Microtubule-mediated peroxisomal saltations.
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Animals↗
Biomedical subjects
W Ansorge
Publications and source records attributed to W Ansorge.
Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis.
The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum-deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E- and cyclin D1-dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin-dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum-starved cells. Further, in isoleucine-deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.
3T3 Cells↗
Sequencing and analysis of 51 kb on the right arm of chromosome XV from Saccharomyces cerevisiae reveals 30 open reading frames.
We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.
Base Sequence↗
"Doublex" fluorescent DNA sequencing: two independent sequences obtained simultaneously in one reaction with internal labeling and unlabeled primers.
The novel "doublex" DNA sequencing technique that makes it possible to obtain simultaneously two independent sequences from one sequencing reaction with the use of unlabeled primers and internal labeling is described. The different sequencing products are labeled in parallel with fluorescein-15-dATP and Texas red-5-dCTP present in the same tube. The characteristics of T7 DNA polymerase are exploited to ensure that only either of the labeled dNTPs is incorporated into the corresponding sequencing products. Specificity of labeling is ensured by the selection of primers. One of the unlabeled primers is chosen to be followed by an "A," the other by a "C" to be incorporated immediately downstream from the primer binding site. The doublex sequencing technique is applicable to the simultaneous sequencing of either the same DNA template/strand or a mixture of different templates. Combinations of unlabeled and labeled primers in the same sequencing reaction are also possible. The two sequences can be determined in parallel and on-line in the same lanes of a gel with a novel automated DNA sequencer, which was previously described for use with labeled primers.
Autoanalysis↗
Microtubule-based peroxisome movement.
The association of peroxisomes with cytoskeletal structures was investigated both by electron microscopy and by kinetic analysis of peroxisome movement. The morphological studies indicated distinct interactions of peroxisomes with microtubules and frequently revealed multiple contact sites. The kinetic approach utilised microinjection and import of fluorescein-labeled luciferase in order to mark and track peroxisomes in vivo. Peroxisomal motility was analysed by time-lapse imaging and fluorescence microscopy. According to their movement peroxisomes were classified into two groups. Group 1 peroxisomes comprising the majority of organelles at 37 degrees C moved slowly with an average velocity of 0.024 +/- 0.012 micron/second whereas the movement of group 2 peroxisomes, 10-15% of the total population, was saltatory exhibiting an average velocity of 0.26 +/- 0.17 micron/second with maximal values of more than 2 microns/second. Saltations were completely abolished by the microtubule-depolymerising drug nocodazole and were slightly reduced by about 25% by cytochalasin D which disrupts the actin microfilament system. Double fluorescence labeling of both peroxisomes and microtubules revealed peroxisome saltations linked to distinct microtubule tracks. Cellular depletion of endogenous levels of NTPs as well as the use of 5'-adenylylimidodiphosphate, a nonhydrolysable ATP analog, applied to a permeabilised cell preparation both completely blocked peroxisomal movement. These data suggest an ATPase dependent, microtubule-based mechanism of peroxisome movement. Both the intact and the permeabilised cell system presented in this paper for the first time allow kinetic measurements on peroxisomal motility and thus will be extremely helpful in the biochemical characterisation of the motor proteins involved.
Actins↗
Reducing "double sequences" in automated DNA sequencing with T7 DNA polymerase and internal labeling.
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Automation↗
Sequencing 39,350 bp of Saccharomyces cerevisiae chromosome XII utilizing ordered shotgun libraries.
A 39,350 bp cosmid containing DNA of Saccharomyces cerevisiae chromosome XII was sequenced by making use of ordered sub-clones of 1 kb insert-length selected from a physical clone map. In a first analysis, 96 clones were sequenced from both ends (10 gels) with two standard sequencing primers covering 91% of the total sequence (49% double-stranded). After selection of another eight clones six gaps of a total of 1.8 kb and several single-stranded stretches remained. These gaps were closed by 86 primer walks leading to an overall redundancy of 4.4 per base and a total of 292 sequencing reactions. The number of walking primers can be reduced significantly by more uniform clone lengths and longer sequencing reads, thus, the total amount of sequencing reactions can approach the minimum value achieved with primer walking strategies, with only very few walking primers needed for gap closure.
Chromosomes, Fungal↗
Efficient low redundancy large-scale DNA sequencing at EMBL.
An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.
Animals↗
Simultaneous on-line DNA sequencing on both strands with two fluorescent dyes.
We describe an automated DNA-sequencing technique which allows both the simultaneous sequencing from the two strands of double-stranded templates and the subsequent detection of the sequencing products online and in parallel. The technique is based on hardware technology also used in the ALF DNA sequencer (Pharmacia, Uppsala). A helium-neon laser was mounted into the sequencing device additionally to the standard argon laser. Two different primers, labeled with either fluorescein or Texas red, are used in a single sequencing reaction resulting in an output of two sequences. Both sequencing products are then analyzed on-line in the same lanes of a gel. This technique is especially useful for the complete sequencing of DNA fragments up to 1 kb. High accuracy sequencing of PCR products in double-stranded form can now be accomplished in only one sequencing reaction.
Fluorescent Dyes↗
Nucleotide sequence and analysis of the centromeric region of yeast chromosome IX.
We have determined the nucleotide sequence of a cosmid (pIX338) containing the centromere region of yeast (Saccharomyces cerevisiae) chromosome IX. The complete nucleotide sequence of 33.8 kb was obtained by using an efficient directed sequencing strategy in combination with automated DNA sequencing on the A.L.F. DNA sequencer. Sequence analysis revealed the presence of 17 open reading frames (ORFs), four of them previously known yeast genes (sly12, pan1, sts1 and prl1), a tRNA gene and the centromere motif. Exhaustive database searches detected sequence homologues of known function for as many as 14 of the 17 ORFs. These include a mammalian tyrosine kinase substrate; the Escherichia coli cell cycle protein MinD; the human inositol polyphosphate-5-phosphatase (gene OCRL) involved in Lowe's syndrome, a developmental disorder; and helicases, for which the new yeast member defines a distinct DEAD/H-box subfamily. A surprisingly large fraction of the ORFs (at least six out of 17) in the centromeric region are apparently involved in RNA or DNA binding.
Adenosine Triphosphatases↗
Five new microsatellite polymorphisms at the q21 region of human chromosome 21.
Five clones, containing polymorphic CA-repeat sequences, have been isolated from a specific human chromosome 21 phage library and have been localised to band q21 of chromosome 21 using a somatic cell hybrid panel. These highly repetitive sequences (D21S1263, D21S1264, D21S1415, D21S1417 and D21S1420) have been characterised in the CEPH reference parents and have heterozygosities ranging from 0.30 to 0.81 and an average polymorphism information content (PIC) of 0.62. The relative order of these markers, based on the somatic cell hybrid panel, is cen-D21S1417, D21S1420-D21S1263, D21S1415-D21S1264-tel. The most polymorphic marker (D21S1264) has been included in the chromosome 21 genetic map. They have also been localised in the CEPH/Généthon YAC panel, providing a refined localisation of these polymorphic sequences. These five CA-repeat markers should provide a better characterisation of the q21 region of chromosome 21.
Animals↗
Identification of two highly polymorphic CA-repeats (D21S1224 and D21S1261) on human chromosome 21q22.3.
Two highly polymorphic CA-repeat microsatellites (D21S1224 and D21S1261) are reported. The clones containing these CA-repeats (ABM-C60 and ABM-C29) have been isolated from a human chromosome-21-specific library (LA21NS01) and have been localised to the q22.3 band using a specific chromosome 21 somatic cell hybrid panel. Both polymorphisms showed heterozygosities of 0.83 in the unrelated reference parents from the Centre d'Etude du Polymorphisme Humain. These new markers should improve the map of the 21q22.3 region, which is believed to contain a large number of genes.
