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At least 523 records · Page 29Linked to original sources
PubMed1997

Mapping replication origins, pause sites, and termini by neutral/alkaline two-dimensional gel electrophoresis.

Neutral/alkaline two-dimensional gel electrophoresis is a robust, easily interpretable, sensitive technique that has yielded insights about the in vivo replication of many types of DNA, from multicopy yeast plasmids to single-copy chromosome regions in unsynchronized mammalian cells. It can provide information about directions of replication fork movement and locations of replication origins, termini, and pause sites. Especially when combined with its partner technique, neutral/neutral two-dimensional gel electrophoresis, it is a method of choice for investigation of unknown situations.

Animals↗
PubMed1992

[Toward a new definition of malignant histiocytosis in children].

The anaplastic large cell, CD30 positive lymphomas represent a heterogeneous group of lymphomas in which immunocytochemical and molecular investigations have demonstrated the existence of malignancies of T, B or undetermined origin. The recent identification, in a few cases of this group of a chromosomal 5q35 breakpoint may allow the individualization of a peculiar disease entity. In these cases, the 5q35bp has been found to be a permanent abnormality, present in five human permanent cell lines and associated with various translocations including t(2;5), t(1;5) and t(5;6). A primitive myelo-monocytic origin of these 5q35bp cell is suggested on the basis of the following arguments: they express the c-fms proto-oncogene which encodes the macrophage growth receptor (CSF-1-R) the c-fms is mapped on 5q33,34 close to the 5q35bp the express spontaneously CD68 the treatment by phorbol-diester of a 5q35bp cell line (DEL cell line) induces an immunodependent phagocytosis and a modulation of expression of c-fms, CSF-1 and TNF alpha. Because some 5q35bp cell lines also presents rearrangements for TCR-bêta, or IgF, these data suggest an ancestral stem cell origin, prior to the T, B and myelomonocytic differentiation. Whatever its origin, the 5q35bp abnormality has been mainly encountered in children's malignancies. It has been constantly associated with clinical and biological manifestations of a condition recognized by paediatricians as malignant histiocytosis. For this reason, the 5q35bp may today represent the best criterion for the identification of malignant histiocytosis in childhood.

Child↗
PubMed1996

Genomic characterization of human HMGIC, a member of the accessory transcription factor family found at translocation breakpoints in lipomas.

HMGIC, a member of the HMGI family of high mobility group proteins, is disrupted in 3/3 lipomas characterized by 12q14-q15 rearrangements. To define the genomic structure of the HMGIC gene, YACs from the HMGIC locus were identified and subcloned in lambda FIXII, and genomic lambda clones containing the HMGIC exons were isolated. The HMGIC gene consists of five exons that span at least 60 kb and encodes a 4.1-kb transcript. The coding region is 330 bp, the 5'UTR is 854 bp, and the 3'UTR is 2966 bp. Intron 3, which separates the DNA-binding domains from the acidic domain, is unusually large ( > 25 kb) and is the site of disruption in lipomas with 12q14-q15 translocations. The genomic organization of HMGIC should further our understanding of the 12q14-q15 region, where other neoplasms of mesenchymal origin are also mapped.

Animals↗
PubMed1981

Retrograde and anterograde axonal transport demonstrated by intracerebral injection of a labeled protein-acylating agent.

Following injection of the tritium-labeled protein-acylating agent, N-succinimidyl propionate, into the neostriatum in rats, retrograde labeling of nigrostriatal cells in the pars compacta of the subtantia nigra and anterograde labeling of the striatonigral terminal field in the nigral pars reticulata were demonstrated by autoradiography, at 18 hr survival. Labeling of the corticopontine terminal field and of corticothalamic cell bodies in cortical layer 6 were also seen, deriving from corticofugal fibers of passage through the striatal injection site. In comparison, neostriatal injection of horseradish peroxidase was found to yield nigral cell and terminal field labeling, but the corticopontine terminal field was not labeled, and very few cortical cells in layer 6 were labeled. These results show that the retrograde and anterograde axonal transport of endogenous proteins can be profitably studied by this in vivo protein-acylating technique, and suggest that the method may prove useful as a connection-tracing technique, especially because of the novel attribute of labeling terminal fields by anterograde axonal transport following injections into white matter axonal bundles, with concomitant labeling od the cell bodies of origin of these axons.

Animals↗
PubMed1982

Construction of a genetic map for Caulobacter crescentus.

RP4-mediated conjugation has been used to transfer large fragments of chromosomal material in Caulobacter crescentus. In this system, conjugation proceeds from multiple origins, and haploid recombinants are recovered at frequencies of 10(-6) and 10(-7) per donor cell. The data from five-factor crosses were subjected to computer-assisted crossover analyses as a rapid method to determine marker order. Using this information and data from additional two- and three-factor crosses mediated by RP4 or the generalized transducing bacteriophage phi Cr30, we constructed the first genetic map for C. crescentus.

Bacteria↗
PubMed1979

Different chromosome banding procedures to distinguish between mouse and rabbit chromosomes in mouse-rabbit somatic cell hybrids.

The discrimination of the parental chromosomes in interspecific somatic cell hybrids, is a powerful tool in gene mapping studies. We report the methods used to distinguish between murine and rabbit chromosomes in mouse-rabbit somatic cell hybrids. By means of G-banding patterns it is possible to determine the murine or rabbit origin of the chromosomes, as well as the single chromosome identification in the same hybrid metaphase. To rapidly establish whether the chromosome are of murine or rabbit origin we use C-banding and Hoechst staining procedures, which staining or elongating respectively and preferentially the centromeric area of the mouse chromosomes allowed indubitable species assignment.

Animals↗
PubMed1998

Evidence for MAP kinase activation during mitotic division.

MAP kinases have been implicated in the control of a broad spectrum of cellular events in many types of cells. In somatic cells, MAP kinase activation seems to be triggered after exit from a quiescent state (in G0 or G2) only and then inactivated by entry into a proliferative state. In oocytes of various species, a one-time activation of MAP kinase that is apparently not repeated during the succeeding mitotic cycles occurs after meiotic activation. However, several reports suggest that a myelin basic protein (MBP) kinase activity, unrelated to that of maturation promoting factor, can sometimes be detected during mitotic divisions in various types of cells and oocytes. We have reinvestigated this problem in order to determine the origin and the role of MBP kinase that is stimulated at time of mitosis in the fertilized eggs of the sea urchin Paracentrotus lividus. We used anti-ERK1 antibodies or substrates specific for different MAP kinases, and performed in-gel phosphorylation experiments. Our results suggest that an ERK1-like protein was responsible for part of the MBP kinase activity that is stimulated during the first mitotic divisions. Furthermore, we observed that wortmannin, an inhibitor of PI 3-kinase that arrests the fertilized sea urchin eggs at the prometaphase stage, inhibited the inactivation of MAP kinase normally observed when the eggs divide, suggesting a role for PI 3-kinase in the deactivation process of MAP kinase. We also discuss how the activities of MPF and MAP kinase may be interconnected to regulate the first mitotic divisions of the early sea urchin embryo.

Activating Transcription Factor 2↗
PubMed1984

On the development of a standard two-dimensional polypeptide map of the human X chromosome.

Two-dimensional gel electrophoresis analysis of a rodent-human somatic cell hybrid containing the X as the only human chromosome reveals three polypeptides that are absent in the parental cell line. The presence of these spots in human female fibroblasts indicates their human origin. The polypeptides have molecular weights and isoelectric points of 30,000/5.8,37,000/5.4, and 57,000/4.7 and are designated as PFHG 1, PFHG 2, and PFHG 3. Comparison of their molecular characteristics with those of polypeptides assigned to the human X in two other investigations shows that some but not all polypeptides are similar. Factors are discussed that might interfere with the rapid development of a standardized polypeptide map of the human X.

Animals↗
PubMed1996

Monoclonal antibody U36, a suitable candidate for clinical immunotherapy of squamous-cell carcinoma, recognizes a CD44 isoform.

