scholarly journals The configuration of DNA replication sites within the Trypanosoma brucei kinetoplast.

1994 ◽  
Vol 126 (3) ◽  
pp. 641-648 ◽  
Author(s):  
D R Robinson ◽  
K Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
S Phase ◽  
Immunogold Labeling ◽  
Kinetoplast Dna ◽  
Dna Molecules ◽  
Circular Dna ◽  
Replication Sites ◽  
Or Organization

The kinetoplast is a concatenated network of circular DNA molecules found in the mitochondrion of many trypanosomes. This mass of DNA is replicated in a discrete "S" phase in the cell cycle. We have tracked the incorporation of the thymidine analogue 5-bromodeoxyuridine into newly replicated DNA by immunofluorescence and novel immunogold labeling procedures. This has allowed the detection of particular sites of replicated DNA in the replicating and segregating kinetoplast. These studies provide a new method for observing kinetoplast DNA (kDNA) replication patterns at high resolution. The techniques reveal that initially the pattern of replicated DNA is antipodal and can be detected both on isolated complexes and in replicating kDNA in vivo. In Trypanosoma brucei the opposing edges of replicating kDNA never extend around the complete circumference of the network, as seen in other kinetoplastids. Furthermore, crescent-shaped labeling patterns are formed which give way to labeling of most of the replicating kDNA except the characteristic midzone. The configuration of these sites of replicated DNA molecules is different to previous studies on organisms such as Crithidia fasciculata, suggesting differences in the timing of replication of mini and maxicircles and/or organization of the replicative apparatus in the kinetoplast of the African trypanosome.

scholarly journals Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

1999 ◽  
Vol 19 (12) ◽  
pp. 8191-8200 ◽  
Author(s):  
Philippe Bastin ◽  
Thomas H. MacRae ◽  
Susan B. Francis ◽  
Keith R. Matthews ◽  
Keith Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Reporter ◽  
Mutant Proteins ◽  
Green Fluorescent ◽  
A Minor ◽  
Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

scholarly journals Differences in cell cycle characteristics among patients with acute nonlymphocytic leukemia

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1647-1653 ◽  
Author(s):  
A Raza ◽  
Y Maheshwari ◽  
HD Preisler
Keyword(s):  
Cell Cycle ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Total Cell ◽  
Acute Nonlymphocytic Leukemia ◽  
Cell Cycle Time ◽  
Myeloid Leukemias ◽  
History Of

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post- BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.

Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei

1990 ◽  
Vol 95 (1) ◽  
pp. 49-57 ◽  
Author(s):  
R. Woodward ◽  
K. Gull
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Unit Cell ◽  
Kinetoplast Dna ◽  
Interphase Cell ◽  
Body Formation ◽  
Timing Of Initiation ◽  
Single Mitochondrion

We have used immunofluorescent detection of 5-bromo-2-deoxyuridine-substituted DNA in order to determine the timing of initiation and the duration of nuclear and kinetoplast S-phases within the procyclic stage of the Trypanosoma brucei cell cycle. Both nuclear and kinetoplast S-phases were shown to be periodic, occupying 0.18 and 0.12 of the unit cell cycle, respectively. In addition, initiation of both of these S-phases were in approximate synchrony, differing by only 0.03 of the unit cell cycle. We have also used a monoclonal antibody that recognises the basal bodies of T. brucei in order to visualise cells possessing a new pro-basal body and hence determine the time of pro-basal body formation within the cell cycle. Pro-basal body formation occurred within a few minutes of the initiation of nuclear S-phase, at 0.41 of the unit cell cycle. This provides detection of the earliest known cell cycle event in T. brucei at the level of the light microscope. Cell cycle events including initiation of nuclear and kinetoplast DNA replication and pro-basal body formation may be strictly coordinated in T. brucei in order to maintain the precise single-mitochondrion (kinetoplast), singleflagellum status of the interphase cell.

Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication

2001 ◽  
Vol 114 (10) ◽  
pp. 1861-1866 ◽  
Author(s):  
J. Postberg ◽  
S.A. Juranek ◽  
S. Feiler ◽  
H. Kortwig ◽  
F. Jonsson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Matrix ◽  
Binding Protein ◽  
Specific Interaction ◽  
Eukaryotic Cells ◽  
Dna Molecules ◽  
Telomere Binding Protein ◽  
The Matrix

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8)individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

scholarly journals Alteration of the proliferative rate of acute myelogenous leukemia cells in vivo in patients

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2600-2603 ◽  
Author(s):  
HD Preisler ◽  
A Raza ◽  
RA Larson
Keyword(s):  
Cell Cycle ◽  
Acute Myelogenous Leukemia ◽  
S Phase ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Cell Cycle Time ◽  
Proliferative Rate ◽  
Acute Myelogenous ◽  
Bioactive Agents

Abstract Ten patients with active acute myelogenous leukemia (AML) received either 13 cis retinoic acid (RA) + alpha interferon (IFN) or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 3 days. Cell cycle measurements were performed before and at the conclusion of administration of the bioactive agent(s). The proliferative rate of the leukemia cells in vivo decreased in four of five patients receiving RA+IFN whereas in one patient proliferation accelerated. The proliferative rate of AML cells accelerated in three of the five patients who received rhGM-CSF and slowed in two patients. These data show that while the proliferative rate of AML cells can be altered in vivo, the effect produced by bioactive agents may be the opposite of the desired effect. Furthermore, the studies described here demonstrate the usefulness of marrow biopsies for measuring the percent S-phase cells and the importance of measuring the duration of S phase so that the effects of bioactive agents on the cell cycle time of the leukemia cells can be determined.

scholarly journals Kinetoplast maxicircle DNA replication in Crithidia fasciculata and Trypanosoma brucei.

1995 ◽  
Vol 15 (12) ◽  
pp. 6794-6803 ◽  
Author(s):  
L R Carpenter ◽  
P T Englund
Keyword(s):  
Electron Microscopy ◽  
Trypanosoma Brucei ◽  
Topoisomerase Ii ◽  
Repetitive Sequences ◽  
Variable Region ◽  
Kinetoplast Dna ◽  
Crithidia Fasciculata ◽  
Interstrand Cross Links ◽  
Replication Intermediates

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.

scholarly journals Functional Interactions between Herpesvirus Oncoprotein MEQ and Cell Cycle Regulator CDK2

1999 ◽  
Vol 73 (5) ◽  
pp. 4208-4219 ◽  
Author(s):  
Juinn-Lin Liu ◽  
Ying Ye ◽  
Zheng Qian ◽  
Yongyi Qian ◽  
Dennis J. Templeton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Phosphorylation Site ◽  
Fibroblast Cell ◽  
S Phase ◽  
Morphological Transformation ◽  
Necrosis Factor Alpha ◽  
Coiled Bodies ◽  
Cdk Phosphorylation

ABSTRACT Marek’s disease virus, an avian alphaherpesvirus, has been used as an excellent model to study herpesvirus oncogenesis. One of its potential oncogenes, MEQ, has been demonstrated to transform a rodent fibroblast cell line, Rat-2, in vitro by inducing morphological transformation and anchorage- and serum-independent growth and by protecting cells from apoptosis induced by tumor necrosis factor alpha, C2-ceramide, UV irradiation, or serum deprivation. In this report, we show that there is a cell cycle-dependent colocalization of MEQ protein and cyclin-dependent kinase 2 (CDK2) in coiled bodies and the nucleolar periphery during the G1/S boundary and early S phase. To our knowledge, this is the first demonstration that CDK2 is found to localize to coiled bodies. Such an in vivo association and possibly subsequent phosphorylation may result in the cytoplasmic translocation of MEQ protein. Indeed, MEQ is expressed in both the nucleus and the cytoplasm during the G1/S boundary and early S phase. In addition, we were able to show in vitro phosphorylation of MEQ by CDKs. We have mapped the CDK phosphorylation site of MEQ to be serine 42, a residue in the proximity of the bZIP domain. An indirect-immunofluorescence study of the MEQ S42D mutant, in which the CDK phosphorylation site was mutated to a charged residue, reveals more prominent cytoplasmic localization. This lends further support to the notion that the translocation of MEQ is regulated by phosphorylation. Furthermore, phosphorylation of MEQ by CDKs drastically reduces the DNA binding activity of MEQ, which may in part account for the lack of retention of MEQ oncoprotein in the nucleus. Interestingly, the localization of CDK2 in coiled bodies and the nucleolar periphery is observed only in MEQ-transformed Rat-2 cells, implicating MEQ in modifying the subcellular localization of CDK2. Taken together, our data suggest that there is a novel reciprocal modulation between the herpesvirus oncoprotein MEQ and CDK2.

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro

1996 ◽  
Vol 8 (6) ◽  
pp. 935 ◽  
Author(s):  
AW Schuetz ◽  
DG Whittingham ◽  
R Snowden
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Granulosa Cells ◽  
Dna Content ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Ovarian Follicles ◽  
S Phase

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.

scholarly journals Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

2007 ◽  
Vol 28 (4) ◽  
pp. 1313-1325 ◽  
Author(s):  
Meredith E. K. Calvert ◽  
Kristin M. Keck ◽  
Celeste Ptak ◽  
Jeffrey Shabanowitz ◽  
Donald F. Hunt ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
S Phase ◽  
Bud Formation ◽  
Evolutionarily Conserved ◽  
Assembly Factor ◽  
And Function ◽  
Cycle Regulation

ABSTRACT In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.

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