N is a TIR-NB-LRR R gene that confers resistance to Tobacco mosaic virus, specifically the p50 helicase domain. An intriguing question is how plant R proteins recognize the presence of pathogen-derived Avirulence (Avr) elicitor proteins. We have used biochemical cell fraction and immunoprecipitation in addition to confocal fluorescence microscopy of living tissue to examine the association between N and p50. Surprisingly, both N and p50 are cytoplasmic and nuclear proteins, and N's nuclear localization is required for its function. We also demonstrate an in planta association between N and p50. Further, we show that N's TIR domain is critical for this association, and indeed, it alone can associate with p50. Our results differ from current models for plant innate immunity that propose detection is mediated solely through the LRR domains of these molecules. The data we present support an intricate process of pathogen elicitor recognition by R proteins involving multiple subcellular compartments and the formation of multiple protein complexes.Plant innate immunity is mediated by Resistance (R) proteins, which bear a striking resemblance to animal molecules of similar function. Tobacco  Resistance (R) gene encodes a protein that specifically recognizes and protects against one pathogen or strain of a pathogen carrying a corresponding Avirulence (Avr) gene. In tobacco and its relatives, the N resistance protein confers resistance to infection by the Tobacco mosaic virus (TMV).Each year, up to 10% of world agricultural production is lost to pests and diseases caused by a variety of pathogens including bacteria, fungi, nematodes, and viruses. Scientists have understood for nearly a century that plants carry their own immune system that actively engages pathogens and prevents many infections. One aspect of the plant immune system is defined by the gene-for-gene hypothesis: a plant We have used N, and the TMV elicitor, p50, to investigate the mechanism of gene-for-gene resistance. We show that N and p50 associate in the cytoplasm and nucleus of plant cells and that this association is mediated by N's TIR domain, which is structurally similar to animal innate immunity molecules. Our findings provide novel insight into how R proteins recognize pathogen Avr proteins, and should help in long-term efforts to enhance crop yield. How do plant resistance gene recognize the cognate pathogen? These authors dissect the interaction of a resistance gene and a viral pathogen. Resistance (R) genes, and each R protein typically initiates a defense response in the presence of one pathogen-derived elicitor protein that is termed the Avirulence (Avr) determinant [Plants, like animals, are able to launch successful defense responses against invading microorganisms. For this purpose, plants have developed a variety of strategies that include molecular, chemical, and physical barriers to infection. One of the most important of these defense systems relies on germline-encoded molecules that can specifically recognize a particular pathogen or strain of a given pathogen. These molecules are encoded by erminant . The generminant , and thiR genes have been cloned [R genes are structurally similar to each other and contain a few, conserved domains. The leucine-rich repeat (LRR) domain is the most common domain among R proteins, and it is also found in animal innate immunity molecules, including Toll from Drosophila and Toll-like receptors (TLRs), and nucleotide binding-oligomerization domain proteins (NODs) from mammals [Over the last several years, approximately 40 n cloned . These g mammals ,4. Membe mammals ,5. The N mammals .R-Avr systems. One interesting aspect of R protein function is its localization. These proteins have been found in a variety of cellular locations, depending on the localization of the eliciting pathogen or its Avr determinant. For example, the tomato Cf proteins, which recognize extra-cellular Cladosporium fulvum Avr proteins, are localized to the plasma membrane [Arabidopsis RPM1 and RPS2 are associated with cellular membranes although they do not possess any canonical membrane-targeting domains [Solanaceae R protein Bs2 [In recent years, extensive molecular and genetic analyses have been performed in a number of membrane . Interes domains ,9. This  domains . Apart f domains . Howevertein Bs2  and for tein Bs2 . One cavArabidopsis, both RPM1 and RPS2 and their cognate Avr elicitors interact with a host protein RIN4 [Arabidopsis RPS5 and PBS1 [In addition to studies on localization, researchers have also identified some of the host proteins that are involved in R protein activation and signaling downstream of the activation event. Recent work from several groups has attempted to address a central issue in plant innate immunity: how R proteins recognize pathogen-derived Avr proteins and initiate a defense response. Early models of the R-Avr relationship proposed a direct interaction between the host and pathogen proteins based on the gene-for-gene hypothesis. However, a direct interaction has been demonstrated for few R-Avr pairs: ,14–18. Iein RIN4 ,20. RIN4ein RIN4 , as welland PBS1 . Thus, dand PBS1 . Interesand PBS1 ,To date, an in vivo association between an R protein and its corresponding Avr protein has not been demonstrated. This is true even for cases in which a direct interaction has been demonstrated in yeast two-hybrid assays or in vitro. There is no obvious biological explanation for this seeming anomaly. One possibility is that detection has been technically challenging, presumably because R proteins are present at relatively low levels in plant cells . AttemptNicotiana tabacum (tobacco) plants. Tobacco and other Nicotiana species carrying the N gene are resistant to infection by all strains of TMV, except the Ob strain [N gene has been cloned, and it encodes a TIR-NB-LRR R protein [N-containing plants by TMV is the formation of necrotic lesions at the infection sites [R gene–dependent defense response that is called the hypersensitive response (HR). The helicase domain of the TMV replicase proteins, termed the p50 protein, is necessary and sufficient to elicit an HR in N-containing Nicotiana plants [N–p50 interaction is generally assumed to indicate N function [One of the classic model systems for studying plant-virus interactions involves Tobacco mosaic virus (TMV) infection of b strain . The N g protein . The firon sites ,29. Thesa plants ,31. The N-mediated responses even when expressed exclusively in the cytoplasm. Once we determined that N and its elicitor are present in the same subcellular compartments, we then tested the association between N and p50. We show by co-immunoprecipitation and fluorescence microscopy-based assays in intact, living tissue that N and p50 associate in planta. This is the first report of R-Avr association occurring in living tissue undergoing a defense response. Another interesting finding is that the TIR domain of N is critical for this association to occur. These results propose additional functions for the TIR domain in addition to its known role as an adaptor for signaling in animal innate immunity. We propose that the N TIR domain acts as an adaptor between the pathogen Avr protein and the signaling function of the R protein.In this work, we have used the N-TMV system to investigate how an R protein recognizes its pathogen-derived elicitor protein. We set out to determine the subcellular localization of N and its Avr elicitor, p50. Using biochemical approaches and fluorescence microscopy, we show that N and p50 are both cytoplasmic and nuclear proteins. We also investigated whether nuclear localization was important for a defense response and found that N's presence in the nucleus was indeed required for its function. However, p50 could elicit Nicotiana benthamiana leaves with Agrobacterium cultures expressing gN-TAP and/or p50-U1-HA. Tissue co-expressing the two proteins exhibited cell death typical of the HR 