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PubMed2002

CCR5 and HIV infection.

Chemokines and chemokine receptors play a crucial role in the trafficking of leukocyte populations across the body, and are involved in the development of a large variety of human diseases. CCR5 is the main coreceptor used by macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2, which are responsible for viral transmission. CCR5 therefore plays an essential role in HIV pathogenesis. A number of inflammatory CC-chemokines, including MIP-1 alpha, MIP-1 beta, RANTES, MCP-2, and HCC-1[9-74] act as CCR5 agonists, while MCP-3 is a natural antagonist of the receptor. CCR5 is mainly expressed in memory T-cells, macrophages, and immature dendritic cells, and is upregulated by proinflammatory cytokines. It is coupled to the Gi class of heterotrimeric G-proteins, and inhibits cAMP production, stimulates Ca2+ release, and activates PI3-kinase and MAP kinases, as well as other tyrosine kinase cascades. A mutant allele of CCR5, CCR5 delta 32 is frequent in populations of European origin, and encodes a nonfunctional truncated protein that is not transported to the cell surface. Homozygotes for the delta 32 allele exhibit a strong, although incomplete, resistance to HIV infection, whereas heterozygotes display delayed progression to acquired immunodeficiency syndrome (AIDS). Many other alleles, affecting the primary structure of CCR5 or its promoter have been described, some of which lead to nonfunctional receptors or otherwise influence AIDS progression. CCR5 is considered as a drug target in the field of HIV, but also in a growing number of inflammatory diseases. Modified chemokines, monoclonal antibodies and small chemical antagonists, as well as a number of gene therapy approaches have been developed in this frame.

Amino Acid Sequence↗
PubMed2002

Initiation sites are distributed at frequent intervals in the Chinese hamster dihydrofolate reductase origin of replication but are used with very different efficiencies.

Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred. However, high-resolution analysis by a PCR-based nascent strand abundance assay of the 12-kb subregion encompassing ori-beta has suggested the presence of a relatively small number of fixed, highly efficient initiation sites distributed at infrequent intervals that correspond to genetic replicators. To attempt to reconcile these observations, two different approaches were taken in the present study. In the first, neutral-neutral 2-D gel analysis was used to examine replication intermediates in 31 adjacent and overlapping restriction fragments in the spacer, ranging in size from 1.0 to 18 kb. Thirty of 31 fragments displayed the complete bubble arcs characteristic of centered origins. Taking into account overlapping fragments, these data suggest a minimum of 14 individual start sites in the spacer. In the second approach, a quantitative early labeled fragment hybridization assay was performed in which radioactive origin-containing DNA 300 to 1,000 nucleotides in length was synthesized in the first few minutes of the S period and used to probe 15 clones distributed throughout the intergenic spacer but separated on average by more than 1,000 bp. This small nascent DNA fraction hybridized to 14 of the 15 clones, ranging from just above background to a maximum at the ori-beta locus. The only silent region detected was a small fragment lying just upstream from a centered matrix attachment region--the same region that was also negative for initiation by 2-D gel analysis. Results of both approaches suggest a minimum of approximately 20 initiation sites in the spacer (two of them being ori-beta and ori-gamma), with ori-beta accounting for a maximum of approximately 20% of initiations occurring in the spacer. We believe that the results of all experimental approaches applied to this locus so far can be fitted to a model in which the DHFR origin consists of a 55-kb intergenic zone of potential sites that are used with very different efficiencies and which are separated in many cases by a few kilobases or less.

Animals↗
PubMed1996

CDKN2A (p16INK4A) somatic and germline mutations.

