bluuebunny/crossref_metadata_2025_split huggingface dataset:
Membrane Fractions from the Outer Layers of Spores of Bacillus thuringiensis with Toxicity to Lepidopterous Larvae
Authors: SCHERRER Peter S. , SOMERVILLE Hugh J. 
Published: March 2005

Two membrane fractions, F1 and F2, have been purified from the outer layers of spores of Bacillus thuringiensis. Both fractions contain 6‐7% cysteine and appear to be similar in composition. Amino acids account for about 75% of the dry weight, carbohydrate for about 2% and lipids for about 25%. The fractions are both toxic to Pieris brassicae and the toxicity is inactivated by antiserum to the toxic crystal of Bacillus thuringiensis. The fractions can be distinguished by examination under the electron microscope; both fractions show similar hexagonal patterns but with different spacings. The same fractions from an acrystaliferous mutant (cr) were prepared. These were identical in density and in appearance under the electron microscope; the amino acid analysis of fraction F2 from both strains was identical. However, the spores and fractions F1 and F2 from this strain lacked toxicity.Fraction F2 from the cr strain was used to prepare antiserum specific to fraction F2. Using this antiserum and anticrystal serum, crystal and F2 antigens were shown to appear simultaneously in sporulating cultures. Crystal and F2 antigens appeared some time before the maximum rate of uptake of [35S]cysteine.It is concluded that fraction F2 is derived from the exosporium and that fraction F1 probably originates from the spore coat. The exosporium in Bacillus thuringiensis appears to be synthesised during stages II and III of sporulation although uptake of [35S]cysteine occurs much later.
DNA Synthesis in HeLa Cells and Isolated Nuclei after Treatment with an Inhibitor of Spermidine Synthesis, Methyl Glyoxal Bis(guanylhydrazone)
Authors: KROKAN Hans , ERIKSEN Arne 
Published: March 2005

Addition of methyl glyoxal bis(guanylhydrazone) to HeLaS3 suspension cultures resulted in increased putrescine levels and decreased spermidine and spermine levels preceding a drop in incorporation of [3H]thymidine, [3H]uridine and [14C]leucine into macromolecules. When putrescine, spermidine, spermine or cadaverine was added simultaneously with methyl glyoxal bis(guanylhydrazone), the drug had no detectable effect on the synthesis of macromolecules. In nuclei isolated from cells treated with methyl glyoxal bis(guanylhydrazone) the reduction in the rate of DNA synthesis was equal to the reduction of [3H]thymidine incorporation in the corresponding whole cells. The capability of the nuclei to synthesize DNA could not be restored by adding spermidine or spermine to the system in vitro.The rate of DNA chain elongation was only reduced slightly by methyl glyoxal bis(guanylhydrazone) indicating that decreased levels of spermidine and spermine lead to a decrease in the number of replication units active in DNA synthesis within each cell.
Bacteriophage‐T7‐Induced DNA‐Priming Protein
Authors: SCHERZINGER Eberhard , LANKA Erich , MORELLI Giovanna , SEIFFERT Detlef , YUKI Atsushi 
Published: March 2005

The T7 gene‐4protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA‐priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initiate DNA synthesis on various circular single‐stranded DNA templates by a mechanism which involves the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3′ : 5′‐phosphodiester bond, starts with pppA‐C and terminates predominantly with AMP. When only ATP and CTP are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA‐C‐C‐A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initiators, T7 DNA‐priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA‐priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template‐primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2–4‐fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from initiation of DNA chains de novo.Incubation of three proteins: T7 DNA‐priming protein, T7 DNA polymerase, and T7 DNA‐binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with θX174 DNA as template leads to the generation of «rolling circle‐like’ structures as visualized in the electron microscope. Single‐stranded regions at the tail‐circle junction indicate that initiations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of ‘lagging’ strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo.
Forms AI and AII DNA‐dependent RNA Polymerases as Components of Two Defined Pools of Polymerase Activity in Mammalian Cells
Authors: KELLAS Beatrice L. , AUSTOKER Joan L. , BEEBEE Trevor J. C. , BUTTERWORTH Peter H. W. 
Published: March 2005

Two species of form A (or I) RNA polymerase have been identified in eucaryotic cells and there is evidence that this α‐amanitin‐insensitive activity exists as two discrete pools: a pool of ‘free’ activity, which is identified by its ability to transcribe poly[d(A‐T)] in the presence of actinomycin in vitro, and a pool of enzyme in the form of a transcription complex (‘engaged’) which is unaffected by inhibitors of initiation of RNA synthesis.

