scholarly journals MOLECULAR FATE OF HETEROLOGOUS BACTERIAL DNA IN COMPETENT BACILLUS SUBTILIS. I. PROCESSING OF B. PUMILUS AND B. LICHENIFORMIS DNA IN B. SUBTILIS

Genetics ◽  
1982 ◽  
Vol 101 (2) ◽  
pp. 179-188
Author(s):  
Hein P J te Riele ◽  
Gerard Venema
Keyword(s):  
Bacillus Subtilis ◽  
Dna Synthesis ◽  
Gradient Centrifugation ◽  
Single Strand ◽  
Bacterial Dna ◽  
Cscl Gradient ◽  
Donor Moiety ◽  
Three Components

ABSTRACT Competent Bacillus subtilis cells were exposed to radioactive and density labeled donor DNA extracted from B. pumilus and B. licheniformis. The DNA from these strains hybridized with B. subtilis DNA in vitro at a rate of 24% and 11%, respectively. After entry the vast majority of heterologous DNA was found at the single-strand DNA position in CsCl gradients, and was gradually degraded during incubation. Much less donor DNA than expected from the hybridization values participated in the formation of the donorrecipient complex (DRC). By subjecting the heterologous DRC to sonication and alkaline CsCl gradient centrifugation, it was established that the DRC consisted of three components: (1) recipient DNA in which breakdown products of donor DNA were incorporated through DNA synthesis, (2) recipient DNA in which donor DNA was covalently integrated and (3) recipient DNA in which the donor moiety was not covalently integrated.

scholarly journals Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

1982 ◽  
Vol 152 (1) ◽  
pp. 275-283
Author(s):  
J Van Randen ◽  
K Wiersma ◽  
G Venema
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gradient Centrifugation ◽  
Cross Linking ◽  
Dna Fragments ◽  
Dna Complexes ◽  
Cscl Gradient ◽  
Donor Moiety

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.

scholarly journals Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1973 ◽  
Vol 136 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Daniel B. Ellis ◽  
Glenn H. Stahl
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sialic Acids ◽  
Equilibrium Density ◽  
Agarose Gels ◽  
Cscl Gradient ◽  
Tracheal Explants

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-3H]fucose and l-[U-14C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.

scholarly journals Resolution of Parvovirus Dimer Junctions Proceeds through a Novel Heterocruciform Intermediate

2003 ◽  
Vol 77 (11) ◽  
pp. 6245-6254 ◽  
Author(s):  
Susan F. Cotmore ◽  
Peter Tattersall
Keyword(s):  
Dna Synthesis ◽  
Crucial Role ◽  
Dna Amplification ◽  
Single Strand ◽  
Minute Virus Of Mice ◽  
Initiator Protein ◽  
Resolution Model ◽  
Minute Virus ◽  
Single Sequence

ABSTRACT The minute virus of mice initiator protein, NS1, excises new copies of the left viral telomere in a single sequence orientation, dubbed flip, during resolution of the junction between monomer genomes in palindromic dimer intermediate duplexes. We examined this reaction in vitro using both 32P-end-labeled linear substrates and similar unlabeled templates labeled by incorporation of [α-32P]TTP during the synthesis. The observed products suggest a resolution model that explains conservation of the hairpin sequence and in which a novel heterocruciform intermediate plays a crucial role. In vitro, NS1 initiates two replication pathways from OriLTC, the single active origin embedded in one arm of the dimer junction. NS1-mediated nicking liberates a base-paired 3′ nucleotide to prime DNA synthesis and, in a reaction we call “read-through synthesis,” forks established while the substrate is a linear duplex synthesize DNA in the flop orientation, leading to DNA amplification but not to junction resolution. Nicking leaves NS1 covalently attached to the 5′ end of the DNA, where it can serve as a 3′-to-5′ helicase, unwinding the NS1-associated strand. In the second pathway, resolution substrates are created when such unwinding induces the palindrome to reconfigure into a cruciform prior to fork assembly. New forks can then synthesize DNA in the flip orientation, copying one cruciform arm and creating a heterocruciform intermediate. Resolution proceeds via hairpin transfer in the extended arm of the heterocruciform, which releases one covalently closed duplex telomere and a partially single-stranded junction intermediate. We suggest that the latter intermediate is finally resolved via an NS1-induced single-strand nick at the otherwise inactive origin, OriLGAA.

Trans-species transfer of Wolbachia: microinjection of Wolbachia from Litomosoides sigmodontis into Acanthocheilonema viteae

Parasitology ◽  
2003 ◽  
Vol 126 (6) ◽  
pp. 503-511 ◽  
Author(s):  
N. HARTMANN ◽  
H. STUCKAS ◽  
R. LUCIUS ◽  
W. BLEIß ◽  
F. THEURING ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Meriones Unguiculatus ◽  
Intracellular Bacteria ◽  
Rodent Host ◽  
Bacterial Dna ◽  
Litomosoides Sigmodontis ◽  
Acanthocheilonema Viteae ◽  
Filarial Nematodes ◽  
Ftsz Gene