The cell cycle is composed of a series of steps that can be negatively or positively regulated by various factors. A group of low-molecular-weight proteins have recently been identified that specifically inhibit the function of cyclin-dependent kinases in mammalian cells. Inactivation of the CDKN2A gene (also known as p16INK4A and MTS1) attracted considerable interest after it was mapped to 9p21, a locus for familial melanoma. In an effort to standardize the information regarding human CDKN2A mutations detected in cancers, a database with information of 146 point mutations has been created. Cancer type, origin of cells, specific mutation, amino acid change, literature citation, and other data are provided for each mutation entry. Studies of biochemical and biological functions of both wild-type and mutant proteins are central to our understanding of the role of p16INK4a mutations in tumorigenesis, a summary of these studies is also included in the present update.

Carrier Proteins↗
PubMed1983

Claustroneocortical projections studied in the cat by means of multiple retrograde fluorescent tracing.

The topographical interrelations of claustroneocortical cells and their degree of divergent collateralization were investigated in cat by means of retrograde fluorescent double labeling. The tracers Fast Blue (FB) and Nuclear Yellow (NY) were injected in several combinations in two different cortical fields. FB-labeled and NY-labeled cells were found in all cases in the insular portion of the ipsilateral claustrum (CII). The distributions of labeled cells confirmed the organization reported in previous studies. In some cases the two labeled cell populations were segregated in different parts of the CII; in other cases there was more or less overlap in the two distributions. The degree of overlap was not simply related to the topographic proximity of the injected cortical territories. In all cases the vast majority of cells were single labeled. In one case both FB and NY were injected in the same cortical area in order to control that claustral axons can retrogradely transport two tracers back to the same parent cell bodies. In this control case CII cells were double labeled. All together these data indicate that widely divergent claustroneocortical projection take origin mainly from separate cells.

Animals↗
PubMed1996

Quantitative X-ray imaging of labelled molecules in tissues and cells.

Using cisplatin as a model system, we have been able to demonstrate the feasibility of studying the cellular and subcellular distribution of a labelled molecule containing a single atom of platinum per molecule in bone marrow. An X-ray imaging system consisting of a microcomputer, a 4pi system and a software package was interfaced with an electron microscope enabling the computer to control the beam movements as well as receive signals from the STEM and EDS X-ray detectors. X-ray imaging is useful for both tissue and samples in which the population of cells is not homogeneous. Imaging permits elemental distributions to be measured throughout the sample and not in just randomly selected areas as previously done in X-ray microanalysis. Images are created for not only the element labelling the molecule of interest but also other specified elements present. Three types of maps for imaging labelled molecules are compared and discussed. When the original (collected) data are mapped, the elements of interest are obscured by the continuum. The maps calculated using an internal standard give a concentration distribution on the basis of volume (mmol L(-1) of packed cells). The maps calculated using the continuum normalization method according to Hall produces concentration distribution on the basis of mass (mmolk(-1) dry weight). By recalculating using the 'Peak' or 'Hall' method the continuum problem is removed yielding quantitative images of the intracellular distribution of labelled molecules present in low concentrations.

Animals↗
PubMed1997

Identification and analysis of a lytic-phase origin of DNA replication in human herpesvirus 7.

Human herpesvirus 7 (HHV-7) DNA sequences colinear with the HHV-6 lytic-phase origin of DNA replication (oriLyt) were amplified by PCR. Plasmid constructs containing these sequences were replicated in HHV-7-infected cord blood mononuclear cells but not in HHV-6-infected cells. In contrast, plasmids bearing HHV-6 oriLyt were replicated in both HHV-6- and HHV-7-infected cells. Finally, the minimal HHV-7 DNA element necessary for replicator activity was mapped to a 600-bp region which contains two sites with high homology to the consensus binding site for the HHV-6 origin binding protein. At least one of these binding sites was shown to be essential for replicator function of HHV-7 oriLyt.

Base Sequence↗
PubMed1995

cis-Acting components of human papillomavirus (HPV) DNA replication: linker substitution analysis of the HPV type 11 origin.

