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M H Linskens

Publications and source records attributed to M H Linskens.

At least 19 recordsLinked to original sources
PubMed2001

Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts.

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.

Blotting, Northern↗
PubMed2000

Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells.

We established a tetracycline-regulated gene expression system that tightly controls expression of genes in Dictyostelium discoideum. The control elements are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator protein (tTA(s)(*)). The second component is an extrachromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promoter contains a tetracycline-responsive element, which is the binding site for tTA(s)(*). Tetracycline prevents tTA(s)(*) from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTA(s)(*) binds to its target sequence and strongly induces gene expression. The kinetics of activation and repression of the system were monitored using luciferase as a reporter. The results reveal efficient inhibition of gene expression by low concentrations of tetracycline and an induction of gene expression by several orders of magnitude within a few hours after removal of tetracycline. Green fluorescent protein (GFP) provided information about the effects of modulation of the tetracycline concentration on gene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population express the reporter gene.

Actins↗
PubMed2000

Optimizing heterologous expression in dictyostelium: importance of 5' codon adaptation.

Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-beta gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5-17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5'-sequence of the hCG gene to the Dictyostelium 'Kozak'-like sequence increased expression levels approximately 1.5-fold. Thus, using both codon optimization and 'Kozak' adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG.

Amino Acid Sequence↗
PubMed2000

Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation.

Worldwideattention is presently focused on proliferating populations of neural precursor cells as an in vitro source of tissue for neural transplantation and brain repair. However, successful neuroreconstruction is contingent upon their capacity to integrate within the host CNS and the absence of tumorigenesis. Here we show that human neural precursor cells express very low levels of telomerase at early passages (less than 20 population doublings), but that this decreases to undetectable levels at later passages. In contrast, rodent neural precursors express high levels of telomerase at both early and late passages. The human neural precursors also have telomeres (approximately 12 kbp) significantly shorter than those of their rodent counterparts (approximately 40 kbp). Human neural precursors were then expanded 100-fold prior to intrastriatal transplantation in a rodent model of Parkinson's disease. To establish the effects of implanted cell number on survival and integration, precursors were transplanted at either 200,000, 1 million, or 2 million cells per animal. Interestingly, the smaller transplants were more likely to extend neuronal fibers and less likely to provoke immune rejection than the largest transplants in this xenograft model. Cellular proliferation continued immediately post-transplantation, but by 20 weeks there were virtually no dividing cells within any of the grafts. In contrast, fiber outgrowth increased gradually over time and often occupied the entire striatum at 20 weeks postgrafting. Transient expression of tyrosine hydroxylase-positive cells within the grafts was found in some animals, but this was not sustained at 20 weeks and had no functional effects. For Parkinson's disease, the principal aim now is to induce the dopaminergic phenotype in these cells prior to transplantation. However, given the relative safety profile for these human cells and their capacity to extend fibers into the adult rodent brain, they may provide the ideal basis for the repair of other lesions of the CNS where extensive axonal outgrowth is required.

Animals↗
PubMed1999

Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum.

The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism. Furthermore, both follicle stimulation hormone and choriogonadotropin produced by D. dictyostelium bind to their human receptors and elicit a biological response comparable to the wild-type hormones. We also show that structure-function analysis using random mutagenesis and screening of recombinant glycoprotein hormones is feasible. Thus, expression of gonadotropins in D. dictyostelium opens the way to the engineering of potential new therapeutic analogues.

Animals↗
PubMed1998

Identification and cloning of a sequence homologue of dopamine beta-hydroxylase.

We have identified and cloned a cDNA encoding a new member of the monooxygenase family of enzymes. This novel enzyme, which we call MOX (monooxygenase X; unknown substrate) is a clear sequence homologue of the enzyme dopamine beta-hydroxylase (DBH). MOX maintains many of the structural features of DBH, as evidenced by the retention of most of the disulfide linkages and all of the peptidyl ligands to the active site copper atoms. Unlike DBH, MOX lacks a signal peptide sequence and therefore is unlikely to be a secreted molecule. The steady-state mRNA levels of MOX are highest in the kidney, lung, and adrenal gland, indicating that the tissue distribution of MOX is broader than that of DBH. Antisera raised to a fusion protein of MOX identifies a single band of the expected mobility by Western blot analysis. MOX mRNA levels are elevated in some fibroblast cell strains at replicative senescence, through this regulation is not apparent in all primary cell strains. The gene for MOX resides on the q arm of chromosome 6 and the corresponding mouse homolog has been identified.

Amino Acid Sequence↗
PubMed1998

Expression of a bioactive, single-chain choriogonadotropin in Dictyostelium discoideum.

Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptor-binding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.

