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At least 541 records · Page 30Linked to original sources
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↗
PubMed2003

Transcriptional regulation of the elafin gene in human keratinocytes.

Elafin (also known as skin-derived anti-leukoproteinase/trappin-2) is an epithelial host-defense protein that is absent in normal skin but highly induced in keratinocytes of inflamed skin (e.g., psoriasis), in epidermal skin tumors, and after wounding. Previously, it was shown that in cultured keratinocytes, elafin expression is induced by serum or tumor necrosis factor-alpha, and that expression is suppressed by retinoids, dithranol, and p38 mitogen-activated protein kinase inhibitors. Here we have studied the regulation of elafin gene expression in epidermal keratinocytes at the molecular level. First we determined the transcription start site of the elafin gene and found that the elafin mRNA possesses an unusually short 5'-untranslated region. Using transient transfection of luciferase reporter constructs of the elafin promoter, we mapped a 440 bp region upstream of the translation start site that conferred high-level expression in keratinocytes, but not in A431 cells or cells of mesenchymal origin. We observed that the promoter constructs were not subjected to the same regulation as the endogenous elafin gene as these constructs were highly active independent of keratinocyte stimulation. When elafin promoter constructs were stably transfected in the HaCaT keratinocyte cell line, tumor necrosis factor-alpha inducible expression of both the endogenous elafin gene and the transgene was observed, suggesting that regulation of the elafin gene is also dependent on chromatin structure. We found, however, that a stably transfected 4 kb elafin promoter fragment did not confer retinoid sensitivity indicating that additional sequences are required for proper regulation. This study reveals the complex regulation of a gene that can be used as a paradigm for the specific differentiation program of activated epidermal keratinocytes.

5' Untranslated Regions↗
PubMed2003

Differential use of multiple replication origins in the ribosomal DNA episome of the protozoan parasite Entamoeba histolytica.

The factors that control the initiation of eukaryotic DNA replication from defined origins (oris) on the chromosome remain incompletely resolved. Here we show that the circular rDNA episome of the human pathogen Entamoeba histolytica contains multiple potential oris, which are utilized in a differential manner. The primary ori in exponentially growing cells was mapped close to the promoter of rRNA genes in the upstream intergenic spacer (IGS) by two-dimensional gel electrophoresis. Replication initiated predominantly from the upstream IGS and terminated in the downstream IGS. However, when serum-starved cells were allowed to resume growth, the early oris which became activated were located in other parts of the molecule. Later the ori in the upstream IGS became activated, with concomitant silencing of the early oris. When the upstream IGS was located ectopically in an artificial plasmid, it again lost ori activity, while other parts of the rDNA episome could function as oris in this system. Therefore, the activation or silencing of the ori in this episome is context dependent, as is also the case with many eukaryotic replicons. This is the first replication origin to be mapped in this primitive protozoan and will provide an opportunity to define the factors involved in differential ori activity, and their comparison with metazoans.

Animals↗
PubMed2006

Mechanisms of gonadal morphogenesis are not conserved between chick and mouse.

To understand mechanisms of sex determination, it is important to know the lineage relationships of cells comprising the gonads. For example, in mice, the Y-linked gene Sry triggers differentiation of Sertoli cells from a cell population originating in the coelomic epithelium overlying the nascent gonad that also gives rise to uncharacterised interstitial cells. In contrast, little is known about origins of somatic cell types in the chick testis, where there is no Sry gene and sex determination depends on a ZZ male/ZW female mechanism. To investigate this, we performed fate mapping experiments in ovo, labelling at indifferent stages the coelomic epithelium by electroporation with a lacZ reporter gene and the underlying nephrogenous (or mesonephric) mesenchyme with chemical dyes. After sex differentiation, LacZ-positive cells were exclusively outside testis cords and were 3betaHSD-negative, indicating that the coelomic epithelium contributes only to non-steroidogenic interstitial cells. However, we detected dye-labelled cells both inside and outside the cords. The former were AMH-positive while some of the latter were 3betaHSD-positive, showing that nephrogenous mesenchyme contributes to both Sertoli cells and steroidogenic cells. This is the first demonstration via lineage analysis that steroidogenic cells originate from nephrogenous mesenchyme, but the revelation that Sertoli cells have different origins between chick and mouse suggests that, during evolution, mechanisms of gonad morphogenesis may diverge alongside those of sex determination.

Animals↗
PubMed1991

Mouse chromosome-specific markers generated by PCR and their mapping through interspecific backcrosses.

We have utilized an oligonucleotide primer from the 3' end of the mouse L1 repeat element for amplification of mouse-specific inter-repeat PCR products from Chinese hamster/mouse somatic cell hybrids. PCR of a Chinese hamster/mouse somatic cell hybrid (96AZ2), containing only mouse chromosome 16, produced a range of mouse-specific bands. Two of the mouse-specific PCR products, of 250 and 580 bp, have been confirmed as originating from mouse chromosome 16 by somatic cell hybrid analysis. Both the 250- and 580-bp PCR products have been sequenced and demonstrate the expected sequence organization. Furthermore, both the 250- and 580-bp markers have been genetically mapped in detail to mouse chromosome 16 by direct hybridization to inter-repeat PCR products of progeny DNAs from Mus domesticus/Mus spretus interspecific backcrosses.

Animals↗
PubMed2001

A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection.

The discovery of dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN) as a DC-specific ICAM-3 binding receptor that enhances HIV-1 infection of T cells in trans has indicated a potentially important role for adhesion molecules in AIDS pathogenesis. A related molecule called DC-SIGNR exhibits 77% amino acid sequence identity with DC-SIGN. The DC-SIGN and DC-SIGNR genes map within a 30-kb region on chromosome 19p13.2-3. Their strong homology and close physical location indicate a recent duplication of the original gene. Messenger RNA and protein expression patterns demonstrate that the DC-SIGN-related molecule is highly expressed on liver sinusoidal cells and in the lymph node but not on DCs, in contrast to DC-SIGN. Therefore, we suggest that a more appropriate name for the DC-SIGN-related molecule is L-SIGN, liver/lymph node-specific ICAM-3-grabbing nonintegrin. We show that in the liver, L-SIGN is expressed by sinusoidal endothelial cells. Functional studies indicate that L-SIGN behaves similarly to DC-SIGN in that it has a high affinity for ICAM-3, captures HIV-1 through gp120 binding, and enhances HIV-1 infection of T cells in trans. We propose that L-SIGN may play an important role in the interaction between liver sinusoidal endothelium and trafficking lymphocytes, as well as function in the pathogenesis of HIV-1.

Animals↗
PubMed2000

Postnatal development of the rat intrinsic cardiac nervous system: a confocal laser scanning microscopy study in whole-mount atria.

We used confocal laser scanning microscopy and fluorescent immunohistochemistry to study the developmental pattern and distribution of specific neuronal phenotypes within the intrinsic cardiac nervous system in whole-mount atrial preparations from newborn to 5 week old rats. Individual ganglia and neuronal cell bodies were localized by means of two general neuronal markers: protein gene product 9.5 (PGP) and microtubule-associated protein two (MAP). In rats < or =2 weeks old there were two main subpopulations of intrinsic neurons located in the intraatrial septum and around the origin of the superior vena cava. The more abundant was a population of strongly tyrosine hydroxylase (TH) immunoreactive (IR) neurons (10-40 microm in diameter) most of which were also PGP-IR. The second, less numerous (approximately 60-70% than the TH-IR group) type of neurons exhibited ChAT-IR which colocalized with MAP-IR. Towards the end of the second postnatal week and during the third, the ganglia containing these neurons became more numerous and their localization also included tissues around the origins of the inferior vena cava and the pulmonary veins, as well as both atrial walls close to the AV junction. During the second and third postnatal weeks, when the extrinsic innervation of the adrenergic and cholinergic phenotypes largely increases, the intrinsic innervation also changed greatly, and around the 21st postnatal day it appeared to acquire mature characteristics. The TH-IR neurons changed their characteristics and formed two types of ganglia. The larger ganglia containing large cells (20-40 microm in diameter) expressed TH-IR mostly close to their inner body surface (approximately 80-90% of identified neurons). Most of these neurons also expressed neuropeptide Y (NPY)-IR, specifically around their nuclei. The second type of small strongly TH-IR neurons (approximately 10% of all identified neurons) were contained in smaller groups (20-50 cells) which were usually embedded into much larger ganglia (100-400 cells), containing large (20-50 microm) neurons. Unlike all other intrinsic neurons, these small TH-IR cells did not exhibit any PGP-IR or MAP-IR. The number of ChAT-IR neurons increased at this stage, reaching approximately 90% of the neurons identified by the general neuronal markers. These neurons were surrounded by a rich network of cholinergic varicose nerve fibers, some of which were likely of an extrinsic origin. We have also identified relatively small ganglia expressing immunoreactivity to vasoactive intestinal polypeptide (VIP), and to substance P (SP). The presented data indicate that the phenotypes of intrinsic neurons in the rat heart change greatly during the first month of postnatal development. This may be at least partially related to the development and maturation of functional extrinsic nervous control of the heart.

