Differential sensitivity of assays for determining vein endothelial cell senescence.

In vivo endothelialization of polymer-based cardiovascular implant materials is a promising strategy to reduce the risk of platelet adherence and the subsequent thrombus formation and implant failure. However, endothelial cells from elderly patients are likely to exhibit a senescent phenotype that may counteract endothelialization. The senescence status of cells should therefore be investigated prior to implantation of devices designed to be integrated in the blood vessel wall. Here, human umbilical vein endothelial cells (HUVEC) were cultivated up to passage (P) 4, 10 and 26/27 to determine the population doubling time and the senescence status by four different methods. Determination of the senescence-associated ß-galactosidase activity (SA-ß-Gal) was carried out by colorimetric staining and microscopy (i), as well as by photometric quantification (ii), and the expression of senescence-associated nuclear proteins p16 and p21 as well as the proliferation marker Ki67 was assessed by immunostaining (iii), and by flow cytometry (iv). The population doubling time of P27-cells was remarkably greater (103±65 h) compared to P4-cells (24±3 h) and P10-cell (37±15 h). Among the four different methods tested, the photometric SA-ß-Gal activity assay and the flow cytometric determination of p16 and Ki67 were most effective in discriminating P27-cells from P4- and P10-cells. These methods combined with functional endothelial cell analyses might aid predictions on the performance of implant endothelialization in vivo.

The bovine papillomavirus E2 transactivator is stimulated by the E1 initiator through the E2 activation domain.

Bovine papillomavirus type 1 (BPV-1) encodes two regulatory proteins, E1 and E2, that are essential for viral replication and transcription. E1, an ATP-dependent helicase, binds to the viral ori and is essential for viral replication, while the viral transcriptional activator, E2, plays cis-dominant roles in both viral replication and transcription. At low reporter concentrations, E1 stimulates E2 enhancer function, while at high reporter concentrations, repression results. An analysis of cis requirements revealed that neither replication nor specific E1-binding sites are required for the initiators' effect on E2 transactivator function. Though no dependence on E1-binding sites was found, analysis of E1 DNA binding and ATPase mutants revealed that both domains are required for E1 modulation of E2. Through the use of E2 fusion-gene constructs we showed that a heterologous DNA-binding domain could be substituted for the E2 DNA-binding domain and this recombinant protein remained responsive to E1. Furthermore, E1 could rescue activation domain mutants of E2 defective for transactivation. These data suggest that E1 stimulation of E2 involves interactions between E1 and the E2 activation domain on DNA. We speculate that E1 may allosterically interact with the E2 activation domain, perhaps stabilizing a particular structure, which increases the enhancer function of E2.

Assembly of functionally active Drosophila origin recognition complex from recombinant proteins.

In eukaryotes the sites for the initiation of chromosomal DNA replication are believed to be determined in part by the binding of a heteromeric origin recognition complex (ORC) to DNA. We have cloned the genes encoding the subunits of the Drosophila ORC. Each of the genes is unique and can be mapped to discrete chromosomal locations implying that the pattern and developmental regulation of origin usage in Drosophila is not regulated solely by a large family of different ORC proteins. The six-subunit ORC can be reconstituted with recombinant proteins into a complex that restores DNA replication in ORC-depleted Drosophila or Xenopus egg extracts.

Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes.

A regulatory system for the in-depth study of gene functions in higher eukaryotic cells has been developed. It is based on the tetracycline-controlled transactivators and reverse tTA, which were remodeled to discriminate efficiently between two different promoters. The system permits one to control reversibly the activity of two genes, or two alleles of a gene, in a mutually exclusive way, and also allows one to abrogate the activities of both. This dual regulatory circuit, which can be operated by a single effector substance such as doxycycline, overcomes limitations of conventional genetic approaches. The conditional mutants that can now be generated will be useful for the study of gene function in vitro and in vivo. In addition, the system may be of value for a variety of practical applications, including gene therapy.

Competition for DNA binding sites between the short and long forms of E2 dimers underlies repression in bovine papillomavirus type 1 DNA replication control.

Papillomaviruses establish a long-term latency in vivo by maintaining their genomes as nuclear plasmids in proliferating cells. Bovine papillomavirus type 1 encodes two proteins required for viral DNA replication: the helicase E1 and the positive regulator E2. The homodimeric E2 is known to cooperatively bind to DNA with E1 to form a preinitiation complex at the origin of DNA replication. The virus also codes for two short forms of E2 that can repress viral functions when overexpressed, and at least one copy of the repressor is required for stable plasmid maintenance in transformed cells. Employing a tetracycline-regulated system to control E1 and E2 production from integrated loci, we show that the short form of E2 negatively regulates DNA replication. We also found that the short form could repress replication in a cell-free replication system and that the repression requires the DNA binding domain of the protein. In contrast, heterodimers of the short and long forms were activators and, by footprint analysis, were shown to be as potent as homodimeric E2 in loading E1 to its cognate site. DNA binding studies show that when E1 levels are low and are dependent upon E2 for occupancy of the origin site, the repressor can block E1-DNA interactions. We conclude that DNA replication modulation results from competition between the different forms of E2 for DNA binding. Given that heterodimers are active and that the repressor form of E2 shows little cooperativity with E1 for DNA binding, this protein is a weak repressor.

Stimulation of cyclin-dependent kinase activity and G1- to S-phase transition in human lymphocytes by the human T-cell leukemia/lymphotropic virus type 1 Tax protein.

