Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.
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PMID:Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I. 7 87

Plasmid DNA released from bacteria by boiling in the presence of lysozyme and Triton x-100 and without further purification can be sequenced by the dideoxy method using T7 DNA polymerase, when conditions during alkali denaturation and subsequent ethanol precipitation are adjusted to remove contaminants. The samples remain in the same microcentrifuge tubes from the harvesting of the bacteria until the splitting of the sample into four aliquots for the termination reactions. Less background label is observed with end-labelled primers (radioactivity or fluorescence), but even when radioactive nucleotides are incorporated during the sequencing reactions, 250 bases or more can be read from template prepared from 1.5 ml bacterial culture. The DNA can also be cut by restriction enzymes; the purification procedure described thus provides the rapid preparation of plasmids for a variety of purposes.
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PMID:Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes. 180 39

In our attempts to establish a cell-free DNA replication system for the yeast Saccharomyces cerevisiae, we have observed that recombinant DNA plasmids purified from Escherichia coli by a common procedure (lysozyme-detergent lysis and equilibrium banding in cesium chloride ethidium bromide gradients) often serve as templates for DNA synthesis by elongation enzymes. The templates could be elongated equally well by enzymes present in the yeast cell-free extracts, by the large proteolytic fragment of E. coli DNA polymerase I or by T4 DNA polymerase. The template activity of the purified plasmids was dependent on the presence of heterologous DNA segments in the bacterial vectors. The template activity could be diminished by treatment with alkali. We propose that the ability of recombinant plasmids isolated from bacterial hosts to serve as elongation templates may lead to erroneous conclusions when these plasmids are used as templates for in vitro replication or transcription reactions.
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PMID:DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli. 388 51

Previous work from this laboratory has shown that the cytosine-containing T4 deoxyribonucleic acid (DNA) made by deoxycytidine triphosphatase (dCTPase) amber mutants is extensively degraded, and that nucleases controlled by genes 46 and 47 participate in this process. In this paper, we examine other consequences of a defective dCTPase. Included are studies of DNA synthesis and phage production, and of the control of both early and late protein synthesis after infection of Escherichia coli B with various T4 mutants defective in genes 56 (dCTPase), 42 (dCMP hydroxymethylase), 1 (deoxynucleotide kinase), 43 (DNA polymerase), 30 (polynucleotide ligase), 46 and 47 (DNA breakdown) or e(lysozyme). By varying the temperature of infection with a temperature-sensitive dCTPase mutant, we have been able to control intracellular dCTPase activity, and thus vary the cytosine content of the phage DNA. We have produced and characterized viable T4 phage in which cytosine replaces 20% of the 5-hydroxymethylcytosine (HMC) in the DNA. We present evidence which suggests that intact, cytosine-containing T4 DNA is much less efficient than is normal T4 DNA in directing the synthesis of tail-fiber antigen. Lysozyme production is much less affected by progressively decreasing dCTPase activity; however, complete substitution of cytosine is correlated with a depression of lysozyme synthesis greater than expected from the defective synthesis of DNA. Low but significant lysozyme synthesis is observed late after infection of E. coli B with T4 amber mutants defective in a number of genes controlling DNA synthesis. The "20% cytosine" T4 phage, once produced, can initiate an apparently normal infection at permissive temperatures; the synthesis of early enzymes, DNA, and phage does not appear to be impaired. Two roles for HMC in T4 DNA have been indicated previously: (i) involvement in host-controlled restriction of the phage, in which glucosylation of the hydroxymethyl group plays a crucial role (16, 29, 53, 58), and (ii) protection of vegetative DNA against phage-controlled nucleases, a protection not dependent on glucosylation (41, 66, 67). A third role is suggested by our present results: transcription of at least some late genes can occur only from HMC-containing DNA and not from cytosine-containing DNA.
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PMID:Biological effects of substituting cytosine for 5-hydroxymethylcytosine in the deoxyribonucleic acid of bacteriophage T4. 430 78

During nonpermissive infection by a T7 amber mutant in gene 1 (phage RNA polymerase-deficient), synthesis of the products of the phage genes 3 (endonuclease), 3, 5 (lysozyme), 5 (DNA polymerase), and 17 (serum blocking power) was shown to occur at about half the rate as during wild-type infection. This relatively high rate of expression of "late" genes (transcribed normally by the phage RNA polymerase) seems to be a general feature of all T7 mutants in gene 1 from our collection. In contrast, T3 gene 1 mutants and a T7 gene 1 mutant from another collection showed late protein synthesis at very reduced rates. Synthesis of the gene 3 endonuclease by T7 gene 1 mutants was very sensitive to the addition of rifampin 2 min after infection, conditions under which there was very little inhibition during wild-type infection. This supports the notion that late gene expression during nonpermissive infection by gene 1 mutants is dependent on the transcription of the T7 genome by the host RNA polymerase. In contrast to T3 gene 1 mutants, the T7 gene 1 mutants of our collection directed the synthesis of phage DNA during nonpermissive infection. This DNA accumulated as a material sedimenting faster than mature T7 DNA.
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PMID:Synthesis of bacteriophage-coded gene products during infection of Escherichia coli with amber mutants of T3 and T7 defective in gene 1. 457 63