2 d after co-infiltration  tag containing nine copies of the MYC epitope (9xMYC) to the C terminus of N . The fulltration A. Tissueltration A. The exltration B, lane 1ltration C, lane 1The subcellular localization of N and p50 was first examined by cell fractionation. Tissue transiently expressing gN-TAP was collected, and protein extracts were ultra-centrifuged to produce crude soluble (S100) and membrane (P100) fractions. The proteins in the fractions were separated by SDS-PAGE and analyzed by Western blot with anti-MYC antibodies. gN-TAP was found in the S100 soluble fraction of protein extracts in the absence of TMV or p50-U1  to geneN. benthamiana leaves by agroinfiltration. Sections were cut from the infiltrated leaves and observed under the confocal microscope. As expected, Citrine alone, driven by the strong 35S promoter, was localized to the cytoplasm and nuclei of cells . Therefore, we decided to use a chimeric p50 containing the first 192 amino acids from p50-U1 and the remaining sequence from p50-Ob (p50-U1-Ob) . The p50sistance . To inveanalysis C, lane 2mut) in which critical leucine residues have been substituted with alanine, fails to prevent N's nuclear localization  to the C-terminus of gN-Citrine. The NES sequence was derived from the human immunodeficiency virus-1 (HIV-1) Rev protein . As expemut and p50-U1-HA and examined whether an HR occurred. As expected, gN-Citrine and p50-U1-HA produced an HR . These results suggest that the nuclear localization of p50-U1 is not required for recognition by N and a subsequent defense response. Taken together, our data indicate that recognition may occur in the cytoplasm of plant cells, supporting the hypothesis that nuclear N has another function in addition to pathogen recognition.Similarly, we wanted to determine whether the nuclear localization of p50 was necessary for a defense response. Using the same strategy we had employed to investigate the function of nuclear N, we attached a C-terminal NES to p50-U1-Cerulean to determine its intra-cellular distribution and examine whether an HR still occurred in the absence of nuclear p50-U1. As expected, the NES prevented the nuclear accumulation of p50-U1-Cerulean-NES . Therefore, we allowed sufficient time for Agrobacterium to establish a successful infection and T-DNA integration, and then collected samples over a time course from 16 to 48 hpi. Extracts were tumbled with anti–green fluorescent protein (GFP) antibodies to immunoprecipitate p50-U1-Cerulean or Cerulean. Isolated immunocomplexes were analyzed by Western blot, and gN-TAP was detected with anti-MYC antibodies. We found that at 46 hpi, we were able to detect gN-TAP in immunoprecipitated p50-U1-Cerulean complexes  assay was used. BiFC, as originally described, splits a fluorescent molecule into two parts that are then individually fused to two proteins whose association is being investigated . If the 155), and the other contained the remaining Citrine sequence (YC155). As a control to show that the two portions of Citrine could reconstitute fluorescence, published interactions using the 14-3-3 protein, T14-3c, which is known to homodimerize, were repeated [We used Citrine for the BiFC analysis of N and p50-U1′s association. One tag contained the amino-terminal 155 amino acids . Samples were collected from plants expressing gN-YC and/or p50-U1-YC at 46 hpi, and observed under the confocal microscope. As expected, gN-YC and p50-U1-YC did not produce any fluorescence when expressed individually (155 (GUS-YC), we were not able to detect any fluorescence , was then tagged with YNrescence B and S1Crescence D. This i155 was fused to p50-U1-Ob to produce p50-U1-Ob-YC, and as expected, it did not produce fluorescence when expressed alone ng assay . A recenN. benthamiana leaves. At 46 hpi, tissue was collected and protein extracts prepared. Anti-GFP antibodies were used to immunoprecipitate p50-U1-Cerulean from extracts, and the precipitate was probed with anti-MYC antibodies after separation by SDS-PAGE. Surprisingly, mutants missing the P-loop of the NB, the entire NB, or the LRR retained the ability to co-immunoprecipitate with p50-U1-Cerulean . Each tagged mutant was then co-expressed with p50-U1-YC or GUS-YC as a control, and tissue was monitored for fluorescence at 46 hpi. The loss of the NB or LRR domain did not disrupt the ability of N and p50-U1 to associate, and fluorescence was detected in those samples  and N(W141S), disrupt sistance . TAP-tagN. benthamiana plants. Tissue was collected 46 hpi, and protein extracts were prepared. Co-immunoprecipitation using anti-GFP antibodies was performed, and the precipitate probed with anti-MYC antibodies. N(TIR)-TAP was found in complexes containing p50-U1-Cerulean -TAP. N(TIR)-TAP and p50-U1-Cerulean or p50-U1-Ob-Cerulean were co-transfected into Cerulean A, lane 1Cerulean A, lane 2155 to produce N(TIR)-YN, which was then co-expressed with p50-U1-YC or p50-U1-Ob-YC. Consistent with the co-immunoprecipitation results, fluorescence was detected when N(TIR)-YN complemented p50-U1-YC -YN and RPP5(TIR), respectively. The expression of these constructs was confirmed by Western blot (unpublished data). No Citrine fluorescence was observed in tissue co-expressing BS4(TIR)-YN and p50-U1-YC (We then investigated the specificity of the N(TIR)-p50-U1 association. To do this, we chose the TIR domains from two R proteins closely related to N, tomato BS4 and R domain , whereasR domain . The TIR6-p50-HA purified from E. coli. N(TIR) did not precipitate p50-U1 in this assay (We examined whether the association we observed between N(TIR) and p50-U1 was a direct interaction by pull-down assays, using in vitro transcribed and translated N(TIR) and (HIS)is assay , and thiR genes and several studies of direct interactions by yeast two-hybrid assays, we expected the LRR domain of N to mediate the association with p50. Surprisingly, we found that the TIR domain was not only necessary for association with p50, but also sufficient. We propose a new model for how N may recognize the presence of its elicitor in a plant cell . Consistent with this idea, host proteins like Arabidopsis RIN4 that interact with both an R protein and its corresponding Avr elicitor have been identified [Despite the availability of cloned entified ,20. IndeL alleles, which encode TIR-NB-LRR R proteins, have pointed to the LRR as being critical for determining the specificity of L-Avr recognition [L alleles that differ solely in the sequence of their LRRs confer resistance to different Avr determinants. This suggests that the specificity is derived from the ability of the LRR to associate with, and hence recognize, an Avr protein. This is supported by a recent report of direct L–AvrL interactions in yeast [R–Avr interaction. Interestingly, results from other R proteins support a possible role for domains other than the LRR in association with pathogen-derived elicitors. For example, Arabidopsis RPM1 interacts with the host protein RIN4 through its amino-terminus [Genetic analyses of flax ognition . L allelin yeast . The LRRin yeast  and, intin yeast . It is iin yeast . Our stuterminus ; RIN4 interminus . Pto andterminus . Thus, iAt first glance, our findings appear to contradict two recent papers that investigate the N-p50 association. The first suggests that N and p50 do not associate in plant tissue , whereasR–Avr interactions. Although most of these findings were unexpected, they are consistent with the emerging view that pathogen recognition is a complex process involving players other than the R and elicitor proteins. An intricate recognition system allows for the fine control of the output of this initial event in defense, an important consideration when the outcome for most cells that detect the presence of a pathogen-derived elicitor is death. We expect that our model for N, although differing in the small details, will hold true for other R proteins and their elicitors. It will be interesting