An autoradiographical study of amino acid and nucleoside incorporation during the cell cycle of Amoeba proteus

1981 ◽  
Vol 51 (1) ◽  
pp. 219-228
Author(s):  
K.I. Mills ◽  
L.G. Bell
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Amino Acid ◽  
Rna Synthesis ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Amoeba Proteus ◽  
Macromolecular Synthesis ◽  
Future Studies ◽  
Specific Proteins

The incorporation of tritiated thymidine, uridine and leucine, into the acid-precipitable material of DNA. RNA and proteins, respectively, was studied by autoradiography throughout the cell cycle of Amoeba proteus. DNA synthesis occupied the first 17 h of the cycle (57 h long) and 2 peaks between 0.5 and 9.13 h accounted for the majority of the thymidine incorporation. RNA synthesis was represented by a series of peak uridine grain counts, the 3 major peaks occurring at 10, 26–27 and 47–48 h. The incorporation of leucine also followed a pattern of peaks and dips, the main peaks occurring 1–2 h after the major increases in uridine incorporation. The fraction of label present over the nucleus decreased during the cell cycle, and this was probably due to a lowered incorporation of the leucine label by proteins synthesized in the cytoplasm and destined to become nuclear proteins. The incorporation patterns of 6 amino acids (arginine, aspartic acid, leucine, lysine, serine and valine) were studied individually during 3 periods of the cell cycle: 0-10 h (S phase); 20–30 h (early G2); and 40–50 h (mid-late G2). Variations in the intensity and timings of the incorporation maxima of the amino acids were observed, although the timings of increased grain counts of some of the amino acids frequently coincided. “Unique” incorporation peaks (i.e. only observed in one of the amino acids studied) possibly indicate the synthesis of phase-specific proteins. The amino acid and nucleoside incorporation profiles presented in this paper will enable the results obtained from future studies on amoebae to be related to the macromolecular synthesis patterns.

Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells

2010 ◽  
Vol 298 (6) ◽  
pp. R1615-R1626 ◽  
Author(s):  
Neil I. Bower ◽  
Ian A. Johnston
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Amino Acid ◽  
Atlantic Salmon ◽  
Antigen Expression ◽  
Nuclear Antigen ◽  
Quiescent State ◽  
Mrna Levels ◽  
Proliferating Cells

The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon ( Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18°C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression ( R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoD1a transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoD1a and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the G1 and S/G2 phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in the Atlantic salmon lineage.

Timing of nucleolar DNA replication in Amoeba proteus

1976 ◽  
Vol 22 (3) ◽  
pp. 521-530
Author(s):  
I. Minassian ◽  
L.G. Bell
Keyword(s):  
Electron Microscope ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Central Region ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Amoeba Proteus ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
G2 Phase

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.

Control of RNA Synthesis by Amino Acids in Landschutz Ascites Tumor Cells

1974 ◽  
Vol 52 (10) ◽  
pp. 867-876 ◽  
Author(s):  
Paul Jolicoeur ◽  
Fernand Labrie
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Inhibitory Effect ◽  
Amino Acid Starvation ◽  
Cytoplasmic Rna ◽  
Polysomal Mrna ◽  
Deficient Medium ◽  
Synthesis And Processing

Landschutz cells incubated in amino acid-deficient medium for 2.5 h show a markedly reduced incorporation of [3H]uridine into 18 S and 28 S cytoplasmic ribosomal RNA (rRNA) and into 28 S, 32 S, and 36 S nuclear RNA measured during the last 90 min of incubation, whereas the radioactivity associated with 45 S pre-rRNA is not affected. Ten-minute pulse-labeling and 15-min pulse-chase experiments show that amino acid starvation inhibits both the synthesis and processing of 45 S pre-rRNA. Amino acid starvation has no significant effect on the labeling of the nucleotide pools. This effect of amino acids was specific for rRNA since the synthesis of 4 S and 5 S cytoplasmic RNA separated on polyacrylamide gels and of polysomal mRNA analyzed on sucrose gradients was not significantly affected during amino acid starvation. These data also indicate that RNA synthesis is non-coordinated in Landschutz cells. Among the 13 amino acids essential for growth of these cells, arginine and glutamine appear to be mainly responsible for the inhibition of synthesis of 18 S and 28 S rRNA measured during incubation in complete amino acid-deficient medium. The removal of any one of the other amino acids has a small inhibitory effect on the incorporation of [3H]uridine into rRNA and their effect on the synthesis of 18 S rRNA is more pronounced than on that of 28 S rRNA. Such effect results in an unbalanced production of these two ribosomal RNA species.