Papillomavirus DNA replication requires the viral trans-acting factors E1 and E2 in addition to the host cell's general replication machinery. The origins of DNA replication in bovine and human papillomavirus genomes have been localized to a specific part of the upstream regulatory region (URR) which includes recognition sites for E1 and E2 proteins. To fine map cis-acting elements influencing human papillomavirus type 11 (HPV-11) DNA replication and to determine the relative contributions of such sites, we engineered consecutive linker substitution mutations across a region of 158 bp in the HPV-11 origin and tested mutant origins for replication function in a cell-based transient replication assay. Our results both confirm and extend the findings of others. E2 binding sites are the major cis components of HPV-11 DNA replication, and there is evidence for synergy between these sites. Differential capacity of the three E2 binding sites within the origin to affect replication may be attributed, at least in part, to context. At least one E2 binding site is essential for replication. The imperfect AT-rich palindrome of the E1 helicase binding site is not essential since replication occurs even in the absence of this sequence. However, replication is enhanced by the presence of the palindromic sequence in the HPV-11 origin. Sequence components adjacent to the E1 and E2 binding sites, comprising AT-rich and purine-rich elements and the consensus TATA box sequence, probably contribute to the overall efficiency of replication, though they are nonessential. None of the other cis elements of the HPV-11 origin region analyzed seems to influence replication significantly in the system described. The HPV-11 origin of DNA replication therefore differs from those of the other papovaviruses, simian virus 40 and polyomavirus, inasmuch as an intact helicase binding site and adjacent AT-rich components, while influential, are not absolutely essential.

Animals↗
PubMed1990

Genomic regions of neurovirulence and attenuation in Theiler murine encephalomyelitis virus.

Full-length cDNA clones of two Theiler murine encephalomyelitis virus (TMEV) strains, one highly virulent and the other less virulent, were constructed in the bacterial plasmid pGEMR-3. Transfection of BHK-21 cells with RNA transcribed from these cDNAs yielded progeny viruses with the exact in vitro growth phenotype and mouse neurovirulence pattern of the respective parental virus strains. RNA transcripts derived from recombinant chimeras constructed by exchanging corresponding genomic regions [5' noncoding, leader/P1 (L/P1), P2, P3, and 3' noncoding] between the parental cDNAs were infectious and enabled analysis of the growth characteristics in vitro and mouse neurovirulence of the chimeras. A correlation was found between plaque size and temperature sensitivity and the origin of the L/P1 region. Neurovirulence mapped primarily to the L/P1 region encoding the leader and coat proteins. Depending on parental origin, the 5' noncoding region either influenced virus attenuation or augmented virulence.

Animals↗
PubMed1985

Cloned DNA probes regionally mapped to human chromosome 21 and their use in determining the origin of nondisjunction.

A number of unique sequence recombinant DNA clones were isolated from a recombinant DNA library constructed from DNA enriched for chromosome 21 by flow sorting. Of these, five were mapped to chromosome 21 using a somatic cell hybrid. Regional mapping of these probes and of a probe previously assigned to chromosome 21, was carried out with the aid of chromosome 21 rearrangements using both chromosome sorting and a somatic cell hybrid. Three probes were shown to be located on either side of the breakpoint 21q21.2. Two of the probes were shown to identify restriction fragment length polymorphisms (RFLPs) with high rare-allele frequencies (0.46 and 0.43). A Bgl II RFLP revealed the parental origin of non-disjunction in three of ten families with Down's syndrome.

Cell Line↗
PubMed1979

The transforming gene of Moloney murine sarcoma virus.

A cleavage map of the Moloney murine sarcoma viral DNA was constructed and compared with that of a spontaneously occurring deletion mutant. By restriction enzyme analysis, it was shown that a region encompassing over 40% of the viral information was not essential for transformation or rescue of the deletion mutant. The transforming region was further localised by analysis of the transforming activity in tissue culture of isolated restriction fragments of linear duoble-stranded sarcoma viral DNA. In each case, DNA fragments that retained transforming activity preserved the cell-derived insertion sequences of the viral genome. Moreover, such transformants invariably expressed RNA specific to this region. By these two approaches, it was possible to demonstrate that the transforming region of the viral genome begins very near or within the cell-derived insertion sequences. Thus, the transforming gene of this mammalian sarcoma virus originates from within the mouse cell genome.

