However, a new study suggests that Tax preferentially transactivates the 21-bp repeats through CREB1 and not other bZIP proteins. The additional transactivation of Tax-responsive promoters subsequent to initiation is also presented. This result highlights a potentially novel role of Tax following TBP recruitment ( Viruses have long been a source of key scientific discoveries. Historically, they have contributed to our knowledge of transcription, cell cycle, and apoptosis. To date activated transcription in higher eukaryotic cells with or without chromatin is a great area of active research and many researchers use viral activators, including herpes virus VP16, adenovirus E1A, HIV-1 Tat and HTLV-I Tax to not only understand viral, but also basic mechanisms related to host control of vital cellular machineries, including transcription. Eukaryotic transcription has five distinct phases, pre-initiation, initiation, promoter clearance, elongation and termination, and is a tightly regulated and coupled process [et al. [in vivo on the 21-bp repeats  in HTLV-I infected cells through chromatin immunoprecipitation (ChIP) assays [et al. demonstrated that among the various bZIP proteins, CREB1 was clearly favored for Tax transactivation of the 21-bp repeats. Additionally, CREB1 has also been found to primarily bind at the 5' LTR (rather than the 3' LTR) in vivo within HTLV-I infected cells, lending support to the idea that CREB1 is important for HTLV-I activated transcription [In the report by Ching et al.  the authet al. -5. It wa) assays . By usincription .et al. [in vitro and in vivo).If CREB1 is the dominant bZIP protein that is needed for Tax transactivation of the LTR, then what is the purpose of the additional bZIP proteins? Besides contributing to Tax transactivation, could these bZIP proteins help to exclude negative regulators from the LTR? A report by Basbous et al.  suggesteet al. presented the intriguing possibility of Tax enhancing transcription following transcription initiation. To determine whether Tax functioned solely to target TBP to the TATAA-element or if additional events subsequent to TBP (TFIID) recruitment were promoted by Tax, the authors constructed four independent reporters. Each promoter contained the minimal TATAA-element from HTLV-I, HIV-1, SV-40, or E1b promoters, two 21-bp repeats, and five copies of the Gal4-binding site. TBP was artificially targeted to the TATAA-element thru Gal4-TBP. The authors reasoned that if Tax functioned strictly to recruit TBP to the TATAA-element, then additional enhancement of transcription would not be observed when Tax and Gal4-TBP were present. Interestingly, only the Tax-responsive promoters, i.e. HTLV-I and HIV-1, were both synergistically stimulated by the addition of Tax and Gal4-TBP. These results suggest that Tax may control downstream transcription subsequent to the initiation phase.Lastly, Ching 2/cdk4 complexes [Other viral transactivators have been shown to have a role at initiation and downstream events, such as elongation. The most notable of these has been Tat, the viral transactivator of HIV-1. Without cellular stimulation and Tat expression, RNAP II transcriptional elongation was shown to be inefficient, producing only short transcripts . One majomplexes ,14. Thiset al. [hsp70 mRNA. Tax has been shown to associate with BRG1 components of the ATP-dependent chromatin remodeling complex, SWI/SNF, and increase Tax transactivation [et al. are preliminary at this time, Tax transactivation post-initiation is indeed a novel concept. Further detailed analysis of Tax at both the LTR of HTLV-I and downstream of this region will help to resolve many of these questions and provide important insight into the transcription field.Processive elongation is highly dependent on remodeling of chromatin structure ,10. A stet al.  demonstrtivation . DisruptHTLV-I, human T cell leukemia virus, type ICRE, cAMP response elementCREB, cAMP response element binding proteinChIP, chromatin immunoprecipitationRNAP II, RNA polymerase IICTD, C-terminal domainHIV-1, human immunodeficiency virus, type 1LTR, long terminal repeatTBP, TATA binding proteinTxREs, Tax-responsive elementsGTFs, general transcription factorsTAR, transactivation regionNone declared.Both authors contributed equally to the structure and content of the manuscript.


The pulmonary residence time of inhaled glucocorticoids as well as their rate and extend of absorption into systemic circulation are important facets of their efficacy-safety profile. We evaluated a novel approach to elucidate the pulmonary absorption of an inhaled glucocorticoid. Our objective was to monitor and compare the combined process of drug particle dissolution, pro-drug activation and time course of initial distribution from human lung tissue into plasma for two different glucocorticoid formulations.®/Becloforte™ and Ventolair®/Qvar™). Initially we developed a simple dialysis model to assess the transfer of BDP and its active metabolite from human lung homogenate into human plasma. In a novel experimental setting we then administered the aerosols into the bronchus of an extracorporally ventilated and reperfused human lung lobe and monitored the concentrations of BDP and its metabolites in the reperfusion fluid.We chose beclomethasone dipropionate (BDP) delivered by two different commercially available HFA-propelled metered dose inhalers (Sanasthmax®/Becloforte™ and Ventolair®/Qvar™ in both the dialysis as well as in the human reperfusion model. The HFA-BDP formulated as Ventolair®/Qvar™ displayed a more rapid release from lung tissue compared to Sanasthmax®/Becloforte™. We succeeded to explain and illustrate the observed differences between the two aerosols with their unique particle topology and divergent dissolution behaviour in human bronchial fluid.Unexpectedly, we observed differences between the two aerosol formulations Sanasthmax®/Qvar™ are beneficial for high lung deposition, they also yield a less desired more rapid systemic drug delivery. While the differences between Sanasthmax®/Becloforte™ and Ventolair®/Qvar™ were obvious in both the dialysis and lung perfusion experiments, the latter allowed to record time courses of pro-drug activation and distribution that were more consistent with results of comparable clinical trials. Thus, the extracorporally reperfused and ventilated human lung is a highly valuable physiological model to explore the lung pharmacokinetics of inhaled drugs.We conclude that though the ultrafine particles of Ventolair Current asthma management guidelines recommend inhaled glucocorticoids as preferred therapy for control of mild persistent, moderate and severe asthma ,2. GlucoThe time course of inhaled drug absorption has been frequently studied in life animals . For thiin vivo distribution of inhaled glucocorticoids between human lung tissue and plasma has been determined in patients undergoing thoracotomy. During surgery tissue and plasma samples were obtained and analyzed for drug concentrations which were found to be significantly higher in lung tissue compared to plasma  and Ventolair®/Qvar™ 100 μg/dose ) or chlorofluorocarbon (CFC) propellant (Sanasthmax®/Becloforte™) were obtained from a local pharmacy. BDP, beclomethasone-17-propionate (17-BMP), beclomethasone-21-propionate (21-BMP), beclomethasone (B) and fluticasone propionate (FP) were a generous gift from GlaxoSmithKline . Diethylether (HPLC grade) was purchased from Fluka  and acetonitrile  from Fisher Scientific . Water was obtained from a Millipore™ water purification unit. Bovine serum albumin (BSA), dextrane 70000, and N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) were purchased from GERBU , glucose monohydrate from Gruessing GmbH , and stock solution containing 10000 IU/mL penicilline and 10000 μg/mL streptomycine in 0.9% NaCl from Biochrom AG . All other chemicals were obtained from E. Merck .Beclomethasone dipropionate (BDP) pressurized metered dose inhalers (MDI) with hydrofluorocarbon (HFA) propellant . Homogenization was performed under continuous cooling using an Ultraturrax . Before starting the series of dialysis experiments the required amount of human lung homogenate was estimated and subsequently a sufficient amount was prepared and divided into aliquots. Since all aliquots descended from this preparation protein content and enzymatic activity of the homogenate was identical for each experiment.Human lung tissue specimen were obtained from patients with bronchial carcinomas who gave informed consent. Only cancer-free tissue was used for the experiments. None of the patients was treated with glucocorticoids for the last 4 weeks prior to surgery. Tissue samples were shock frozen in liquid nitrogen after resection and