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PubMed1995

Asymmetrical blastomere origin and spatial domains of dopamine and neuropeptide Y amacrine subtypes in Xenopus tadpole retina.

Amacrine cells are located almost exclusively in the inner nuclear layer (INL) of the retina, but they express a variety of neurotransmitters. To begin to elucidate the relative roles of the local environment and cell lineage in determining the different neurotransmitter subtypes of amacrine cells, we combined lineage tracing and immunocytochemical techniques to map the spatial distribution and clonal origin of dopamine (DA) and neuropeptide Y (NPY) amacrine cells in Xenopus tadpole retina. At the earliest period of neurotransmitter expression, both DA and NPY amacrine cells were distributed preferentially in center and intermediate annular regions, and in anterior and dorsal quadrants. Most of the DA and NPY cells first emerged as scattered cells and later as clusters (of 2 or more cells) that increased in number and size up to premetamorphic stages. These results suggest that DA and NPY amacrine subtypes may be influenced by environmental cues localized to specific regions of the retina. Lineage analysis showed that the percentage of DA or NPY amacrine cells produced by most blastomere progenitors is significantly different from that predicted by the number of cells in the retina produced by those blastomeres. Only two blastomeres produced over 90% of the DA amacrine cells and only four produced 97% of the NPY amacrine cells. Some retinal progenitors did not contribute at all to these two amacrine subtypes. There also is a marked asymmetry in the blastomere origin of DA and NPY amacrine cells. Two retinal progenitors produced significant numbers of NPY but very few DA amacrine cells. This analysis provides evidence that blastomere origin restricts the developmental choices of retinal progenitors.

Animals↗
PubMed1999

Intracellular fate mapping in a basal metazoan, the ctenophore Mnemiopsis leidyi, reveals the origins of mesoderm and the existence of indeterminate cell lineages.

Ctenophores are marine invertebrates that develop rapidly and directly into juvenile adults. They are likely to be the simplest metazoans possessing definitive muscle cells and are possibly the sister group to the Bilateria. All ctenophore embryos display a highly stereotyped, phylum-specific pattern of development in which every cell can be identified by its lineage history. We generated a cell lineage fate map for Mnemiopsis leidyi by injecting fluorescent lineage tracers into individual blastomeres up through the 60-cell stage. The adult ctenophore body plan is composed of four nearly identical quadrants organized along the oral-aboral axis. Each of the four quadrants is derived largely from one cell of the four-cell-stage embryo. At the eight-cell stage each quadrant contains a single E ("end") and M ("middle") blastomere. Subsequently, micromeres are formed first at the aboral pole and later at the oral pole. The ctene rows, apical organ, and tentacle apparatus are complex structures that are generated by both E and M blastomere lineages from all four quadrants. All muscle cells are derived from micromeres born at the oral pole of endomesodermal precursors (2M and 3E macromeres). While the development of the four quadrants is similar, diagonally opposed quadrants share more similarities than adjacent quadrants. Adult ctenophores possess two diagonally opposed endodermal anal canals that open at the base of the apical organ. These two structures are derived from the two diagonally opposed 2M/ macromeres. The two opposing 2M/ macromeres generated a unique set of circumpharyngeal muscle cells, but do not contribute to the anal canals. No other lineages displayed such diagonal asymmetries. Clones from each blastomere yielded regular, but not completely invariant patterns of descendents. Ectodermal descendents normally, but not always, remained within their corresponding quadrants. On the other hand, endodermal and mesodermal progeny dispersed throughout the body. The variability in the exact complements of adult structures, along with previously published cell deletion experiments, demonstrates that cell interactions are required for normal cell fate determination. Ctenophore embryos, like those of many bilaterian phyla (e.g., spiralians, nematodes, and echinoids), display a highly stereotyped cleavage program in which some, but not all, blastomeres are determined at the time of their birth. The results suggest that mesodermal tissues originally evolved from endoderm tissue.

Animals↗
PubMed1988

The somitic level of origin of embryonic chick hindlimb muscles.