to see whether other R proteins are also nuclear-targeted and if so, to determine what function these proteins perform in the nucleus. Also, the investigation of the formation of multiple complexes by different R proteins with their cognate elicitors in addition to high-resolution structures for R proteins will be most useful in explaining these interactions and how they culminate in defense. Finally, it will be exciting to investigate the nuclear–cytoplasmic partitioning of N and other R proteins, and determine the role of these proteins in the nucleus.We have proposed a complex model for p50 recognition by the N protein that involves different multiple compartments and different regions of N interacting with p50 at different times during the recognition event. Our model also explains the findings from flax (discussed above) that both the LRR and the TIR domains contribute to the specificity of mut. The N-terminal 465 nucleotides of Citrine sequence were amplified by PCR, and cloned into N and N deletion mutants in place of the TAP tag to generate N-YN and N deletion mutants-YN constructs. To generate the TIR domain fused to the TAP and YN155 tags, the TIR sequence was PCR amplified and cloned between the NcoI and SacI sites of the gN-TAP and gN-YN constructs. The TIR region of BS4 and RPP5 was PCR amplified from tomato and Arabidopsis cDNA, respectively, and cloned between NcoI and SacI sites of gN-TAP. The p50 region of TMV-U1 and Ob replicases was amplified using a primer containing 2xHA sequence, and cloned into pYL400, a T-DNA vector containing the 35S promoter and the NOS terminator cassette. Cerulean and the C-terminal 255 bases of Citrine were cloned into the 3′ end of p50 to generate the p50-Cerulean and p50-YC constructs, respectively. The HIV Rev NES sequence and NES mutant sequence [mut. To produce p50-U1-Ob constructs, p50-U1 sequence (nucleotides 1–576) and p50-Ob sequence  were amplified and inserted in place of p50-U1 sequence in p50-U1-HA, p50-U1-Cerulean, and p50-U1-YC plasmids. PCR-amplified p50-U1-HA was recombined into DEST17 vector  to generate (HIS)6-p50-HA. To generate GUS-YC, the GUS sequence was amplified from pCAMBIA3301 and was inserted in place of p50-U1 in the p50-U1-YC construct. All constructs were confirmed by DNA sequencing.To generate N-TAP and N deletion mutants-TAP constructs, the TAP tag consisting of 9xMYC-3xHis-3C protease cleavage site-2xIgG binding domain from the vector pYL436  was clonsequence  were incAgrobacterium cultures were grown overnight in LB medium containing appropriate antibiotic selections. Cells were pelleted at 3,000 rpm and resuspended in infiltration medium containing 10 mM MgCl2, 10 mM 2-morpholinoethanesulfonic acid (MES), and 150 μM acetosyringone, and incubated at room temperature for 2–3 h. Strains containing N-derived constructs were infiltrated into N. benthamiana leaves at an optical density (OD600) = 1.8, and those containing p50-derived constructs were infiltrated at OD600 = 1.0. For co-infiltration, equal volumes of Agrobacterium were mixed. Cultures were infiltrated into leaves with a 1-ml needleless syringe. N. benthamiana plants were grown on light carts under 24 h of light, and 4–5-wk-old seedlings were used for all assays.Protein was extracted from ground tissue with buffer containing 150 mM NaCl, 20 mM Tris/HCl, 1 mM EDTA, 1% Triton X-100, 0.1% β-mercaptoethanol, 1 mM PMSF, and complete protease inhibitors . Protein concentrations were determined by Bradford assay , and equal amounts were loaded onto polyacrylamide gels. Proteins were transferred to PVDF membrane  for Western blot analysis. Antibodies used were as follows unless otherwise stated: mouse anti-MYC , rat anti-HA , mouse anti-GFP (Covance), anti-mouse horseradish peroxidase conjugate , and anti-rat IgG peroxidase (Roche).×g at 4 °C. Extracts were ultra-centrifuged at 100,000×g at 4 °C for 1 h. The supernatant or soluble fraction (S100) was collected, and the pellet (P100) was washed with extraction buffer before resuspension in an equal volume of buffer as the S100. Western blot analysis was carried out as described above.Samples were ground in liquid nitrogen, and protein was extracted in buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 500 mM sucrose, 10 mM EDTA, 1 mM DTT, 1 mM PMSF, and complete protease inhibitors (Roche). Cell debris was spun down at 10,000×g, and extracts were incubated with 50-μl Protein A bead slurry  equilibrated in IP buffer. Samples were tumbled at 4 °C for at least 1 h. Protein A beads were spun down at 600×g and the supernatant collected. A total of 1-μl rabbit anti-GFP antibodies  were added to each 1-ml sample, and the mixture was tumbled at 4 °C for 2 h. A total of 50 μl Protein A bead slurry equilibrated in IP buffer was added to each sample, and the antibodies were allowed to couple to the beads with rotation for 1 h at 4 °C. Beads were spun down at 600×g and the supernatant discarded. Beads were washed three times with IP buffer. A total of 25-μl 4xSDS loading buffer was added to each sample and boiled for 4 min. Immunoblotting was carried out as described previously.Plant tissue expressing proteins of interest was collected and ground in liquid nitrogen. Protein was extracted with IP buffer containing 100 mM NaCl, 20 mM Tris/HCl (pH 7.5), 1 mM EDTA, 0.1 % Triton X-100, 10 % glycerol, 1 mM DTT, 2 mM NaF, 1 mM PMSF, and complete protease inhibitors (Roche). Cell debris was pelleted at 20,0006-p50-HA protein was produced in C43(DE3) cells  and affinity purified using nickel-NTA resin . Approximately 1 μg of purified protein was used to pull down 35S-Met-labeled in vitro–translated  N and N(TIR) as described in [(HIS)ribed in . TNT mixN. benthamiana leaves at approximately 46 hpi and infiltrated with water. The 458-nm and 514-nm laser lines of a 25-mW argon laser  and the 543-nm laser line of a 1-mW helium neon laser  with appropriate emission filters were used to image Cerulean, Citrine, and chloroplast autofluorescence, respectively. In some instances, 488-nm and 568-nm laser lines of a 15-mW argon:krypton laser (Coherent) were used for Citrine and chloroplast autofluorescence. All images were acquired in fastline switch mode and processed with the Zeiss LSM 510 (Ver. 3.2) channel unmixing algorithm to eliminate crosstalk.Live plant imaging was performed on a Zeiss Axiovert 200M light microscope equipped with a LSM 510 NLO confocal microscope  using either a 40× or 63× C-Apochromat water immersion objective lens . Tissue samples were cut from Figure S1(A) The YN and YC tags are functional when used to test known protein–protein interactions of the 14-3-3 protein, T14-3c (column 1). As expected, by themselves, they do not produce signal (columns 2 and 3). Fluorescence was imaged with the 514-nm laser line of a 15-mW argon laser. Scale bar represents 20 μm.(B) GUS-YC used in BiFC is functional as shown by blue color from enzymatic assay (left panels), whereas control infiltrated tissue does not show blue color (right panels).(C) GUS-YC carrying a single HA tag is detected with anti-HA antibodies (lane 1). p50-HA is shown for comparison (lane 2). M is the size marker, and protein size is shown in kDa.(D) When expressed alone, GUS-YC does not produce fluorescence. Scale bar represents 20 μm.(7.6 MB TIF)Click here for additional data file.Figure S2E. coli–purified (HIS)6-p50-U1-HA (lanes 1 and 2).(A) Western blot analysis of 35S-Met–labeled full-length N (lane 2) and 35S-Met–labeled N(TIR) (lane 4) . Five percent of input 35S-Met–labeled full-length N (lane 1) and 5% of input 35S-Met labeled N(TIR) (lane 2) are shown.(B) p50 fails to pull-down (1.6 MB TIF)Click here for additional data file.Figure S3N(TIR)-YC (column 1), BS4(TIR)-YC (column 3), and RPP5(TIR)-YC (column 5) do not produce fluorescence when co-expressed with GUS-YC. BS4(TIR)-YC (column 2) and RPP5(TIR)-YC (column 4) also do not exhibit BiFC with p50-U1-Ob-YC.(5.9 MB TIF)Click here for additional data file.