The principles underlying and the practical application of the technique used to define the pool of ‘free’ RNA polymerase activity have been analysed in considerable detail. On the basis of actinomycin titrations of poly[d(A‐T)]dependent activity in isolated organelles, it is concluded that a pool of ‘free’ RNA polymerase A activity exists in mammalian nuclei which, under certain circumstances, is lost from nuclei during their isolation. The evidence presented suggests that nucleoli, resolved from nuclei by the classical sonication technique, contain form A polymerase exclusively in the transcription complex form.
Different techniques used to solubilise RNA polymerase activity from nucleoli are shown to give rise to different proportions of the two form A RNA polymerase species (AI and AII, as defined by their differential elution from phosphocellulose): whereas low‐ionic‐strength extraction gives rise to form AII, high‐salt, sonication extracts contain predominantly the form AI enzyme. It is shown that the sonication technique results in the conversion of form AII to form AI. By a careful appraisal of the products of these procedures and a novel polymerase solubilisation technique, it is concluded that RNA polymerase AII is the ‘engaged’ form of the enzyme found in the transcription complex.
Making use of the finding that the ‘free’ form of the enzyme is lost to the cytoplasmic fraction on nuclear isolation, this activity has been characterised without the requirement for solubilisation techniques which might result in the conversion of one form to another: the ‘free’ species is shown to be form AI RNA polymerase.
These conclusions that two discrete pools of form A RNA polymerase activity contain different species of the enzyme are briefly discussed in the light of other published information concerning their subunit structures and their potential role in the expression of the ribosomal RNA coding sequences.
Purification of an Endonuclease from Adenovirus‐Infected KB Cells
Authors: REIF Ulrike M. , WINTERHOFF Ute , LUNDHOLM Ulla , PHILIPSON Lennart , DOERFLER Walter 
Published: March 2005

This report describes the purification of an endonuclease from extracts of adenovirus‐type‐2‐infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus‐infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus‐infected cells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated.The endonuclease in crude extracts from uninfected or adenovirus‐infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus‐type‐2‐infected KB cells.A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.
5.9‐S RNA, a New RNA Characterized in Several Mammalian Cell Lines
Authors: GALIBERT Francis , HAMPE Annie 
Published: March 2005

A new species of RNA has been isolated from several different cell lines, both oncornavirus producing and non‐producing. This RNA, which we designate 5.9‐S RNA is present in the cellular cytoplasmic fraction at very low concentration (approximately 1% of the quantity of 4‐S RNA), but it accumulates to much higher levels in two murine oncornaviruses, Moloney murine sarcoma leukemia virus complex and Gross leukemia virus, where it represents as much as 10% of the low‐molecular‐weight RNA fraction associated with the 70‐S RNA genome.The electrophoretic mobility and fingerprint analysis of T1 R Nase digest products show that this species of RNA is approximately 160–165‐residues long, and can be unequivocally distinguished from all previously described species of RNA in this size range.
Superoxide Dismutase from Thermus aquaticus
Authors: SATO Showbu , HARRIS J. Ieuan 
Published: March 2005

Superoxide dismutase has been isolated and characterised from the extreme thermophile Thermus aquaticus. The pure enzyme is a reddish‐purple manganese‐containing protein with a molecular weight of ∼ 80000 ± 5000. Combination of gel electrophoresis in dodecylsulphate and amino acid analysis shows that it is composed of four identical subunit polypeptide chains consisting of approximately 186 amino acids. The tetrameric protein contains two atoms of manganese.A stable manganese‐free apoprotein has been prepared by treatment with EDTA in 8 M urea at acidic pH. The apoprotein regains the tetrameric structure in the absence of manganese but is inactive. Reconstitution of active Mn‐enzyme was achieved by addition of Mn2+ to apoprotein in 8 M urea at acid pH. Reconstitution was monitored by absorption spectroscopy, manganese analysis and regain of activity and by these criteria the reconstituted enzyme with two atoms Mn per mole is indistinguishable from the native enzyme. The enhanced stability of the thermophile apoenzyme and Mn‐enzyme is of advantage for studies of the structure and mechanism of action of superoxide dismutase.The N‐terminal amino acid sequence to the 40th residue of the subunit was determined by automated Edman degradation. The sequence has a close resemblance to that of the dimeric Mnenzyme from another thermophile, Bacillus stearothermophilus.
Comparative Analysis of Three Guinea Pig Satellite DNAs by Restriction Nucleases
Authors: ALTENBURGER Werner , HÖRZ Wolfram , ZACHAU Hans G. 
Published: March 2005