Studies of avian chimeras made by transplanting groups of quail somites into chick embryos have consistently shown that the muscle cells of the hindlimb are derived from the adjacent somites, however, the pattern of cell distribution from individual somites to individual hindlimb muscles has not been characterized. I have mapped quail cell distribution in the chick hindlimb after single somite transplantation to determine if cells from an individual somite populate discrete limb muscle regions and if there is a spatial correspondence between a muscle's somitic level of origin and the known spinal cord position of its motoneuron pool. At stages 15-18 single chick somites or equivalent lengths of unsegmented somitic mesoderm adjacent to the prospective hindlimb region were replaced with the corresponding tissue from quail embryos. At stages 28-34, quail cell distribution was mapped within individual thigh muscles and shank muscle regions. A quail-specific antiserum and Feulgen staining were used to identify quail cells. Transplants from somite levels 26-33 each gave rise to consistent quail cell labeling in a unique subset of limb muscles. The anteroposterior positions of these subsets corresponded to that of the transplanted somitic tissue. For example, more anterior or anteromedial thigh muscles contained quail cells when more anterior somitic tissue had been transplanted. For the majority of thigh muscles studied and for shank muscle groups, there was also a clear correlation between somitic level of origin and motoneuron pool position. These data are compatible with the hypothesis that motoneurons and the muscle cells of their targets share axial position labels. The question of whether motoneurons from a specific spinal cord segment recognize and consequently innervate muscle cells derived from the same axial level during early axon outgrowth is addressed in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 408-419). Quail cell distribution was also mapped in chick embryos in which quail somites or unsegmented mesoderm had been placed 2-3 somites away from their position of origin. In all cases donor somitic tissues contributed to muscles in accord with their host position. These results indicate that muscle cell precursors within the somites are not specified to migrate to a predetermined target region.

Animals↗
PubMed2006

Direct thrombin inhibitors built on the azaphenylalanine scaffold provoke degranulation of mast cells.

The main structural feature of direct thrombin inhibitor LK-732 responsible for the appropriate interaction at the thrombin active site is a strong basic group. A possibility that a strong basic group of LK-732 might contribute to the mast cell degranulation effect and consequent reduction of tracheal air flow (TAF) and fall of mean arterial blood pressure (MAP) in rats was investigated in the present study. At doses up to 5 mg/kg (i.v.), LK-732 did not cause significant changes of TAF and MAP. At 7 mg/kg (i.v.), a sudden reduction of TAF and a fall of MAP was observed within 5 min after LK-732 administration (75% mortality, p = 0.007). A less basic direct thrombin inhibitor LK-658 (21 mg/kg, i.v.) did not significantly disturb TAF and MAP. A reduction of TAF and a fall of MAP caused by LK-732 (7 mg/kg, i.v.) was almost completely abolished in rats with degranulated mast cells (0% mortality, p = 0.008). LK-732 concentration-dependently degranulated rat peritoneal mast cells in vitro (pEC(50) = 1.92 +/- 0.05 muM). A structure-activity relationship (SAR) study revealed that the terminal basic groups attached to the aromatic ring are responsible for the mast cell degranulation effect. A good correlation was observed between mast cell degranulation and pK(b) of analogues of LK-732 (R(2) = 0.49), but not between mast cell degranulation and thrombin K(i) (R(2) = 0.23). LK-732-induced reduction of TAF, the fall of MAP and high mortality originate from LK-732-induced mast cell degranulation. As judged by the SAR study, this effect could be overcome by reducing the basicity of LK-732.

Animals↗
PubMed2004

Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences.

A functional origin of replication was mapped to the transcriptional promoter and exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the mouse and human genomes. This origin was lost in mouse embryonic stem (ES) cells with a spontaneous deletion (approximately 36 kb) at the 5' end of the HPRT locus. Restoration of HPRT activity by homologous recombination with human/mouse chimeric sequences reconstituted replication origin activity in two independent ES cell lines. Quantitative PCR analyses of abundance of genetic markers in size-fractionated nascent DNA indicated that initiation of DNA replication coincided with the site of insertion in the mouse genome of the 335 bp of human DNA containing the HPRT exon 1 and a truncated promoter. The genetic information contained in the human sequence and surrounding mouse DNA was analyzed for cis-acting elements that might contribute to selection and functional activation of a mammalian origin of DNA replication.

Animals↗
PubMed1999

Early-replicating DNA from mosquito cells is associated with a distinct EcoRI fragment.