Therefore, we investigated the cellular distribution of different forms of E6 with reference to E6AP. E6 and E6*I proteins, expressed from a wild type E6 gene cassette, were dispersed in the nucleus and the cytoplasm. Whereas, the E6 splice donor mutant (E6MT) was primarily localized to the nucleus. E6*I protein and E6AP were found to co-localize mainly to the cytoplasm, whereas the co-localization of full-length E6 protein and E6AP, if at all, was found mainly at the perinuclear region. These results suggest a functional relationship between the E6*I and full-length E6 protein which correlates with their localization and likely is important in regulation of the E6-E6AP complex.High-risk Human Papillomavirus (HPV) is the etiological agent associated with the majority of anogenital cancers. The primary HPV oncogenes, E6 and E7, undergo a complex splicing program resulting in protein products whose purpose is not fully understood. Previous mouse studies have confirmed the existence of a translated product corresponding to the E6*I splice product. In terms of function, the translated E6*I protein has been shown to bind to E6 protein and to E6 associated protein (E6AP). E6*I has an inhibitory effect on E6-mediated p53 degradation in E6 expressing cells. In order to analyze the relationship between E6*I and full-length E6 in relation to localization, we created a series of green fluorescent protein (GFP) fusion products. The localization of these proteins with reference to E6AP  Human papillomavirus (HPV) is a ubiquitous sexually transmitted DNA virus. A subset of mucosal HPVs are termed "high-risk"  because of an increased association with cervical cancer . HoweveXhoI and KpnI sites. From previous studies [To overcome the difficulty in E6 protein detection by use of antibodies, GFP-linked E6 constructs were created Figure . HPV16 E studies ,17, simiWe observed the localization of the different forms of E6 protein in 293T cells and HaCaT cells. The results are shown in figure in vitro [As the E6 protein has been shown to associate with E6AP in HPV-induced cervical cancers, the location of these functional complexes is of importance. From results in figure in vitro ,15 and tin vitro , E6AP-inin vitro  or, in fIn addition, we observed stronger fluorescent signals with E6-transfected cells than from E6*I or E6MT-transfected cells (data not shown). More intense signals might suggest interaction with and stabilization by cellular proteins. This could involve the ability of E6 protein to oligomerize with E6 or E6*I protein . In otheIn conclusion, our results suggest a functional role for expression of E6*I protein in high-risk HPV-infected cancer cells. We propose a model as shown in figure Human papillomavirus (HPV), E6-associated protein (E6AP), 4',6-Diamidino-2-phenylindole (DAPI), green fluorescent protein (GFP)The author(s) declare that they have no competing interests.K. V. designed and created the GFP fusion constructs, performed all of the described experiments and interpreted the data. S. C. created the E6MT mutant. P. M. and P. C. A. provided logistical, financial and material support and helped in data interpretation and manuscript preparation.


Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such asDrosophila Nipped-B, a regulator ofcut andUltrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs ofCaenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation inC. elegans embryos. Using antisense morpholino oligonucleotides to knock downXenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development. A complex previously found only in yeast is described in metazoa, where it functions both in chromatid cohesion and in migration during development. Sister chromatid cohesion in eukaryotes occurs by a well-conserved mechanism that depends on cohesins, protein complexes that bind to multiple sites on chromosome arms but are highly enriched at centromeres. At the heart of cohesins are tripartite SMC-kleisin complexes that form ring-like structures   were incubated overnight at 4 °C, using gentle agitation with anti-FLAG M2 affinity gel . The resin was collected by centrifugation, washed with 10 volumes of 0.25-HEGN Buffer  glycerol, 0.1% (v/v) NP-40, 1 mM DTT, 0.1 mM PMSF). Bound proteins were eluted with 0.25-HEGN containing 0.5 mg/ml FLAG peptide . Fractions of the eluate were separated on 8% SDS-PAGE gels, and protein was visualized by silver stain. FLAG-Nipped-B was identified in the eluate by Western blot using an anti-FLAG M2 antibody , goat anti-mouse secondary  and the Protoblot AP System  for detection . A molecular mass range up to twice the apparent molecular weight (as estimated from electrophoretic relative mobility) was covered, with a mass accuracy restriction of less than 35 ppm, and maximum one missed cleavage site allowed per peptide. To confirm PMF results with scores ≤ 40, mass spectrometric sequencing of selected peptides was done by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples, using the UltraFlex instrument in “LIFT” mode. Fragment ion spectra were taken to search NR using the MASCOT MS/MS Ion Search program , 10 mM KCl, 1.5 mM MgClmM PMSF . Cells whttp://psort.hgc.jp. Protein structure prediction used the PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred) and the position-specific scoring matrix of Jones et al.  andCGAGUGUGUAUAUACGGGAAGGAAA [M2]) and delangin . Negative control oligonucleotides were from Eurogentec. Transient transfection of HeLa cells used 200 nM oligonucleotides and LipofectamineTM 2000 (Invitrogen) according to the manufacturer's instructions. After 24 h following transfection, the cells were washed in PBS, fresh medium was added, and a second round of siRNA transfection was carried out using the same protocol as for the first round. Knockdown efficiency was validated at 48 and 72 h after the second transfection by immunoblotting with human MAU-2- and delangin-specific antibodies (see above).Test siRNA oligonucleotides  included ones designed to knock down production of human MAU-2 . The cells were re-centrifuged and re-suspended in fix solution again before dropping onto slides. The cells were then stained with DAPI, and a total of 300 metaphases were microscopically analyzed for PSCS. To provide an objective assessment, individual chromosome pairs were scored for evidence of PSCS.HeLa cells subjected to MAU-2-specific siRNA knockdown, or to negative control siRNA, were synchronized in G2/M by nocodazole treatment (0.1 μg/ml as above) and then released to progress into the cell cycle. Chromatin fractions were prepared from aliquots collected at 0, 1.5, 2.5, and 3.5 h following release of the nocadazole block using a slight modification of the method of Méndez and Stillman , i.e., cTM fluorescence-activated cell sorter .HeLa cells were subjected to siRNA-mediated gene knockdown, synchronization in G2/M by nocodazole, and released to progress into the cell cycle as above. Aliquots were collected at 0 and 3.5 h, cells were washed in PBS, and then fixed in . For analysis, cells were re-hydrated and permeabilized in PBS [0.1% Triton X-100], treated with RNase A (0.2 mg/ml), and stained with propidium idodide (0.02 mg/ml). The samples were analyzed using a FACSCaliburC. elegans [scc-3, mau-2, orpqn-85 function was generated by in vitro transcription of a gel-purified PCR product flanked by T7 and T3 promoter sequences as described [scc-3F: 5′ –AATTAACCCTCACTAAAGGACAGCGACTTTTTGCGCTAT – 3′;scc-3R: 5′ –TAATACGACTCACTATAGGTCGATGAAGAACGTCGTGAG – 3′;mau-2F: 5′ -AATTAACCCTCACTAAAGGAGCGCCTTTCAAAAATCAAA – 3′;mau-2R: 5′ –TAATACGACTCACTATAGGCGCCAAAAATGTGAAAACTG – 3′;pqn-85F: 5′ –AATTAACCCTCACTAAAGGCCGCCAATAATTCAAGCAGT – 3′;pqn-85R: 5′ –TAATACGACTCACTATAGGTGTGACCCAAAGAAATGCTG – 3′. All of the above PCR reactions were carried out usingC. elegans genomic DNA as a template. Worms were dissected in water to remove early stage eggs from the gonad 48 h after injection of RNA. The released eggs were transferred to a fresh agarose pad (2%) and the histone::GFP was visualized using a Zeiss Axiophot microscope.Standard methods were used for maintaining and manipulatingelegans . A histoelegans ) was usescribed . The RNAX. tropicalis priming and fertilizations were carried out as described [X. tropicalis Nipbl andKIAA0892 mRNAs, respectively) were modified by addition of lissamine fluorochromes. One-cell embryos were injected with 10 ng of test or control (GeneTools standard control) morpholino in 1 nl H2O. Embryos were observed, photographed, and fixed in 4% formaldehyde at gastrula (stages 10–12) and tailbud (stages 24–28).scribed . AntisenNIPBL for mutations inKIAA0892, the human ortholog ofC. elegans mau-2. Exons and immediately neighboring intronic sequences were amplified using the primers detailed inWe screened a panel of 18 CdLS patients that had not previously been found to have mutations inhttp://www.ncbi.nlm.nih.gov) as follows: human KIAA0892 protein (Q9Y6X3),D. melanogaster CG4203-PA protein (Q9VFC0),S. purpuratus (XP_784063),X. tropicalis (NP_001015927), and chick (XP_425908). Note that the human sequence Q9Y6X3 contains eight incorrectly predicted amino acids at the N-terminal end, which were omitted for the purpose of sequence comparison, and the chick sequence was amended to remove sequence from two incorrectly predicted exons.The protein sequences of various members of the metazoan MAU-2 family used for sequence comparisons had accession numbers listed at the US National Center for Biotechnology Information (NCBI) Web site (http://www.ncbi.nlm.nih.gov/OMIM) accession number for Cornelia de Lange syndrome is 122470.The Online Mendelian Inheritance in Man (OMIM) (


EGFR are common in lung adenocarcinoma. The presence of mutations correlates with tumor sensitivity to the EGFR inhibitors erlotinib and gefitinib, but the transforming potential of specific mutations and their relationship to drug sensitivity have not been described.Somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene EGFR active site mutants are oncogenic. Mutant EGFR can transform both fibroblasts and lung epithelial cells in the absence of exogenous epidermal growth factor, as evidenced by anchorage-independent growth, focus formation, and tumor formation in immunocompromised mice. Transformation is associated with constitutive autophosphorylation of EGFR, Shc phosphorylation, and STAT pathway activation. Whereas transformation by most EGFR mutants confers on cells sensitivity to erlotinib and gefitinib, transformation by an exon 20 insertion makes cells resistant to these inhibitors but more sensitive to the irreversible inhibitor CL-387,785.Here, we demonstrate that EGFR mutants causes differential sensitivity to gefitinib and erlotinib. Treatment of lung cancers harboring EGFR exon 20 insertions may therefore require the development of alternative kinase inhibition strategies.Oncogenic transformation of cells by different  Different EGFR mutations are associated with lung cancer. All of the classes can transform fibroblasts and lung epithelial cells, most are sensitive to erlotinib and gefininib, but exon 20 mutations are only sensitive to an irreversible EGFR inhibitor. The human epidermal growth factor receptor gene product (EGFR), a member of the ErbB family of receptor tyrosine kinases, is an integral component of signaling in epithelial cell proliferation. Stimulation of the receptor with EGF or other cognate ligands induces receptor dimerization and autophosphorylation, providing docking sites for SH2-containing adaptor proteins that mediate the activation of intracellular signaling pathways –3.EGFR variants with deletions in the extracellular domain, including the v-erbB oncogene of avian erythroblastosis virus and the vIII mutant found in human cancers, transforms vertebrate cells in the absence of exogenous EGF [EGFR gene can transform NIH-3T3 cells only upon EGF addition [Consistent with a role in proliferative signaling, the oncogenic potential of nous EGF –7. In coaddition . Kinase addition ,10.EGFR kinase domain mutations observed in human lung adenocarcinomas has recently been described [EGFR deletion mutations or substitution mutations [EGFR exons 18 through 21 reported a total of 18 exon 20 insertions out of 350 EGFR mutations identified in 1,108 non-small-cell lung cancers [A series of novel escribed –16. Thesutations ,12,13,15 cancers –18. Pati cancers ,20.EGFR are dependent on EGFR function for survival [EGFR kinase domain mutations to constitutively activate EGFR signaling and contribute to tumorigenesis in model cell culture systems.Although gefitinib treatment and small interfering RNA experiments suggest that cells expressing mutant survival ,21,22, tNIH-3T3 cells obtained fromATCC  were maintained in DMEM  supplemented with 10% calf serum  and penicillin/streptomycin (Gibco/Invitrogen). NCI-H3255 cells were maintained in ACL-4 media as previously described . Unless EGFR was amplified from a cDNA template with the PCR primers 5′-GATGATATCATGCGACCCTCCGGGAC-3′ and 5′-ATCGATATCTCATGCTCCAATAAATTC-3′, digested with EcoRV, and inserted into the SnaB1 site of pBabe-Puro. Point mutations, insertions, and deletions were made using the Quick-Change Mutagenesis XL kit  with the following oligonucleotide primers: 5′-AAGATCACAGATTTTGGGAGGGCCAAACTGCTGGGTG-3′ and 5′-CACCCAGCAGTTTGGCCCTCCCAAAATCTGTGATCTT-3′ for L858R; 5′-AAGATCAAAGTGCTGAGCTCCGGTGCGTTCG-3′ and 5′-CGAACGCACCGGAGCTCAGCACTTTGATCTT-3′ for G719S; 5′-GGTGCACCGCGCCCTGGCAGCCA-3′ and 5′-TGGCTGCCAGGGCGCGGTGCACC-3′ for D837A; 5′-GTCGCTATCAAGGAACCAACATCTCCGAAA-3′ and 5′-TTTCGGAGATGTTGGTTCCTTGATAGCGAC-3′ for L747_E749del, A750P; 5′-GGCCAGCGTGGACAACCCCGGCAACCCCCACGT-3′ and 5′-ACGTGGGGGTTGCCGGGGTTGTCCACGCTGGCC-3′ for D770_N771insNPG. All constructs were fully sequenced.Replication incompetent retroviruses were produced from pBabe-Puro-based vectors either by cotransfection of 293T cells with pCL-Ampho  or by transfection into the Phoenix 293T packaging cell line  using Lipofectamine 2000 (Invitrogen). Cells were infected with these retroviruses in the presence of polybrene. Two days after infection, puromycin  was added and pooled stable cell lines were selected, from which clonal cell lines were derived.5 cells in triplicate in 0.4% Noble agar. Colonies of hTBE cells were counted microscopically 6–8 wk after plating with the MultiImage imaging counter .EGFR-expressing NIH-3T3 cells were suspended in a top layer of DMEM containing 10% calf serum and 0.4% Select agar (Gibco/Invitrogen) and plated on a bottom layer of DMEM containing 10% calf serum and 0.5% Select agar. EGF, gefitinib, or erlotinib was added as described to the top agar. NIH-3T3 colonies were counted in triplicate wells from ten fields photographed with a 10× objective. Growth of hTBE cells in soft agar was determined by plating 1 × 10NIH-3T3 cells infected with EGFR retrovirus diluted 1:1, 1:10, or 1:100 were split 2 d after infection into 10-cm plates with and without puromycin selection. Cells for the focus assay were maintained without passage as a monolayer in the absence of puromycin for 3 wk, after which foci were stained with crystal violet  and scored. Numbers of foci were multiplied by the viral dilution factor and normalized to the relative number of infectious units in each viral stock, as determined by a WST assay  on cells after 3 d of puromycin selection.6 cells were injected subcutaneously into immunocompromised mice, three injections per animal, as described [In this assay, 2 × 10escribed . Tumors Cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM EDTA, 1% Triton X-100, and 0.25% IPEGAL. Protease inhibitors (Roche) and phosphatase inhibitors  were added prior to use. Samples were normalized for total protein content unless otherwise indicated. Lysates were boiled in sample buffer, separated by SDS-PAGE on 8% or 10% polyacrylamide gels, transferred to nitrocellulose, and probed as described. Antibodies used for immunoblotting were: anti-EGFR , anti-phospho-EGFR Y1173 , anti-phospho-EGFR Y1068 , anti-phospho-EGFR Y845 , anti-phospho-EGFR Y1045 , anti-actin , anti-Shc , anti-phospho-Shc Y317 , anti-Stat3 , and anti-phospho-Stat3 Y705 , anti-phospho-Akt S473 , and anti-Akt .Cells were lysed as described above. Anti-EGFR conjugated beads  were used for immunoprecipitation. Beads (25 μl) were incubated with fresh lysate in 300 μl of lysis buffer for 1 h, washed twice with lysis buffer, eluted in 1% SDS at room temperature for 20 min, and boiled in sample buffer.The STAT3  reporter m67 pTATA TK-luc  was kindEGFR kinase domain mutants, tumor-derived mutations were introduced into the wild-type human EGFR cDNA by site-directed mutagenesis. The resulting wild-type and mutant EGFR cDNAs were then cloned into the pBabe-Puro retroviral vector and transferred into NIH-3T3 cells by retroviral infection. We initially examined two representative substitution mutations: G719S, observed in exon 18, and L858R, observed in exon 21 .Representative exon 19 deletion and exon 20 insertion mutations of l, A750P  and D770 antigen D. ExpresEGFR to transform a more physiologically relevant cell type, retroviruses expressing the L858R and G719S mutant forms of EGFR were introduced into hTBE cells expressing the SV40 early region T antigens and the human telomerase catalytic subunit hTERT [EGFR genes conferred enhanced anchorage-independent growth upon such hTBE cells, with colony numbers approximately 15-fold above the background level of microscopic colonies observed in hTBE cells expressing wild-type EGFR or a control vector  of these agents than was required to inhibit colony formation by cells expressing the EGFR missense mutants or deletion mutant , or in combination with chemotherapy . These results suggest that cancers harboring distinct activating kinase domain mutations of EGFR may exhibit a differential sensitivity to specific EGFR inhibitors.Consistent with this result, all three lung adenocarcinoma patients with known exon 20 insertion mutants of EGFR, completely inhibiting transformation at 0.003 μM (unpublished data). These effects are also observed upon assessment of receptor autophosphorylation  inhibitors have shown promising results in some patients. As the name suggests, these drugs inhibit a protein called EGFR, which is altered in a subset of lung cancers.The aim of this study was to understand in more detail what role the different EGFR alterations played in the tumor and which ones made the tumor responsive to EGFR inhibitor treatment. First, the researchers introduced different altered EGFR versions into human cells and studied their behavior. The EGFR protein, which can stimulate cell growth, is normally tightly regulated and active only when a cell receives a signal from its neighbors. However, the alterations made the EGFR protein always active, regardless of a stimulating signal, and caused them to “grow out of control.” They then treated the cells expressing the various alterations with different EGFR inhibitor drugs and showed that the specific alteration determined whether cell growth could be stopped by a specific drug.EGFR inhibitors are still considered to be experimental treatment. Researchers are making progress in understanding how genetic alterations in EGFR cause abnormal growth in some lung cancers and also which specific alterations cause the tumor to be responsive to a particular drug. The goal is to match the tumor and the drug to maximize anti-tumor response and avoid giving a drug that doesn't work with a particular tumor.The following Web sites contain information on lung cancer, the role of EGFR mutations, and the EGFR class of drugs.Lung Cancer Online :http://www.lungcanceronline.org/index.htmUS National Cancer Institute information page on lung cancer:http://www.nci.nih.gov/cancertopics/types/lungOngoing clinical trials of EGFR inhibitors:http://www.clinicaltrials.gov


CTLA-4 encodes cytotoxic T lymphocyte-associated antigen-4, a cell-surface molecule providing a negative signal for T-cell activation. CTLA-4 gene polymorphisms have been widely studied in connection with genetic susceptibility to various autoimmune diseases, but studies have led to contradictory results in different populations. This case-control study sought to investigate whether CTLA-4 CT60 and/or +49A/G polymorphisms were involved in the genetic predisposition to primary Sjögren syndrome (pSS). We analysed CTLA-4 CT60 and +49A/G polymorphisms in a first cohort of 142 patients with pSS (cohort 1) and 241 controls, all of Caucasian origin. A replication study was performed on a second cohort of 139 patients with pSS (cohort 2). In cohort 1, the CTLA-4 +49A/G*A allele was found on 73% of chromosomes in patients with pSS, compared with 66% in controls  1.41, 95% confidence interval (CI) 1.02 to 1.95). No difference in CTLA-4 CT60 allelic or genotypic distribution was observed between patients (n = 142) and controls (n = 241). In the replication cohort, the CTLA-4 +49A/G*A allele was found on 62% of chromosomes in patients with pSS, compared with 66% in controls . Thus, the CTLA-4 +49A/G*A allele excess among patients from cohort 1 was counterbalanced by its under-representation in cohort 2. When the results from the patients in both cohorts were pooled (n = 281), there was no difference in CTLA-4 +49A/G allelic or genotypic distribution in comparison with controls. Our results demonstrate a lack of association between CTLA-4 CT60 or +49A/G polymorphisms and pSS. Premature conclusions might have been made if a replication study had not been performed. These results illustrate the importance of case-control studies performed on a large number of patients. In fact, sampling bias may account for some contradictory results previously reported for CTLA-4 association studies in autoimmune diseases. CTLA-4, the gene encoding cytotoxic T lymphocyte-associated antigen-4, have been widely studied in connection with genetic susceptibility to various autoimmune diseases [Polymorphisms in diseases , but stuCTLA-4 gene polymorphisms, a G to A transition at position 49 (+49A/G) of exon 1 leads to an alanine to threonine amino acid substitution at codon 17 in the leader peptide (A17T), and a C to T transition at position 60 (CT60) is located within the 3'-untranslated region [CTLA-4 polymorphisms. The CT60 G allele has been reported to increase susceptibility to several autoimmune diseases [Among d region . The G adiseases , and a fdiseases .CTLA-4 +49A/G*A allele and of the CTLA-4 +49A/G*A allele carrier haplotypes with primary Sjögren syndrome (pSS), especially in patients with anti-SSA or anti-SSB antibodies, in a study including 111 Australian patients with pSS and 156 controls [Downie-Doyle and colleagues have recently reported a significant association of the controls .CTLA-4 CT60 and/or +49A/G SNPs were involved in genetic predisposition to pSS in French patients.The aim of our study was to investigate in a large case-control study whether CTLA-4 CT60 and +49A/G polymorphisms. A second independent cohort of 139 patients with pSS was further genotyped for CTLA-4 +49A/G polymorphisms in a replication study. The geographical origin and the clinico-biological characteristics of the patients in this second cohort were the same as those in the first. In this second cohort, 27% of patients were anti-SSA and anti-SSB negative, 35% had anti-SSA only and 38% had both anti-SSA and anti-SSB. All patients and controls were Caucasians and provided informed consent.A first cohort of 142 unrelated patients with pSS diagnosed in accordance with the European American consensus group criteria [CTLA-4 CT60 and +49A/G polymorphisms were genotyped by restriction fragment length polymorphism with the use of BbvI (+49A/G) and NlaIII (CT60).After the isolation of genomic DNA from peripheral blood mononuclear cells, CTLA-4 CT60 and +49A/G polymorphisms were compared between patients and controls by using a two-sided χ2 test. All genotyped SNPs were in Hardy–Weinberg equilibrium. CTLA-4 (+49A/G or CT60) haplotypes, constructed with the PHASE program, were also examined for association with pSS. P < 0.05 was considered significant.Allelic