scholarly journals PERIODIC CHANGES IN RATE OF AMINO ACID UPTAKE DURING YEAST CELL CYCLE

1973 ◽  
Vol 58 (2) ◽  
pp. 401-409 ◽  
Author(s):  
B. L. A. Carter ◽  
H. O. Halvorson
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Amino Acid ◽  
Uptake Rate ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Uptake System ◽  
Transport Capacity ◽  
Initial Uptake Rate ◽  
Initial Uptake

Uptake of amino acids is a complex process but in cells growing with ammonia as sole nitrogen source the initial uptake rate of amino acids is a measure of the transport capacity of the uptake system (permease). In synchronous cultures of Saccharomyces cerevisiae amino acids were transported at all stages of the cell cycle. However, for any one amino acid the initial uptake rate was constant for most of the cycle and doubled during a discrete part of the cycle. Thus, for a variety of amino acids the functioning amino acid transport capacity of the membrane doubles once per cycle at a characteristic stage of the cycle. Arginine, valine, and phenylalanine exhibit periodic doubling of uptake rate at different stages of the cell cycle indicating that the transport of these amino acids is mediated by three different systems. Serine, phenylalanine, and leucine exhibit periodic doubling of the uptake rate at the same stage of the cycle. However, it is unlikely that serine and phenylalanine share the same transport system since the uptake of one is not inhibited by the other amino acid. This phenomenon is analogous to the periodic synthesis of soluble enzymes observed in S. cerevisiae.

Effect of hypophysectomy on amino acid, thymidine, and orotic acid incorporation into the mucosa of the small bowel

1970 ◽  
Vol 48 (7) ◽  
pp. 828-830 ◽  
Author(s):  
Reuven Levitan ◽  
Eli Havivi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Small Bowel ◽  
Intestinal Mucosa ◽  
Orotic Acid ◽  
Tritiated Thymidine ◽  
Dna And Rna ◽  
Acid Incorporation

After hypophysectomy, the incorporation of tritiated thymidine and orotic acid into DNA and RNA, respectively, decreased in the intestinal mucosa. Hypophysectomy lowered the incorporation of amino acids into protein in vitro, yet the proportion of polysomes remained unaltered. After hypophysectomy, the polysomes were impaired in their ability to incorporate amino acids in vitro.

Kinetic Studies of Cultured Human Keratinocytes: A Comparison between 5-Bromodeoxyuridine and Tritiated Thymidine Incorporation

1988 ◽  
Vol 16 (1) ◽  
pp. 27-31
Author(s):  
Luisa Benassi ◽  
Giovanna Zambruno ◽  
Paola Massimi ◽  
Fabrizio Fantini ◽  
Alberto Gianetti
Keyword(s):  
Cell Cycle ◽  
Monoclonal Antibodies ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Kinetic Studies ◽  
Human Keratinocytes ◽  
S Phase ◽  
Radioactive Material ◽  
Immunological Method ◽  
Pyrimidine Analogue

5-Bromodeoxyuridine (5-BrdU), a pyrimidine analogue of thymidine, is incorporated into the DNA which is actively synthesised during the S-phase of the cell cycle. In the present study, the labelling indices of cultured human keratinocytes, marked by monoclonal antibodies to 5-BrdU and by tritiated thymidine (3H-TdR), were compared. The proportion of cells found to be labelled by the two methods was similar. The immunological method has advantages over 3H-TdR autoradiography, because it does not require radioactive material, and is both faster and less expensive.

scholarly journals Investigating the Specific Interactions Between Microtubule Stabilising Agents and β-Tubulin

2021 ◽  
Author(s):  
◽  
Benjamin Jones
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Binding Site ◽  
Drug Class ◽  
Efflux Pumps ◽  
Peloruside A ◽  
Mutant Cells ◽  
Β Tubulin

