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PubMed2005

An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism.

X-linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber-FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome-specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands.

Animals↗
PubMed1998

Multirhythmic bursting.

A complex modeled bursting neuron [C. C. Canavier, J. W. Clark, and J. H. Byrne, J. Neurophysiol. 66, 2107-2124 (1991)] has been shown to possess seven coexisting limit cycle solutions at a given parameter set [Canavier et al., J. Neurophysiol 69, 2252-2259 (1993); 72, 872-882 (1994)]. These solutions are unique in that the limit cycles are concentric in the space of the slow variables. We examine the origin of these solutions using a minimal 4-variable bursting cell model. Poincare maps are constructed using a saddle-node bifurcation of a fast subsystem such as our Poincare section. This bifurcation defines a threshold between the active and silent phases of the burst cycle in the space of the slow variables. The maps identify parameter spaces with single limit cycles, multiple limit cycles, and two types of chaotic bursting. To investigate the dynamical features which underlie the unique shape of the maps, the maps are further decomposed into two submaps which describe the solution trajectories during the active and silent phases of a single burst. From these findings we postulate several necessary criteria for a bursting model to possess multiple stable concentric limit cycles. These criteria are demonstrated in a generalized 3-variable model. Finally, using a less direct numerical procedure, similar return maps are calculated for the original complex model [C. C. Canavier, J. W. Clark, and J. H. Byrne, J. Neurophysiol. 66, 2107-2124 (1991)], with the resulting mappings appearing qualitatively similar to those of our 4-variable model. These multistable concentric bursting solutions cannot occur in a bursting model with one slow variable. This type of multistability arises when a bursting system has two or more slow variables and is viewed as an essentially second-order system which receives discrete perturbations in a state-dependent manner. (c) 1998 American Institute of Physics.

Journal Article↗
PubMed2005

A bidirectional origin of replication maps to the major noncoding region of human mitochondrial DNA.

In solid tissues of vertebrates, initiation of mitochondrial DNA replication encompasses a broad zone downstream of the major noncoding region (NCR). In contrast, analysis with two-dimensional agarose gel electrophoresis of mitochondrial DNA replication intermediates in cultured human cells revealed initiation concentrated in the NCR. Mapping of prominent free 5' ends on the heavy strand of mitochondrial DNA identified two clusters of potential start sites. One mapped to the previously assigned origin of strand-asynchronous replication (O(H)); the other lay several hundred nucleotides away from O(H), toward the other end of the NCR. The latter cluster is proposed to be the major site of bidirectional replication initiation on the basis of the following: its prominence is enhanced in cells amplifying mitochondrial DNA after experimentally induced mitochondrial DNA depletion; free 5' ends are found in corresponding positions on the opposite strand; it is transient in nature; and it is associated with bubble arcs.

Base Sequence↗
PubMed1999

Physical and genetic mapping of mammalian replication origins.

The neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques are sensitive physical mapping methods that have been used successfully to identify replication initiation sites in genomes of widely varying complexity. We present detailed methodology for the preparation of replication intermediates from mammalian cells and their analysis by both neutral/neutral and neutral/alkaline two-dimensional gel approaches. The methods described allow characterization of the replication pattern of single-copy loci, even in mammalian cells. When applied to metazoans, initiation is found to occur at multiple sites scattered throughout zones that can be as long as 50 kb, with some subregions being preferred. Although these observations do not rule out the possibility of genetically defined replicators, they offer the alternative or additional possibility that chromosomal context may play an important role in defining replication initiation sites in complex genomes. We discuss novel recombination strategies that can be used to test for the presence of sequence elements critical for origin function if the origin lies in the vicinity of a selectable gene. Application of this strategy to the DHFR locus shows that loss of sequences more than 25 kb from the local initiation zone can markedly affect origin activity in the zone.

Animals↗
PubMed1995

Precise mapping of t(12;14) leiomyoma breakpoint on chromosome 14 between D14S298 and D14S540.

Uterine leiomyoma is a common tumor of smooth muscle cell origin often characterized by the presence of a balanced t(12;14)(q13-15;q24.1) chromosomal translocation. This breakpoint on chromosome 14 had previously been placed between the markers SPTB and D14S77, a region estimated to span 7 cM. In this study we have used a meiotic breakpoint mapping panel to construct a high resolution genetic map of this interval. Markers that mapped within this interval were used to analyze DNA from a somatic cell hybrid containing the t(12;14) translocated chromosome. The results of this analysis localize the t(12;14) breakpoint on chromosome 14 between D14S298 and D14S540, between which no meiotic recombination was detected. This sets the stage for identifying the gene(s) disrupted by the chromosomal translocation by defining the markers that flank the translocation breakpoint.

Base Sequence↗
PubMed2000

Identification of a 1-megabase consensus region of deletion at 1p36.3 in primary neuroblastomas.

BACKGROUND: Deletion of the distal short arm of chromosome 1 occurs frequently in neuroblastoma. In addition, neuroblastoma has been described in children with constitutional deletions within 1p36, supporting the existence of one or more neuroblastoma suppressor genes within this region. PROCEDURE: We have pursued a 1p36 tumor suppressor gene identification strategy that has included deletion mapping of 566 primary neuroblastomas and 46 neuroblastoma-derived cell lines, and have determined the parental origin of the deleted 1p homologue in 44 cases to determine whether there is evidence for genomic imprinting within this region. RESULTS AND CONCLUSIONS: We have identified a 1-Mb consensus region of deletion within 1p36.3 defined by primary tumor deletions, constructed a physical map of the region that is being sequenced to completion, and have identified and prioritized candidate genes within this region for further analyses.

Alleles↗
PubMed1985

In vivo proteolytic activity of the mammary gland. Contribution to the origin of secretory component, beta 2-microglobulin and bovine-associated mucoprotein (BAMP) in cows milk.

Milk samples were collected from Holstein-Friesian cows at various times after milking (10-30 min; 30 min-10 hr) and treated with a protease inhibitor or control solution. Samples were then fractionated into whole, skimmed and cell-free skimmed milk aliquots. Some animals were treated with E. coli endotoxin prior to sample collection. The concentrations of three membrane-associated proteins (MAP), beta 2-microglobulin (beta 2M), secretory component (SC) and bovine-associated mucoprotein (BAMP) as well as albumin were measured in each aliquot to determine if in vivo proteolysis of milk elements could explain the origin of these MAP in milk. All three MAP could be localized on milk fat globules (MFG) and alveolar epithelial cells of the gland. Data revealed that all BAMP in milk can be accounted for by in vivo proteolytic degradation of MFG while most beta 2M is derived by similar degradation, from cellular elements in milk, presumably monocytes. Experiments with endotoxin which elevate PMN levels, failed to influence the release of any MAP while elevating albumin levels by greater than 10-fold. Based on these studies, SC release into milk cannot be ascribed to a protease-dependent mechanism.

Animals↗
PubMed1990

Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division.

Neurogenesis in the adult avian brain is restricted to the telencephalon. New neurons originate in the ventricular zone (VZ) from cells that have not been identified. We mapped the position of [3H]thymidine-labeled cells in the walls of the ventricles of the adult canary brain. Labeled VZ cells were restricted to the telencephalon (lateral ventricles) and concentrated in "hot spots". The coincidence of these hot spots with regions rich in radial cells suggested that radial cells may be the cells undergoing mitosis. We used smears prepared from fragments of the VZ containing the hot spots to show directly that radial cells accumulate [3H]thymidine. In addition, grain counts at different survival times demonstrated that these cells divide. Hot spots of VZ cell division also coincided with sites of neuronal origin. We suggest that radial cell division may give rise to new neurons.

Animals↗
PubMed1999

A 450-kb contig of defensin genes on human chromosome 8p23.

Defensins are a large family of host defense peptides expressed in leukocytes and epithelia. Using P1 and BAC clones, we have determined the organization of the human alpha-defensin genes and the beta-defensin gene HDEFB1 on chromosome 8p23. From the telomere, the order of the genes (with encoded peptides in parentheses) is HDEFA5 (HD-5), HDEFA1/1A (HNP-1/3), HDEFA4 (HNP-4), HDEFA6 (HD-6), and HDEFB1 (HBD-1). These genes span a region of approximately 450kb. Genes encoding intestinal Paneth cell defensins (HDEFA5 and HDEFA6) flank the myeloid defensin gene cluster (HDEFA1, HDEFA1A, HDEFA4). Based on our previous studies, the remaining known defensin gene, HDEFB2 (HBD-2), is about 400kb centromeric to HDEFB1. This map supports the hypothesis, originally proposed because of sequence similarities, that myeloid alpha-defensin genes evolved by reduplication and divergence from Paneth cell defensin genes, and identifies regions and clones, which should be useful in the search for new defensin genes.

Base Sequence↗
PubMed1995

The Chinese hamster dihydrofolate reductase origin consists of multiple potential nascent-strand start sites.

Previous two-dimensional gel replicon-mapping studies on the amplified dihydrofolate reductase (DHFR) domain in CHOC 400 cells suggested that replication can initiate at any of a large number of sites scattered throughout a 55-kb region lying between two convergently transcribed genes. It could be argued that this unusual distributive initiation mode is unique to amplified chromosomal loci. In this paper, we report the first application of the two-dimensional gel techniques to the analysis of a single-copy locus in mammalian cells. Results obtained with both synchronized and exponentially growing CHO cells suggest that (i) initiation can also occur at any of a large number of sites distributed throughout the intergenic region in the nonamplified DHFR locus, (ii) initiation is confined to the first 2 to 2.5 h of the S period, and (iii) initiation occurs only in a fraction of the DHFR loci in each cell cycle.

Animals↗
PubMed1984

Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells.

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.

Animals↗
PubMed1998

Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone.

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.

Animals↗
PubMed1990

Genetic mapping of a cloned sequence responsible for susceptibility to ecotropic murine leukemia viruses.

A mouse cDNA that confers susceptibility to ecotropic murine leukemia viruses following transfection into human EJ cells has been cloned and sequenced. We show that this sequence is likely to be Rec-1, the chromosome 5 locus originally defined by studies with somatic cell hybrids as responsible for virus susceptibility, and provide a specific chromosomal map position for this locus by analysis of an interspecies backcross. This locus maps in the distal region of chromosome 5 and is thus not within the cluster of retrovirus-related genes near the centromere.

Animals↗
PubMed1995

The angiogenic potentials of the cephalic mesoderm and the origin of brain and head blood vessels.

We have used two molecular markers to label blood vessel endothelial cells and their precursors in the early avian embryo. One marker, called Quek1, is the avian homologue of the mammalian VEGF receptor flk-1 and the other is the MB1/QH1 monoclonal antibody. Quek1 is expressed in a subset of mesodermal cells from the gastrulation stage. Quek1 positive cells later form blood vessel endothelial cells and express the MB1/QH1 antigen which is specific for endothelial and hemopoietic cells of the quail species. These two markers allowed us first to show that the cephalic paraxial mesoderm has angiogenic potentials which are much more extended than its trunk counterpart (the somites). Secondly, the origin of the endothelial cells lining the craniofacial and head blood vessels was mapped on the 3-somite stage cephalic mesoderm via the quail-chick chimera technique, in which well defined mesodermal territories are exchanged between stage-matched embryos of both species in a strictly isotopic manner. We found that the anterior region of the cephalic paraxial mesoderm is largely recruited to provide the forebrain and the upper face with their vasculature. This means that large volumes of tissues are vascularized by a discrete region of the cephalic mesoderm, the fate of which is otherwise to give rise to muscles. The widespread expansion of the angiogenic cells arising from the anterior paraxial mesoderm must be related to the high growth rate of the anterior region of the neural primordium, yielding the telencephalon and of the neural crest-derived facial structures which are themselves devoid of angiogenic potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution.

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.

DNA, Bacterial↗
PubMed2002

Intracellular signal propagation in a two-dimensional autocatalytic reaction model.

We study a simple reaction scheme in a two-dimensional lattice of particles or molecules with a refractory state. We analyze the dynamics of the propagating front as a function of physical-chemical properties of the host medium. The anisotropy of the medium significantly affects the smoothness of the wave front. Similarly, if particles or molecules may diffuse slowly to neighboring sites, then the front wave is more likely to be irregular. Both situations affect the ability of the whole system to relax to the original state, which is a required feature in the biological cells. Attempts to map this simple reaction scheme to reactions involved in the intracellular pathways suggest that, in some cases, signal transduction might take both connotation of a random walk and a propagating wave, depending on the local density of the medium. In particular, a sufficient condition for the appearance of waves in high-density regions of the media, is the existence of at least one autocatalytic reaction in the chain of reactions characterizing the pathway.

Biophysical Phenomena↗
PubMed1999

Timing regulation in a network reduced from voltage-gated equations to a one-dimensional map.

We discuss a method by which the dynamics of a network of neurons, coupled by mutual inhibition, can be reduced to a one-dimensional map. This network consists of a pair of neurons, one of which is an endogenous burster, and the other excitable but not bursting in the absence of phasic input. The latter cell has more than one slow process. The reduction uses the standard separation of slow/fast processes; it also uses information about how the dynamics on the slow manifold evolve after a finite amount of slow time. From this reduction we obtain a one-dimensional map dependent on the parameters of the original biophysical equations. In some parameter regimes, one can deduce that the original equations have solutions in which the active phase of the originally excitable cell is constant from burst to burst, while in other parameter regimes it is not. The existence or absence of this kind of regulation corresponds to qualitatively different dynamics in the one-dimensional map. The computations associated with the reduction and the analysis of the dynamics includes the use of coordinates that parameterize by time along trajectories, and "singular Poincaré maps" that combine information about flows along a slow manifold with information about jumps between branches of the slow manifold.

Animals↗
PubMed2002

Fish oil slows S phase progression and may cause upstream shift of DHFR replication origin ori-beta in CHO cells.

Fish oils (FOs) have been noted to reduce growth and proliferation of certain tumor cells, effects usually attributed to the content of polyunsaturated fatty acids of the n-3 family, which are thought to modulate cellular signaling pathways. We investigated the influence of FO on cell cycle kinetics of cultured Chinese hamster ovary cells. Exponentially growing cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) and analyzed by flow cytometry after 5-day treatment with exogenous fat. Bivariate BrdU-DNA analysis indicated slower progression through S phase and thus longer S phase duration time in FO- but not corn oil-treated or control cells. We hypothesize that FO treatment might interfere with spatial/temporal organization of replication origins. Therefore, we mapped the well-characterized replication origin ori-beta downstream of the dihydrofolate reductase gene with the nascent strand length assay. Three DNA marker segments with known positions relative to this origin were amplified by PCR. By quantitatively assessing DNA length of the fragments in all fractions containing these markers, the location of ori-beta was established. In control or corn oil-treated cells, the location of ori-beta was consistent with previous studies. However, in FO-treated cells, DNA replication appears to start from a new site located farther upstream from ori-beta, suggesting a different replication initiation pattern. This study suggests novel mechanism(s) by which fats affect cell proliferation and DNA replication in mammalian cells.

Animals↗