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Biomedical subjects

W Ansorge

Publications and source records attributed to W Ansorge.

At least 19 recordsLinked to original sources
PubMed1992

Human casein kinase II subunit alpha: sequence of a processed (pseudo)gene and its localization on chromosome 11.

A human 4.3 kb genomic DNA fragment, containing the information of a processed (pseudo)gene of casein kinase II subunit alpha (CKII alpha) was isolated and sequenced. The genomic CKII alpha sequence is 99% homologous to the CKII alpha cDNA, carries several nucleotide exchanges, a poly(A) stretch at its 3' end and is flanked at both ends by a 16 bp repeat. It has a promoter-like region including two TATA boxes and a CAAT box. Although translation of transcripts would be terminated by a stop codon after two third of the coding region, the resulting protein would still contain the catalytic domains. However, so far Northern blots with a 3' specific probe were negative. The 4.3 kb genomic fragment containing the processed CKII alpha (pseudo)gene was mapped by in situ hybridization to chromosome 11p15.

Base Sequence↗
PubMed1992

Cyclin A is required at two points in the human cell cycle.

Cyclins play a fundamental role in regulating cell cycle events in all eukaryotic cells. The human cyclin A gene was identified as the site of integration of hepatitis B virus in a hepatocarcinoma cell line; in addition, cyclin A is associated with the E2F transcription factor in a complex which is dissociated by the E1A oncogene product. Such findings suggest that cyclin A is a target for oncogenic signals. We have now found that DNA synthesis and entry into mitosis are inhibited in human cells microinjected with anti-cyclin A antibodies at distinct times. Cyclin A binds both cdk2 and cdc2, giving two distinct cyclin A kinase activities, one appearing in S phase, the other in G2. These results suggest that cyclin A defines novel control points of the human cell cycle.

Adenovirus Early Proteins↗
PubMed1991

Structure of the gene encoding human casein kinase II subunit beta.

Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with numerous key functions in cell metabolism and growth. The human CKII has a tetrameric structure; two catalytic subunits (alpha and alpha') form the holoenzyme together with two presumably regulatory subunits (beta). The gene encoding CKII subunit beta was isolated from human genomic DNA and analyzed for its primary structure using exclusively nonradioactive procedures. The gene was found to span 4.2 kilobase pairs and to be composed of seven exons. Exon sizes range from 76 (exon 5) to 329 base pairs (bp) (exon 1), intron sizes from 145 (intron V) to 965 bp (intron II). All exon-intron junctional sequences conform to the canonical GT-AG rule. Primer extension analysis determined three transcription initiation sites, at 951, 919, and (minor) 840 bp upstream of the translation start site. The translation start is located early in the second exon; exon 1 is untranslated. The 3'-cleavage/polyadenylation signal sequence (AA-TAAA) is in the last exon at position 4173 bp relative to the first transcription initiation site. The coding sequence for CKII beta comprises 648 nucleotides identical to the published CKII beta-cDNA sequence (Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem. 183, 227-233). The upstream promoter region of the CKII beta gene contains multiple potential gene regulatory sequence elements, noticeable DNA structures, and the characteristics of a housekeeping gene (more than one transcription initiation site, lack of a TATA-box, presence of a CpG island, occurrence of multiple GC boxes and of nonstandard positioned CCAAT boxes). The CKII beta gene promoter shares common features with that of mammalian protein kinases and is closely related to the regulatory subunit gene promoter of cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1991

In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.

The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo.

Animals↗
PubMed1991

The cytoplasmic domain of alphavirus E2 glycoprotein contains a short linear recognition signal required for viral budding.

Intracellular alphavirus nucleocapsids express a binding site for the cytoplasmic domain of the viral E2 spike glycoprotein. This binding site is recognized by the anti-idiotype monoclonal antibody, F13. The monoclonal anti-anti-idiotype antibody, raised against F13 and designated 3G10, recognizes the carboxy-terminal eight residues of the E2 cytoplasmic domain in Semliki Forest virus (SFV), identifying this as the signal for nucleocapsid interaction. F13 binding to cells infected with SFV or a second alphavirus, Sindbis virus, is inhibited by a synthetic peptide corresponding to the entire 31 residue cytoplasmic domain (E2c), and also by a synthetic peptide corresponding to the eight residue epitope recognized by 3G10. Both E2c and the eight residue peptide inhibited viral budding in microinjection experiments and when conjugated to colloidal gold are bound specifically to nucleocapsids in infected cells. These results identify a short linear signal in the E2 cytoplasmic domain required for the interaction with nucleocapsids which leads to budding of at least two alphaviruses from infected cells.

Amino Acid Sequence↗
PubMed1991

Cell growth stimulation by EGF: inhibition through antisense-oligodeoxynucleotides demonstrates important role of casein kinase II.

Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active subunits (alpha and/or alpha') and two presumably regulatory subunits (beta). CKII has numerous cellular functions including a possible role in mitogenic signaling. To address this question, growth-arrested primary human fibroblasts (IMR-90) were exposed prior growth stimulation by epidermal growth factor (EGF) to oligodeoxynucleotides complementary to the translation start region of mRNAs coding for CKII alpha and beta subunits. A significant inhibition of growth stimulation (up to 60%) was observed with both antisense-alpha and antisense-beta. The inhibition was reversible, became decreased with mutated antisense-oligodeoxynucleotides, and neutralized by simultaneous presence of respective sense-oligodeoxynucleotides. The expected down-regulation of CKII protein due to hybrid formation of antisense-oligodeoxynucleotides with target mRNAs was investigated by determination of the intracellular protein level of CKII beta-subunit by immunofluorescence and quantitative image analysis. The protein was revealed to be localized predominantly in the nucleus and to become significantly decreased due to antisense-beta treatment of cells. The maximum decrease coincided with the early phase (first several hours) of growth stimulation by EGF when antisense-beta incubation was started 6-2 h before growth stimulation, the period within which application of antisense-alpha and antisense-beta caused the maximum of inhibition of growth stimulation. Thus CKII obviously plays, with both subunit alpha and subunit beta, an important role in the early phase of mitogenic stimulation.

Base Sequence↗
PubMed1991

Single cell assay with an automated capillary microinjection system.

An automated capillary microinjection system with computer controlled positioning of the cells and of the capillary, and its applications and advantages are described. About 1500 injections are possible in one hour, with high reproducibility. In cytoplasmic and nuclear injections more than 90% and 85% of the cells are successfully injected. Using FITC-Dextran at a concentration of 0.5% as a fluorescently labelled coinjection marker, 99% of the cells can be retrieved in culture medium even 48 hours after injection. The coordinates of the cells are stored in the computer and accuracy in statistical evaluation of experiments is improved in comparison to the manual techniques. Methods for preparation and handling of glass capillaries were developed resulting in reproducible form and significantly reduced clogging rate. The improved characteristics offered by this system are demonstrated in studies leading to the confirmation of existence of mRNA(s) inhibiting cell proliferation. Functional screening by cell injections of cDNA libraries and of size fractionated mRNA molecules can be performed efficiently with the automated microinjection system.

Animals↗
PubMed1990

Cell proliferation inhibited by MyoD1 independently of myogenic differentiation.

Cell growth and differentiation are usually mutually exclusive. Transformation of myoblasts by retroviruses containing the myc oncogene inhibits differentiation, preventing cells from withdrawing from the cell cycle. If cell-cycle withdrawal is a prerequisite for myoblast differentiation, it is probably an early event in terminal cell differentiation, but this has not yet been established. MyoD1 regulates myogenesis. It is expressed only in skeletal muscle, but can convert other cells to muscle cells. The MyoD1 protein, a nuclear phosphoprotein in part similar to the myc family of proteins, is a DNA-binding protein binding to the enhancer sequences of the muscle-specific creatine phosphokinase gene. Thus, introduction of MyoD1 into cells provides a simple approach to study the effect of induction of differentiation on cell growth. In cultured NIH 3T3 cells, inhibition of cell proliferation occurs within 18 hours, and expression of myosin starts after 72 hours. Furthermore, injection of MyoD1 into quiescent NIH 3T3 cells inhibit cell proliferation independently of induction of differentiation. Deletion of the myc-like domain in the MyoD1 gene eliminates the inhibition of DNA synthesis, but substitution of the basic domain with the analogous domain from the E12 transcription factor inhibits growth yet fails to induce differentiation. Inhibition of DNA synthesis, therefore, seems to be controlled separately from myogenic differentiation.

Animals↗
PubMed1990

Automated DNA sequencing of the human HPRT locus.

The complete sequence of 57 kb of the human HPRT locus has been determined using automated fluorescent DNA sequencing. The strategy employed increasingly directed sequencing methods: A randomly generated M13 library was sequenced to generate contiguous overlapping sets of sequences (contigs). M13 clones at the ends of these contigs were further sequenced using M13 (universal and reverse) and custom oligonucleotide primers to order the contigs and to complete the sequencing project. The human HPRT sequence includes 1676 bp 5' and 15,238 bp 3' to exons 1 and 9, respectively. The sequence contains 49 representatives of the Alu repeat, along with several other types of repetitive sequences. The Alu sequences exhibit a biased orientation, with those sequences in the first half of the locus oriented in the minus direction relative to transcription of the gene (3'----5' = 77%, P less than 0.005) and those sequences in the latter half of the locus oriented randomly (5'----3' = 67%, P less than 0.5). The development and performance of the sequencing strategy and the features of the human HPRT gene are presented.

Amino Acid Sequence↗
PubMed1990

Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells.

MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E.H.K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M.H. Bré, G. Griffiths, and E. Karsenti. 1990. 110:1123-1136). In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced double-immunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4 microns/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a t1/2 of less than 30 min, whereas in confluent monolayers, a large population of microtubules had a t1/2 of greater than 2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.

Animals↗
PubMed1990

Automated sequencing of fluorescently labelled DNA by chemical degradation.

A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.

Automation↗