stored at -70°C until usage. To collect sufficient material for the experiments, tissue samples of three or more patients were pooled. Tissue was thawed and cut into small pieces. One part of the tissue pieces was homogenized in two parts of Krebs-Ringer-HEPES buffer , wedged into a subsegment bronchus. Small bronchial fluid aliquots of four patients were collected and pooled. The specimen was frozen and stored at -70°C until usage.Six patients with a bronchial tumour undergoing standard thoracotomy, were included in the study. None of the patients was treated with glucocorticoids for the last 4 weeks prior to surgery. Only patients with tumours that were located peripherally within the lung lobe were included. Each patient signed a written informed consent before surgery, and a local Ethics Committee approved the use of resected human lungs for perfusion. Immediately after perfusion, the lung preparations were examined as usually by a pathologist.Dialysis was performed with a dialysis unit (designed by our working group) consisting of two individual tightly fitting Teflon chambers separated by a dialysis membrane Figure . One chaIn initial experiments we evaluated the functionality and reliability of the experimental setting with respect to the convection, appropriate sample recovery and absence of trapped air. Therefore, the upper chamber was replaced by a chamber with a top made of acrylic glass instead of Teflon to monitor the processes within the unit. After filling the lower chamber with buffer the dialysis membrane was placed on this chamber and the dialysis unit was closed by attaching the second chamber. The complete and air bubble free filling of the chamber by smooth negative pressure was visually controlled. To check whether there was sufficient convection for a homogenous distribution of the analyzed compounds in the plasma and whether replacement of the sample volume was readily achieved, buffer in the reservoir was stained with a green dye. Then sampling was performed as described above. The appropriate moment for closing the valve was defined as the point of time where no visible flow of stained buffer into the chamber could be observed. It was also determined if any air bubbles gained access through the sampling aperture during sampling or after removing the pipette. Control experiments with the dye verified that the sample was not adulterated during the process of sampling and synchronous replacement of the sample volume with fresh solution. The degree of convection was controlled by following the distribution of the green dye into the clear buffer after sampling. A visually homogenous solution was obtained within a few minutes under experimental conditions. Accurate and reproducible sample volume was assured by weighing ten replicates of samples drawn under experimental conditions. Parameters for accuracy and reproducibility were within the specifications of the manufacture of the used pipettor.ex valve was applied to the homogenate employing a dosing device  was filled into one chamber. 500 μg BDP 18 column . Typically, 20 μL of sample were injected, a flow rate of 1 mL/min was used, and detection wavelength was set to 254 nm. Mobile phase consisted of water containing 0.2 % (v/v) acetic acid (A) and acetonitrile (B). The gradient elution started at 62 % eluent A, decreasing nonlinearly to 53 % A by 22 min and finally decreasing linearly to 28 % eluent A by 18 min. The lower limit of quantification of the assay was 20 ng/mL for all glucocorticoids.Samples of the dialysis experiments were mixed with 50 μL internal standard solution (3 μg/mL FP in methanol) and extracted twice with 2 ml diethylether for 20 min using a roller mixer, followed by centrifugation at 3000 rpm  for 5 min. The combined organic phases were evaporated to dryness under a gentle stream of nitrogen at 25°C. The resulting residue was reconstituted in 0.2 mL methanol and analyzed by liquid chromatography using a Waters HPLC  consisting of a 1525 binary pump, a 717plus autosampler and 2487 dual wavelength absorbance detector. Data collection and integration were accomplished using Breeze™ software version 3.30. Analysis was performed on a Symmetry Ci of drug related to the parent compound found in whole plasma at i-th sample was calculated by the following equations:The amount of the parent compound BDP and its metabolites 17-BMP (active metabolite), 21-BMP and beclomethasone that were distributed from the lung tissue into plasma were determined and calculated as percentage of the applied dose. Amount AP V= r2• π • h(1) i Concentration of the compound at the i-th sample [m/V]ch Inner height of the plasma containing chamberMW Molecular weight of the compoundr Inner radius of the plasma containing chamberP Calculated volume of plasma in the dialysis unitVi the release of the drug into plasma Di at the i-th sample can be expressed as percentage of applied dose using equation (3) or (4):On basis of the calculated amount Aj-1 Amount of drug related to the parent compound found in whole plasma at the (j-1)-th sampleAapplied Actually applied dose to the human lung homogenateD2 × 2 H2O, 1.18 mM MgCl2 × 6 H2O, 2.5 mM KH2PO4, 20 mM NaHCO3, 5.5 mM glucose, 5 % bovine serum albumin and 2 % dextran; 100 μl of a stock solution containing 10,000 IU penicilline and 10,000 μg/mL streptomycine in 0.9% NaCl were added to 1 L perfusion buffer and pH was adjusted to 7.4 by addition of 10% NaHCO3), it was placed within the perfusion apparatus in a tempered water bath (37°C) and ventilated using a respirator  with air  and registered via a computer system. By addition of CO2 using a conventional oxigenator, perfusate pH was maintained within the physiological range of about 7.4. Lung preparations were weighed before and after perfusion to check for oedema formation during perfusion.During lung perfusion, pH, pOexvalve was 500 μg BDP for each MDI. Samples of 5 mL were drawn from the main venous output and the sample volume was replaced with fresh perfusion buffer. Samples were immediately frozen and transported on dry ice, and stored at -20°C until further analysis. In order to calculate the fraction of dose reaching the human lung lobe the spacer and all fittings were rinsed quantitatively and analyzed for BDP. The fraction of BDP that was determined in the spacer and connecting fittings was subtracted from the dose of 500 μg applied ex valve. The amount of the parent compound BDP and its metabolites 17-BMP (active metabolite), 21-BMP and beclomethasone that were distributed from the lung tissue into perfusion fluid were determined and calculated as percentage of the applied dose analogous to the dialysis experiments.After ventilation and perfusion were established the system was equilibrated for 5 min, and a 5 mL sample (blank sample) was drawn before administration of the glucocorticoid. Dosing was performed using a glass spacer (constructed by our group) that was placed between the respirator and the ventilated lung tissue. During application the tidal volume was increased 1.5-fold in comparison to the volume during perfusion. The dose applied 18 column . The mobile phase was a mixture of water containing 0.1 % (v/v) formic acid (A) and acetonitrile (B), the flow rate was 0.6 mL/min. A gradient elution started at 50 % eluent B, increasing linearly to 65 % B by 8 min and then increasing linearly to 80 % B by 8 min. A total sample volume of 25 μL was injected. The mass spectrometer was operated in selective reaction monitoring observing the transitions from 465 m/z to 355 m/z, 501 m/z to 313 m/z, and 521 m/z to 411 m/z for 17-BMP, FP  and BDP, respectively. The lower limit of quantification of the assay was 400 pg/mL for BDP and 17-BMP.Perfusion samples of 1 mL were mixed with 25 μl internal standard solution (300 ng/mL FP in methanol). Glucocorticoids were extracted twice with 3 mL diethylether for 20 min using a roller mixer, followed by centrifugation at 3000 rpm  for 5 min. The organic layers were evaporated to dryness under a gentle stream of nitrogen at 25°C. The residue was reconstituted in 50 μl methanol and analyzed by HPLC – MS/MS using an Agilent 1100 HPLC system  consisting of a binary pump, a vacuum degasser, a temperature-controlled autosampler and a variable wavelength absorbance detector coupled with an Agilent LC/MSD Trap SL mass sensitive detector. An ESI interface was used in the positive ionization mode. Data collection and integration were accomplished using ChemStation-for-LC-3D™ and LC/MSD-Trap™ version 4.2 software. Analysis was performed on a Symmetry C3. The aerosol device was actuated and the glucocorticoid particles were allowed to alight on the glass slide. Number and distribution of BDP particles was visually controlled by light microscopy . After BDP application onto human bronchial fluid the glass microscopy slide was sealed with silicone paste and a cover slide and incubated at 37°C for one hour. Thereafter the cover slide was removed and the fluid was evaporated under a gentle stream of nitrogen. Subsequently the slide was carefully washed twice with 50 μL purified water to remove bronchial fluid proteins. The water was evaporated under a stream of nitrogen.Beclomethasone dipropionate (BDP) from different devices was either directly applied on regular glass microscopy slides or on microscopy slides with incubation slots of 15 × 2 mm  containing human bronchial fluid. For application of BDP the glass microscopy slide was placed in a plastic spacer of about 900 cmA Zeiss DSM 962 scanning electron microscope  was used to obtain the SEM photographs. The samples mounted on the glass microscopy slides were coated with palladium/coal for 3 min using a Baltec SCD 005 sputter-coater in an argon atmosphere (45 Pa and 50 mA).