoplasts showed many qualitative and quantitative differences. Labelling of proteins with [³⁵S]methionine in vivo showed that there was active protein synthesis throughout ripening, but there was a change in the plastid proteins made as ripening proceeded. The cellular location of synthesis of specific proteins has yet to be established.Abbreviations CS citrate synthase - EDTA ethylenediaminetetraacetic acid,-acetate - GAPDH NADP⁺-glyceraldehyde-3-phosphate dehydrogenase - rRNA ribosomal RNA - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - Tris 2-amino-2(hydroxymethyl)-1,3-propanediol     

Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae

RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with ¹²⁵I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.     

Escherichia coli persister cells suppress translation by selectively disassembling and degrading their ribosomes

Bacterial persisters are rare, phenotypically distinct cells that survive exposure to multiple antibiotics. Previous studies indicated that formation and maintenance of the persister phenotype are regulated by suppressing translation. To examine the mechanism of this translational suppression, we developed novel methodology to rapidly purify ribosome complexes from persister cells. We purified His‐tagged ribosomes from Escherichia coli cells that over‐expressed HipA protein, which induces persister formation, and were treated with ampicillin to remove antibiotic‐sensitive cells. We profiled ribosome complexes and analyzed the ribosomal RNA and protein components from these persister cells. Our results show that (i) ribosomes in persisters exist largely as inactive ribosomal subunits, (ii) rRNAs and tRNAs are mostly degraded and (iii) a small fraction of the ribosomes remain mostly intact, except for reduced amounts of seven ribosomal proteins. Our findings explain the basis for translational suppression in persisters and suggest how persisters survive exposure to multiple antibiotics.     

Synaptosomal protein synthesis in a cell-free system

—Synaptosomes were isolated from cerebral cortex of young rats and incubated with ¹⁴C-labelled l -leucine in vitro. Amino acid incorporation into proteins of the synaptosomal cytoplasm, mitochondria and membrane components was observed. There was no incorporation into proteins of the vesicles. The protein-synthesizing system was not stimulated by the addition of either ATP or an ATP-generating system. ATP at all concentrations was inhibitory. Two different protein-synthesizing systems operate in the synaptosome. One, sensitive to inhibition by chloramphenicol and related antibiotics, is found in the mitochondrial subfraction and the other, inhibited by cycloheximide, is located either in the membrane components or the synaptosomal cytoplasm. This second system resembles the eukaryotic ribosomal system in its sensitivity to cycloheximide. Both the synaptosomal soluble fraction and the synaptosomal membrane fractions were shown previously to contain RNA. This RNA could function in protein-synthesizing mechanisms in the synaptosome. These results deomonstrate that protein is synthesized in axonal components and show that it is unnecessary to postulate that all axonal protein is supplied by somato-axonal flow.     

Satellite DNA associated with heterochromatin in Rhynchosciara

The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm³. The larger of the two minor bands has a buoyant density of 1.680 g/cm³ while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm³. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA.