Strain distribution in Si capping layers on SiGe islands: influence of cap thickness and footprint in reciprocal space.

We present investigations on the strain properties of silicon capping layers on top of regular SiGe island arrays, in dependence on the Si-layer thickness. Such island arrays are used as stressors for the active channel in field-effect transistors where the desired tensile strain in the Si channel is a crucial parameter for the performance of the device. The thickness of the Si cap was varied from 0 to 30 nm. The results of high resolution x-ray diffraction experiments served as input to perform detailed strain calculations via finite element method models. Thus, detailed information on the Ge distribution within the buried islands and the strain interaction between the SiGe island and Si cap was obtained. It was found that the tensile strain within the Si capping layer strongly depends on its thickness, even if the Ge concentration of the buried dot remains unchanged, with tensile strains degrading if thicker Si layers are used.

Unit cell parameters of wurtzite InP nanowires determined by x-ray diffraction.

High resolution x-ray diffraction is used to study the structural properties of the wurtzite polytype of InP nanowires. Wurtzite InP nanowires are grown by metal-organic vapor phase epitaxy using S-doping. From the evaluation of the Bragg peak position we determine the lattice parameters of the wurtzite InP nanowires. The unit cell dimensions are found to differ from the ones expected from geometric conversion of the cubic bulk InP lattice constant. The atomic distances along the c direction are increased whereas the atomic spacing in the a direction is reduced in comparison to the corresponding distances in the zinc-blende phase. Using core/shell nanowires with a thin core and thick nominally intrinsic shells we are able to determine the lattice parameters of wurtzite InP with a negligible influence of the S-doping due to the much larger volume in the shell. The determined material properties will enable the ab initio calculation of electronic and optical properties of wurtzite InP nanowires.

Polyomavirus small T antigen transactivates genes by its ability to provoke the synthesis and the stabilization of MYC.

DNA tumor viruses are capable of driving quiescent cells into the cell cycle. In case of polyomaviridae, two viral proteins, the large and the small (ST) T antigens are responsible for this outcome. ST interacts with the protein phosphatase PP2A and with chaperons of the dnaK type and leads to the transactivation of several genes, which play a role in S-phase induction. One of these is the transcription factor myelocytomatosis (MYC), which by itself is an important regulator of growth. Microarray analysis has revealed several ST-induced genes, which are also targets of MYC; hence, ST may induce these genes via MYC. Experiments shown here are in line with this assumption. MYC-regulated genes are induced by ST at later times than MYC and a MYC responsive promoter is stimulated by ST. Regulation of MYC occurs through signal transduction pathways, which are co-ordinated by PP2A suggesting that they may be targets of ST. Here, we show that this is the case as important kinases involved in these pathways appear in the active phosphorylated form in the presence of ST. Inhibition of these kinases interferes with MYC induction and inhibition of MYC activity blocks ST-mediated transactivation.

Transactivation of murine cyclin A by polyomavirus large and small T antigens.

Polyomavirus large and small T antigens cooperate in the induction of S phase in serum-deprived Swiss 3T3 cells. While the large T antigen is able to induce S phase-specific enzymes, we have recently shown that both T antigens contribute to the production of the cyclins E and A and that the small T antigen is essential for the induction of cyclin A-dependent cdk2 activity (S. Schüchner and E. Wintersberger, J. Virol. 73:9266-9273, 1999). Here we present our attempts to elucidate the mechanisms by which the large and the small T antigens transactivate the murine cyclin A gene. Using Swiss 3T3 cells carrying the T antigens and various mutants thereof under the hormone-inducible mouse mammary tumor virus promoter, as well as transient-cotransfection experiments with the T antigens and cyclin A promoter-luciferase reporter constructs, we found the following. The large T antigen activates the cyclin A promoter via two transcription factor binding sites, a cyclic AMP responsive element (CRE), and the major negative regulatory site called CDE-CHR. While an intact binding site for pocket proteins is required for the function of this T antigen at the CDE-CHR, its activity at the CRE is largely independent thereof. In contrast, an intact J domain and an intact zinc finger are required at both sites. The small T antigen also appears to have an influence on the cyclin A promoter through the CRE as well as the CDE-CHR. For this an interaction with protein phosphatase 2A is essential; mutation of the J domain does not totally eliminate but greatly reduces the transactivating ability.

