Cyclin‐dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re‐replication. Here we demonstrate ...that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N‐terminal Cy‐motif that is similar to the cyclin binding regions in p21WAF1/SDI1 and E2F‐1. The in vivo phosphorylation of CDC6 was dependent on three N‐terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re‐replication during S phase and G2.
To develop and evaluate a liquid phase immunoassay for accurate determination of allergen-specific IgE (sIgE) as a useful tool in the diagnosis of allergy patients.
A fully automated, quantitative ...sIgE assay was developed for the ADVIA Centaur technology platform using a unique calibration method based on a recombinant reference allergen. Compared to most other IgE-assays, the assay employs a reverse sandwich architecture using monoclonal mouse anti-human IgE antibody covalently bound to paramagnetic particles in the solid phase and capturing the sample IgE. Bound sIgE reacts with liquid biotin-labeled allergen, which is detected as chemiluminescence using acridiniumester-labeled streptavidin.
The ADVIA Centaur sIgE assay (Centaur assay) has exclusive reactivity to human IgE and performs with excellent linearity in the assay range 0.35–100 kU/L and high precision (imprecision within-run <2.6%, between-run <4.9%, and total imprecision <7.1%). The analytical sensitivity is <0.10 kU/L. Using Pharmacia CAP system FEIA (CAP) as a comparative method, positive/negative concordance was 94% at 0.35 kU/L cut-off, and the Centaur assay has a sensitivity of 90% and a specificity of 98%. Validation of the assay in a general population sample (The Copenhagen allergy study) revealed that sIgE was highly associated with a clinical diagnosis of inhalation allergy.
The Centaur assay is an allergen-specific assay for measurement of IgE without interference from other types of immunoglobulins or nonspecific IgE. The assay performs with a linear reaction, high assay range, and good reproducibility. The assay correlates well with the CAP system and is in agreement with clinical diagnosis.
CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated ...subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G(1). A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G(1) and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent proteolysis of CDC6 in early G(1) and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G(1).
Based on the sequences of mammalian RNase L inhibitors (RLI), homologues were identified in
Arabidopsis thaliana and
Nicotiana plant species. RLI homologue (RLIh) cDNAs were cloned from
Nicotiana ...benthamiana and
Nicotiana tabacum and used to induce post-transcriptional gene silencing (PTGS) in
Nicotiana species. Downregulation of RLIh in
N. benthamiana plants by virus-induced gene silencing had severe effects on plant morphology and plant growth but did not affect the susceptibility of the plant to infection by
Potato virus Y (PVY). Upon transformation of
N. benthamiana and
N. tabacum with an inverted repeat of RLIh only one transformant, X#2.1, displayed an abnormal phenotype characterized by reduced growth and distorted leaves with white spots. Northern blotting revealed the presence of RLIh-specific small interfering RNAs in X#2.1 but not in 14 other transformants with a normal phenotype. Based on our observations we propose that RLIh has an essential role in plant growth and development.
RNA silencing is a sequence-specific RNA degradation mechanism found in most eukaryotes, where small cleavage products (siRNAs) of double stranded RNA (dsRNA) mediate silencing of genes with sequence ...identity to the dsRNA inducer. In several systems, silencing has been found to spread from the dsRNA inducer sequence into upstream or downstream regions of the target RNA, a phenomenon termed transitive silencing. In nematodes, silencing spreads only in the 3'-5' direction along the target mRNA by siRNAs serving as primers for cRNA synthesis by RNA-dependent RNA polymerase. In plants, transitive silencing is seen in both directions suggesting that at least some cRNA synthesis occurs by un-primed initiation at the 3' end of mRNAs. Replicating plant viruses trigger an RNA silencing defence response that degrades the viral RNA, thus tempering the virus infection. Likewise, fragments of plant genes inserted into a virus will become targets for degradation, leading to virus-induced gene silencing (VIGS) of the homologous plant mRNAs. We have analyzed the spreading of gene silencing in VIGS experiments using a transgene and two endogenous genes as targets. In Nicotiana benthamiana plants expressing a β-glucuronidase (GUS) transgene, a Potato virus X vector carrying a 5' fragment of the GUS gene induced silencing which spread to downstream regions of the transgene mRNA including the 3'-untranslated region. Conversely, silencing induced by a 3' fragment spread only for a limited distance in the 3'-5' direction. Silencing induced by a central GUS gene fragment spread only into downstream regions. Similar analyses using the endogenous plant genes, magnesium chelatase subunit I (ChlI) and an RNase L inhibitor homologue (RLIh), revealed no spreading along target sequences. This implies that transitive silencing in plants occurs by un-primed cRNA synthesis from the 3' end of targeted (transgene) transcripts, and not by siRNA-primed cRNA synthesis.