and genotypic frequencies of CTLA-4 +49A/G polymorphism was found on 73% of chromosomes in patients with pSS, in comparison with 66% in controls  1.41, 95% confidence interval (CI) 1.02 to 1.95; Table CTLA-4 +49A/G*A allele frequencies was observed among subgroups of patients according to their anti-SSB and/or anti-SSA status  and controls (n = 241). CTLA-4 (+49A/G or CT60) haplotype distribution mirrored the CTLA-4 +49A/G*A allele excess among patients with pSS , leading to an excess of +49A/G*A allele carrier haplotypes among patients .In the first cohort of patients with pSS, the A allele of the CTLA-4 +49A/G*A with pSS as a result of the somewhat small sample size of our first cohort, and because the CTLA-4 +49A/G*A allele has been only marginally associated with autoimmune diseases compared with the CTLA-4 +49A/G*G allele [CTLA-4 +49A/G*A allele was found on 62% of chromosomes in patients with pSS, compared with 66% in controls , there was no difference in CTLA-4 +49A/G polymorphism allelic or genotypic distribution in comparison with controls , thus excluding any possible gender effect.To avoid the possibility of a false positive association of G allele , we perfCTLA-4 CT60 or +49A/G polymorphisms and pSS among Caucasians.Our results therefore demonstrate a lack of association between CTLA-4 +49A/G*A), was actually more probably due to the statistical weight of the CTLA-4 +49A/G*A allele than to a true functional effect of two different haplotypes, bearing either CTLA-4 CT60*C or CTLA-4 CT60*T alleles, each having opposite functional effects on CTLA-4 mRNA expression [The results from our first cohort were very close to those from the study of Downie-Doyle and colleagues , with a pression .CTLA-4 +49A/G*A allele with pSS in the first cohort of patients. When data were pooled (cohorts 1 and 2), no significant association was found with the CTLA-4 +49A/G polymorphism in our Caucasian population of patients with pSS. This was not the consequence of different origin or different clinico-biological characteristics of the patients from the two cohorts and could only be the result of a sampling bias. Indeed, the findings observed in our first cohort of patients, as those from Downie-Doyle and colleagues [CTLA-4A/G*A allele with autoimmune diseases [In fact, our results suggest a false positive association of lleagues , were undiseases -7. ConseCTLA-4 association studies in autoimmune diseases.Our study illustrates the necessity to include a large number of patients in genetic case-control studies. In fact, sampling bias may partly account for some contradictory results previously reported for CI = confidence interval; CTLA-4 = cytotoxic T lymphocyte-associated antigen-4; OR = odds ratio; pSS = primary Sjögren syndrome; SNP = single nucleotide polymorphism.The authors declare that they have no competing interests.JEG contributed to the study design, performed the statistical analysis and drafted the manuscript. PL and DC supervised genotyping and contributed to DNA samples collection. MA and CC performed genotyping. NC performed PHASE analyses. EH, XP, and JS contributed to DNA samples collection. XM and CMR supervised the study design and gave valuable advice to JEG and PL. All authors read and approved the final manuscript.


The cellular distribution of 4-borono-2-[18F]fluoro-L-phenylalanine , a potential agent for boron neutron capture therapy (BNCT), and [6-3H]thymidine  in murine two B16 melanoma sublines and FM3A mammary carcinoma was studied in vivo using double-tracer microautoradiography. Tumour volume, tumour age, cell density in the tissues and the proportion of S phase cells in the cell cycle were the same in the three tumour models. Volume doubling time, which represents tumour growth rate, was fastest in B16F10, followed by B16F1 (P < 0.05), the slowest being in FM3A (P < 0.001). The rate of DNA synthesis in S phase cells corresponded to the volume doubling time. The greatest amount of [18F]FBPA was observed in S phase melanocytes and the lowest amount was found in non-S phase non-melanocytes. The [18F]FBPA accumulation was primarily related to the activity of DNA synthesis and, secondarily, to the degree of pigmentation in melanocytes. The therapeutic efficacy of BNCT with p-boronophenylalanine may be greater in melanoma that exhibits greater DNA synthesis activity and higher melanin content.


Chromosome shaping and individualization are necessary requisites to warrant the correct segregation of genomes in either mitotic or meiotic cell divisions. These processes are mainly prompted in vertebrates by three multiprotein complexes termed cohesin and condensin I and II. In the present study we have analyzed by immunostaining the appearance and subcellular distribution of condensin I in mouse mitotic and meiotic chromosomes. Our results demonstrate that in either mitotically or meiotically dividing cells, condensin I is loaded onto chromosomes by prometaphase. Condensin I is detectable as a fuzzy axial structure running inside chromatids of condensed chromosomes. The distribution of condensin I along the chromosome length is not uniform, since it preferentially accumulates close to the chromosome ends. Interestingly, these round accumulations found at the condensin I axes termini colocalized with telomere complexes. Additionally, we present the relative distribution of the condensin I and cohesin complexes in metaphase I bivalents. All these new data have allowed us to propose a comprehensive model for meiotic chromosome structure. Xenopus egg extracts  protein family, and three non-SMC proteins, CAP-G, CAP-D2 and the kleisin CAP-H  is considered the earliest event defining the onset of mitotic condensation . In ordeDuring meiosis I, CAP-H was first detected in pachytene spermatocytes. In these spermatocytes, several large CAP-H accumulations, about five, were observed scattered in the nucleoplasm . One of A double immunolabeling of CAP-H and pH3 on meiosis I spermatocytes showed that pH3 was first detected at the heterochromatic chromocentres in diplotene spermatocytes as previously reported , while CIn late interkinesis nuclei, CAP-H appeared as nucleoplasmic accumulations representing nucleoli, and as small spots . AfterwaA double immunolabeling of CAP-H and pH3 on meiosis II spermatocytes showed that CAP-H was detected at nucleoli and small spots in late interkinesis nuclei, when their chromatin was completely labeled by pH3  of the synaptonemal complex (SC), and the unsynapsed sex axial elements (AEs) . By contIn either the autosomal or sex metaphase I bivalents, RAD21 was observed at the so-called interchromatid domain, and at centromeres . An accuWe have found that the non-SMC subunits of the condensin I complex CAP-G, CAP-D2, and CAP-H, become associated to chromosomes in mouse spermatogonial cells during prometaphase. Consistently, our results show that these subunits are recruited after the phosphorylation of histone H3 at serine 10, a landmark of the initiation of mitotic condensation . Thus, oXenopus  containing 10% fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin and 1% 0.2 M L-glutamine , at 37°C in a humidified atmosphere containing 5% CO2HPO4, 1.7 mM KH2PO4, [pH 7.4]). Then, they were lysed in boiling SDS sample buffer .HeLa cells extracts were prepared by harvesting cultured cells, which were washed twice with PBS . Then, testes were homogenized on ice in a Potter homogenizer. The extract was placed in boiling water bath for 5 minutes. The appropriate quantity of extract was diluted with 5X SDS-lysis buffer  and boiled for 5 minutes.SDS-PAGE was carried out in 10% polyacrylamide gels. Gels were electrically transferred to Trans-Blot sheets  for 1.5 hour at 4°C and 310 mA. Sheets were blocked for 1 hour with 4% non-fat dry milk in PBS, followed by an overnight incubation at 4°C with an anti-hCAP-H antibody at a 1:10,000 dilution in 4% non-fat dry milk in PBS. Immunoreactive bands were visualized by incubation for 1 hour at room temperature with horseradish peroxidase-conjugated