®/Becloforte™ and 5 × 100 μg Ventolair®/Qvar™) ex valve were applied to each 4.8 mL human lung tissue homogenate  were analyzed by SEM. To visualize the particle topology the devices were actuated and the particles were allowed to alight directly on regular glass microscopy slides. Representative HFA-BDP particles delivered by Sanasthmax®/Becloforte™ were about 2 μm  particles delivered by pressurized metered dose inhalers (MDI) with hydrofluorocarbon (HFA) propellant  into a reperfused and ventilated lung lobe and measured the rate and extend of pulmonary absorption. We succeeded to explain kinetic differences we observed between the two aerosols with unique particle topology and divergent dissolution behaviour in human bronchial fluid.In the present study we successfully demonstrated that the intial distribution of beclomethasone dipropionate (BDP) from lung tissue into extrapulmonary circulation can be assessed employing a human lung perfusion model. Therefore, we applied BDP delivered by two different commercially available HFA-propelled aerosols  and the distribution of BDP and 17-BMP from lung tissue into plasma or perfusion fluid, respectively.We evaluated novel approaches to elucidate pulmonary absorption of an inhaled glucocorticoid max) of BDP and its ester metabolites after inhalation of HFA-BDP in various clinical studies [We used the newly developed dialysis model to monitor the kinetics of drug diffusion over six hours. During this time we observed constant drug transfer into plasma and continuous generation of the active BDP metabolite. The latter suggests that the non-specific esterases responsible for pro-drug activation were functional within the chosen time interval. We ventilated and reperfused the resected human lung lobe over about one hour to be as close as possible to physiological conditions and avoid problems such as oedema formation that might be associated with prolonged perfusion times. The time interval of one hour is consistent with the time to reach maximal plasma levels  of 17-BMP or beclomethasone esters, respectively, after inhalation of HFA-BDP compared to CFC-BDP [max of 17-BMP after inhalation of CFC-BDP was not significantly altered when activated charcoal was co-administered and thus gut absorption was prevented [max of 17-BMP after inhalation of CFC-BDP is the particle size as it should be expected that bigger drug crystals dissolve more slowly. HFA-BDP with its smaller particles exhibited a faster and more efficient systemic drug delivery [In contrast to the unanticipated results of our absorption experiments with two HFA formulations, differences in pulmonary absorption after administration of CFC-BDP compared to HFA-BDP would have been expected based on the results of clinical studies. After inhalation of HFA-propelled BDP higher lung deposition rates were achieved and higher plasma concentrations of 17-BMP were observed in adults ,26 and c CFC-BDP ,26. The revented . A plaus®/Becloforte™ or Ventolair®/Qvar™, respectively. Scanning electron microscopic experiments illustratively confirmed this postulate. Particles of BDP delivered by Ventolair®/Qvar™ were significantly smaller and displayed faster dissolution in human bronchial fluid compared to particles delivered by Sanasthmax®/Becloforte™. Additionally, for the first time we provide evidence that BDP particles delivered by HFA-propelled aerosols are subjected to a re-crystallization process once they come in contact with the bronchial fluid. Obviously, the particles as delivered from the device dissolve rapidly due to their large surface area. Subsequently, locally oversaturated solutions tend to crystallize and crystals with a thermodynamically preferred smaller surface area are formed. It seems very likely that this might also occur in the life human lung since the volume of bronchial fluid is very limited. This re-crystallization of particles with high surface area to solid cubic particles with smaller surface area would be a highly desired effect as it slows down the further dissolution and therefore prolongs the pulmonary residence time.Based on the comprehension of the interrelation of aerosol particle size and rate of pulmonary absorption we suspected that the different time courses of distribution into extrapulmonary compartments we observed were due to different particle sizes of HFA-BDP formulated as Sanasthmax®/Qvar™ with its smaller BDP particles. The fact that we also detected the active metabolite 17-BMP in the perfusion fluid of the extracorporally perfused and ventilated lung lobe after only 2–3 min allows the conclusion that the hydrolysis of BDP is an extremely rapid process. This is in excellent agreement with the results of earlier in vitro assays with human lung tissue [The notion that BDP delivered by HFA-propelled inhalers initially rapidly dissolves and then locally re-crystallizes would also explain the detection of BDP in reperfusion fluid of the human lung preparation after only 2–3 min. It seems plausible that a certain amount of dissolved drug is instantly distributed into extrapulmonary compartments since the human lung has a high inner surface area and is the organ with the highest blood flow of about 15 L/min/kg . The detg tissue . Hence, ®/Qvar™ displayed a rapid release from lung tissue while Sanasthmax®/Becloforte™ exhibited a prolonged residence time in tissue. We succeeded to explain the observed differences in lung tissue residence time on the basis of the particle size and dissolution behaviour in human bronchial fluid. We conclude that though the ultrafine particles of Ventolair®/Qvar™ are beneficial for high lung deposition [®/Becloforte™ and Ventolair®/Qvar™ were obvious in both the dialysis and lung perfusion experiments, the latter allowed to record time courses of pro-drug activation and distribution that were more consistent with results of comparable clinical trials. Though it is not clear whether the kinetic differences observed between Sanasthmax®/Becloforte™ and Ventolair®/Qvar™ might be of clinical relevance the detection of divergent absorption kinetics confirm the functionality and sensitivity of our experimental setting. Thus, the extracorporally perfused and ventilated human lung is a highly valuable physiological model to explore the comparative lung pharmacokinetics of inhaled drugs.To summarize, we employed two different models to monitor the distribution kinetics of two HFA-propelled BDP aerosols from human lung tissue into extra-pulmonary compartments. With both the dialysis setting as well with the human lung perfusion model we detected significant differences between the two BDP formulations. The HFA-BDP formulated as Ventolairposition ,35, they


Checkpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired. In fission yeast, checkpoint mechanisms are known to regulate entry into mitosis, but so far no checkpoint inhibiting S phase entry has been identified.Schizosaccharomyces pombe spores to UV irradiation in G1. When germinating spores are irradiated in early G1 phase, entry into S phase is delayed. We argue that the observed delay is caused by two separate mechanisms. The first takes place before entry into S phase, does not depend on the checkpoint proteins Rad3, Cds1 and Chk1 and is independent of Cdc2 phosphorylation. Furthermore, it is not dependent upon inhibiting the Cdc10-dependent transcription required for S phase entry, unlike a G1/S checkpoint described in budding yeast. We show that expression of Cdt1, a protein essential for initiation of DNA replication, is delayed upon UV irradiation. The second part of the delay occurs after entry into S phase and depends on Rad3 and Cds1 and is probably due to the intra-S checkpoint. If the germinating spores are irradiated in late G1, they enter S phase without delay and arrest in S phase, suggesting that the delay we observe upon UV irradiation in early G1 is not caused by nonspecific effects of UV irradiation.We have studied the response of germinating S. pombe spores to UV irradiation in G1 and shown that S phase entry is delayed by a mechanism that is different from classical checkpoint responses. Our results point to a mechanism delaying expression of proteins required for S phase entry.We have studied the response of germinating  They prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired . Checkpoand cds1 -13 The cewed in . Rad3 i protein ,16. Rad3 protein  The Rad1 protein -21. Rad1 protein , a compl2 in a Rad3 dependent manner [There are two known effector kinases downstream of the checkpoint Rad proteins, Chk1 and Cds1. Chk1 is phosphorylated in response to DNA damage induced in late S or Gt manner ,23,24. Pt manner  and is ot manner ,26 Activt manner -29.Crb2 and Mrc1 act upstream of Chk1 and Cds1, respectively. Crb2 shares homology with the budding yeast RAD9 protein , which icdc10 arrest point was shown to depend on Chk1 [orp1 mutant cells depends on the checkpoint Rad proteins and on Chk1 [In addition to the G2/M, S/M and intra-S checkpoints, three papers have reported the existence of G1 checkpoints that inhibit mitosis when the cells are arrested in G1 using cell cycle mutants. Arrest at the  on Chk1  and Rum1 on Chk1 . Arrest  on Chk1 . It shouS. pombe. The drop of CDK activity at the M/G1 transition allows the assembly of the pre-Replication Complex, preRC, which is the first step leading to initiation of S phase. The preRC consists of the ORC (Origin Recognition Complex), Cdc18, Cdt1 and the MCM proteins. Expression of Cdc18 and Cdt1 is cell cycle regulated, thus providing one of the means to regulate initiation of S phase [Xenopus that the intra-S phase checkpoint cannot be engaged until polα-primase is loaded and replication begins [A G1/S checkpoint has so far not been detected in  S phase ,38 Once n begins ,40 This S. pombe due to poor nutrient availability. We decided to use several approaches to synchronise the cells and/or to extend G1. Recently we reported the existence of a mechanism that delays entry into S phase when cycling cells are UV-irradiated in G1, using cdc10 and cdc25 mutants to synchronise the cells or growing the cells in medium where G1 is extended [G1 in fission yeast is very short under standard laboratory growth conditions, rendering the investigation of a G1/S checkpoint(s) difficult. However, G1 might be much extended in the natural habitat of extended .S. pombe spores delay entry into S phase upon UV irradiation in early but not late G1. We demonstrate that there is a G1/S delay that is not dependent on any of the known checkpoint proteins and does not target Cdc2 phosphorylation. We argue that the delay is due to a novel mechanism that leads to delayed expression of Cdt1 and possibly other replication proteins.Here we show that germinating 2, which gave a cell survival of about 30% in wild type cells (data not shown). At the time of irradiation, the majority of germinating spores had a 1C DNA content. The timing of S phase was measured, by flow cytometry, as an increase in cellular DNA content from 1C to 2C. The decrease of the 1C population was plotted against time, and the graphs for unirradiated control and UV irradiated cells were compared at the point where 50% of the cells had 1C DNA content.Spores made from diploid cells were allowed to germinate for 3.5 h at 30°C before UV irradiation. At this time point, 1 – 2 hours before S phase entry, the spores showed visible signs of germination by phase contrast microscopy. The dose of UV light was 1200 J/mAn inherent problem in the present experiments is that the time of germination varies both within each population of spores (low degree of synchrony) and between the different preparations (experiment-to-experiment variation). Thus, the time from resuspension in medium until the cells enter S phase is variable, and it is difficult to ensure that irradiation occurs at exactly the same time point relative to S phase entry. Therefore the experiments were repeated at least twice and the averages were determined Table . The expde novo synthesis of Cdc18 and Cdt1, which in turn are required for MCM loading. We investigated Cdt1 levels in germinated spores treated as above to establish the timing of the 1C delay relative to Cdt1 expression. Wild type spores carrying myc-tagged Cdt1 were UV irradiated as described above and samples of irradiated and control cells were removed for analysis by flow cytometry and by immunoblotting. Cdt1 expression was induced already at 30 minutes in the control cells, but not until 55 minutes later in the UV irradiated cells . We conclude that Rad26, Rad1, Rad9, Hus1 and Rad17 are required for at least a part of the 1C delay.We have investigated whether the other checkpoint Rad proteins are involved in the G1/S delay. cds1 and chk1 are both known downstream targets of the Rad3 protein kinase and they are required for Cdc2 phosphorylation in the DNA damage and replication checkpoints. We irradiated germinating spores carrying mutations of cds1, chk1 or both. In cds1 spores the delay was reduced to 45 minutes . We conclude that Mrc1 and Cds1 are required for part of the delay, while Crb2 and Chk1 are not required.Crb2 and Mrc1 are required for activation of Chk1 and Cds1, respectively. We irradiated germinating 1 spores . ConsistCdc2 kinase activity is required for the initiation of S phase and is inhibited by phosphorylation on Tyr15 as DNA replication commences ,47. We ires2 mutant should override the UV-induced G1 delay. We have irradiated germinating res2Δ spores as described above and found that S phase entry was not delayed compared to unirradiated control cells . We conclude that Cdc10 dependent transcription is not the mechanism of the delay.The above result indicates that either constitutive expression of Cdc10 dependent genes required for S phase entry can override the delay or inhibiting Cdc10 dependent transcription might be the mechanism of the delay. A prediction of the latter alternative is that cells arrested with 1C DNA content upon UV irradiation should not have performed the Cdc10-dependent transcriptional events, including induction of the We have provided evidence for the existence of a mechanism in germinating fission yeast spores that delays entry into S phase upon UV irradiaton in early G1. Germinating wild type spores displayed a pronounced delay in entering S phase after UV irradiation. The delay was observed only when irradiation was carried out in early but not in late G1. We have investigated the dependence of the delay on classical checkpoint proteins and showed that they are required for some but not all of the delay with 1C DNA content. We argue that the observed delay is caused by two separate mechanisms, the first taking place before entry into S phase, and the second in early S phase (see below).rum1 mutant, which presumably loses the G1 part of the delay. Third, mutants lacking Mrc1 or Cds1, which are essential for S-phase checkpoints reported so far in fission yeast [res2 mutant, which can not turn off the Cdc10 dependent transcription signal. The finding that a mutation affecting expression of proteins crucial for preparation for S phase abolishes the delay argues that the wild type cells first stop in G1 and only later stop in S phase.The delay in exit from the 1C population was demonstrated by means of flow cytometry, which does not allow us to distinguish between a G1/S and an early S delay. The following data represent strong evidence that part of the delay takes place before entry into S phase. First, the irradiated cells delay expression of Cdt1. In the absence of Cdt1 the cells cannot form preRCs and thus cannot initiate S phase. Second, the irradiated cells express Rum1 longer than unirradiated control cells. Since Rum1 expression is cell cycle regulated such that it is only expressed in G1, , this obon yeast , still don yeast . Fourth,on yeast ,47, argurad, cds1 and mrc1 mutants delay 30–50 minutes and rum1Δ cells lose ca 40 minutes of the delay, compared to wild type cells. The remaining ca 40 minutes of the total delay requires the checkpoint Rads, Mrc1, Cds1 and Cdc2 is phosphorylated. We argue that this part of the delay is brought about by the intra-S checkpoint. However, the resolution of our experiments is not high enough to exclude the possibility that some of the checkpoint Rad- and Cds1-dependent part of the delay occurs in late G1. We consider this possibility unlikely for two reasons; first, previous work has shown