The Saethre-Chotzen syndrome (SCS) is an autosomal dominant craniosynostosis syndrome with uni- or bilateral coronal synostosis and mild limb deformities. It is caused by loss-of-function mutations ...of the TWIST 1 gene. In an attempt to delineate functional features separating SCS from Muenke's syndrome, we screened patients presenting with coronal suture synostosis for mutations in the TWIST 1 gene, and for the Pro250Arg mutation in FGFR3. Within a total of 124 independent pedigrees, 39 (71 patients) were identified to carry 25 different mutations of TWIST 1 including 14 novel mutations, to which six whole gene deletions were added. The 71 patients were compared with 42 subjects from 24 pedigrees carrying the Pro250Arg mutation in FGFR3 and 65 subjects from 61 pedigrees without a detectable mutation. Classical SCS associated with a TWIST 1 mutation could be separated phenotypically from the Muenke phenotype on the basis of the following features: low-set frontal hairline, gross ptosis of eyelids, subnormal ear length, dilated parietal foramina, interdigital webbing, and hallux valgus or broad great toe with bifid distal phalanx. Functional differences were even more important: intracranial hypertension as a consequence of early progressive multisutural fusion was a significant problem in SCS only, while mental delay and sensorineural hearing loss were associated with the Muenke's syndrome. Contrary to previous reports, SCS patients with complete loss of one TWIST allele showed normal mental development.
The transcription factor complex E2F-1/DP-1 regulates the G1-to-S-phase transition and has been associated with sensitivity to the S-phase-specific anticancer agents camptothecin and etoposide, which ...poison DNA topoisomerase I and II, respectively. To investigate the relationship between E2F-1 and drug sensitivity in detail, we established human osteosarcoma U-20S-TA cells expressing full-length E2F-1/ DP-1 under the control of a tetracycline-responsive promoter, designated UE1DP-1 cells. Topoisomerase I levels and activity as well as the number of camptothecin-induced DNA single- and double-strand breaks were unchanged in UEIDP-1/tc- cells with >10-fold E2F-1/DP-1 overexpression. However, UE1DP-1/tc- cells were hypersensitive to camptothecin in both a clonogenic assay and four different apoptotic assays. This indicates that camptothecin-induced toxicity in this model is due to the activation of an E2F-1/ DP-1-induced post-DNA damage pathway rather than an increase in the number of replication forks caused by the S-phase initiation. In contrast, topoisomerase IIalpha levels (but not topoisomerase IIbeta levels), together with topoisomerase IIalpha promoter activity, increased 2--3-fold in UE1DP-1/tc-cells. Furthermore, the number of etoposide-induced DNA single- and double-strand breaks increased in UE1DP-1/tc-cells together with a rise in clonogenic sensitivity to etoposide, but an equal apoptotic sensitivity to etoposide. The increase in topoisomerase IIalpha promoter activity in UE1DP-1/tc--cells was shown to be due to S-phase initiation per se because it was blocked by ectopic expression of dominant negative cyclin-dependent kinase 2. In conclusion, overexpression of E2F-1/DP-1 in U-20S-TA cells is sufficient to increase clonogenic sensitivity to both topoisomerase I- and II-targeted anticancer drugs. However, the mechanism by which this occurs appears to be qualitatively different. The UE1DP-1 cell model may be used to elucidate post-DNA damage mechanisms of cell death induced by topoisomerase I-directed anticancer agents.
The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and ...cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. TheCDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G
1
controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication.
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