The human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) induces a malignant lymphocytic disease. The HTLV-1 transactivator protein, Tax, is believed to be crucial for the development of the disease since it is transforming in vitro and induces tumors in transgenic animals. Although the transcriptional modulation of viral and cellular gene expression by Tax has been analyzed thoroughly, it has remained unclear how the Tax functions act on the cell cycle of primary T cells. To investigate the mechanism of Tax-mediated T-cell stimulation, we transduced primary human cord blood T cells with a conditional, tetracycline repressor-based tax expression system. Permanent Tax expression results in an abnormal proliferation of T cells which closely resemble HTLV-1-infected lymphocytes. Suppression of Tax synthesis stopped lymphocyte growth and caused cell cycle arrest in the G1 phase. Upon reinduction of tax expression, the arrested cells entered the S phase. This showed that Tax has mitogenic activity, which is required for stimulating the G1- to S-phase transition of immortalized lymphocytes. In mammalian cells, the G1-phase progression is controlled by the serial activation of several cyclin-dependent kinases (Cdks), starting with Cdk4 and Cdk6. In the presence of Tax, both Cdk4 and Cdk6 were activated. The suppression of Tax synthesis, however, resulted in a significant reduction of the Cdk4 and Cdk6 activities but did not influence the expression of Cdk4, Cdk6, or cognate D-type cyclin proteins. These data suggest that Tax induces Cdk4 and Cdk6 activity in primary human T lymphocytes; this Cdk activation is likely to account for the mitogenic Tax effect and for the abnormal T-cell proliferation of HTLV-1-infected lymphocytes.

Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential.

Several tetracycline-controlled transactivators (tTA) were generated which differ in their activation potential by >3 orders of magnitude. The transactivators are fusions between the Tet repressor and minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). By reducing the VP16 moiety of the previously described tTA to 12 amino acids, potential targets for interactions with various cellular transcription factors were eliminated, as were potential epitopes which may elicit a cellular immune response. When compared with the originally described tTA, these new transactivators are tolerated at higher intracellular concentrations. This will facilitate establishment of tet regulatory systems under a variety of conditions, but particularly when cell type-restricted tetracycline-controlled gene expression is to be achieved in transgenic organisms via homologous recombination.

Characterization of the rat spinocerebellar ataxia type 3 gene.

Machado-Joseph disease (MJD) belongs to a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by progressive cerebellar ataxia. The disease-causing mutation has recently been identified as an unstable and expanded (CAG)n trinucleotide repeat in a novel gene of unknown function. In Caucasians, repeat expansions in the MJD1 gene have also been found in patients with the clinically distinct autosomal dominant spinocerebellar ataxia type 3 (SCA3). In order to gain insight into the biology of the MJD1/SCA3 gene we cloned the rat homologue and studied its expression. The rat and human ataxin-3 genes are highly homologous with an overall sequence identity of approximately 88%. However, the C-terminal end of the putative protein differs strongly from the published human sequence. The (CAG)n block in the rat cDNA consists of just three interrupted units suggesting that a long polyglutamine stretch is not essential for the normal function of the ataxin-3 protein in rodents. The expression pattern of the SCA3 gene in various rat and human tissues was investigated by Northern blot analyses. The mature transcript is approximately 6 kb in length. In rat testis, a smaller transcript of 1.3 kb was identified. Transcription of rsca3 was detected in most rat tissues including brain. Analyzing the expression level of the SCA3 gene in several human brain sections revealed no significant higher mRNA level in regions predominantly affected in MJD. Thus additional molecules and/or regulatory events are necessary to explain the exclusive degeneration of certain brain areas.

Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.

cDNA cloning and expression of rsca1, the rat counterpart of the human spinocerebellar ataxia type 1 gene.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by an expanded and unstable (CAG) > 40 repeat within a gene of unknown function. We isolated the complete coding region of the rat SCA1 gene (rsca1), the 5'-untranslated region (UTR) and 1.3 kb of the 3'-UTR. The rat sequence exhibits 90% peptide identity to the human counterpart. In comparison to human, the rat (CAG)n block is reduced to two trinucleotide motifs preceded by three different proline codons not present in man. Furthermore, we investigated the expression of rsca1 in different rat tissues. The rsca1 gene is predominantly expressed in brain throughout all developmental stages. In situ hybridizations reveal high levels of expression in various regions of the adult rat brain, including cerebellum, hippocampus and cortex.

Separation of origin recognition complex functions by cross-species complementation.

Transcriptional silencing at the HMRa locus of Saccharomyces cerevisiae requires the function of the origin recognition complex (ORC), the replication initiator of yeast. Expression of a Drosophila melanogaster Orc2 complementary DNA in the yeast orc2-1 strain, which is defective for replication and silencing, complemented the silencing defect but not the replication defect; this result indicated that the replication and silencing functions of ORC were separable. The orc2-1 mutation mapped to the region of greatest homology between the Drosophila and yeast proteins. The silent state mediated by DmOrc2 was epigenetic; it was propagated during mitotic divisions in a relatively stable way, whereas the nonsilent state was metastable. In contrast, the silent state was erased during meiosis.

A Drosophila homolog of the yeast origin recognition complex.

Genes from Drosophila melanogaster have been identified that encode proteins homologous to Orc2p and Orc5p of the Saccharomyces cerevisiae origin recognition complex (ORC). The abundance of the Drosophila Orc2p homolog DmORC2 is developmentally regulated and is greatest during the earliest stages of embryogenesis, concomitant with the highest rate of DNA replication. Fractionation of embryo nuclear extracts revealed that DmORC2 is found in a tightly associated complex with five additional polypeptides, much like the yeast ORC. These studies will enable direct testing of the initiator-based model of replication in a metazoan.