that the role of Cds1 is specific for S phase [On the basis of these results we conclude that there is a UV-induced G1 delay, which is not checkpoint Rad dependent and is brought about by an as yet undescribed mechanism. This part of the total delay with 1C DNA content is ca 40 minutes, since  S phase  and secoSince the level of synchrony is low in germinating spores, we have not emphasised minor differences in the timing of S phase entry. In spite of poor synchrony, we deem germinating spores a good model system, since spore germination is a natural phenomenon, it involves an extended G1 period and we observed clear-cut effects. Furthermore, this model system allowed us to investigate the effects of a number of mutations that would have not been possible using synchronisation by other methods.rad3 mutation does not abolish all of the G1/S delay. Since we have not identified a checkpoint mutation which abolishes the delay, we attribute the effect of caffeine to another effect than the inhibition of checkpoint proteins. Interestingly, recent data suggest that caffeine inhibits checkpoint responses without inhibiting the ATR and ATM kinases in human cells [We have explored whether the G1/S delay is caused by a checkpoint mechanism. We have shown that caffeine abolishes the delay, but this is not entirely due to inhibition of Rad3 activity, since a an cells ,52.Previously, Rhind and Russell  showed tWe have recently discovered a mechanism that delays entry into S phase in cells irradiated in early G1 in synchronised or in cycling cells  This inhS. pombe, Cds1, is not involved in the present pathway. The budding yeast G1/S checkpoint delays entry into S phase by phosphorylating and thereby downregulating Swi6, the homologue of Cdc10 [In budding yeast there is a G1 DNA damage checkpoint response that depends upon Mec1 ,32,54, aof Cdc10 . In contres2Δ overrides the delay, (3) transcription of Cdc10 dependent genes is not downregulated during the delay and (4) expression of Cdt1 is delayed, suggest that the G1/S delay is caused by delayed expression of Cdt1 and probably also of Cdc18 and Cig2. Since we have shown that transcription of Cdc10 regulated genes is not downregulated, the most likely mechanism of the delay is reduced translation rate of Cdt1 and possibly other proteins required for initiation of DNA replication.Other possible mechanisms for the G1/S delay include inhibition of Cdc2 by Rum1 or an as yet unidentified mechanism such as preventing the formation of Cdc2-cyclin complexes or by restricting the availability of cyclins. We have shown that Rum1 is expressed during the delay and is required for the G1 delay. This observation does not imply that Rum1 is a direct target of the G1/S delay, but this remains an attractive possibility. Another possible mechanism for the delay is delaying expression of proteins required for the initiation of DNA replication. In particular, the findings that (1) irradiation in late G1 does not cause a delayed entry into S phase, (2) increased transcription of Cdc10 dependent genes in Schizosaccharomyces pombe cells to UV irradiation in G1. We used germinating spores to exploit a natural phenomenon where the cells have a long G1. In this paper we provide evidence for the existence of a mechanism in fission yeast that delays entry into S phase upon UV irradiaton in early G1. The G1 delay is independent of classical checkpoint proteins and Cdc2 phosphorylation. Our results point to a mechanism that delays translation of proteins required for S phase entry.We studied the response of Schizosaccharomyces pombe L972h- strain. All basic growth and media conditions were as described [All our strains are derivatives of the escribed .h+ and h- strains carrying the met3 or ade1 complementing markers. The rum1:::ura4+/rum1+ diploid was made by protoplast fusion since rum1Δ is sterile [res2Δ  at 30°C overnight. The spores were washed twice in water and resuspended in EMM2 supplemented with adenine and methionine for germination and UV irradiation.Diploids were made by interrupted mating of  sterile . All dip meiosis ) and rum2. Cell survival was monitored by conventional plating on YE plates. The incident dose does not reflect the dose absorbed by the cells because UV light of this wavelength penetrates poorly into water. However, since irradiation conditions were constant, the incident dose was proportional to the absorbed dose.Cells were irradiated with 254 nm UV light while rapidly stirred in a thin layer (3 mm) of liquid medium. The dose administered was measured with a radiometer (UVP instruments) and an exposure time of 4 minutes gave an incident dose of about 1100 J/mProtein extracts for western blotting were made by TCA extraction, as described previously . For wes32P-labelled RNA probes, generated with T7 RNA polymerase . For cig2 and cdc18, the ORFs were inserted into pGEM-3 MCS to serve as template for producing the RNA probes. For cdt1, a PCR fragment of the ORF with T7 promoter sequence attached to the lower primer was used as template. Hybridisation was carried out using standard procedures and visualised by a STORM 860 Phosphoimager (Molecular Dynamics).Total RNA was isolated as described , resolve7 cells were spun down for each sample and fixed in 70% ethanol before storing at 4°C. Samples were processed for flow cytometry as described [About 10escribed  and staiescribed , and anaEAN showed the existence of the delay and investigated the roles of Rad3, Chk1 and Cds1, Cdc2 phosphorylation and Cdc10-dependent transcription as a potential mechanism. MS, TT and HV investigated the roles of further checkpoint proteins and that of Res2. EB participated in the design and coordination of the study and in writing the manuscript. BG devised the study, analysed Rum1 and Cdt1 expression and drafted the manuscript. All authors read and approved the final manuscript.


Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein , again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times . Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein  This unique "clinical window" has clear safety advantages over in vivo gene delivery since excess vector can be removed from the graft prior to coronary grafting. However, the clinical window is short (likely 10–60 minutes) and therefore necessitates the use of an efficient vector system for gene delivery. Adenoviral vectors have proven efficient for gene delivery in this context  of Ad vectors for 10, 30, 60 mins at 37°C. The cells were washed twice in PBS and the media changed. 24 hours post-infection, the cells were again washed in PBS, lysed in PBS/0.2% Triton-X-100 and transduction quantified using the Wallac 1420 (Victor2) Multilabel Counter with recombinant eGFP  as a standard. Reporter gene expression was normalised for total protein using the bicinchoninic acid (BCA) protein assay  with bovine serum albumin as standard, measured using a VICTOR2 plate reader. Exposure of HSVEC to Ad-CTL or Ad-RGD [50 plaque forming units (pfu) / cell] resulted in a time-dependent increase in the level of transduction  supplemented with 20% (v/v) foetal calf serum . HSVSMC were grown from medial explants from the same material and maintained in Dulbecco's modified Eagle's medium (DMEM) with 4500 mg/l glucose supplemented with 20% (v/v) FCS and 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mmol/l L-Glutamine. All cells were grown in a humidified atmosphere with 5% COn Figure . At eachn Figure . Fluoresn Figure  at all tn Figure . HSVSMC n Figure , consistn Figure . Again, 9 pfu / ring) before being washed twice in PBS and maintained in organ culture for 5 days. Rings were maintained in wash medium supplemented with 30% (v/v) FCS. Vein rings were snap frozen in liquid nitrogen and homogenised using a mortar and pestle for determination of reporter gene expression 5 days post-infection. Ex vivo homogenates were suspended in 100 μL reporter lysis buffer (RLB) and kept on ice for 1 hour before supernatants were analysed for luciferase expression using the Luciferase Assay System (Promega). 96 well plates were prepared using 10 μL/well of homogenate suspension diluted to a total volume of 100 μL with RLB. 100 μL of Luciferase Assay Reagent was added to each well and the plate immediately read for 10 seconds per well. Detection was achieved using a Wallac 1420 (VICTOR2) Multilabel Counter with recombinant luciferase (Promega) as a standard and normalised for total protein. Ad-RGDLuc mediated a time-dependent increase in the level of transgene expression that was evident at all exposure times studied – 10, 30 and 60 minutes . Individual vein rings were incubated with Ad vectors for 10, 30 or 60 minutes  and the British Heart Foundation (PG03/031 to A.H.B.)