The structures of guinea pig satellite DNAs I, II, and III have been analyzed by digestion with seven restriction nucleases. From the cleavage patterns it is obvious that the long‐range periodicities in these three satellites differ rather characteristically.Satellite I is fairly resistant to six nucleases and gives only a number of weak discrete bands which do not show a simple regularity. By the restriction nuclease from Arthrobacter luteus, however, it is cleaved extensively and yields very heterogeneous breakdown products. This is consistent with the high extent of divergence previously found for this satellite, e. g. by sequence analysis.Satellite II is almost completely resistant to all nucleases, indicative of a high degree of sequence homogeneity of this satellite.Satellite III is completely broken by the restriction nuclease from Bacillus subtilis into fragments which form a novel, highly regular series of bands in gel electrophoresis. The patterns show that the satellite is composed of tandem repeats of approximately 215 nucleotide pairs length, each repeat unit containing two cleavage sites for this nuclease. The data are consistent with the assumption that 30–40% of all cleavage sites have been eliminated by a random process. Satellite III DNA yields weak degradation patterns of the same periodicity with a number of other restriction nucleases. Cleavage sites for these nucleases are clustered on separate small segments of the satellite DNA. In this respect, the satellite is similar to others, notably the mouse satellite DNA.The three guinea pig satellites are examples of more general types of satellite structures also found in other organisms. Similarities and differences to other satellites are discussed with special consideration to theories on the evolution of this class of DNA.
The Trypsin and Chymotrypsin Inhibitors in Chick Peas (Cicer arietinum L.)
Authors: BELEW Makonnen 
Published: March 2005

The six isoinhibitors of trypsin and chymotrypsin which were previously purified from a crude extract of chick peas were further studied with the intention of establishing the relationships among them. Exposure of each fraction to active, matrix‐bound trypsin resulted in a decrease of the specific inhibitory activities towards trypsin of fractions P‐1, P‐3 and P‐5 while those of P‐4 and P‐6 were increased. Each of these fractions was separated into several sub‐fractions after chromatography on a DEAE‐Sephadex A‐25 column under conditions identical to those used previously for their purification. The elution positions, specific inhibitory activities towards trypsin and electrophoretic patterns in polyacrylamide gels of the various sub‐fractions were determined. On the basis of the results thus obtained it is concluded that fractions P‐3, P‐4, P‐5 and P‐6 originate from a common inhibitor molecule. From the amino acid compositions and aminoterminal sequences of these four fractions, it is further concluded that P‐3 and P‐4 are identical proteins and that P‐5 and P‐6 are probably derived from P‐3 and P‐4, respectively, as a result of tryptic removal of the amino‐terminal Val‐Lys residues from the latter two fractions. Since the distribution of the products obtained from P‐1 is different from that obtained from the other four fractions, it is also concluded that P‐1 is propably derived from a second inhibitor originally present in chick peas.The inhibitors in chick peas were also isolated by conventional chromatographic procedures. Preliminary results indicate the presence of two inhibitors, both with a molecular weight of around 16000. Since the fractions thus obtained were not homogeneous they were not further characterized. Nevertheless, the findings confirm the conclusions reached above and also show that the inhibitors probably exist as dimers in the seeds of chick peas since each of the isoinhibitors previously isolated had molecular weights of about 10000.
Nucleotide Sequence of a Region in 23‐S RNA Adjacent to Peptidyl Transferase Catalytic Center of Escherichia coli Ribosomes
Authors: YUKIOKA Munehiko , HATAYAMA Takumi , OMORI Koichiro 
Published: March 2005

N‐Iodoacetylphenylalanyl‐tRNAPhe was used as an affinity label to localize the RNA components intimately involved in the catalytic center of Escherichia coli ribosomes. This analogue could alkylate the specific region of 23‐S RNA that was located within 2000 nucleotides from the 3′ terminus of the molecule. Sequence analysis revealed that the alkylation by the active substrate (N‐iodo‐acetylphenylalanyl‐tRNAPhe) was directed to the 5′‐terminal adenosine residue of a heptanucleotide, A‐U‐U‐U‐U‐A‐Gp, which seemed to be derived from a heptadecanucleotide, U‐U‐A‐A‐A‐A‐A‐C‐A‐C‐A‐U‐U‐U‐U‐A‐Gp, in the original 23‐S RNA.The significance of the unique sequence in the ribosomal functions is discussed.
Proton Movements Coupled to Sugar Transport via the Galactose Transport System in Salmonella typhimurium
Authors: VAN THIENEN Gerda M. , POSTMA Pieter W. , VAN DAM Karel 
Published: March 2005