At present, tumor-targeting with monoclonal antibodies (MAbs) is among the most promising novel adjuvant-therapy modalities for the treatment of patients with minimal residual disease of head-and-neck squamous-cell carcinoma (HNSCC). For this purpose we developed MAb U36, recognizing a 200-kDa antigen expressed on the outer cell surface of squamous-cell carcinomas and their normal counterparts. Clinical radioimmunoscintigraphy (RIS) and biodistribution studies have shown that the MAb-U36-defined antigen is a suitable target molecule for antibody-based therapy of head-and-neck cancer. In the present study we further characterized the antigen by cDNA cloning. The cDNA was isolated by expression cloning in COS-7 cells. Sequence analysis and database searching revealed that the MAb-U36-defined antigen is identical to the squamous-cell-specific CD44 splice variant epican. The epitope recognized by MAb U36 was mapped by screening overlapping synthetic peptides of the epican-specific region encoded by exon 7-11 (v3-v7), and appeared to be located in the v6 domain. The applicability of MAb U36 for targeting human tumors of various origin expressing the CD44v6 domain is discussed.

Amino Acid Sequence↗
PubMed2003

Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location.

Heterochromatin proteins are thought to play key roles in chromatin structure and gene regulation, yet very few genes have been identified that are regulated by these proteins. We performed large-scale mapping and analysis of in vivo target loci of the proteins HP1, HP1c, and Su(var)3-9 in Drosophila Kc cells, which are of embryonic origin. For each protein, we identified approximately 100-200 target genes among >6000 probed loci. We found that HP1 and Su(var)3-9 bind together to transposable elements and genes that are predominantly pericentric. In addition, Su(var)3-9 binds without HP1 to a distinct set of nonpericentric genes. On chromosome 4, HP1 binds to many genes, mostly independent of Su(var)3-9. The binding pattern of HP1c is largely different from those of HP1 and Su(var)3-9. Target genes of HP1 and Su(var)3-9 show lower expression levels in Kc cells than do nontarget genes, but not if they are located in pericentric regions. Strikingly, in pericentric regions, target genes of Su(var)3-9 and HP1 are predominantly embryo-specific genes, whereas on the chromosome arms Su(var)3-9 is preferentially associated with a set of male-specific genes. These results demonstrate that, depending on chromosomal location, the HP1 and Su(var)3-9 proteins form different complexes that associate with specific sets of developmentally coexpressed genes.

Animals↗
PubMed2006

Distinct expression pattern of microtubule-associated protein/microtubule affinity-regulating kinase 4 in differentiated neurons.

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate tau protein and related microtubule-associated proteins (MAPs), and their role in the establishment of cell polarity in different contexts. Recent papers have indicated that microtubule affinity-regulating kinase 4 (MARK4) is a gene that is finely regulated at transcriptional level and expressed in two spliced isoforms called MARK4L and MARK4S. We here describe the characterization of the mouse orthologue of the human MARK4 gene. Interestingly, MARK4S is predominantly expressed in the brain, whereas MARK4L shows lower transcript levels in this organ. Using MARK4 antibodies specific for each isoform, we found that both isoforms have an identical expression pattern in the mouse CNS, and are present in a number of neuronal populations. We also found that human microtubule affinity-regulating kinase 4S (hMARK4S), whose expression is not detectable in human neural progenitor cells (HNPCs) and NTera2 (NT2) cells, is up-regulated in both cell systems from the very early stages of neuronal differentiation. This indicates that neuronal commitment is marked by MARK4S up-regulation. In conclusion, this study provides the first direct evidence suggesting that MARK4 is a neuron-specific marker in the CNS, and the up-regulation of MARK4S during neuronal differentiation suggests that it plays a specialized role in neurons.

Animals↗
PubMed2004

Mutations in SLC6A19, encoding B0AT1, cause Hartnup disorder.

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis. Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B(0)AT1 (ref. 4). We isolated the human homolog of B(0)AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.