Why is there late replication?

It has been known for about 40 years that the S phase of the cell cycle is regulated and that parts of the genome are replicated early, while others are replicated late. Numerous studies in the past two decades have revealed that while expressed genes, such as those coding for housekeeping proteins, are usually replicated early, genes not expressed in a particular cell and heterochromatic regions of the genome, such as the centromeres or the inactivated X chromosome of females, are usually replicated late. As details of the mechanisms leading to the formation of replication complexes were worked out, in particular for the budding yeast, Saccharomyces cerevisiae, new insights into the control of the order of replication of genes were obtained that indicate that this process is highly regulated. It is coordinated with transcription, epigenetic changes in chromatin structure, regulation of precursor pools and surveillance mechanisms.

Binding of polyomavirus small T antigen to protein phosphatase 2A is required for elimination of p27 and support of S-phase induction in concert with large T antigen.

Although polyomavirus large T antigen readily transactivates S-phase-specific enzymes in serum-starved Swiss 3T3 mouse fibroblasts, it is incapable by itself to efficiently drive such cells into S phase. We describe here that this inability correlates with a weak proficiency of the viral protein to induce the synthesis of cyclin A and cyclin E and to stimulate the respective cyclin/cdk activities. Polyomavirus small T antigen, which together with the large T protein supports S-phase induction, strongly contributes to the synthesis of cyclin A. In addition, small T antigen causes a dramatic induction of cyclin A- and, together with large T antigen, of cyclin E-specific protein kinase activity. This latter function of polyomavirus small T antigen correlates with its competence to provoke the elimination of the kinase inhibitor p27(Kip1). An interaction of the small T antigen with the protein phosphatase 2A is essential for this activity. Hence, the ability to drive quiescent Swiss 3T3 cells into S phase results from the capacity of large T antigen to transactivate DNA synthesis enzymes by its interaction with retinoblastoma-type proteins and from the potential of the large and the small T antigens together to stimulate cyclin A synthesis and cyclin A- and cyclin E-dependent protein kinase activity.

Sp1 and NF-Y are necessary and sufficient for growth-dependent regulation of the hamster thymidine kinase promoter.

Thymidine kinase (TK) genes from different species are growth- and cell cycle-regulated in a very similar manner; still, the promoter regions of these genes show little homology to each other. It was previously shown that the murine TK gene is growth-regulated by Sp1 and E2F. Here we have characterized cis-regulatory elements in the hamster promoter that are essential and sufficient to confer efficient and serum-responsive expression. The TK promoter was isolated from baby hamster kidney cells. DNase I protection experiments revealed a protected region from positions -24 to -99 relative to the transcription start site. Within this region, binding sites for the transcription factor Sp1 and a CCAAT box, which interacts with the transcription factor NF-Y, were identified. An E2F-like sequence was found not to bind protein, and its removal did not affect promoter activity. This was supported by the observation that cotransfection of a hamster TK reporter gene construct with E2F-1 does not lead to transactivation of the promoter. A 122-base pair region that contains a single Sp1 site, a CCAAT box, and a TATA element was found to be sufficient for serum-responsive expression of a reporter gene. Mutations that inactivate any one of these three elements caused a strong reduction or a loss of promoter activity.

Histone deacetylase 1 can repress transcription by binding to Sp1.

The members of the Sp1 transcription factor family can act as both negative and positive regulators of gene expression. Here we show that Sp1 can be a target for histone deacetylase 1 (HDAC1)-mediated transcriptional repression. The histone deacetylase inhibitor trichostatin A activates the chromosomally integrated murine thymidine kinase promoter in an Sp1-dependent manner. Coimmunoprecipitation experiments with Swiss 3T3 fibroblasts and 293 cells demonstrate that Sp1 and HDAC1 can be part of the same complex. The interaction between Sp1 and HDAC1 is direct and requires the carboxy-terminal domain of Sp1. Previously we have shown that the C terminus of Sp1 is necessary for the interaction with the transcription factor E2F1 (J. Karlseder, H. Rotheneder, and E. Wintersberger, Mol. Cell. Biol. 16:1659-1667, 1996). Coexpression of E2F1 interferes with HDAC1 binding to Sp1 and abolishes Sp1-mediated transcriptional repression. Our results indicate that one component of Sp1-dependent gene regulation involves competition between the transcriptional repressor HDAC1 and the transactivating factor E2F1.

