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 Metabolic and contractile influence of carbonic anhydrase III in skeletal muscle is age dependent

1999 ◽  
Vol 276 (2) ◽  
pp. R559-R565 ◽  
Author(s):  
Claude H. Côté ◽  
Fabrisia Ambrosio ◽  
Guylaine Perreault
Keyword(s):  
Skeletal Muscle ◽  
Data Analysis ◽  
Carbonic Anhydrase ◽  
Soleus Muscle ◽  
Type I ◽  
Carbonic Anhydrase Iii ◽  
Age Dependent ◽  
Old Rats ◽  
Birth Data

Carbonic anhydrase (CA) III is very abundant in type I skeletal muscle, but its function is still debated. Our aims were to examine CA III expression during growth and determine whether the effects of CA inhibition previously observed in adult muscles could be seen in younger rats in which CA III levels are lower. CA III content and activity were measured in soleus muscles from 10- to 100-day-old rats, and the influence of CA inhibitor on fatigue and hexosemonophosphate content was quantified in vitro. CA III activity and content increased fivefold between 10 and 100 days of age. Data analysis revealed that the influence of CA inhibitor on fatigue was to some extent positively and linearly related to the level of CA III activity. Hexosemonophosphate accumulation with CA inhibition also became more significant with age. In conclusion, CA III level in soleus muscle does not stabilize before 3 mo after birth; data also confirm that the effects of CA inhibitors are due to inhibition of the CA III isoform.

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

Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro

1989 ◽  
Vol 9 (2) ◽  
pp. 469-476
Author(s):  
J D Roberts ◽  
B D Preston ◽  
L A Johnston ◽  
A Soni ◽  
L A Loeb ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Leukemia Virus ◽  
Dna Polymerases ◽  
Moloney Murine Leukemia Virus ◽  
Avian Myeloblastosis Virus ◽  
Single Base ◽  
Reverse Transcriptases ◽  
Template Strand ◽  
Cellular Dna

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.

scholarly journals DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs

2007 ◽  
Vol 189 (15) ◽  
pp. 5652-5657 ◽  
Author(s):  
Kazuo Tori ◽  
Megumi Kimizu ◽  
Sonoko Ishino ◽  
Yoshizumi Ishino
Keyword(s):  
Dna Synthesis ◽  
Proliferating Cell Nuclear Antigen ◽  
Pyrococcus Furiosus ◽  
Dna Polymerases ◽  
Nuclear Antigen ◽  
Hyperthermophilic Archaeon ◽  
Sliding Clamp ◽  
Replication Fork Progression ◽  
Proliferating Cell

ABSTRACT Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

scholarly journals The separate roles of glucose and insulin in the induction of glucokinase in hepatocytes isolated from neonatal rats

1981 ◽  
Vol 196 (2) ◽  
pp. 383-390 ◽  
Author(s):  
M J Wakelam ◽  
D G Walker
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Actinomycin D ◽  
Neonatal Rats ◽  
Dibutyryl Cyclic Amp ◽  
Subsequent Effect ◽  
Old Rats ◽  
Effect Of Glucose

1. The specificity of the effect of glucose on the induction of glucokinase activity that occurs when hepatocytes freshly isolated from 13-day-old rats are incubated in Medium 199 together with insulin [Wakelam & Walker (1980) FEBS Lett. 111, 115-119] was examined. A pattern that is different from other known effects of glucose is found, and metabolism of this compound is not necessarily to account for this particular effect. 2. The effects of a raised glucose concentration and of insulin on the induction can be separated. The hexose initiates the process in the absence of insulin in a manner that is sensitive to actinomycin D but not to cycloheximide. The subsequent effect of insulin is dependent on the prior effect of glucose or other positive analogue, does not require the presence of glucose and is inhibited by cycloheximide but not by actinomycin D. 3. Induction of glucokinase in vitro in hepatocytes from neonatal animals is inhibited by adrenaline, glucagon and dibutyryl cyclic AMP, but not by vasopressin or angiotensin II. The inhibition by cyclic AMP is on the stage requiring insulin and is comparatively specific, because total protein synthesis is not apparently diminished. 4. The implications of these results are discussed with reference to possible mechanisms of induction and to the situation in vivo.

scholarly journals Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

2004 ◽  
Vol 24 (7) ◽  
pp. 2734-2746 ◽  
Author(s):  
Atsuko Niimi ◽  
Siripan Limsirichaikul ◽  
Shonen Yoshida ◽  
Shigenori Iwai ◽  
Chikahide Masutani ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Pyrimidine Dimer ◽  
Replication Fidelity ◽  
Dna Polymerase Α ◽  
Replication Errors ◽  
Translesion Dna

ABSTRACT We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

scholarly journals Group A Rotavirus Infection and Age-Dependent Diarrheal Disease in Rats: a New Animal Model To Study the Pathophysiology of Rotavirus Infection

2002 ◽  
Vol 76 (1) ◽  
pp. 41-57 ◽  
Author(s):  
Max Ciarlet ◽  
Margaret E. Conner ◽  
Milton J. Finegold ◽  
Mary K. Estes
Keyword(s):  
Animal Model ◽  
Small Intestine ◽  
Virus Antigen ◽  
Neonatal Rats ◽  
Histopathological Changes ◽  
Rotavirus Infection ◽  
Age Dependent ◽  
Group A Rotaviruses ◽  
Group A ◽  
Old Rats

ABSTRACT Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (ECwt) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not ≥21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naÏve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine ECwt rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.

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