<p>Microtubule stabilising agents are a class of cytotoxic compounds that cause mitotic arrest through inhibition of microtubule function. They specifically target β-tubulin subunits promoting tubulin polymerisation, which eventually leads to cell death. Members of this drug class include the cancer chemotherapeutics paclitaxel and ixabepilone. However, like many cytotoxic agents, tumour cells often develop multi-drug resistance phenotypes limiting the effectiveness of such compounds. This results from the expulsion of these drugs from cells by efflux pumps, as well as mutation of their binding site. Much effort has been focused on improving the utility of this important drug class in the ongoing fight against cancer.  The microtubule stabilising agents peloruside A and laulimalide originate from marine sponge species native to the South Pacific. They have similar pharmacological profiles to paclitaxel and ixabepilone, however with several unique properties. They are poor substrates for efflux pumps and target a different region on β-tubulin subunits, giving them the potential for treatment of resistant tumours. This represents a novel mechanism of action that may be exploited for drug development, and further characterisation of the binding site is warranted.  The aim of this study is to investigate the contribution of two amino acids of human βItubulin to the interactions with peloruside A and laulimalide. Specifically, glu127 and lys124 have been predicted by computational modelling and analogue studies to form hydrogen bonds and other associations with the two compounds. These amino acids are located on β-tubulin subunits adjacent to the main binding pocket of peloruside A and laulimalide, and represent a potential inter-protofilament interaction that does not occur with other microtubule stabilising agents. This binding mechanism has not yet been shown by crystallography and is hence based solely on in silico work, requiring biological validation.  HEK293 cells were transfected with βI-tubulin with these amino acids mutated to alanines to prevent hydrogen bond formation. Cell proliferation assays, flow cytometry, and immunoblotting were used to study the effect loss of the inter-protofilament interaction has on the bioactivity of peloruside A and laulimalide. These mutations did not significantly alter the concentration-response of cells to either drug in the cell proliferation assay. However, accumulation of cells in the G2/M phase of the cell cycle and the proportion of transfected cells showing signs of mitotic arrest significantly decreased for E127A mutant cells compared to wild type βI-tubulin transfected control cells treated with peloruside A. Furthermore, a similar reduction in cell cycle block was also seen in E127A mutant cells treated with the negative control ixabepilone, which binds to a different site on β-tubulin.  No evidence seen in this study suggests that either amino acid plays a major role in peloruside A or laulimalide target binding. However, the amino acid E127 is important for inter-protofilament associations independent of drug treatment, as its mutation appeared to reduce global stability of microtubule structures. This information requires further validation, it may be useful in the design of future analogue syntheses as development of these promising drug candidates continues.</p>

Growth and Changes in Pool and Macromolecular Components of Schizosaccharomyces Pombe During the Cell Cycle

1971 ◽  
Vol 9 (3) ◽  
pp. 701-717
Author(s):  
NOWELL STEBBING
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Minimal Medium ◽  
Linear Growth ◽  
Dry Weight ◽  
Total Cell ◽  
Complex Medium ◽  
Synchronous Cultures

Amino acids, nucleotide and carbohydrate material were found to account for 46% of the total dry weight of pool material in Schizosaccharomyces pombe growing in minimal medium. The composition of the amino acid pool was also determined by autoanalysis and was found to be unaltered during growth in 2 M sorbitol, indicating that pool amino acids are not important in the osmoregulation of the cell. Kinetic analysis of the amino acid pool using 14C-labelled amino acids showed that amino acids accumulated from the medium enter an ‘expandable’ pool distinct from the ‘internal’ pool which is maintained during growth on minimal medium. Total RNA, protein, pool amino acid and pool ‘nucleotide’ material were estimated in synchronous cultures grown in minimal medium. All these components appeared to accumulate in an exponential manner during the cell cycle. Direct estimation of total cellular dry weight and the total pool in synchronous cultures showed that total cell dry weight increased exponentially and the pool did not fluctuate during growth in minimal medium. This contrasts with previous work on single cells of S. pombe grown in complex medium which showed that the dry weight of the pool fluctuates during the cell cycle and total cell dry weight increased linearly. Linear growth of S. pombe in malt extract broth can be accounted for by the presence of the second (‘expandable’) pool of amino acids formed during growth in complex medium. The phenomenon of linear growth during the cell cycle is shown to occur generally only in cells growing in complex medium. The phenomenon is considered in relation to mechanisms for controlling the size of the pool during growth in complex media.

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