Polyomavirus large T antigen binds the transcriptional coactivator protein p300.

Using coimmunoprecipitation and glutathione S-transferase pulldown experiments, we found that polyomavirus large T antigen binds to p300 in vivo and in vitro. The N-terminal region of the viral protein, including the pRB binding motif, was dispensable for this interaction, which involved several regions within the C-terminal half of the large T antigen. Interestingly, anti-T antibody coimmunoprecipitated a subspecies of p300 which has high histone acetyltransferase activity.

Growth-regulated antisense transcription of the mouse thymidine kinase gene.

The expression of the salvage pathway enzyme thymidine kinase (TK) is very low in resting mammalian cells, but increases dramatically when growth-stimulated cells enter S phase. The 30-fold rise in TK mRNA levels in response to growth factors is due to a well-characterized transcriptional activation and less defined post-transcriptional mechanisms. A minigene containing the murine TK promoter and the TK cDNA showed a 3-fold increase in TK mRNA levels after growth induction in stably transfected mouse TK-deficient L fibroblasts. Introduction of the first three TK introns resulted in a 10-fold regulation of TK expression which was predominantly due to repressed TK mRNA levels in serum-deprived cells. Removal of intron 3 from this construct or replacement of the TK promoter by a constitutive SV40 promoter led to a reduced, but still significant increase in TK mRNA levels during the onset of proliferation. These results indicate that both the TK promoter and specific TK introns contribute independently to the growth-dependent regulation of TK mRNA expression. To examine the regulatory mechanisms in more detail we analyzed TK transcription rates and steady-state levels of nuclear transcripts from an SV40 promoter-driven minigene that contains introns 2 and 3 of the TK gene. Using a set of single-stranded probes we detected TK-specific antisense transcription that was up-regulated in resting cells. Similarly, antisense transcription of the endogenous TK gene in Swiss 3T3 cells rose during serum deprivation while sense transcription was regulated in the opposite way. Luciferase reporter assays revealed the presence of a putative antisense promoter in intron 3 of the murine TK gene. These results suggest a negative role for intron-dependent antisense transcription in the regulation of TK mRNA expression in mouse fibroblasts.

Mouse thymidine kinase stability in vivo and after in vitro translation.

Using a combination of centrifugal elutriation and recultivation of synchronised cell populations we could show that murine thymidine kinase (TK) is rapidly degraded during mitosis in polyoma virus-transformed mouse fibroblasts, in parallel to the time-course for loss of cyclin A. Transformation is no prerequisite for the instability phenotype since artificial overexpression of TK under the control of a constitutive promoter in normal mouse fibroblasts also resulted in rapid turnover of TK during mitosis. The decay of TK protein could be partially mimicked in vitro with enzymatically active protein translated in a rabbit reticulocyte lysate: full length polypeptide was lost slightly more rapidly in the presence of G2/M cytosolic extracts than with G1/S preparations. In addition, an enzymatically active C-terminal truncation of 37 amino acids at Gln-196 was completely stable under the conditions tested, confining the instability domain between residues 196 to 233. These experiments also indicated the border for intact TK since translation products up to Tyr-189 or less were completely inactive. This was also confirmed by a mutant TK protein from mouse F9tk- teratocarcinoma cells which harboured a similar deletion.

Polyomavirus large T antigen-dependent DNA amplification.