In an effort to define an origin of bi-directional DNA replication (OBR) in mosquito genomic DNA, we applied methods that take advantage of characteristic features of single-stranded DNA to methotrexate-resistant Aedes albopictus cells. The Mtx-5011-256 cells contained approximately 1000 copies of a 200 kb amplicon containing the dihydrofolate reductase locus, which likely contained one or more replication origins. When Mtx-5011-256 cells were synchronized by treatment with hydroxyurea, released into the S phase of the cell cycle, and labeled in vivo with tritiated DNA precursors, a 1.9 kb EcoRI fragment was preferentially labeled in EcoRI-digested genomic DNA. Similarly, we detected a 1.9 kb EcoRI fragment in DNA from wild type cells after cell cycle synchronization and in vivo labeling. In a complementary method, unlabeled single-stranded DNA was isolated from Mtx-5011-256 cells, labeled in vitro, and hybridized to EcoRI-digested genomic DNA from mosquito cells. The labeled probe hybridized preferentially to a 1.9 kb fragment. Finally, a 1.9 kb EcoRI fragment was detected when nascent DNA was recovered from unsynchronized cells, made double-stranded by in vitro labeling, and digested with EcoRI. Taken together, these results suggest that in Aedes albopictus mosquito cells, many replication origins used at different times during S are flanked by EcoRI sites that define a 1.9 kb fragment, which has become more abundant in Mtx-5011-256 cells because it occurs in the dhfr amplicon. Tentative mapping of this origin to amplicon DNA remains ambiguous, further suggesting that a repeated sequence element occurs at or near the origin of replication.

Aedes↗
PubMed1997

Site-related specificities of the control by androgenic status of adipogenesis and mitogen-activated protein kinase cascade/c-fos signaling pathways in rat preadipocytes.

In rats, castration induces a complete defective adipose conversion of preadipocytes from the epididymal fat depots (Lacasa, D., B. Agli, D. Noynarol, and Y. Giudicelli, 1995, Endocrine 3: 789-793). The aim of this study was to establish the eventual site-specificity of this effect as well as the mechanisms involved. Therefore, the influence of androgenic status on the Fos protein induction and the Raf/mitogen-activated protein (MAP) kinase kinase (MEK)MAP cascade, which are all required for adipose conversion of preadipocytes, was compared in proliferating and differentiated preadipocytes from femoral sc and deep intraabdominal (epididymal and perirenal) fat depots. In epididymal and perirenal proliferating preadipocytes, increased proliferation due to castration is associated with increased MAP kinase activity. However, higher immunoreactive levels of the upstream activators of MAP kinase, Raf-1 and MEK, were observed only in epididymal cells. Moreover, in vivo testosterone treatment corrected the effects of castration on Raf-1 but not on MEK and MAP kinase. MAP kinase activity was decreased during the course of adipogenesis. In differentiated cells, MAP kinase activity showed variations according to the anatomical origin of preadipocytes but not to the androgenic status. In contrast, MEK and Raf-1 immunoreactive levels were both sensitive to androgenic status but were differently affected depending on cell origin. Finally, the defective adipogenesis seen in epididymal preadipocytes from castrated rats was associated with reduced Fos protein induction in these cells, an alteration which was partly corrected by testosterone-treatment. Taken together, these results suggest that androgenic status affects adipogenesis from deep intraabdominal preadipocytes through alterations of some components of the MAP kinase cascade/Fos signaling pathways.

Adipocytes↗
PubMed1997

Three types of serotonin-containing amacrine cells in tadpole retina have distinct clonal origins.

In the Xenopus tadpole there are three different serotonin-containing amacrine cells: large, brightly fluorescent (LB), and small, dimly fluorescent (SD) cells in the inner nuclear layer and displaced (DIS) cells in the ganglion cell layer. To reveal the potential roles of regional cues and lineage factors in their determination, quantitative maps were made of the spatial distribution and blastomere origin of each cell type. LB and SD cells were evenly distributed across the four retinal quadrants, arguing against a hypothesis that these cells are induced differentially by quadrant-specific cues. Blastomere progenitors of the 32-cell embryo are biased to produce only subsets of serotonin amacrine cells: 1) all nine progenitors of one retina produced some SD cells, but only eight produced LB, and only five produced DIS cells; and 2) there are overlapping but distinct subsets of blastomere progenitors for each serotonin subtype. This bias is not simply a reflection of the size of a clone in the retina; significant quantitative differences were observed between the proportion of serotonin progeny and the proportion of the entire retina produced by six of the nine retinal progenitors. This bias also is not simply a reflection of the spatial distribution of a blastomere clone in the retina; the number of LB descendants in each retinal quadrant was statistically different from its progenitor's total contribution to the quadrant. These results indicate that the development of the three different serotonin-containing amacrine cells in the retina is biased by membership in specific blastomere clones.

