Characterization of the Bacillus subtilis bacteriophage PBS2-induced DNA polymerase and its associated exonuclease activity.
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The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 has a Stokes radius of 7.2 in buffers of high ioninc strength, suggesting a molecular weight in the range 145,000 to 195,000. The polypeptide bands observed on gel electrophoresis in dodecyl sulfate have apparent molecular weights of 78,000 and 69,000 (and possibly another 27,000) in equimolar amounts. In buffers of low ionic strength, the enzyme appears to form large aggregates and even precipitates, with about 90% loss of activity. A nuclease activity co-purifies with the PBS2 DNA polymerase and shows similar responses to changes in pH, MgCl2, N-ethylmaleimide, temperature, and dextran sulfate levels. The nuclease produces deoxyribonucleoside 5'monophosphates from denatured DNA containing thymine or uracil. No endonuclease activity is detectable on supercoiled DNA. The inhibition of nuclease activity by added deoxyribonucleoside triphosphates, the DNA-dependent turnover of triphosphates, to free monophosphates during DNA polymerization, the inhibition of nuclease activity by 3'-phosphates on the DNA template-primer, and the pattern of digestion of 5'-[32P]phosphate-labeled DNA all indicate that the PBS2 DNA polymerase-associated hydrolytic activity is a 3' leads to 5'-exonuclease. (+info)
Bacillus subtilis bacteriophage PBS2-induced DNA polymerase. Its purification and assay characteristics.
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The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 (whose DNA contains uracil instead of thymine) has been purified and characterized for its specificity. The enzyme requires a high ionic strength for optimal stability and activity and is sensitive to various anions and to sulfhdryl reagents. Both dUTP and dTTP are incorporated efficiently as substrates and are competitive inhibitors at the same active site. The apparent Km and Ki values are about 6 micrometers for dTTP and 15 micrometers for dUTP, when denatured, uracil-containing B. subtilis or salmon sperm DNA (3.9 micrometers for dUTP and 2.6 micrometers for dTTP). The PBS2 enzyme works best on denatured DNA, on double-stranded DNA activated by DNase to produce gaps, or on primed homopolymeric DNA. Using denatured DNA preparations of average molecular weight 6.2 million, the apparent Km values are 270 micrograms/ml for B. subtilis DNA and 360 micrograms/ml for PBS2 DNA; the Vmax value for denatured PBS2 DNA containing uracil is 7-fold greater than that for denatured B. subtilis DNA containing thymine. However, lower molecular weight DNAs have 10-fold lower apparent Km values and show similar Vmax values for both B. subtilis and PBS2 DNAs. Thus, the PBS2 phage-induced DNA polymerase (which likely replicates only uracil-containing phage DNA using dUTP in vivo) has little selectivity for uracil- versus thymine-containing deoxyribonucleotides or DNA in vitro. (+info)
Xf phage invading the host cells with their protein coats.
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The Xf phage coat protein associated with infected cells could not be removed by washing with antiserum and tris-EDTA buffer. Although the infected cells were consecutively washed 6 times with tris-EDTA buffer, the ratios of parental phage 6H-DNA to 14C-protein were not changed. A considerable amount of the parental 14C-protein and 3H-DNA in the original ratio were detected in the membrane and the soluble cytoplasmic fractions of infected cells. The studies of the change in Xf 14C-protein and 3H-DNA incorporation into the host cells and their release showed that DNA and protein penetrate together into the host cells during the first 60 min after infection (p.i.). While virtually all parental DNA was conserved, re-utilized and released from the infected cell 60 min p.i., no apparent release of parental protein was observed. Approx. 40% of the parental protein became degraded and could be washed from the infected cell after 90 min; the rest of the parental protein remained and probably was re-utilized by the host. (+info)
Transcription of the genome of the filamentous bacteriophage cf from both plus and minus DNA strands.
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The filamentous bacteriophage cf infects the bacterium Xanthomonas campestris pv. citri. Northern blot analysis with probes derived from various restriction fragments of cf replicative form (RF) DNA has revealed the presence of five major phage-specific transcripts in infected cells. Four of these transcripts were shown to be derived from the region of the cf genome extending from gene II to gene VIII and are consistent with the cascade model of transcription proposed for Ff coliphages. These transcripts overlap with each other and terminate upstream of an efficient Rho-independent transcription terminator. Unlike the well-characterized Ff phages, in which only the minus strand of viral DNA serves as a transcription template, both strands of the RF DNA of phage cf appeared to be transcribed. Thus one of the five major cf transcripts was shown to be derived from a region of the viral minus strand that contains an open reading frame encoding a putative polypeptide of 165 amino acids. Primer extension analysis mapped the transcriptional initiation site of this RNA to a cytosine residue at position 870. A partial transcription map of phage cf revealed two independent regions of transcriptional activity. The region with the highest activity coincides with that encoding the polypeptides required in the largest amounts during the cf infection cycle. (+info)
Generating FSH antagonists and agonists through immunization against FSH receptor N-terminal decapeptides.