Age Factors↗
PubMed1994

DNA damage-inducible replication of the Escherichia coli chromosome is initiated at separable sites within the minimal oriC.

When Escherichia coli cells are subjected to genetic stress by exposure to agents or conditions that transiently block DNA replication, the mode of DNA replication is profoundly altered. One of the alterations is the induction of inducible stable DNA replication (iSDR) that does not require the initiator protein, DnaA, and occurs despite the presence of rifampin and chloramphenicol, which inhibit the initiation of usual chromosome replication at oriC. It has been demonstrated that iSDR starts primarily from both the oriC and terC regions of the chromosome. To precisely map the iSDR origin (oriM1) located in the oriC region, various oriC fragments were inserted into a plasmid vector derived from pSC101, and the copy number of these plasmid constructs was measured in the presence of rifampin and chloramphenicol after cells were induced for the SOS response by thymine starvation. The results indicated that there are at least two origins for iSDR within the minimal oriC; one (oriM1A) is located between the BamHI (coordinate +1) and the AvaII(155) sites, and the other (oriM1B) between the AvaII(155) and the HindIII(244) sites. Furthermore, a 263 bp fragment containing oriM1, which was placed at the att lambda site of the chromosome, was found to initiate chromosome replication in the presence of the drugs when cells were starved of thymine. Introduction of additional copies of oriM1 into a cell stimulated initiation of iSDR at oriM1 on the chromosome. The result supported the model that iSDR starts from D-loops created between oriM1 sequences and that the amount of D-loops determines the level of the iSDR activity.

Bacterial Proteins↗
PubMed1999

An origin of bidirectional DNA replication is located within a CpG island at the 3" end of the chicken lysozyme gene.

We previously identified a broad initiation zone of DNA replication at the chicken lysozyme gene locus. However, the existence of a highly preferred origin of bidirectional replication (OBR), often found in initiation zones, remained elusive. In order to re-examine this issue we used a competitive PCR assay to determine the abundance of closely spaced genomic segments in a 1 kb size fraction of nascent DNA. A sharp peak of nascent strand abundance occurred at the 3" end of the gene, where initiation events were 17 times more frequent than upstream of the gene. This primary initiation site, active in lysozyme expressing myelomonocytic HD11 cells and non-expressing hepatic DU249 cells, was found to reside within an unusually located CpG island. While most CpG islands are found at the 5" end of genes, the lysozyme gene island extends from the 3" end of the second intron and includes approximately 1.2 kb of 3" flanking DNA. As diagnosed by methylation-sensitive restriction enzymes, the island is largely non-methylated in HD11 cells, DU249 cells and inactive chicken erythrocytes. Furthermore, a DNase I hypersensitive site (HS) that is composed of two subsites separated by approximately 100 bp, was localised very close to the segment with the highest initiation activity. Our results suggest that the non-methylated CpG island and the HS provide an accessible chromatin structure for the lysozyme gene origin of replication.

Animals↗
PubMed1994

A method for constructing radiation hybrid maps of whole genomes.