Animals↗
PubMed1978

Field potential and single unit analyses of the avian dorsal motor nucleus of the vagus and citeria for identifying vagal cardiac cells of origin.

Field potentials evoked by mid-cervical vagal stimulation were systematically mapped in the dorsal motor and solitary nuclei of the pigeon. Since responses varied predictably with microelectrode position, they could be used for localization in the dorsal medulla. By varying stimulus intensity and monitoring the vagal compound action potential, contributions of the different compound action potential waves were then established. Activation of the Bl-wave, which includes cardioinhibitory fiber activity, has its most prominent effect in the intermediate rostrocaudal zone of the dorsal motor nucleus in the region of subnuxleus b. This is where the cells of origin of the vagal cardiac fibers have previously been anatomically localized. Single unit experiments then established that (a) vagal motoneurons with axonal conduction velocities in the cardioinhibitory fiber range (8.0-14.5 m/sec) are primarily localized to the intermediate rostrocaudal zone of the dorsal motor nucleus in the region of subnucleus b, and (b) motoneurons in this zone that conduct at 8.0-14.5 m/sec distribute their axons in the cardiac branches. Furthermore, no error is introduced by identifying such neurons with mid-cervical rather than midthoracic vagal stimulation. Thus, the following criteria establish a neuron as giving rise to a vagal cardioinhibitory fiber: (a) localizing it to the intermediate rostrocaudal zone of the dorsal motor nucleus on the basis of the field potential evoked by mid-cervical vagal stimulation; (b) antidromically activating it with mid-cervical vagal stimulation; and (c) demonstrating that its axon conducts at 8.8-14.5 m/sec.

Animals↗
PubMed2000

Activation of MAP kinase after reversible ATP depletion in LLC-PK 1 cells.

We examined the effect of recovery following reversible ATP depletion on MAP kinase activity in cultured renal cells of proximal tubular origin (LLC-PK1). We induced ATP depletion by 0.1 micromol/l antimycin A in combination with substrate deprivation, and obtained recovery by restoration of substrate supply. MAP kinase activity increased from 374+/-45 pmol/mg protein/mm during ATP depletion to 768 +/- 77 pmol/mg protein/mm after 15 min of recovery. We used ATP to activate a representative G-protein coupled receptor, or epidermal growth factor (EGF) to activate receptors with intrinsic tyrosine kinase activity, and measured the effect of these manipulations on MAP kinase activity during ATP depletion or following recovery. ATP and EGF stimulated MAP kinase activity under control conditions, but not during ATP depletion or after recovery. This shows that two distinct signal transduction pathways represented by ATP and EGF are blocked during ATP depletion and recovery. The lack of energy during ATP depletion and the already maximally stimulated MAP kinase during recovery is likely to be the reason for these results. In summary, these findings suggest that MAP kinase may be involved in the physiological response of cells injured by hypoxia.

Adenosine Triphosphate↗
PubMed1998

The distribution of murine 115-kDa epithelial microtubule-associated protein (E-MAP-115) during embryogenesis and in adult organs suggests a role in epithelial polarization and differentiation.

In interphase cells microtubules play fundamental roles in the intracellular distribution and movement of organelles and vesicles and thereby contribute to cellular polarization and differentiation. The organization of microtubules varies with the cell type and is presumably controlled by tissue-specific microtubule-associated proteins (MAPs). The 115-kDa epithelial MAP (E-MAP-115) has been identified as a microtubule-stabilizing protein predominantly expressed in cell lines of epithelial origin. To assess a putative function of E-MAP-115 in epithelial morphogenesis in vivo, we have cloned the cDNA encoding the murine protein and studied the cellular distribution of E-MAP-115 mRNA and protein during murine embryogenesis and in adult organs. Analysis of the predicted amino acid sequence of murine E-MAP-115 revealed 81% sequence identity with its human homolog, the best-conserved part of the protein being the microtubule-binding site. Our data indicate that E-MAP-115 is expressed in several epithelia from 9.5 days of embryogenesis onwards and that its expression levels increase during development. From 14.5 days onwards, E-MAP-115 mRNA is found in some neuronal cells as well. In adult organs, E-MAP-115 is most abundant in epithelial cells of kidney tubules, in absorptive cells of the intestine and is widely distributed in the testis. E-MAP-115 expression correlates with the differentiation of certain epithelial cell types: in the adult intestine, for example, E-MAP-115 mRNA and protein are more abundant in the differentiating than in the proliferative cell compartment. Moreover, E-MAP-115 expression clearly correlates with the degree of cellular apicobasal polarity. In the developing kidney, E-MAP-115 mRNA is detected in the cuboidal cells of S-shaped bodies, of primitive tubules and glomerula, whereas, E-MAP-115 mRNA and protein are absent from mature podocytes which have lost their initial apico-basal polarity. The pattern of distribution of E-MAP-115 in vivo is so far unique for a MAP. Taken together, our results provide support for a role of E-MAP-115 in reorganizing the microtubule cytoskeleton during epithelial cell polarization and differentiation.