Base Sequence↗
Primer design for automated DNA sequencing utilizing T7 DNA polymerase and internal labeling with fluorescein-15-dATP.
We have identified additional criteria for the walking primer design that improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and T7 DNA polymerase. These criteria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used. This led us to evaluate primer performance relative to (i) the template sequence immediately downstream of the primer binding site and (ii) the primer sequence itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence. The closer to the primer that the first 'A' is incorporated, the stronger the peak intensities are. An additional feature of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the primer by the 3'-->5' exonuclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity.
Binding Sites↗
Complete DNA sequence of yeast chromosome XI.
The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.
Base Sequence↗
Casein kinase II is required for transition of G0/G1, early G1, and G1/S phases of the cell cycle.
Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with many cellular functions, including participation in mitogenic signaling by cytoplasmic nuclear translocation (Lorenz, P., Pepperkok, R., Ansorge, W., and Pyerin, W. (1993) J. Biol. Chem. 268, 2733-2739). To examine whether cell compartment-specific availability is a requirement for CKII function during cell cycle progression, antibodies against CKII beta, the regulatory subunit of CKII, were microinjected into the cytoplasm or the nucleus of G0-synchronized human primary fibroblasts (IMR-90) at the time of mitogenic stimulation or at various intervals thereafter. Significant inhibition of the stimulation was obtained with both cytoplasmic and nuclear injections. The inhibition was reversible, was not observed with control antibodies, and was abolished by co-injection of purified CKII holoenzyme. The inhibition differed, however, in extent, duration, and cell cycle phase between cytoplasmic and nuclear injections. After cytoplasmic injection, inhibition reached 45-50% and was effective at two intervals within the first 2 h and at 12-16 h post-stimulation, i.e. at G0/G1 phase transition and at the G1/S phase boundary of the cell cycle. After injection into the nucleus, the inhibition was considerably stronger, reaching 80-85%, and was effective for the first 6 h post-stimulation, i.e. for the transition of G0/G1 phase and the adjoining first part of G1 phase. Cytoplasmic or nuclear injections within S phase affected neither DNA synthesis nor cell division. The data suggest that cell cycle transition from G0 to S phase requires the presence of a certain functional level of CKII at defined times and at defined cellular locations as follows: for transition of G0/G1 at both the nucleus and the cytoplasm, for transition of early G1 at the nucleus, and for transition of G1/S at the cytoplasm.
Animals↗
The human gene (CSNK2A1) coding for the casein kinase II subunit alpha is located on chromosome 20 and contains tandemly arranged Alu repeats.
We have isolated and characterized a 18.9-kb genomic clone representing a central portion of the human casein kinase II (CKII) subunit alpha gene (CSNK2A1). Using the whole clone as a probe, the gene was localized on chromosome 20p13. The clone contains eight exons whose sequences comprise bases 102 to 824 of the coding region of the human CKII alpha. The exon/intron splice junctions conform to the gt/ag rule. Three of the nine introns are located at positions corresponding to those in the CKII alpha gene of the nematode Caenorhabditis elegans. The introns contain eight complete and eight incomplete Alu repeats. Some of the Alu sequences are arranged in tandems of two or three, which seem to originate from insertions of younger Alu sequences into the poly(A) region of previously integrated Alu sequences, as indicated by flanking direct repeats.
Animals↗
Three CA/GT repeat polymorphisms from loci D21S414 and D21S1234 on human chromosome 21.
We report three new polymorphic CA repeat microsatellites (ABM-21, ABM-C37A, and ABM-C37B) in two different loci (D21S414 and D21S1234), located in bands q21 and q11.2 of human chromosome 21 (HC21) and that were isolated from a HC21 phage library (LA21NS01). Heterozygosities for ABM-21, ABM-C37A, and ABM-C37B were 0.74, 0.50, and 0.67 respectively. These three CA repeat markers should be useful in the construction of a high resolution genetic map of this region of HC21.
Base Sequence↗
A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division.
Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccharomyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2. Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bromodeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.
3T3 Cells↗