Intracellular bacteria of the genus Wolbachia are found in most filarial nematodes, but are lacking in some species like Acanthocheilonema viteae. Due to their symbiotic nature and their role in the pathology of filarial infections they are considered to be potential targets for intervention against filarial infections in man. Infection of A. viteae (a species which does not naturally carry Wolbachia) with Wolbachia bacteria could allow comparative studies on the effect of the endobacterium on the parasite and on the host's immune systems. As a step towards such studies we microinjected adult female A. viteae with Wolbachia obtained from Litomosoides sigmodontis. The bacteria were isolated from L. sigmodontis by density-gradient centrifugation, microinjected into A. viteae worms and bacterial DNA detected by PCR with Wolbachia specific primers (ftsZ gene). Microinjected worms were cultured in vitro, and 81% survived for 10 days. Implantation of microinjected worms into Meriones unguiculatus, the rodent host of A. viteae resulted in 38% survival. The DNA of the microinjected worms recovered from jirds 8 weeks after implantation contained Wolbachia DNA as shown by PCR, suggesting that Wolbachia of L. sigmodontis can be horizontally transmitted to A. viteae.

scholarly journals Multiple GTPases Participate in the Assembly of the Large Ribosomal Subunit in Bacillus subtilis

2006 ◽  
Vol 188 (23) ◽  
pp. 8252-8258 ◽  
Author(s):  
Laura Schaefer ◽  
William C. Uicker ◽  
Catherine Wicker-Planquart ◽  
Anne-Emmanuelle Foucher ◽  
Jean-Michel Jault ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Protein Composition ◽  
Large Ribosomal Subunit ◽  
Sucrose Density Gradient Centrifugation ◽  
Direct Role

ABSTRACT GTPases have been demonstrated to be necessary for the proper assembly of the ribosome in bacteria and eukaryotes. Here, we show that the essential GTPases YphC and YsxC are required for large ribosomal subunit biogenesis in Bacillus subtilis. Sucrose density gradient centrifugation of large ribosomal subunits isolated from YphC-depleted cells and YsxC-depleted cells indicates that they are similar to the 45S intermediate previously identified in RbgA-depleted cells. The sedimentation of the large-subunit intermediate isolated from YphC-depleted cells was identical to the intermediate found in RbgA-depleted cells, while the intermediate isolated from YsxC-depleted cells sedimented slightly slower than 45S, suggesting that it is a novel intermediate. Analysis of the protein composition of the large-subunit intermediates isolated from either YphC-depleted cells or YsxC-depleted cells indicated that L16 and L36 are missing. Purified YphC and YsxC are able to interact with the ribosome in vitro, supporting a direct role for these two proteins in the assembly of the 50S subunit. Our results indicate that, as has been demonstrated for Saccharomyces cerevisiae ribosome biogenesis, bacterial 50S ribosome assembly requires the function of multiple essential GTPases.

scholarly journals Studies on gastric mucoproteins. The production of radioactive mucoproteins by pig gastric mucosal scrapings in vitro

1972 ◽  
Vol 127 (3) ◽  
pp. 577-587 ◽  
Author(s):  
D. Snary ◽  
A. Allen
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sedimentation Velocity ◽  
Water Soluble ◽  
Equilibrium Density ◽  
Optimum Conditions ◽  
Ph Values ◽  
Cscl Gradient ◽  
Sepharose 4B

1. Optimum conditions, including the effect of media of different pH values, were determined for the incorporation of radioactive precursors into mucoproteins by pig gastric mucosa in vitro. 2. Mucosal scrapings incorporated radioactivity from [U-14C]-glucose and from [G-3H]threonine or [G-3H]serine solely into the carbohydrate and protein portions respectively of the mucoprotein molecules. 3. Of the radioactive mucoprotein 22% was water-soluble and up to 80% of the remainder was soluble in other solvents. 4. Pronase was the most successful proteolytic enzyme tested for making the mucoprotein water-soluble, up to 94% dissolving after digestion. 5. The Pronase digestion products of the mucoproteins were separated from protein by equilibrium-density-gradient centrifugation in a CsCl gradient. 6. These Pronase-digested mucoproteins were further fractionated on Sepharose 4B and the isolated fractions analysed by chemical and sedimentation-velocity methods. 7. Pronase digestion and solvent extraction of mucosal scrapings labelled with 14C in the carbohydrate and 3H in the protein showed that one type of mucoprotein was the only non-diffusible biosynthetic product of the scrapings in vitro, and that this mucoprotein was the only mucoprotein constituent of the water-soluble and water-insoluble mucus.

scholarly journals Single-strand binding protein enhances fidelity of DNA synthesis in vitro.

1979 ◽  
Vol 76 (12) ◽  
pp. 6331-6335 ◽  
Author(s):  
T. A. Kunkel ◽  
R. R. Meyer ◽  
L. A. Loeb
Keyword(s):  
Dna Synthesis ◽  
Binding Protein ◽  
Single Strand

Use of Antibody to aid Visualization of Protein at the Termini of Bacteriophage Ø29 DNA

1980 ◽  
Vol 38 ◽  
pp. 540-541
Author(s):  
Dwight Anderson ◽  
Charlene Peterson ◽  
Gursaran Notani ◽  
Bernard Reilly
Keyword(s):  
Electron Microscopy ◽  
Bacillus Subtilis ◽  
Dna Synthesis ◽  
Restriction Endonuclease ◽  
Protein Complex ◽  
Protein Product ◽  
Viral Dna ◽  
Small Proteins ◽  
Antibody Labeling ◽  
Subtilis Bacteriophage

The protein product of cistron 3 of Bacillus subtilis bacteriophage Ø29 is essential for viral DNA synthesis and is covalently bound to the 5’-termini of the Ø29 DNA. When the DNA-protein complex is cleaved with a restriction endonuclease, the protein is bound to the two terminal fragments. The 28,000 dalton protein can be visualized by electron microscopy as a small dot and often is seen only when two ends are in apposition as in multimers or in glutaraldehyde-fixed aggregates. We sought to improve the visibility of these small proteins by use of antibody labeling.

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