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Follicle-stimulating hormone (FSH) via interaction with G-protein coupled specific receptors plays a central role in the control of gametogenesis in mammals of both sexes. In females, FSH is crucial for follicle growth, follicle maturation and ovulation. FSH receptors, together with luteinizing hormone-chorionic gonadotropin and thyrotropin receptors belong to a subfamily of structurally related receptors within the seven transmembrane receptor family. Among several other regions, the N-terminus of these receptors is believed to be responsible for important specific hormone-receptor contact sites. Recombinant filamentous phages displaying at their surface three overlapping N-terminal decapeptides of the FSH receptor, peptides A18-27, B25-34 and C29-38 were constructed. Ewes and female mice were immunized against the three FSH receptor (FSHR) recombinant phages. Immunoglobulins purified from immunized animals were analyzed for their biochemical properties on a Chinese hamster ovary cell line expressing the porcine FSH receptor. AntiA and antiB immunoglobulins (IgGs) behave as antagonists for 125I-FSH binding and for FSH-dependent cAMP production, while antiC IgGs did not compete for hormone binding. By contrast, antibodies against the C29-38 peptide displayed FSH agonist activity and stimulated the FSH receptor, whereas antiA and antiB IgGs did not. Furthermore, when the FSHR phages were used as peptidic vaccines, they induced a reversible inhibition of ovulation rate in ewes, and impaired fertility in female mice. (+info)
Cooperative interaction of CI protein regulates lysogeny of Lactobacillus casei by bacteriophage A2.
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The temperate bacteriophage A2 forms stable lysogens in Lactobacillus casei. The A2-encoded cI product (CI), which is responsible for maintaining the A2 prophage in the lysogenic state, has been purified. The CI protein, which is a monomer of 25.3 kDa in solution, specifically binds to a 153-bp DNA fragment that contains two divergent promoters, PL and PR. These promoters mediate transcription from cI and a putative cro, respectively. Three similar, although not identical, 20-bp inverted repeated DNA segments (operator sites O1, O2, and O3) were found in this segment. CI selectively interacts with O1, which is placed downstream from the transcription start point of the cro gene, and with O2 and O3, which overlap with the -35 region of the two promoters. Using a heterologous RNA polymerase, we have determined the transcription start points of PL and PR. CI exerts a negative effect on the in vitro transcription of PR by repositioning the RNA polymerase in a concentration-dependent manner. CI, when bound to O1 and O2, enhances the positioning of the RNA polymerase with the PL promoter. Our data indicate that the CI protein regulates the lytic and lysogenic pathways of the A2 phage. (+info)
Staphylococcus aureus expresses a cell surface protein that binds both IgG and beta2-glycoprotein I.
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The existence of a second IgG-binding protein, protein Sbi, in Staphylococcus aureus has been reported previously. Later data indicated that protein Sbi also bound another serum component. This component has now been affinity-purified on immobilized protein Sbi and identified as beta2-glycoprotein I (beta2-GPI), also known as apolipoprotein H. The minimal beta2-GPI-binding domain was identified by shotgun phage display and the binding was shown to be mediated by a region of 57 amino acids, clearly separated from the IgG-binding domain. It is also shown that protein Sbi, and thus the beta2-GPI-binding activity, is expressed on the staphylococcal cell surface at levels varying between strains. (+info)
Isolation of human tumor-specific antibodies by selection of an antibody phage library on melanoma cells.
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A human single-chain Fv (scFv) library as fusion to phage was constructed from donors with a high titer of autoantibodies. The library was subjected to three rounds of positive selection on human melanoma cells and negative selection on human peripheral blood mononuclear cells. Two scFv clones, B3 and B4, were isolated that bound melanoma cells in cell ELISA and fluorescence-activated cell sorting. The scFvs were characterized further by immunohistochemistry on a large number of normal human tissues. No cross-reactivity with normal tissues was observed. On the other hand, the target antigens were expressed in sections from several different melanoma patients and in some breast cancer and basal cell carcinoma sections. The unusually high tumor specificity of the B3 and B4 antigens makes them attractive targets for the specific therapy of melanoma. The selection strategy used should be generally applicable to the identification of novel cell surface antigens by antibody phage display. (+info)