DNA amplification is a readily measurable indicator for genome destabilization. Contrary to normal senescing cells, those of most immortal or transformed cell lines are karyotypically unstable and permissive for amplification. Permissivity for amplification can be generated by gene products of several DNA tumor viruses whereby their interaction with the tumorsuppressor protein p53 is important. p53 is the major protein involved in check point control of DNA damage. Polyomavirus large T antigen is also involved in immortalization and transformation of cells but it does not interact with p53. We, therefore, examined whether this protein could still make the non-permissive cell line REF52 permissive for gene amplification. To this end REF52 cell lines were constructed which conditionally expressed the wild type polyomavirus large T antigen or a mutant form unable to bind the retinoblastoma protein. Using the inhibitor of de novo pyrimidine biosynthesis, phosphonoacetyl-L-aspartate (PALA), as selective agent we found that PALA resistant cells arise with a frequency of about 5 x 10(-5) and that the interaction of polyomavirus large T protein with the retinoblastoma protein or another related pocket protein is important for this to occur. PALA resistant cells have an increased number of chromosomes and dicentric chromosomes which are considered as starting point for DNA structures characteristic for amplified DNA. Such structures were indeed found with the help of fluorescence in situ hybridization. PALA resistant cells appear normal with respect to p53. Our data indicate that PALA induces a G1 block which can be partially overcome by polyomavirus large T protein by its interaction with E2F-pocket protein complexes providing further evidence that these complexes are downstream targets of p53.

Carboxy-terminal residues of mouse thymidine kinase are essential for rapid degradation in quiescent cells.

The expression of murine thymidine kinase (TK) is highly dependent on the growth state of the cell. The enzyme is nearly undetectable in resting (G0) cells, but TK protein levels rise dramatically when serum-stimulated cells reach the G1/S boundary. To study post-transcriptional regulation of TK expression, Ltk- cells were stably transfected with the coding region of the TK cDNA under the control of a constitutive SV40 promoter. While TK mRNA levels were growth independent in this cell line, TK protein expression and enzyme activity were low in resting cells but increased strongly after growth stimulation by serum. Measurements of translation efficiency and protein stability by immunoprecipitation and pulse-chase experiments indicated that a fourfold change in protein synthesis rate and a sevenfold rise in protein stability are responsible for the increase of TK expression. Progressive deletion of three, six, ten and 20 carboxy-terminal residues of the enzyme resulted in a stepwise loss of its growth-dependent regulation. In addition, a truncated protein lacking the last 30 amino acid residues was expressed at a level tenfold higher than the full-length polypeptide. Further analysis showed that removal of the C-terminal 30 residues did not affect the translation rate, but resulted in the drastic increase in protein half-life. These results demonstrate that residues at the carboxy terminus of the murine enzyme are essential for the growth-dependent regulation of TK protein stability.

Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F.

Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase gene carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter protein appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion proteins that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of E2F1, a domain also present in E2F2 and E2F3 but absent in E2F4 and E2F5, were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was estabilished that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin-organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle regulated tethering factor between Sp1 and the basic transcription machinery.

Overexpression of thymidine kinase mRNA eliminates cell cycle regulation of thymidine kinase enzyme activity.

Expression of thymidine kinase (TK) enzyme activity and mRNA is strictly S phase-specific in primary cells. In contrast, DNA tumor virus-transformed cells have enhanced and constitutive levels of TK mRNA during the whole cell cycle. Their TK protein abundance, however, still increases at the G1-S transition and stays high throughout G2 until mitosis. Therefore, post-transcriptional control must account for the decoupling of TK mRNA from protein synthesis in G1. To characterize the underlying mechanism, we studied the consequences of TK mRNA abundance on the cell cycle-dependent regulation of TK activity in nontransformed cells. Constitutive as well as conditional human and mouse TK cDNA vectors were stably transfected into mouse fibroblasts, which were subsequently synchronized by centrifugal elutriation. Low constitutive TK mRNA expression still resulted in a fluctuation of TK activity with a pronounced maximum in S phase. This pattern of cell cycle-dependent TK activity variation reflected the one in primary cell but is caused by post-transcriptional control. Increasing overexpression of TK transcripts after hormonal induction compromised this regulation. At the highest constant mRNA levels, regulation of enzyme activity was totally abolished in each phase of the cell cycle. These data indicate that post-transcriptional regulation of TK is tightly coupled to the amount of mRNA; high concentrations apparently titrate a factor(s) required for repressing TK production during G1 and presumably also G2.

A common regulation of genes encoding enzymes of the deoxynucleotide metabolism is lost after neoplastic transformation.