Cell Transformation, Viral↗
PubMed1992

Patterns of cell motility in the organizer and dorsal mesoderm of Xenopus laevis.

In a companion paper (Shih, J. and Keller, R. (1992) Development 116, 901-914), we described a sequence of cell behaviors, called mediolateral intercalation behavior (MIB), that produces mediolateral cell intercalation, the process that drives convergence and extension of the axial and paraxial mesoderm of Xenopus. In this paper, we describe the pattern of expression of MIB in the mesoderm during gastrulation, using video image processing and recording of cell behavior in 'shaved', open-faced explants of the marginal zone. At midgastrula stage (10.5), MIB begins at two dorsolateral sites in the prospective anterior mesoderm and progresses medially along two arcs that lengthen toward and meet at the midline to form a single arc of cells expressing MIB, called the vegetal alignment zone (VgAZ). The notochordal-somitic mesodermal boundary forms within the VgAZ at stage 11, and then progresses animally and laterally, along the prospective anterior-posterior axis, eventually bounding a trapezoidal area the shape of the fate-mapped notochord. Meanwhile, from its origin in the VgAZ, MIB spreads in the prospective posterior direction along the lateral boundaries of both the notochordal and somitic mesoderm. From there it spreads medially in both tissues. Subsequently, vacuolation of notochord cells, and segmentation and expression of a somite-specific marker repeat the progression of mediolateral intercalation behavior. Thus cells in the posterior, medial regions of the notochordal and the somitic territories are the last to express mediolateral intercalation behavior and subsequent tissue differentiations. In explants that do not converge, these cells neither express mediolateral intercalation behavior nor differentiate. These facts suggest that progressions of MIB in the anterior-posterior and lateral-medial directions may be organized by signals emanating from the lateral somitic and notochordal boundaries. These signals may have limited range and may be dependent on convergence, driven by mediolateral cell intercalation, to bring cells within their range. In the embryo, the posterior progression of MIB results in arcs of convergence, anchored in the vegetal endoderm at each end, acting on the inside of the blastoporal lip to produce involution of the IMZ.

Animals↗
PubMed2002

The acidic activation domain of the baculovirus transactivator IE1 contains a virus-specific domain essential for DNA replication.

IE1 is a potent transcriptional transactivator of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and has been shown to be essential for viral DNA replication. IE1 contains an acidic activation domain (AAD) at the N terminus that is essential for transcriptional transactivation, but its role in viral DNA replication is unknown. In this study the role of the IE1 AAD in DNA replication is investigated. We have determined that deletion of the AAD eliminates the ability of IE1 to support DNA replication, showing that the AAD is essential for DNA replication as well as transcriptional transactivation. Replacement of the AAD with the archetype domain from herpesvirus VP16 and the evolutionarily related domain from Autographa californica MNPV (AcMNPV) IE1 produces chimeric proteins that are potent transactivators. Surprisingly, however, these chimeric proteins were unable to support DNA replication, indicating that there is a host- or virus-specific replication subdomain in the AAD that was not functionally replaced by the VP16 or AcMNPV AAD. Using N- and C-terminal deletion mutants, the region of the AAD that was essential for DNA replication was mapped to amino acids 1 to 65. AAD deletion mutants also showed that an IE1 that is functional for transcriptional transactivation is not required for viral DNA replication. The IE1 AAD therefore contains an essential replication domain that is separable from the transcriptional activation domains. Our results suggest that IE1 specifically interacts with a component of the viral replication complex, supporting the view that it acts as a nucleating factor by binding to the viral replication origins.