Synthesis of many T7 proteins is prevented in F' episome-containing cells. In order to quantitate the degree of inhibition, we measured the activity of several T7 proteins in extracts prepared from T7-infected F(-) and F' cells and cells containing F factors mutant in phage inhibition [F'(PIF(-)2A) and F'(PIF(-)2A,2B)]. In addition, we were able to assign specific T7 proteins to the three translational units previously defined by polyacrylamide gel analysis of T7 proteins made in F(-) and episome-containing cells. After T7 infection, the presence of the wild-type F' (PIF(+)) episome led to greater than 90% inhibition of T7 DNA polymerase (product of gene 5), T7 lysozyme (gene 3.5), and gene 10 capsid protein synthesis. Nearly normal amounts of T7 RNA polymerase (gene 1) were made in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A) episome led to normal synthesis of T7 RNA polymerase and T7 DNA polymerase; T7 lysozyme was synthesized at 30% of the maximal level in these cells; T7 gene 10 capsid protein synthesis was inhibited by 90%, and T7 DNA synthesis was arrested in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A,2B) episome led to synthesis of normal levels of the enzymes assayed.
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PMID:T7 protein synthesis in F' episome-containing cells: assignment of specific proteins to three translational groups. 458 55

These data demonstrate that actinomycin D inhibits only 75-80% of DNA synthesis in cells of E. coli treated by lysozyme and ethyl enediaminetetraacetate. The residual synthesis is not the result of untemplated polymerization of dNTP. The DNA synthesis in spheroplasts does not correlate with replication of chromosomal DNA of E. coli catalyzed by DNA polymerase III sensitive to sulfhydryl blocking agents. N-ethylmaleimide does not inhibit this synthesis. No ATP stimulation of DNA synthesis is observed. The enzyme(s) responsible for DNA synthesis on endogenous template is (are) concentrated in interphase (D-fraction) as revealed by high speed centrifugation of spheroplasts lysate and they are absent in the chromosomal DNA fraction. dTTP 4-(N-2-chloroethyl-N-mehylamino) benzylamide suppresses completely the insensitive to actinomycin D action DNA synthesis and practically does not act on the sensitive one. It is suggested, that the DNA synthesis stable to the action of the antibiotic is catalyzed by RNA dependent DNA polymerase.
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PMID:[Location of RNA-dependent DNA polymerase within the endogenous template complex and discrimination of RNA- and DNA-dependent synthesis of DNA in Escherichia coli cells by alkylating dTTP gamma-amide]. 619 21

Blast cells in acute leukemia and lymphoma appear to be "frozen" at various stages of lymphoid cell differentiation. The enzymatic and antigenic phenotypes expressed by these cells often correspond to the gene products of their normal precursors. We have used various immunocytochemical and enzymatic techniques to identify membrane, nuclear, and cytoplasmic markers associated with the prolactin-dependent Nb2 lymphoma cell line. The Nb2 cells, whether stationary or in log-phase growth, did not express any surface immunoglobulin. However, 100% of the Nb2 cells bound both a monoclonal antibody raised to rat thymocyte W3/25-HLK, which specifically binds an antigenic determinant on rat T-helper cells, and second monoclonal antibody OX8-HL, which identifies rat nonhelper T-cells. Transmission electron microscopy showed no evidence of phagocytic vacuoles, and activity of the lysosomal enzyme muramidase was also absent. There was no evidence of the DNA polymerase enzyme terminal deoxynucleotidyl transferase. alpha-Naphthyl acetate esterase activity was indicated in about 50% of the Nb2 cells by a faint particulate cytoplasmic staining similar to that found in thymocytes. Rosette formation with guinea pig erythrocytes, a property of mature rat thymocytes, was not observed with Nb2 cells. The data suggest that the Nb2 tumor may have arisen from a thymocyte at an intermediate stage of differentiation. The presence of Thy-like alpha-naphthyl acetate esterase pattern and the binding of both W3/25-HLK and OX8-HL support the thymic origin and relative immaturity of these lymphoid cells. It is becoming increasingly apparent that a significant proportion of lymphomas and leukemias also originate in undifferentiated thymic cels.
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PMID:Thymic origin of the prolactin-dependent Nb2 lymphoma cell line. 704 17

Twenty-one independent thymidylate synthase deficient (td) mutants were isolated after proflavin mutagenesis of T4D0 phage. A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5' splice site of this intron-containing gene. This region comprises only 14% of the total td exon sequence. RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3' end of exon I of the td gene. The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3' ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine. Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phosphodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA. Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis. The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4.
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PMID:A proflavin-induced frameshift hotspot in the thymidylate synthase gene of bacteriophage T4. 768 30

An instrument for continuous-flow quasielastic light scattering is described that allows the translational diffusion coefficient of macromolecules to be determined as a function of time after the initiation of some time-dependent process by mixing. Control experiments are carried out using the proteins lysozyme and BSA to verify that flow of the solution does not lead to erroneous results. The instrument is used to determine the lifetime of the histone octamer. A solution of octamer that is artificially stabilized in 2.0 M NaCl is rapidly diluted to physiological ionic strength, and the Stokes diameter is determined as a function of the time, delta t, after mixing. We find that the octamers dissociate into their component H2A-H2B heterodimers and H(3)2H4 tetramers on a time scale that is faster than the earliest time point for which data were obtained, 1 s after mixing. This result argues against a simple mechanism for the progression of RNA or DNA polymerase through chromatin.
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PMID:Lifetime of the histone octamer studied by continuous-flow quasielastic light scattering: test of a model for nucleosome transcription. 834 88


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