Aging↗
PubMed2003

Microtubule-associated epithelial protein E-MAP-115 is localized in the spermatid manchette.

A microtubule-associated protein E-MAP-115 has been originally isolated and characterized from HeLa cells. Because of its predominant expression in cultured cells of epithelial origin, it has been suggested to be involved in the regulation of cell polarization. The present immunocytochemical, Northern blot and in situ hybridization analysis of E-MAP-115 in the mouse and rat seminiferous epithelium indicates its distinct association with the spermatid manchette, a unique microtubular structure which appears in the cytoplasm of spermatids at step 8 when nuclear polarization and elongation starts. At steps 15-16 when manchette has been disassembled, immunoreactivity for E-MAP-115 disappeared. At immunoelectron microscopical level, E-MAP-15 was associated with the microtubules of the manchette. In the Western and Northern blot analysis, a distinct stage-dependent expression of a single E-MAP-115 polypeptide and two mRNA species (3.4 and 2.4 kb) could be identified. MTEST 60, a spermatid-specific transcript, showed a 100% homology over region of 68-193 bp of E-MAP-115 sequence. The reported specific localization of E-MAP-115 to the spermatid manchette strongly supports its role as a regulator of cell polarization. This, in turn, supports the hypotheses concerning the dynamic function of the manchette during spermiogenesis.

Animals↗
PubMed2000

Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways.

Butyrate induces cytodifferentiation in many tumor cells of different origin, suggesting that an as yet unidentified common mechanism inherent to malignant cells is the target of butyrate action. This study determined the role of different mitogen-activated protein (MAP) kinase signal transduction pathways in butyrate-induced erythroid differentiation of K562 human leukemia cells. Using a panel of anti-ERK, JNK, and p38 phosphospecific antibodies, the study showed that phosphorylation of ERK and JNK is decreased following treatment of cells with butyrate, whereas phosphorylation of p38 is increased. In contrast, a K562 subline defective in butyrate-mediated induction of erythroid differentiation did not reveal these changes in phosphorylation patterns. Inhibition of ERK activity by UO126 induces erythroid differentiation and acts synergistically with butyrate on hemoglobin synthesis and inhibition of cell proliferation, whereas inhibition of p38 activity by SB203580 completely abolished induction of hemoglobin expression by butyrate. Taken together, our data suggest a model in which butyrate induces erythroid differentiation of K562 cells by inhibition of ERK and activation of p38 signal transduction pathways.

Butyrates↗
PubMed1990

Developmental maps of acetylcholinesterase and G4-antigen of the early chicken brain: long-distance tracts originate from AChE-producing cell bodies.

After approaching the outer surface of the neuroepithelium, postmitotic cell bodies abruptly start to synthesize acetylcholinesterase (AChE). Their easy histochemical detection allows us to trace sensitively spatiotemporal patterns of differentiation processes of the chicken nervous system. To investigate the relationship between postmitotic AChE production and the first formation of neurites, AChE histochemistry is combined here with immunohistochemistry using the neurite-specific G4-antibody. Spatial computer reconstructions from double-stained serial sections of whole brains of H.H. stages 10-20 demonstrate that G4-neurite expression spatio-temporally follows the expression of AChE in its complex polycentric pattern closely, the details of which have been described earlier. By comparing both differentiative steps at the single cell level reveals that a great majority (if not all) of the G4-positive neurites originate from AChE-positive cell bodies. Based on both the computer reconstructions as well as single cell analysis, including [3H]-thymidine pulse-experiments followed by autoradiography, we conclude, that AChE expression precedes formation of G4-neurites by about 15 h. In addition, the reconstructions provide the first detailed maps of G4-fiber tract formation and shows that G4-neurites form fascicles, most of which travel over long distances to targets within or without the central nervous system (CNS). This is the first demonstration for the entire young chicken brain which verifies that AChE-expressing cells, generally, are those that will establish efferents to distant targets.