Acids↗
PubMed2000

Genetic characterization of a human skin carcinoma progression model: from primary tumor to metastasis.

The type and number of genetic aberrations required for a fully malignant tumor are still unclear. This study describes the genetic analysis of a series of skin squamous cell carcinomas, representing the primary tumor, two recurrences, and a metastatic lesion from a single patient and cell lines established therefrom (MET-1 to MET-4). Comparative genomic hybridization demonstrated that: (i) most of the gains and losses were common for tumors and cell lines and affected chromosomes 3 (3p loss, 3q gain), 5 (5p gain, 5q loss), 7 (7p gain), 8 (8p loss, 8q gain), 11 (11q gain), and 17 (17p loss), and (ii) only one aberration was present in a tumor but not in the cell line (10 loss in tumor 4); and only few aberrations were cell line specific. From these, 10p loss and 17q gain were shared by all lines and tumor 4, suggesting that they were already present in all tumors, although in only a subpopulation of cells, whereas 20q gain (shared by all lines), 4q loss (MET-2), and 18p gain/18q loss (MET-3) seem to be culture derived. In agreement, multiplex fluorescence in situ hybridization demonstrated a set of common translocations for all lines thereby further confirming their common origin. In addition, each cell line, exhibited one or more individual translocation chromosomes, which suggested that MET-1 was a precursor of MET-4, whereas MET-2 and MET-3 developed in parallel. Whereas MET-1 to MET-3 were hypodiploid or hyperdiploid, MET-4 was characterized by polyploidization, a set of specific aberrations (t(3;7), t(X;2), i(10q)), and increased heterogeneity (varying translocations in individual metaphases). Using sequencing and expression studies, cells from all lines were wild type for p53, did not exhibit mutations in any of the ras genes (Harvey, Kirsten, or N-ras), and expressed wild-type fragile histidine triad gene (FHIT; mapped to 3p14.2, a locus underrepresented in all cells) transcripts. Thus, with the MET cell lines we present an in vivo skin carcinoma progression model that was genetically well defined, and which, despite originating from a sun-exposed site, is wild type for p53.

Carcinoma, Squamous Cell↗
PubMed2000

Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei.

As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G(1)-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-beta) and (beta') that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-(beta/(beta)' appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G(1)-phase of the cell division cycle, and their appearance did not depend on the presence of Xenopus Orc proteins. Therefore, hamster pre-replication complexes appear to be assembled at specific chromosomal sites during G(1)-phase. Third, selective activation of ori-(beta) in late G(1)-nuclei depended on the ratio of Xenopus egg extract to nuclei, revealing that epigenetic parameters such as the ratio of initiation factors to DNA substrate could determine the number of origins activated.

Animals↗
PubMed1998

Concurrent replication and methylation at mammalian origins of replication.

Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-myc origin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.

Animals↗
PubMedDate not supplied

Transduction of the human insulin gene via retroviral vectors fails to yield spliced transcripts.

Previous reports on retroviral vectors have shown them to be useful for transferring genes into animal cells. Genes placed under the retroviral long terminal repeat (LTR) act as dominant loci in recipient cells and can permanently alter their genotype and phenotype. Previous reports have shown that recombinant retroviruses containing genomic sequences with both introns and exons display a high frequency of deletion and abnormal kinetics of splicing of intron sequences. We report here our findings when a 2.9-kb fragment containing the entire human insulin gene was inserted into a Moloney-derived retroviral vector in the same transcriptional orientation as the LTRs. RNA transcripts synthesized in cells containing such constructs remain unspliced, as assessed by both RNA blot analysis and S1 mapping. Ten subclones derived following viral passage showed no splicing, and failure to splice was observed regardless of cell type or species of origin, or number of viral passages. Thus, genomic sequences containing introns when situated within the context of a retroviral transcript do not in all instances exhibit expected kinetics of splicing.

Animals↗