We determined the cell cycle-dependent fluctuation of mRNAs that encode different enzymes of the deoxynucleotide metabolism in permanent cell lines of human and murine origin. In normal growing cells, dihydrofolate reductase, thymidine kinase, and both subunits of ribonucleotide reductase all show exactly the same variation. The mRNAs rise near the G1-S boundary, peak in early S phase, and return in G2 to approximately the level of early G1. Deoxycytidine kinase mRNA does not follow this pattern, but remains essentially unchanged. Conversely, in DNA tumor virus-transformed cells, the levels of all these mRNAs remain relatively constant throughout all phases. These data provide evidence that DNA tumor viruses suppress a transcriptional down-regulation common to enzymes responsible for the DNA precursor pathway. The usefulness of analysis of mRNA levels of these genes for the detection of DNA tumor virus transformation is indicated.

Different regulation of thymidine kinase during the cell cycle of normal versus DNA tumor virus-transformed cells.

We compared the cell cycle regulation of thymidine kinase (TK) after centrifugal elutriation in normal human and mouse cells (primary cells, diploid fibroblasts) with its expression in cells transformed with different DNA tumor viruses. Normal cells showed a rise of TK enzyme activity near the G1/S boundary, which peaked in S phase, and in G2 returned approximately to the level of G1. Conversely, in cells derived from viral transformation, TK activity remained high throughout S and G2 phases, although it was induced to a comparable extent at the onset of DNA replication. In addition, transformed cells exhibited much more enzyme activity during all phases of the cell cycle. The observed differences in expression were due neither to different rates of protein turnover nor to differences in enzyme stability. Enzyme activity was always totally paralleled by the protein level. In all normal cells, the pattern of TK mRNA variation during the cell cycle was similar to that of enzyme activity. In all transformed lines, however, mRNA levels were higher and did not fluctuate throughout the cell cycle. Recently we showed (Ogris et al., 1993) that the E2F binding site present in the TK promoter is a target for trans activation of the TK gene by polyoma virus large T antigen. Using cells expressing this antigen under the control of a hormone-inducible promoter, we were able to switch TK cell cycle expression from the normal to the transformed status. Obviously, DNA tumor viruses suppress transcriptional down-regulation of the endogenous DNA precursor pathway enzyme TK during the eukaryotic cell cycle, maybe to improve conditions for their own replication.

DNA amplification: new insights into its mechanism.

DNA amplification is a process whereby a limited part of the genome is increased in copy number with various consequences for the cell. It is frequently observed in cancer cells and it can be induced in mammalian cells grown in culture as well as in tumor cells when these are subjected to growth inhibiting drugs. In recent years new insights into the mechanisms involved in DNA amplification have been obtained; discussion of these will form the major subject of this short review.

Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein.

Previously constructed Swiss mouse 3T3 fibroblasts producing polyomavirus large T antigen after addition of dexamethasone were used to study the transcriptional activation by the viral protein of five genes coding for enzymes involved in DNA synthesis and precursor production, namely, dihydrofolate reductase, thymidine kinase, thymidylate synthase, DNA polymerase alpha, and proliferating-cell nuclear antigen. It was found that all these genes, whose expression is stimulated at the G1/S boundary of the cell cycle after growth stimulation by serum addition, are coordinately trans activated when T antigen is induced in cells previously growth arrested by serum withdrawal. Cell lines carrying the information for a mutant form of large T antigen, in which a glutamic acid residue in the binding site for the retinoblastoma protein was changed into aspartic acid, were constructed to test the involvement of an interaction of T antigen with the retinoblastoma protein in this reaction. It was found that the mutated T protein is incapable of stimulating transcription of any one of the genes. The promoter of three of the genes (dihydrofolate reductase, thymidine kinase, and DNA polymerase alpha) unequivocally carries binding sites for transcription factor E2F, suggesting that complexes forming with this growth- and cell cycle-regulating transcription factor are the targets for T antigen. Although there is so far no evidence that thymidylate synthase and proliferating cell nuclear antigen are regulated via E2F, our data indicate that the retinoblastoma protein still is involved in the control of these genes. mRNA for E2F itself increases in amount at the G1/S border in serum-stimulated cells but not during polyomavirus T antigen-induced transcriptional activation of DNA synthesis enzymes in arrested cells.