Amino Acid Sequence↗
PubMed1995

Cataloging altered gene expression in young and senescent cells using enhanced differential display.

Recently, a novel PCR-based technique, differential display (DD), has facilitated the study of differentially expressed genes at the mRNA level. We report here an improved version of DD, which we call Enhanced Differential Display (EDD). We have modified the technique to enhance reproducibility and to facilitate sequencing and cloning. Using EDD, we have generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts (HDF). From 168 genetags that were identified initially, 84 could be sequenced directly from PCR amplified bands. These sequences represent 27 known genes and 37 novel genes. By Northern blot analysis we have confirmed the differential expression of a total of 23 genes (12 known, 11 novel), while 19 (seven known, 12 novel) did not show differential expression. Several of the known genes were previously observed by others to be differentially expressed between young and senescent fibroblasts, thereby validating the technique.

Base Sequence↗
PubMed1995

p34Cdc28-mediated control of Cln3 cyclin degradation.

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.

Amino Acid Sequence↗
PubMed1990

Ambiguities in results obtained with 2D gel replicon mapping techniques.

Recently, two 2-dimensional (2D) gel techniques, termed neutral/neutral and neutral/alkaline, have been developed and employed to map replication origins in eukaryotic plasmids and chromosomal DNA (1-11). The neutral/neutral technique, which requires less DNA for analysis, has been preferentially used in recent studies. We show here that the signal predicted for an origin is not detected using the neutral/neutral technique if the origin is located near the end of the analyzed restriction fragment. We also demonstrate that analysis of the same batch of DNA by the two different mapping techniques can generate apparently contradictory results: in some situations where neutral/alkaline 2D analysis indicates that a certain origin is always used, neutral/neutral 2D analysis suggests that the origin is not always used. Several possible explanations for this type of disagreement between the two techniques are discussed, and we conclude that it is important to use both techniques in combination in order to minimize possible misinterpretations.

DNA, Fungal↗
PubMed1989

HL-R5 and HL-D4: two differentiation resistant HL-60 variants.

A wide range of different agents are capable of inducing the onset of HL-60 differentiation along the myeloid lineage. The diversity of these agents has made the analysis of the molecular mechanisms involved in the regulation of the onset and progress of terminal differentiation in these cells difficult. We have adapted the standard soft-agar cloning procedure to enable the single-step selection of clonal populations of spontaneously arising differentiation resistant HL-60 variants. This simple procedure which obviates the need for mutagenesis, long-term exposure to inducing agents or complex manipulations, optimises the chance of obtaining variants with a single lesion blocking the onset of differentiation. The analysis of two variants, HL-R5 and HL-D4, selected by this procedure for resistance to retinoic acid and DMSO, respectively, suggests the existence of different pathways used by the two agents which converge before the onset of terminal differentiation.

Cell Differentiation↗
PubMed1989

DMSO induced modulation of c-myc steady-state RNA levels in a variety of different cell lines.

The nuclear proto-oncogene c-myc is believed to play a regulatory role in eukaryotic cellular growth and differentiation. Several studies have demonstrated rapid down-regulation of the steady-state levels of c-myc RNA during DMSO induced differentiation of the human 'promyelocytic' cell line, HL-60. However, little is known about the effect of DMSO on c-myc regulation in cells which are not induced to differentiate by DMSO. We have examined the effect of DMSO on c-myc RNA levels in a number of cell lines with different lesions in the c-myc gene, including those which do not differentiate in response to DMSO treatment. Here we demonstrate that DMSO induces a rapid, but transient, reduction in the steady-state level of c-myc RNA in human 'erythroid' K562 cells, Burkitt lymphoma lines Daudi and Raji, human T cell lymphoblastoid line CEM and mouse lymphoma line L1210. However, DMSO treatment does not produce a similar effect in the human colon adenocarcinoma cell line COLO 320 or HeLa epithelial cervical carcinoma cells. These observations demonstrate that the DMSO induced modulation of c-myc RNA levels is a more common phenomenon than previously recognised, and is not necessarily correlated with either the induction of cellular differentiation or growth arrest.

Cell Differentiation↗
PubMed1988

Organization of replication of ribosomal DNA in Saccharomyces cerevisiae.

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

Chromosome Mapping↗
PubMed1987

Comparative analysis of DNA loop length in nontransformed and transformed hamster cells.

The size of the looped, matrix-attached DNA domains was estimated in both nontransformed and transformed hamster cells. In BHK cells it was observed that in interphase as well as during mitosis, transformed cells, on the average, have shorter loops than nontransformed cells. In CHO cells, however, no alteration of the length of the looped DNA domains was found to accompany transformation. The implications of these observations are discussed in relation to the process of the process of transformation.

Animals↗