Using data from model organisms, the authors have generated a large-scale human protein-protein interaction map. The map can be used to predict the function of human proteins. Protein-interaction maps are powerful tools for suggesting the cellular functions of genes. Although large-scale protein-interaction maps have been generated for several invertebrate species, projects of a similar scale have not yet been described for any mammal. Because many physical interactions are conserved between species, it should be possible to infer information about human protein interactions (and hence protein function) using model organism protein-interaction datasets.Here we describe a network of over 70,000 predicted physical interactions between around 6,200 human proteins generated using the data from lower eukaryotic protein-interaction maps. The physiological relevance of this network is supported by its ability to preferentially connect human proteins that share the same functional annotations, and we show how the network can be used to successfully predict the functions of human proteins. We find that combining interaction datasets from a single organism (but generated using independent assays) and combining interaction datasets from two organisms (but generated using the same assay) are both very effective ways of further improving the accuracy of protein-interaction maps.The complete network predicts interactions for a third of human genes, including 448 human disease genes and 1,482 genes of unknown function, and so provides a rich framework for biomedical research. Physical interactions between proteins underpin most biological processes. For this reason, large-scale protein-interaction mapping projects have been initiated in several model organisms -6. UnforTo transfer information on gene function between two genomes requires the identification of orthologous genes in the two genomes . However, the identification of gene orthologs is often not a trivial problem; gene duplications can result in a single gene having multiple potential orthologs in a second species. In addition, it is necessary to distinguish true gene orthologs from 'out-paralogs'  . One metSaccharomyces cerevisiae [Drosophila melanogaster [Caenorhabditis elegans [Protein interactions are often evolutionarily conserved between orthologous proteins from different species . Hence wrevisiae -4,10,11,nogaster  and Caen elegans . For eac elegans . A human elegans .In the absence of a comprehensive set of verified human protein interactions, we required another method to assess the accuracy of the interaction network. Proteins that interact physiologically are expected to have related functions. Therefore high-quality interaction datasets should predict a greater proportion of interactions between functionally related proteins than low quality datasets. The functions of human proteins can be systematically described using the Gene Ontology (GO) annotations  availablWe then used the same strategy to compare the accuracy of human interactions predicted by data from the three different model organisms. If the interactions from a particular model organism dataset predict fewer interactions between functionally related human proteins than the other datasets, then this dataset should be considered less reliable as a source of candidate human protein interactions. As shown in Table Next we asked how the confidence in the assignment of gene orthologs affects the accuracy of an interaction. For each predicted interaction, an orthology confidence score was calculated by summing the InParanoid orthology confidence scores for the two human and two model organism proteins . Of the predicted interactions, 24,897 have the maximum possible confidence score of 4. Of these interactions, 28%, 24% and 13% connect proteins that share GO terms at depths of 3, 5 or 7 in the GO hierarchy (excluding proteins without GO annotation). In contrast, for interactions with an orthology confidence score less than 4, these figures are 24%, 20% and 10%. Hence we conclude that the predicted human interactions with high-confidence orthology assignments can be considered more reliable than those interactions with less confidence in their orthology assignments. This confirms that the confidence scores assigned using InParanoid are indeed likely to be useful predictors of functional conservation.The worm and fly interaction mapping projects both defined a subset of high-confidence 'core' interactions that have the greatest experimental support Figure . For theIt has been shown previously that protein interactions detected by more than one high-throughput interaction assay are more accurate . We findBecause physiologically interacting proteins often have similar functions Figure , it shouThe ability to provide a reasonably accurate prediction of a gene's GO terms means that we can use the interaction network to provide probabilistic gene function predictions for novel human proteins and also to predict additional functions for proteins with some known functions. The core interaction map contains 864 proteins with no functional annotations. About 10% of these proteins interact with two or more proteins that share GO terms. The probabilistic predictions of the functions of these novel proteins are listed in Additional data file 4. Often these predicted functions are also supported by the known functions of the protein domains predicted to be encoded by these novel genes . For example, ENSG00000028310 encodes a bromodomain and interacts with six proteins annotated as 'GO:0006355 regulation of transcription, DNA-dependent', ENSG00000080608 encodes an RNA-binding domain and interacts with five proteins annotated as 'GO:0006364 rRNA processing', and ENSG00000104863 encodes a PDZ domain and interacts with three proteins with the annotations 'GO:0005887 integral to plasma membrane, GO:0007242 intracellular signaling cascade' . The complete and core interaction maps also predict interactions for 448 and 292 human disease genes , of which 55 interact with two or more proteins in the core interaction network that share a GO annotation. The functional predictions for these 55 genes are listed in Additional data file 6.We report here the use of data from model organism protein-interaction mapping projects to predict a network of human protein interactions. This network consists of over 70,000 interactions that connect over one-third of all the predicted human proteins, including 1,482 proteins of unknown function and 448 proteins encoded by human disease genes. The physiological relevance of this network is supported by its ability to preferentially connect human proteins that share biological functions Figure . As suchThe accuracy and coverage of the interactions predicted in this network depend primarily on two parameters: the quality of the original model organism interaction datasets; and the ability to identify the human orthologs of a model organism protein. Our analysis suggests that the raw yeast and worm protein-interaction datasets are currently slightly more accurate than the raw fly interaction dataset, but that when filtered for high-confidence interactions the three interaction maps are of very similar accuracy . Table The human orthologs of yeast, worm and fly genes were identified using the InParanoid algorithm, which is designed to distinguish true orthologs from out-paralogs that arose from gene duplications before the divergence of two species . The InPCore interactions were defined as those predicted by worm interactions identified more than once or that reconfirmed when retested in the two-hybrid assay , by fly Human GOs (at levels 3 or deeper in the GO hierarchy) were obtained from Ensembl (v19.34a.1) ,15 usingx or more of its interaction partners (where x varied from 1 to 6). To validate the accuracy and coverage of this approach we predicted GO terms for genes that already have associated GO terms. The accuracy was calculated as the total number of correct GO term predictions divided by the total number of GO term predictions. The coverage was calculated as the total number of correct GO term predictions divided by the total number of known GO terms. This analysis was repeated, but only considering individually GO terms at depths of 3 to 8 and greater than 8 in the GO hierarchy . To avoid biasing the results we again ignored self-interactions. For the same reason, we also only counted once GO terms associated with more than one interaction partner predicted by the same source interaction from a model organism. The InterPro protein domains [To predict the GO terms of a protein, we identified all the GO terms associated with  domains  encoded  domains .Saccharomyces cerevisiae [Drosophila melanogaster [Caenorhabditis elegans [The following additional data files are available with the online version of this article: Additional data file revisiae -4,10,11,nogaster  and Caen elegans .et al. [et al.'s confidence assignment; and whether the interaction was previously known in the literature. For more information, please see [Additional data file et al.  and contease see .Additional data file A complete list of predicted human protein interactionsClick here for additional data fileA list of all core human protein interactionsClick here for additional data fileThe accuracy and coverage of GO term predictions at different levels in the GO hierarchyClick here for additional data fileGene function predictions for 85 human genes of unknown functionClick here for additional data fileHuman disease genes with predicted protein interactionsClick here for additional data fileGene function predictions for 55 human disease genesClick here for additional data file


Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp) promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes.2C+1TY(T)2(Y)3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point.A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y)This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes. Ribosomes are vital organelles, which catalyze protein synthesis in all living organisms. Eukaryotic ribosomes consist of four RNA molecules (rRNAs) and 79 different proteins. The mammalian genes encoding the rRNAs are multicopy and clustered at a few loci, whereas those encoding the ribosomal proteins (rp genes) are single copy and scattered throughout the genome . In addiCoordinated expression of the rp genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. Transcriptional run-on measurements with nuclei of rapidly proliferating cells indicated equivalent loading of RNA polymerases on three unlinked mouse rp genes , consistDespite the obvious importance of rp gene expression for cell viability, there have been very few experimental studies of rp promoter architecture and transcriptional regulation in higher eukaryotes. To date, efforts to identify functionally relevant cis-acting regulatory elements and transcription factor binding sites have been made for only 9 mammalian ,10-23 anBecause the previous experimental studies were limited to a small subset of rp genes, it is important to know whether the results of those studies can be used to predict special features of promoter architecture that characterize the entire set of rp genes or that could be used to sort the rp genes into classes with similar features. This applies not only to transcription factor-binding sites, but also to the polypyrimidine tract that spans the tsp. As an initiator element, this tract is atypical because the tsp of initiators is normally an adenine residue, flanked on both sides by pyrimidines .To address these issues, I have applied the principle of "phylogenetic footprinting", which holds that important regulatory sequences will have a tendency to be evolutionarily conserved and thus revealed by a sequence comparison of corresponding regions of orthologous genes . Some reTo make a study of the evolutionarily conserved features of ribosomal protein gene promoters, I first had to assemble a set of orthologous human and mouse rp gene sequences, in which the transcription start sites are reasonably well defined. For all of the human rp genes, annotated sequences are now available owing to the studies of Kato, Kenmochi and coworkers ,33, who In contrast to the situation with the human rp genes, accurate determinations of the tsp's of mouse rp genes have been made in only a few cases, and therefore the annotations of rp genes in the mouse genome databases are generally incomplete. To overcome this problem, I used the mVISTA alignment program  to comparpS16, rpL30 and rpL32 ). These genes were selected because the mouse orthologues have been previously studied extensively in my laboratory. Scans with a 50 bp window indicated that the conserved (> 75 % identity) flanking sequences are largely confined to regions within a few hundred base pairs of the tsp. There was very little alignment of the upstream region sequences and many gaps caused by the presence of numerous insertion elements. With this analysis, I detected only one short block of conserved sequence at -2 kb in rpL30 (77 % identity over 84 bp), one short block at -1.5 kb in rpL32 (82 % identity over 60 bp), and none in rpS16 . Neither of the conserved blocks contained any recognizable transcription factor binding sites or other remarkable characteristics. The lack of any credible long-range regulatory elements is consistent with earlier conclusions based on transient transfection experiments with these genes [In a preliminary analysis, I used the mVISTA sequence alignment program to examine six to ten kb of 5' flanking sequence of three pairs of orthologus rp genes  in the 5' flanks of a very high proportion of the rp genes see . Indeed,To evaluate the quality of TATA box motifs, I established criteria based on rules derived from a crystallographic analysis of TBP-DNA complexes . With thEmploying stringent criteria Figure , I used rpL13a (panel d) and within exon 2 of rpL30 (panel b), whereas in rpS18 (panel a) and rpS4 (panel c), it is at the extreme 3' end of exon 1. Many rp genes have this latter feature, which may be relevant to the evolution of vertebrate rp promoters (see below).Illustrative examples of four annotated promoter sequence comparisons are shown in Figure rpS18 promoter contains a conserved TATA (+) motif, while the TATA motifs of rpS4 and rpL13a were scored as (+/-) and that of rpL30 as (-). Well-conserved  consensus binding sites for GABP and/or Sp1 are located upstream of the tsp in all four examples; downstream YY1 sites are evident in rpL30 and rpL13a . The rpS18 and rpL13a promoters also contain conserved consensus binding sites for the ubiquitous AP1/ATF factors. The promoter elements that were found to have functional significance in experimental studies of the mouse rpL30 gene [rpL30 and rpL13a .The L30 gene  are all rpL29 ). In 29 % of the rp genes the AUG codon is at the extreme 3' end of exon 1 and in 47 % it is in exon 2. Thus, in 3/4 of the rp genes, the genetic elements involved in transcriptional/translational regulation are spatially separated from those with protein encoding function.The results of this type of analysis for the full set of rp promoters are tabulated in Table Contrary to previous impressions based on an incomplete set of rp genes, the rp promoters cannot generally be classified as "TATA-less". Thirty-five percent of the promoters contain, in the -25 region, a TATA box that should theoretically bind TBP with high affinity. An additional 25 % have an A/T-rich tract in this region, which might bind TBP with lower affinity, and the remainder would not be predicted to bind TBP without help from other proteins. For the most part, the TATA box quality is well conserved between the two mammalian species: the assessed quality of human and mouse TATA motifs was the same in 82 % of the rp promoters.The prevalence in rp promoters of evolutionarily conserved GABP, Sp1 and YY1 binding sites is readily apparent from these results. Conserved upstream GABP and Sp1 sites are present in 54 % and 48 % of the rp promoters, respectively. Conserved downstream YY1 sites are present in 52 % of the rp promoters. The ratio of aligned to unaligned sites in the human rp promoters is approximately 2:1 for GABP, 1:1 for Sp1 and 4:1 for YY1. The occurrence of unaligned sites in the mouse rp promoters is similar to that in the human rp promoters see . It is n2C+1TY(T)2(Y)3. A striking result is that T's are strongly preferred over C's at positions +2, +4 and +5. This preference, which is graphically illustrated in Figure The existence of a novel polypyrimidine initiator sequence in which the conventional A residue at the tsp is replaced by a C residue is well known. Moreover, the roles of this sequence in rp gene transcription and rp mRNA translation have been previously demonstrated experimentally for a few rp genes ,40-42. WrpS3  for mammals and lower vertebrates. Yet, the particular C residue within the initiator that is used as the major tsp may differ slightly. Also the adjudged quality of the TATA box for TBP binding is not always the same. Nevertheless, except for the location of upstream transcription factor-binding sites, the general features of rp promoter architecture are usually well conserved over large evolutionary distances.To determine the features of rp promoter architecture that are conserved over large evolutionary periods, I compared the promoter sequences of five chicken, six amphibian and five fish rp genes to their mammalian orthologues using the Clustal W multiple sequence alignment program. An example of such an alignment for 3 Figure  shows st3 Figure  indicategas5 and U17HG ). Annotated aligned sequences of the nine human and mouse non-rp TOP genes are presented on pages 1 through 9 of a supplementary file [see It is of interest to know which features of rp promoter architecture are specific and which might be common to other ubiquitously expressed genes. To this end, I examined the promoters of two additional sets of genes. One set consisted of non-rp genes that also produce translationally controlled mRNAs with 5'-terminal oligopyrimidine tracts (non-rp TOP genes). At the