Amino Acid Sequence↗
PubMed2004

Mutant ras-induced proliferation of human thyroid epithelial cells requires three effector pathways.

Ras mutations occur as an early event in many human tumours of epithelial origin, including thyroid. Using primary human thyroid epithelial cells to model tumour initiation by Ras, we have shown previously that activation of both the MAP kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) effector pathways are necessary, but even when activated together are not sufficient, for Ras-induced proliferation. Here, we show that a third effector, RalGEF, is also activated by Ras in these cells, that this activation is necessary for Ras-induced proliferation, and furthermore that in combination with the MAPK and PI3K effectors, it is able to reproduce the proliferative effect of activated Ras. The requirement for three effector pathways indicates a more robust control of cell proliferation in this normal human epithelial cell type than has been displayed in previous similar studies using rodent and human cell lines. Our findings highlight the importance of the appropriate cellular context in models of Ras-induced tumour development.

Cell Division↗
PubMed2002

Leaf stomatal and epidermal cell development: identification of putative quantitative trait loci in relation to elevated carbon dioxide concentration in poplar.

Genetic variation in stomatal initiation and density, and epidermal cell size and number were examined in a hybrid pedigree of Populus trichocarpa T. & G. and P. deltoides Marsh in both ambient ([aCO2]) and elevated ([eCO2]) concentrations of CO2. We aimed to link anatomical traits with the underlying genetic map of F2 Family 331, composed of 350 markers across 19 linkage groups. Leaf stomatal and epidermal cell traits showed pronounced differences between the original parents. We considered the following traits in the F2 population: stomatal density (SD), stomatal index (SI), epidermal cell area (ECA) and the number of epidermal cells per leaf (ECN). In [eCO2], adaxial SD and SI were reduced in the F2 population, whereas ECA increased and ECN remained unchanged. In [aCO2], four putative quantitative trait loci (QTL) with logarithm of the odds ratio (LOD) scores greater than 2.9 were found for stomatal traits on linkage group B: adaxial SI (LOD scores of 5.4 and 5.2); abaxial SI (LOD score of 3.3); and SD (LOD score of 3.2). These results imply that QTL for SI and SD share linkage group B and are under genetic control. More moderate LOD scores (LOD scores >/= 2.5) suggest QTL for SI on linkage groups A and B and for SD on linkage groups B, D and X with a probable co-locating quantitative trait locus for SI and SD on linkage group D (position 46.3 cM). The QTL in both [aCO2] and [eCO2] for adaxial SD were co-located on linkage group X (LOD scores of 3.5 and 2.6, respectively) indicating a similar response across both treatments. Putative QTL were located on linkage group A (position 89.2 cM) for both leaf size and ECN in [aCO2] and for ECA at almost the same position. The data provide preliminary evidence that leaf stomatal and cell traits are amenable to QTL analysis.

Carbon Dioxide↗
PubMed1986

A time-comparison circuit in the electric fish midbrain. II. Functional morphology.

The weakly electric fish Eigenmannia is able to detect temporal disparities as small as 400 nsec between two signals from different parts of the body surface (Carr et al., 1986). The elements of this time-comparison circuit have been identified by EM reconstruction of its component cells. Information about the timing of the zero-crossing of signals on each area of the body surface is coded in phase-coder receptors, a subset of tuberous electroreceptors. Electroreceptors on the body surface are innervated by primary afferents with their central termination on the spherical cells of the medullary electrosensory lateral line lobe. These cells project to lamina VI of the midbrain torus, a structure similar to the inferior colliculus. Afferents entering lamina VI form a very restricted terminal arbor in which they synapse on the three cell types of this lamina. Each afferent makes gap-junction synapses on one or two giant cell somata and morphologically mixed synapses on the distal dendrites of two types of small cell. The afferent terminals thus encode the timing of the electric signal on a local patch of the body surface, forming a somatotopic map of the body surface in lamina VI. The giant cells are adendritic and their axonal arbor is such as to distribute timing information originating from one part of the body surface throughout lamina VI, so that each region of lamina VI receives information about the timing of zero-crossings from the entire body surface from giant cells, as well as information from a local portion of the body surface from the afferent terminals. The giant cells terminate exclusively on the cell bodies of the small cells of lamina VI, shown to be sensitive to small temporal disparities by Heiligenberg and Rose (1985). Thus, each small cell receives a single synapse on its soma from a giant cell that conveys phase-coding information from some portion of the body surface and receives local phase-coding input onto its dendrites from spherical cell afferents. The sensitivity of the small cells to temporal disparities appears to be conferred by their segregation of inputs from two different parts of the body surface onto dendrites and soma, respectively. We propose that the dendritic input acts as a delay line, and the small cell fires maximally when the inputs from the dendrites and the giant cell input onto the soma coincide.

Animals↗
PubMed1994

Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication.

Adenovirus preterminal protein (pTP) exists as a heterodimer with the viral DNA polymerase (AdPol) and becomes covalently linked to a dCMP residue during initiation of DNA replication. The in vivo phosphorylation of pTP could be demonstrated when pTP is overproduced using recombinant vaccinia viruses, or by a large scale metabolic labeling of adenovirus 2 (Ad2)-infected HeLa cells. Phosphoserine was the only phosphoamino acid obtained by acid hydrolysis of 32P-labeled pTP immunoprecipitated from metabolically labeled HeLa cells infected with either Ad2 or recombinant vaccinia virus. Tryptic peptide maps of pTP expressed using recombinant vaccinia virus system in HeLa cells revealed that phosphorylation of pTP occurred on multiple sites. Dephosphorylation of pTP with calf intestinal alkaline phosphatase resulted in a significant decrease in its activity in the in vitro DNA replication initiation assays. Further characterization of the phosphatase-treated pTP indicated that although dephosphorylation did not affect its interaction with AdPol, the specific recognition of the DNA replication origin by pTP was significantly reduced as determined by gel electrophoresis-based DNA mobility shift assays.

Adenoviruses, Human↗
PubMed1994

Three ARS elements contribute to the ura4 replication origin region in the fission yeast, Schizosaccharomyces pombe.

The ura4 replication origin region, which is located near the ura4 gene on chromosome III of the fission yeast, Schizosaccharomyces pombe, contains multiple initiation sites. We have used 2D gel electrophoretic replicon mapping methods to study the distribution of these initiation sites, and have found that they are concentrated near three ARS elements (stretches of DNA which permit autonomous plasmid replication). To determine the roles of these ARS elements in the function of the ura4 origin region, we deleted either one or two of them from the chromosome and then assessed the consequences of the deletions by 2D gel electrophoresis. The results suggest that each of the three ARS elements is responsible for the initiation events in its vicinity and that the ARS elements interfere with each other in a hierarchical fashion. It is possible that the large initiation zones of animal cells are similarly composed of multiple mutually interfering origins.

Chromosome Mapping↗
PubMed2000

Characterization and chromosomal mapping of a novel human gene, ANKHZN.

Ankhzn (ankyrin repeats hooked to a zinc finger motif) was originally isolated by means of the gene trap method, as a novel cytoplasmic protein on mouse embryonic stem cells. The Ankhzn protein is ubiquitously expressed in a spatiotemporal-specific manner and is located on endosomes. In the present study, we have cloned human ANKHZN cDNA by PCR using candidate EST clones exhibiting a high homology to mouse Ankhzn cDNA. The human ANKHZN cDNA encoded a 1166aa protein exhibiting 84.9% identity to the mouse one. The size of the transcript was found to be about 7kb on a Northern blot analysis, and ANKHZN mRNA was found to be ubiquitously expressed in human tissues on RT-PCR analysis. Western blot analysis showed that a 130kDa protein was detected at various levels in human tissues and also present in both membrane and soluble fractions obtained on subcellular fractionation. Human ANKHZN is a single copy gene consisting of predicted 25 exons in the human genome, and has been mapped to human chromosome 17p13 by radiation hybrid panel and fluorescence in-situ hybridization.

Amino Acid Sequence↗