We have studied proton movements associated with substrate transport via the galactose transport system in Salmonella typhimurium.The addition of galactose to lightly buffered suspensions of anaerobic, non‐metabolizing cells of Salmonella typhimurium, specifically induced for the galactose transport system, causes an increase in extracellular pH as galactose and protons enter the cell together.Other substrates for this transport system, D‐fucose, 2‐deoxygalactose, glucose and 2‐deoxyglucose similarly cause an influx of protons when transported.In contrast, transport via the other major transport system for galactose, the methylgalactoside transport system, is not coupled to H+ influx.Comparison of kinetic data obtained from pH measurements with data obtained from measurement of active transport of galactose via the galactose transport system suggests that the apparent Km of the galactose transport system for this sugar differs under energized and non‐energized conditions.At pH 7.2 the permeant anion SCN− increases both the rate and extent of galactose‐induced proton influx; at pH 6 the rate, but not the extent is increased by SCN−.
β‐d‐Galactoside Transport in Escherichia coli
Authors: ALTENDORF Karlheinz , MÜLLER Clemens R. , SANDERMANN Heinrich 
Published: March 2005

At relatively low concentrations (< 3 M) the aprotic solvents: dimethylsulfoxide, N‐methyl‐pyrrolidone, tetramethylurea and hexamethylphosphoric triamide, inhibited β‐D‐galactoside transport by whole cells, and the derived membrane vesicles of Escherichia coli. Inhibition was not due to gross leakiness of the membrane and could be largely reversed by a simple washing procedure.At high concentrations of aprotic solvent (9 parts hexamethylphosphoric triamide or N‐methyl‐pyrrolidone/1 part buffer, 100 mM LiCl), 50–80% of the total protein of transport‐positive membrane vesicles from E. coli ML 308‐225 were solubilized. When these extracts were added, with constant sonication, to intact transport‐negative vesicles derived from E. coli ML 35, lactose uptake was reconstituted according to the following criteria.a) Uptake (12 nmol/mg protein) required an energy source, provided by oxidation of d‐lactate or ascorbate/phenazine methosulfate or by a K+‐diffusion potential established with the aid of valinomycin.b) Uptake was completely abolished in the presence of uncoupler (carbonyl cyanide m‐chlorophenylhydrazone, 1 μM) or of high‐affinity substrate (thio‐β‐d‐digalactoside, 5 mM).
Specificity Profiles of the Membrane‐Bound γ‐d‐Glutamyl‐(l)meso‐diaminopimelate Endopeptidase and ld‐Carboxypeptidase from Bacillus sphaericus 9602
Authors: ARMINJON François , GUINAND Micheline , VACHERON Marie‐Jeanne , MICHEL Georges 
Published: March 2005

Membrane‐bound peptidases from Bacillus sphaericus 9602 were tested upon various peptides in order to determine the substrate requirements of each enzyme. Ld‐Carboxypeptidase and γ‐d‐glutamyl‐meso‐diaminopimelate endopeptidase were never both found to be active on the same substrate. Ld‐Carboxypeptidase splits the l‐Lys‐d‐Ala linkage of lysine‐containing substrates and the msA2pm‐d‐Ala linkage of meso‐diaminopimelic‐acid‐containing substrates which have an amide group on the ω‐carboxyl. The endopeptidase hydrolyses the linkage of meso‐diaminopimelic‐acid‐containing peptides and derivatives with free ω‐NH2 and ω‐COOH groups. The presence or the absence of an α‐amide group on glutamic acid, the second residue of peptides, has no influence on the specificities of either enzyme. Likewise, N‐substitution of N‐terminal l‐alanine does not modify the enzymic specificities.Kinetic studies have shown that and are good substrates for Ld‐carboxypeptidase (apparent Km= 0.8 mM and 1.25 mM, respectively). is the best substrate for the endopeptidase (apparent Km= 0.33 mM). An amide group on glutamic acid gives a decrease of 80% of both Ld‐carboxypeptidase and endopeptidase activities.Various inhibitors of endopeptidase were studied:DD and meso‐diaminopimelic acid isomers are very good inhibitors. Dicarboxylic d‐amino acids also show an inhibition which is a function of the length of the side chain: the maximum is observed for n= 4 (d‐α‐aminopimelic acid).
The Reactivity of One Essential Cysteine as a Conformational Probe in Escherichia coli Tryptophanase
Authors: RAIBAUD Olivier , GOLDBERG Michel E. 
Published: March 2005

It is first shown that the inactivation of Escherichia coli apotryptophanase by N‐ethylmaleimide results from the labeling of a single particularly reactive cysteine per protomer.The reactivity of this cysteine under various conditions is investigated and the results indicate that the protein can exist under two classes of conformation: one, corresponding to inactive protein, in which the cysteine is reactive, and a second, corresponding to active enzyme, where the cysteine is masked. The rate of the isomerization step involved in this change in conformation is measured by the stopped‐flow technique (τ= 0.4 s).Finally, the reactivity of the cysteine is used to characterize the conformation of dimeric holotryptophanase (i.e. a dissociated form of the enzyme obtained as a transient species between dimeric apoenzyme and the natural tetrameric holoenzyme). By this criterion, it is shown that dimeric holotryptophanase falls in the class of ‘inactive’ conformations. These results are used to discuss the influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations.