scholarly journals Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase

2021 ◽  
Author(s):  
Erika Schaudy ◽  
Jory Lietard ◽  
Mark M. Somoza
Keyword(s):  
Dna Synthesis ◽  
De Novo ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sequence Preference

Abstract 396: Regulation of Cardiomyocyte Proliferation by the Hippo Pathway and Dystrophin Complex

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Yuka Morikawa ◽  
John Leach ◽  
Todd Heallen ◽  
Ge Tao ◽  
James F Martin
Keyword(s):  
Cell Cycle ◽  
Muscular Dystrophy ◽  
Dna Synthesis ◽  
De Novo ◽  
Hippo Pathway ◽  
Myocardial Damage ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Heart Repair ◽  
The Hippo Pathway

Regeneration in mammalian hearts is limited due to the extremely low renewal rate of cardiomyocytes and their inability to reenter the cell cycle. In rodent hearts, endogenous regenerative capacity exists during development but is rapidly repressed after birth, at which time growth is by hypertrophy. During the developmental and neonatal periods, heart regeneration occurs through proliferation of pre-existing cardiomyocytes. Our approach of activating heart regeneration is to uncover the mechanisms responsible for repression of cardiomyocyte proliferation. The Hippo pathway controls heart size by repressing cardiomyocyte proliferation during development. By deleting Salv , a modulator of the Hippo pathway, we found that myocardial damage in postnatal and adult hearts was repaired both anatomically and functionally. This heart repair occurred primary through proliferation of preexisting cardiomyocytes. During repair, cardiomyocytes reenter the cell cycle; de novo DNA synthesis, karyokinesis, and cytokinesis all take place. The dystrophin glycoprotein complex (DGC) is essential for muscle maintenance by anchoring the cytoskeleton and extracellular matrix. Disruption of the DGC results in muscular dystrophies, including Duchenne muscular dystrophy, resulting in both skeletal and cardiac myopathies. Recently the DGC was shown to regulate cardiomyocyte proliferation and we found that the DGC and the Hippo pathway components directly interact. To address if the DGC and the Hippo pathway coordinately regulate cardiomyocyte proliferation, we conditionally deleted Salv in the mouse model of muscular dystrophy, the mdx line. We found that simultaneous disruption of both the DGC and Hippo pathway leads an increased de novo DNA synthesis and cytokinesis in cardiomyocytes after heart damage. Our findings provide new insights into the mechanisms leading to heart repair through proliferation of endogenous cardiomyocytes.

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

2007 ◽  
Vol 293 (3) ◽  
pp. G552-G559 ◽  
Author(s):  
Lawrence A. Scheving ◽  
Renee Buchanan ◽  
Michael A. Krause ◽  
Xiuqi Zhang ◽  
Mary C. Stevenson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Transforming Growth Factor ◽  
De Novo ◽  
Rat Hepatocytes ◽  
Immunological Methods ◽  
High Dose ◽  
Transforming Growth Factor Α ◽  
High Dose Dexamethasone ◽  
Cultured Rat Hepatocytes

Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10−6 M) on DNA synthesis. When we added transforming growth factor-α at the time of plating, 10−6 M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24–48 h after plating, 10−6 M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10−8 M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10−6 M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.

scholarly journals An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila

2012 ◽  
Vol 23 (11) ◽  
pp. 2213-2225 ◽  
Author(s):  
I-Ting Lin ◽  
Ju-Lan Chao ◽  
Meng-Chao Yao
Keyword(s):  
Dna Repair ◽  
Dna Cleavage ◽  
De Novo ◽  
Tetrahymena Thermophila ◽  
Chromosome Breakage ◽  
Protective Role ◽  
Terminal Deoxynucleotidyl Transferase ◽  
End Joining ◽  
Dna Rearrangements ◽  
Conjugation Process

Programmed DNA rearrangements are important processes present in many organisms. In the ciliated protozoan Tetrahymena thermophila, DNA rearrangements occur during the sexual conjugation process and lead to the deletion of thousands of specific DNA segments and fragmentation of the chromosomes. In this study, we found that the Ku80 homologue, a conserved component of the nonhomologous end-joining process of DNA repair, was essential for these two processes. During conjugation, TKU80 was highly expressed and localized to the new macronucleus, where DNA rearrangements occur. Homokaryon TKU80-knockout mutants are unable to complete conjugation and produce progeny and are arrested at the two-micronuclei/two-macronuclei stage. Analysis of their DNA revealed failure to complete DNA deletion. However, the DNA-cutting step appeared to have occurred, as evidenced by the presence of circularized excised DNA. Moreover, chromosome breakage or de novo telomere addition was affected. The mutant appears to accumulate free DNA ends detectable by terminal deoxynucleotidyl transferase dUTP nick end labeling assays that led to the degradation of most DNA in the developing macronucleus. These findings suggest that Tku80p may serve an end-protective role after DNA cleavage has occurred. Unexpectedly, the large heterochromatin structures that normally associate with DNA rearrangements failed to form without TKU80. Together the results suggest multiple roles for Tku80p and indicate that a Ku-dependent DNA-repair pathway is involved in programmed DNA rearrangements in Tetrahymena.

De Novo DNA Synthesis Using Single-Molecule PCR

2012 ◽  
pp. 35-47 ◽  
Author(s):  
Tuval Ben Yehezkel ◽  
Gregory Linshiz ◽  
Ehud Shapiro
Keyword(s):  
Dna Synthesis ◽  
Single Molecule ◽  
De Novo

Stretch-mediated visceral smooth muscle growth in vitro

1992 ◽  
Vol 262 (5) ◽  
pp. R895-R900
Author(s):  
O. M. Karim ◽  
K. Pienta ◽  
N. Seki ◽  
J. L. Mostwin
Keyword(s):  
Smooth Muscle ◽  
Dna Synthesis ◽  
De Novo ◽  
Specific Activity ◽  
Muscle Growth ◽  
Mechanical Stimulus ◽  
Trophic Factors ◽  
Taenia Coli ◽  
Visceral Smooth Muscle

An in vitro model of smooth muscle stretch was developed to study mechanical stimulus as a possible mediator of visceral smooth muscle growth and differences in the growth response of smooth muscle from young and old animals. De novo DNA synthesis as measured by the aphidicolin-sensitive specific activity of DNA was used as an index of cell growth. Compared with old tissue, the rate of aphidicolin-sensitive DNA synthesis in smooth muscle from young animals was 3-5 and 1.5-2 times greater in bladder and taenia coli, respectively. Stretch of bladder muscle and taenia coli strips from young animals for 6 h increased the aphidicolin-sensitive specific activity of DNA 3-fold (P less than 0.01) and 1.5-fold (P less than 0.01), respectively. Tissue from old animals, however, under the same conditions increased the rate of aphidicolin-resistant DNA synthesis, possibly implying DNA repair. Autoradiography showed only labeled myocyte nuclei. These results indicate that homeostatic mechanisms modulating myocyte growth in visceral smooth muscle can respond to mechanical stimulus in the absence of other trophic factors.

Apoptosis in megaloblastic anemia occurs during DNA synthesis by a p53-independent, nucleoside-reversible mechanism

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3249-3255 ◽  
Author(s):  
Mark J. Koury ◽  
James O. Price ◽  
Geoffrey G. Hicks
Keyword(s):  
Dna Synthesis ◽  
De Novo ◽  
Megaloblastic Anemia ◽  
Experimental Studies ◽  
Hematopoietic Cells ◽  
S Phase ◽  
De Novo Synthesis ◽  
Complete Reversal ◽  
Phase Accumulation

Abstract Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

scholarly journals Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 102 ◽  
Author(s):  
Sebastian Barthel ◽  
Sebastian Palluk ◽  
Nathan J. Hillson ◽  
Jay D. Keasling ◽  
Daniel H. Arlow
Keyword(s):  
Elevated Temperatures ◽  
De Novo ◽  
Fold Increase ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Synthesis Methods ◽  
Transferase Activity ◽  
Oligonucleotide Synthesis ◽  
De Novo Synthesis ◽  
Aqueous Conditions ◽  
Terminal Structure

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3′ terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

scholarly journals MESA: automated assessment of synthetic DNA fragments and simulation of DNA synthesis, storage, sequencing and PCR errors

2020 ◽  
Vol 36 (11) ◽  
pp. 3322-3326
Author(s):  
Michael Schwarz ◽  
Marius Welzel ◽  
Tolganay Kabdullayeva ◽  
Anke Becker ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Web Application ◽  
De Novo ◽  
Supplementary Information ◽  
Dna Fragments ◽  
Synthetic Dna ◽  
Custom Made ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Error Probabilities

Abstract Summary The development of de novo DNA synthesis, polymerase chain reaction (PCR), DNA sequencing and molecular cloning gave researchers unprecedented control over DNA and DNA-mediated processes. To reduce the error probabilities of these techniques, DNA composition has to adhere to method-dependent restrictions. To comply with such restrictions, a synthetic DNA fragment is often adjusted manually or by using custom-made scripts. In this article, we present MESA (Mosla Error Simulator), a web application for the assessment of DNA fragments based on limitations of DNA synthesis, amplification, cloning, sequencing methods and biological restrictions of host organisms. Furthermore, MESA can be used to simulate errors during synthesis, PCR, storage and sequencing processes. Availability and implementation MESA is available at mesa.mosla.de, with the source code available at github.com/umr-ds/mesa_dna_sim. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Detection of terminal deoxynucleotidyl transferase (TdT) by flow cytometry in leukemic disorders.

1989 ◽  
Vol 37 (4) ◽  
pp. 509-513 ◽  
Author(s):  
R H Bardales ◽  
A Carrato ◽  
M Fleischer ◽  
M K Schwartz ◽  
B Koziner
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Biochemical Assay ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Large Numbers ◽  
A New Technique ◽  
T Lymphoblastic Lymphoma

We applied a new technique to the detection of intracellular TdT in 26 leukemic patients, including 16 non-T acute lymphoblastic leukemia (ALL), four T-ALL, one T-lymphoblastic lymphoma in leukemia phase, one undifferentiated leukemia, one de novo lymphoblastic phase of chronic myeloid leukemia, and three acute monocytic leukemias (AMOL). Mononuclear cell suspensions were incubated in saponin to permeabilize the cell membrane. The cells were then stained by indirect immunofluorescence (IF) using anti-human TdT monoclonal antibodies and were analyzed by flow cytometry (FCM). The TdT results were compared with those obtained by biochemical TdT assay (26 cases), immunoperoxidase determination (PAP) (12 cases), and fluorescence microscopy (seven cases). The results obtained by PAP and fluorescence microscopy were 100% concordant with those obtained by FCM and biochemical assay. TdT determination by FCM allows the analysis of large numbers of cells in a fast, objective, and reliable manner, as compared with biochemical assay, PAP, and fluorescence microscopy determinations.

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