In radiation hybrid mapping, chromosomes in human-rodent hybrid cells are fragmented by X-rays and fragments rescued by fusion of the donor cell to a recipient rodent cell. The co-retention frequencies of markers in 100-200 hybrids are used to map individual chromosomes, but mapping the whole genome in this way is impractical. We have reverted to the original protocols of Goss and Harris and have produced a panel of 44 hybrids using irradiated human fibroblasts as donors. This panel has been used to make a map of human chromosome 14 containing 40 ordered markers. The map integrates previously published maps and localizes nine new markers. We suggest that the construction of a high resolution map of the whole human genome is feasible with a single panel of 100-200 hybrids.

Animals↗
PubMed2002

Delimitation of duplicated segments and identification of their parental origin in two partial chromosome 3p duplications.

Two chromosome 3 short arm duplications identified through G-banding were further investigated using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) of microsatellite markers, aiming at mapping breakpoints and disclosing mechanisms of origin of these chromosome aberrations. Patient 1 was found to be a mosaic: a 3p12 --> 3p21 duplication was observed in most of his cells, and a normal cell line occurred with a frequency of about 3% in blood. In situ hybridization of chromosome 3 short- and long-arm libraries confirmed the short-arm duplication. Using FISH of short-arm sequences, the YAC 961_h_3 was shown to contain the proximal breakpoint (3p12.1 or 3p12.2), and the distal breakpoint was located between the YACs 729_c_3 and 806_h_2, which are adjacent in the WC 3.10 contig (3p21.1). In Patient 2, G-banding indicated a 3p21 --> 3p24 duplication, without mosaicism. In situ hybridization of chromosome 3 short- and long-arm libraries confirmed the duplication of short-arm sequences. FISH of chromosome 3 sequences showed that the YAC 749_a_7 spanned the proximal breakpoint (3p21.33). The distal breakpoint mapped to the interval between YACs 932_b_6 (3p24.3) and 909_b_6 (3p25). In both cases, microsatellite genotyping pointed to a rearrangement between paternal sister chromatids.

Adolescent↗
PubMed1997

The Escherichia coli polB locus is identical to dinA, the structural gene for DNA polymerase II. Characterization of Pol II purified from a polB mutant.

Escherichia coli DNA polymerase II (Pol II) is a member of the group B, "alpha-like" family of DNA polymerases. Pol II is encoded by the damage-inducible dinA gene and exhibits SOS induction under the control of Lex A repressor. The polB gene was originally designated as the structural gene for Pol II based on the absence of detectable Pol II activity in cell lysates prepared from a strain containing the mutant polB100 allele. Because polB and dinA mapped at different chromosomal locations, it remained an open question whether polB, in addition to lexA, might be involved in regulating the expression of Pol II. We have cloned and sequenced the polB100 mutant allele, including adjacent surrounding sequences, and have expressed the mutant dinA gene from Pol B100 on a high copy number plasmid. Our sequence data reveal that polB and dinA represent the same gene and that the original transduction mapping of polB was inaccurate. We purified the mutant Pol B100 polymerase and show that it retains 5 to 10% of the wild-type level of polymerase activity. The Pol B100 mutation, Gly401 --> Asp401, is not located within any of the five conserved domains that define group B polymerases. Pol B100 retains a wild-type level of 3' --> 5' exonuclease activity. We suggest that the normal level of exonucleolytic proofreading associated with the mutant Pol B100 enzyme may explain the repeated failures, over the past two decades, to detect phenotypes in polB mutant strains.

Bacterial Proteins↗
PubMed2000

Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay.