Acetylcholinesterase↗
PubMed1991

Temporal analysis and spatial mapping of Lymantria dispar nuclear polyhedrosis virus transcripts and in vitro translation polypeptides.

Genomic expression of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was studied. Viral specific transcripts expressed in cell culture at various times from 2 through 72 h postinfection were identified and their genomic origins mapped through Northern analysis. Sixty-five distinct transcripts were identified in this analysis. Most viral transcripts were expressed late in infection, and originated from throughout the viral genome. Viral polypeptides expressed in infected 652Y cells were labeled with [35S]methionine and identified by autoradiography after separation by SDS polyacrylamide gel electrophoresis. Viral protein synthesis was found to occur in a sequential manner. Four proteins were identified in the early phase of viral replication (4-12 h p.i.), 24 proteins in the intermediate phase (12-24 h p.i.), and 5 proteins during the late phase (greater than 24 h p.i.). Cytoplasmic RNAs were isolated from LdMNPV infected cells at 16, 24, and 48 h p.i., and used for hybrid selections with overlapping DNA fragments that covered the entire LdMNPV genome. The selected RNAs were translated in vitro, and 61 distinct viral polypeptides were identified and their genomic origins mapped. Temporal and spatial transcription and translation maps of the LdMNPV genome were generated with these data, and the expression pattern of the LdMNPV genome was compared to that of the Autographa californica nuclear polyhedrosis virus.

Animals↗
PubMed2007

Mapping yeast origins of replication via single-stranded DNA detection.

Studies in th Saccharomyces cerevisiae have provided a framework for understanding how eukaryotic cells replicate their chromosomal DNA to ensure faithful transmission of genetic information to their daughter cells. In particular, S. cerevisiae is the first eukaryote to have its origins of replication mapped on a genomic scale, by three independent groups using three different microarray-based approaches. Here we describe a new technique of origin mapping via detection of single-stranded DNA in yeast. This method not only identified the majority of previously discovered origins, but also detected new ones. We have also shown that this technique can identify origins in Schizosaccharomyces pombe, illustrating the utility of this method for origin mapping in other eukaryotes.

Chromosome Mapping↗
PubMed2003

Comparison of plastid DNA replication in different cells and tissues of the rice plant.

In a previous study, we mapped replication origin regions of the plastid DNA around the 3' end of the 23S rRNA gene in rice suspension-cultured cells. Here, we examined initiation of the plastid DNA replication in different rice cells by two-dimensional agarose gel electrophoresis. We show for the first time, to our knowledge, that the replication origin region of the plastid DNA differs among cultured cells, coleoptiles and mature leaves. In addition, digestion of the replication intermediates from the rice cultured cells with mung bean nuclease, a single-strand-specific nuclease, revealed that both two single strands of the double-stranded parental DNA were simultaneously replicated in the origin region. This was further confirmed by two-dimensional agarose gel analysis with single-stranded RNA probes. Thus, the mode of plastid DNA replication presented here differs from the unidirectional replication started by forming displacement loops (D-loops), in which the two D-loops on the opposite strands expand toward each other and only one parental strand serves as a template.

Bacterial Proteins↗
PubMed1998

Multiple initiations in the c-myc replication origin independent of chromosomal location.

At supramolecular resolution, DNA synthesis begins at preferred replication origins in the chromosomes of metazoan cells. To characterize one of these origins in detail, the initiation of replication was examined in the HeLa c-myc origin. Polymerase chain reaction (PCR) amplification of size-fractionated nascent chromosomal DNAs revealed multiple replication initiation sites over a 12-kb region spanning the c-myc origin, including the transcribed region and the 5' and 3' flanking DNA of the gene. Two of the start sites for chromosomal replication occurred inside a 2.4-kb region of the origin that exhibits autonomously replicating sequence (ARS) activity. When a plasmid containing the 2.4-kb ARS region was transfected into HeLa cells, PCR mapping of nascent plasmid DNA confirmed that the plasmid replicated semiconservatively and autonomously and that replication did not initiate at random sites but rather began at multiple sites in a limited zone overlapping the c-myc DNA insert. Within the resolution of the PCR assay, the same sites that were used in the chromosomal c-myc origin were used in the 2.4-kb ARS fragment. The locations of replication start sites determined by PCR are considered in the context of other functional and structural elements of the c-myc origin.

Cell Polarity↗