Characteristics of the major DNA polymerases found during early and late maize germination

1988 ◽  
Vol 66 (6) ◽  
pp. 1186-1191 ◽  
Author(s):  
Alejandra Vazquez ◽  
Jorge Vazquez-Ramos
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Absolute Requirement ◽  
Optimum Ph ◽  
Embryo Axes ◽  
The Difference ◽  
Alpha Type ◽  
Degree Of Purification ◽  
Optimum Ph And Temperature

Two types of DNA synthesis have been detected during maize germination (0–24 h). To determine if the difference in these two types of synthesis was due to the presence of distinct DNA polymerases, we partially isolated and characterized the enzymes present at 3 (early) and 24 (late) h of germination. The material used was embryo axes. The result indicates that at both 3 and 24 h, enzymes are similar with regard to optimum pH and temperature, absolute requirement of Mg2+ (12 mM), and stimulation by KCl (50 mM). They are also equally inhibited by N-ethylmaleimide and cytosine-β-D-arabinofuranoside. These facts would classify the enzymes as alpha type; however, the enzymes differ in their sensitivity to aphidicolin and the degree of purification. The nature of the enzymes is discussed.

scholarly journals CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Katerina Zabrady ◽  
Matej Zabrady ◽  
Peter Kolesar ◽  
Arthur W. H. Li ◽  
Aidan J. Doherty
Keyword(s):  
Dna Repair ◽  
Dna Synthesis ◽  
Genome Stability ◽  
Dna Polymerases ◽  
Acquired Immunity ◽  
Strand Displacement ◽  
Genetic Studies ◽  
Type Iiia ◽  
Maintain Genome Stability ◽  
Cas Proteins

AbstractCRISPR-Cas pathways provide prokaryotes with acquired “immunity” against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.

scholarly journals Immobilization of alpha-amylase produced by Bacillus circulans GRS 313

2003 ◽  
Vol 46 (2) ◽  
pp. 167-176 ◽  
Author(s):  
Gargi Dey ◽  
Singh Bhupinder ◽  
Rintu Banerjee
Keyword(s):  
Calcium Alginate ◽  
Immobilized Enzyme ◽  
Fold Increase ◽  
Alginate Beads ◽  
Hydrolysis Kinetics ◽  
Initial Activity ◽  
Reaction Conditions ◽  
Bead Size ◽  
Optimum Ph ◽  
Optimum Ph And Temperature

A maltooligosaccharide-forming amylase from B circulans GRS 313 was immobilized by entrapment in calcium alginate beads. The immobilized activity was affected by the size of the bead and bead size of 2mm was found to be most effective for hydrolysis. Kinetics constants, Km and Vmax were estimated and were found to be affected by the bead size. The catalytic activity of the enzyme was studied in presence of various starchy residues and metal ions. HgCl2, CuSO4 and FeCl3 caused inhibition of the enzyme. The reaction conditions, pH and temperature, was optimized using response surface methodology. At the optimum pH and temperature of 4.9 and 57ºC, the apparent activity was 25.6U/g of beads, resulting in almost 2-fold increase in activity. The immobilized enzyme showed a high operational stability by retaining almost 85% of the initial activity after seventh use.

Effects of Polyamines on In Vitro DNA Synthesis by DNA Polymerases from Calf Thymus

1976 ◽  
Vol 79 (5) ◽  
pp. 895-901 ◽  
Author(s):  
Shonen YOSHIDA ◽  
Shigeo MASAKI ◽  
Teruo ANDO
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Calf Thymus

Cytoplasmic control of DNA synthesis by myocardial nuclei

1980 ◽  
Vol 238 (1) ◽  
pp. H66-H72
Author(s):  
C. J. Limas
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Postnatal Growth ◽  
Neonatal Rats ◽  
Cytoplasmic Factor ◽  
Age Dependent ◽  
Rat Myocytes ◽  
Old Rats ◽  
Isolated Rat

In vitro DNA synthesis by isolated myocardial nuclei declines rapidly during postnatal growth. To study the mechanism(s) responsible for this decline, cytoplasmic extracts (CE) were prepared from isolated rat myocytes at different times after birth. CE from 2-day-old rats stimulated in vitro DNA synthesis by myocardial nuclei from adult (6 mo old) rats (55 +/- 6 pmol[3H]dTMP . mg DNA-1 . 15 min-1 vs. 32 +/- 4 pmol [3H]dTMP . mg DNA-1 . 15 min-1 in untreated controls, P less than 0.01). The ability of cytoplasmic extracts of stimulate DNA synthesis decreased with age, from 73 +/- 9% over controls at age 2 days to 18 +/- 6 at 28 days; adult myocytes were essentially ineffective. Pulse-chase experiments demonstrated that CE-directed DNA synthesis was replicative and discontinuous. CE stimulatory activity was heat-labile, nondialyzable, trypsin-sensitive, and distinct from DNA polymerases. The results indicate that a) adult myocyte nuclei can be induced to synthesize DNA by cytoplasmic extracts from neonatal rats, and b) that absence of regulatory cytoplasmic factor(s) may, in part, explain the age-dependent decline in myocardial DNA synthesis.

scholarly journals Purification and Characterization of a Feruloyl Esterase from the Intestinal Bacterium Lactobacillus acidophilus

2004 ◽  
Vol 70 (4) ◽  
pp. 2367-2372 ◽  
Author(s):  
Xiaokun Wang ◽  
Xin Geng ◽  
Yukari Egashira ◽  
Hiroo Sanada
Keyword(s):  
Lactobacillus Acidophilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Feruloyl Esterase ◽  
Intestinal Bacterium ◽  
Optimum Ph ◽  
Optimum Ph And Temperature

ABSTRACT Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37°C, respectively). The metal ions Cu2+ and Fe3+ (at a concentration of 5 mmol liter−1) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-l-arabinofuranose (FAA) as a substrate, the enzyme exhibited a K m of 0.0953 mmol liter−1 and a V max of 86.27 mmol liter−1 min−1 mg−1 of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-α-l-arabinofuranosyl)-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.

scholarly journals Rolling Circle DNA Synthesis:  Small Circular Oligonucleotides as Efficient Templates for DNA Polymerases

1996 ◽  
Vol 118 (7) ◽  
pp. 1587-1594 ◽  
Author(s):  
Dongyu Liu ◽  
Sarah L. Daubendiek ◽  
Martin A. Zillman ◽  
Kevin Ryan ◽  
Eric T. Kool
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Rolling Circle
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