The constitutive activation of G-protein-coupled receptors is a major new approach to investigating their physiopathology and pharmacology. A large number of spontaneous and site-directed mutations resulting in constitutive activity have been identified, but systematic mapping of the amino acids involved for a given receptor would be extremely useful for complete elucidation of the molecular mechanisms underlying its activation. We carried out such mapping for the angiotensin II type 1A (AT(1A)) receptor by screening a randomly mutated cDNA library after expressing the mutated clones in eukaryotic cells. To test the AT(1A) mutants generated, we developed an original, specific, and highly sensitive assay based on the properties of CGP42112A. This classical AT(2) agonist is a weak partial agonist of the wild-type AT(1A) receptor and becomes a full agonist for constitutively active AT(1A) mutants, as shown experimentally and in allostery-based theoretical models. Activation of the mutated receptors by CGP42112A was monitored by using the bioluminescent protein aequorin, a very sensitive and specific sensor of intracellular calcium mobilization. The screening of 4,800 clones, providing an exhaustive coverage of all of the mutations generated, led to the identification of 16 mutations in sequences encoding the transmembrane domains that were responsible for high sensitivity to CGP42112A. The constitutive activity was confirmed by agonist-independent production of inositol phosphates, which showed that at least half of the clones had significantly increased basal activity. These data demonstrate that this new type of approach is very efficient for the systematic identification of constitutively active mutants of G-protein-coupled receptors.

Amino Acid Sequence↗
PubMed1996

Human enteric defensin genes: chromosomal map position and a model for possible evolutionary relationships.

Defensins, a family of antimicrobial peptides isolated from several mammalian species, have a proposed functional role in innate host defense. In humans, certain defensin genes are expressed in phagocytic cells of hematopoietic origin, while others are expressed in Paneth cells, epithelial cells of the small intestine. In this study, we determined the chromosomal localization of the human defensin (HD) genes expressed in Paneth cells, HD-5 and HD-6. Analysis of a panel of human/hamster hybrids localized both HD-5 and HD-6 to chromosome 8. Southern blot analysis of DNA from cell lines that contain either chromosome 8 deletions or duplications further localized these two genes to 8p21-pter. Fluorescence in situ hybridization analysis of metaphase chromosomes using an HD-5 probe further supported the regional map assignment. Previous studies had localized the hematopoietic genes to chromosome 8p23, and the current work is consistent with both the enteric and the myeloid defensin genes being located at the same cytogenetic region of chromosome 8. In addition, the evolutionary relationships of this gene family were addressed using dot matrix sequence analysis. From this analysis, a model for the possible evolutionary history of the human defensin genes is proposed. According to this model, an early duplication of a primordial defensin gene yielded the ancestral genes of present day HD-5 and HD-6. The model further suggests that a subsequent unequal meiotic crossover event had generated an additional gene, comprised of a hybrid of sequences from the two parental genes, and that this hybrid gene then served as the ancestor to present day hematopoietic defensin genes.

Amino Acid Sequence↗
PubMed2004

Proteomic phenotyping: metastatic and invasive breast cancer.

The adriamycin resistant breast cancer cell line (MCF-7/ADR) is a subject of ongoing debate concerning its origin and or source. Previous studies in our laboratory showed that MCF-7/ADR has a unique cytosolic protein expression pattern when compared to that of the parental MCF-7 cell line and other drug resistant MCF-7 cell lines. Protein expression patterns obtained using two-dimensional gel electrophoresis and mass spectrometry indicated that this MCF-7/ADR cell line shares some similarities with the metastatic breast cancer cell lines MDA-MB. Further comparisons with available two-dimensional gel electrophoresis maps in the literature indicate that MCF-7/ADR has a protein expression signature even closer to of the ductal infiltrating breast carcinoma cell line 8701. These observations suggest that MCF-7/ADR cells might have originated in a selection of ductal infiltrating carcinoma cells, which were present among the original MCF-7 cell population. These ductal infiltrating carcinoma cells may possess an intrinsic adriamycin resistance phenotype.

Antibiotics, Antineoplastic↗
PubMed1991

DNA sequences 3' of the Ig H chain cluster rearrange in mouse B cell lines.

A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to study DNA rearrangements that occur at the Ig H chain locus. One variant, F5.5, has acquired both VH gene and C epsilon gene rearrangements. Through genomic Southern blot analysis initially directed to mapping the C epsilon gene rearrangement, we observed that the VH region rearrangement was linked, through an inversion event, to sequences that originate 3' of the CH cluster, i.e., 3' of the C alpha gene. Subsequent studies have shown that DNA rearrangements within the region 3' of the C alpha gene are detected in several other mouse myeloma and hybridoma cell lines and are not associated with the expression of specific isotypes.

Animals↗