present, there are nine known genes in this category for which the tsp of at least one orthologue has been experimentally determined see . The nonfile see  and the There are some notable differences in the promoter architecture of rp genes and non-rp TOP genes. First, GABP- and YY1-binding motifs, which are prevalent in the rp promoters, are rarely found in the non-rp TOP promoters Table . Second,The second set of ubiquitously expressed genes consisted of 20 housekeeping genes randomly selected from the eukaryotic promoter database see . The proThe foregoing analysis of 79 mammalian ribosomal protein genes has revealed several features of rp promoter architecture, some of which are largely conserved over long periods of vertebrate evolution (about 450 million years) and others that are strongly conserved only in mammals. (about 90 million years). One highly conserved feature, present in over 3/4 of the rp genes, is the separation by an intron of the sequences involved in transcriptional and/or translational regulation from the sequences with protein-encoding function. In 47 % of the rp genes, the AUG translation initiation codon is in exon 2, and in 29 % of the genes, it is at the extreme 3' end of exon 1. It would seem that at an early stage of vertebrate evolution, these regulatory sequences were appended as discrete units to the loci containing the protein-encoding sequences.2C+1TY(T)2(Y)3. Thus, in addition to the C at the tsp, there is a clear preference for T over C at positions +2, +4, and +5. This preference may reflect a structural bias for transcription, e.g., ease of strand separation, or for translation, e.g., affinity of rp mRNAs for a putative repressor.The polypyrimidine tract that spans the tsp is present in all vertebrate rp genes. This tract can function as a transcriptional initiator ,41 and aThe presence of transposable element residues in the 5' flanks of the rp genes is noteworthy. Half of the rp genes contain an element  within 500 bp of the tsp, and over 80 % of the genes contain an element within 900 bp. Some elements are moderately conserved between mouse and human, but most appear to be lineage-specific. These elements are unlikely to have any specific role in rp promoter function. They may be passively tolerated because the vast majority of conserved 5' sequence is confined to within 200 bp of the tsp. Within a segment from -200 to +100, the sequence identity between human and mouse rp orthologues ranges from 50 to 75 % with an average of 61 %, whereas the sequence match beyond -200 is of borderline significance.rpL32 , might bind TBP weakly without help from an additional protein [The observation that 35 % of the rp promoters contain a TATA box motif at -25, which would be predicted to bind TBP with high affinity, and that an additional 25 % possess A/T-rich motifs, which might bind TBP with lower affinity, was unexpected. The assessed quality of these motifs for TBP binding, made according to rules established by a detailed structural study of TBP-DNA complexes , was lar protein ,44, so tWhen human and mouse rp promoter regions from -200 to +100 were scanned for conserved  transcription factor-binding sites with the rVISTA program , which irpL30 and rpL32 genes [rpS14 and rpS6 genes [Xenopus rpL18 gene [rpS16 gene, GABP binding at the initiator decreases transcription activity both in vivo and in vitro [GABP is a heteromeric protein consisting of an ETS family member, which has DNA-binding capability, and an ankyrin repeat-containing subunit, which greatly improves the stability of GABP-DNA interactions ,46. Two-32 genes ,14, the S6 genes ,22, and L18 gene . The GABin vitro . For simrpL32 [rpL30 , a downstream YY1 interaction also contributed positively to transcriptional activity, but only when a strong upstream interaction with GABP was eliminated [Xenopus rpL18 was shown to interact with the frog homologue of YY1, but the functional significance of this interaction was not demonstrable with the reporter constructs that were used in these experiments [YY1 is a zinc finger-containing protein with a variety of gene-specific functions, including transcriptional activation and repression, positioning of RNA polymerase II, and chromatin modification ,48. FiftrpL32 . In mousiminated . Intereseriments . The diveriments , have leBased on the results of the analysis of rp promoter sequences, I have sorted the different rp promoters into eight classes according to whether they possess conserved binding sites for the three prevalent transcription factors, GABP, Sp1 and YY1 Figure . Only 10RACK1 gene is embedded in a polypyrimidine tract that conforms perfectly to the rp initiator consensus sequence. Moreover, the promoter contains a TATA box of +/- quality, an aligned upstream binding motif for Sp1 and an aligned downstream motif for YY1, but no readily detectable motifs for other ubiquitous transcription factors. Interestingly, the features of this promoter resemble those of the rpL13a gene, which also encodes a protein with apparent pleiotropic function.It is worth noting that some ribosomal proteins can also have extraribosomal functions ,51. AmonThis analysis has highlighted features of rp promoter architecture that are shared by a high proportion of the rp genes. The evolutionary conservation of these features lends strong support to their functional relevance. Yet, superimposed on this general design are variations that confer certain idiosyncratic characteristics on each promoter. There does not seem to be a single master switch that co-regulates all rp genes at the transcriptional level. Rather, the rp promoters are tuned to respond to a combination of factors, including components of the general transcription machinery, a relatively small group of sequence-specific transcription factors, and modifiers of chromatin structure. The inherent functional redundancy and lack of dependence on any single factor are useful design features for genes that must be expressed in a broad spectrum of cell types and environmental situations.2C+1TY(T)2(Y)3. Contrary to previous impressions based on studies of a small subset of rp genes, the majority of rp promoters contain a TATA box or an A/T-rich motif at -25 that should theoretically have TBP-binding capability. Similarly, approximately half of the rp promoters contain conserved binding motifs for transcription factors previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Conserved motifs for other ubiquitous factors occurred much less frequently. Transposable element residues within 900 bp of 5'-flanking sequence were present in over 80 % of the rp genes; very little sequence conservation was evident more than 200 bp upstream of the tsp. Some of these architectural features were seen to be specific for rp promoters. From the results of this analysis, it was possible to sort the rp promoters into eight classes according to their possession of putative binding sites for GABP, Sp1 and YY1, and also to specify which promoters should have intrinsic affinity for TBP. This classification might prove useful for interpreting the results of experiments in which cell-specific or physiologically induced variations in the expression of different subsets of rp genes are observed.A sequence comparison of the promoter regions of all 79 orthologous human and mouse ribosomal protein genes has revealed several evolutionarily conserved features that are characteristic of a high proportion of the rp gene set. One such feature, which is also evident in the rp genes of lower vertebrates, is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which in mammals conforms to the consensus (Y)The rp gene sequences were extracted from three database sources see . The vasrRNA, ribosomal RNA; rp, ribosomal protein; tsp, transcriptional start point; TOP, terminal oligopyrimidine sequence; TBP, TATA-binding protein.ST1: Identification of the transcriptional start points (tsp's). Comparison of experimental determinations of the tsp's of human and mouse rp genes.Click here for fileST2: Promoter region sequences of rp genes. Sources of rp sequences, amount of analyzed sequence 5' of tsp, and locations of insertion elements nearest the tsp.Click here for fileAligned promoter sequences of rp genes. Annotated sequence alignments of 79 orthologous human and mouse rp promoters and a key to the annotation.Click here for fileST3: Characterization of the rp initiator. Occupancy matrix for determination of the consensus sequence of the mammalian rp initiator.Click here for fileST4: Promoter region sequences of non-rp TOP genes. Sources of non-rp TOP gene sequences, the amount of analyzed sequence 5' of the tsp, and location of insertion elements nearest the tsp.Click here for fileAligned promoter sequences of non-rp TOP genes. Annotated sequence alignments of 9 orthologous human and mouse non-rp TOP promoters.Click here for fileST5: Housekeeping gene promoters. List of 20 housekeeping gene sequences extracted from the Eukaryotic Promoter Database, which were analyzed for GABP, Sp1 and YY1 binding sites and for TATA box quality.Click here for file