donkey anti-rabbit Ig  at a 1:5,000 dilution in PBS, and subsequent development using an ECL (Enhanced Chemiluminescence) detection system (Amersham Life Science) according to the manufacturer instructions.Adult male C57BL/6 mice were used in the present study. Testis were removed, detunicated, and seminiferous tubules then processed for squashing. For squashing we followed the technique previously described by Parra et al. . BrieflyAfter fixation, the slides were rinsed three times for 5 minutes in PBS, and incubated for 45 minutes at room temperature with primary antibodies. Several subunits of the condensin I complex were detected. CAP-H was detected with a rabbit polyclonal antibody generated against a 211 aa amino-terminal fragment of human CAP-H , at a 1:Observations were performed using an Olympus BH-2 microscope  equipped with epifluorescence optics and the images were recorded with an Olympus DP50 digital camera. Digital images were then treated using the Adobe PhotoShop 7.0 software.Figure S1Immunoblot of HeLa cell extracts (left lane) and mouse testis extracts (right lane) probed with the anti-hCAP-H antibody. The positions of two molecular mass markers are indicated. The antibody specifically recognized a single protein band of about 90 kDa in both extracts.(0.28 MB TIF)Click here for additional data file.Figure S2CAP-D2 distribution in spermatogonial mitosis. Mouse spermatogonia were stained for CAP-D2 (green), kinetochores with an ACA serum (red), and counterstained with DAPI (blue). (A–C) Early prometaphase. Condensing chromosomes present a faint and diffuse CAP-D2 staining, but some bright spots are also observed. (D–F) Metaphase in top view, and (G–I) anaphase. A single diffuse CAP-D2 axis is seen in each chromosome/chromatid, as well as bright accumulations. Bar, 5 µm.(5.93 MB TIF)Click here for additional data file.Figure S3H3 phosphorylation precedes condensin I recruitment to chromosomes in spermatogonial mitosis. Mouse spermatogonia were stained for histone H3 phosphorylated at serine 10 (pH3) (red), CAP-H (green), and counterstained with DAPI (blue). (A–C) Middle prophase. Phosphorylated H3 is present in condensing chromosomes but not CAP-H. (D–F) Late prophase. CAP-H appears as small bright spots (arrowheads) and in the nucleoplasm. (G–I) Metaphase in top view. A single CAP-H axis (arrows) is seen in each chromosome. Bar, 5 µm.(5.91 MB TIF)Click here for additional data file.Figure S4Relative distributions of CAP-H and fibrillarin in prophase I spermatocytes. Mouse spermatocytes were stained for CAP-H (green), fibrillarin (red), and counterstained with DAPI (blue). (A–C) Zygotene spermatocyte. CAP-H is not detected, and fibrillarin appears at nucleoli. (D–F) Early pachytene, (G–I) late pachytene, and (J–L) diplotene spermatocytes. CAP-H and fibrillarin colocalize at the nucleoplasmic nucleoli, and at the nucleolus associated to the sex body (XY). Note that CAP-H and fibrillarin are not present at the round body and/or the fibrillar centre (arrows) inside the sex body-associated nucleolus during pachytene, and the nucleolus in diplotene . Fibrillarin is additionally present in Cajal bodies (arrowheads) lying in the nucleoplasm  or associated to the nucleolus (K). Bar, 5 µm. Supplementary Reference: Knibiehler B, Mirre C, Hartung M, Jean P, Stahl A (1981) Sex vesicle-associated nucleolar orgnizers in mouse spermatocytes: localization, structure, and function. Cytogenet Cell Genet 31: 47–57.(10.16 MB TIF)Click here for additional data file.Figure S5CAP-D2 distribution in spermatocytes. Mouse spermatocytes were stained for CAP-D2 (green), kinetochores with an ACA serum , TRF1 (red in G–I), and counterstained with DAPI (blue). (A–C) Metaphase I spermatocyte. The autosomal bivalents show pairs of bright CAP-D2 spots at their centromeric and distal ends. (D–I) Two selected autosomal metaphase I bivalents. The proximal pair of CAP-D2 signals appears below the closely associated sister kinetochores (D–F). The four pairs of CAP-D2 signals colocalize with the TRF1 signals (G–I). (J–L) Anaphase I, (M–O) metaphase II, and (P–R) anaphase II spermatocytes. Chromosomes/chromatids show a faint CAP-D2 labeling along them, and brighter CAP-D2 dots at their ends. The insets in (J–L) show an enlarged anaphase I half-bivalent (arrows). Bars:  5 µm; (D–I) 3 µm.(1.14 MB JPG)Click here for additional data file.Figure S6H3 phosphorylation at serine 10 precedes condensin I recruitment to chromosomes in meiosis I. Mouse spermatocytes were stained for histone H3 phosphorylated at serine 10 (pH3) (red), CAP-H (green), and counterstained with DAPI blue). (A–C) Diplotene spermatocyte. Phosphorylated H3 is enriched at chromocentres, while CAP-H appears at nucleoli. (D–F) Late diplotene. Phosphorylated H3 appears on all the chromatin, and CAP-H at a nucleolus. (G–I) Prometaphase I spermatocyte. CAP-H appears on bivalents, and as pairs of bright dots (double arrowheads). (J–L) Metaphase I spermatocyte. CAP-H is detected as pairs of bright spots at the centromeric and distal chromosome ends. (M–P) Two focal planes of an anaphase I spermatocyte. In each chromosome, a pair of CAP-H dots is detected at the centromere region (red arrowheads), one spot at the distal end of each chromatid (white arrowheads), and a diffuse axial labeling along chromatids. The inset shows the chromosome indicated in (O), where the CAP-H staining has been pseudocolored in red and superimposed on its corresponding DAPI image. Bar, 5 µm.(9.96 MB TIF)Click here for additional data file.Figure S7H3 phosphorylation precedes condensin I recruitment to chromosomes in meiosis II. Mouse spermatocytes were stained for histone H3 phosphorylated at serine 10 (pH3) (red), CAP-H (green), and counterstained with DAPI blue). (A–D) Two focal planes of a late interkinesis nucleus. Phosphorylated H3 is present on all the chromatin, whereas CAP-H appears at one nucleolus (nu) and at small spots in the nucleoplasm. (E–G) Metaphase II, and (H–K) two focal planes of an anaphase II spermatocyte. Bright CAP-H spots are present at chromosome ends. Bar, 5 µm.(5.26 MB TIF)Click here for additional data file.Figure S8Relative distributions of CAP-H and TRF1 in spermatogonial metaphase chromosomes. (A–D) Mouse metaphase spermatogonia stained for CAP-H (green), TRF1 (red), and counterstained with DAPI (blue). A fuzzy CAP-H axis is visible inside each sister chromatid. The bright CAP-H accumulations at the axes ends colocalize with TRF1. Bar, 5 µm.(2.25 MB TIF)Click here for additional data file.Figure S9Silver staining of metaphase I spermatocytes. (A) Metaphase I spread spermatocyte where the sex bivalent is indicated (XY), and selected autosomal bivalents  and sex (C) bivalent. Two silver-stained structures representing sister kinetochores (arrowheads) are found at the centromeric region of each homologue. Faint silver-stained axes (arrows) are observed along the inner region of the chromatids. (E) Autosomal metaphase I bivalent shown in (5.41 MB TIF)Click here for additional data file.


The human retinoblastoma susceptibility gene encodes a nuclear phosphoprotein RB, which is a negative regulator of cell proliferation. The growth suppression function of RB requires an evolutionarily conserved A/B domain that contains two distinct peptide-binding pockets. At the A/B interface is a binding site for the C-terminal trans-activation domain of E2F. Within the B-domain is a binding site for proteins containing the LxCxE peptide motif.Based on the crystal structure of the A/B domain, we have constructed an RB-K530A/N757F (KN) mutant to disrupt the E2F- and LxCxE-binding pockets. The RB-K530A (K) mutant is sufficient to inactivate the E2F-binding pocket, whereas the RB-N757F (N) mutant is sufficient to inactivate the LxCxE-binding pocket. Each single mutant inhibits cell proliferation, but the RB-KN double mutant is defective in growth suppression. Nevertheless, the RB-KN mutant is capable of reducing etoposide-induced apoptosis.Previous studies have established that RB-dependent G1-arrest can confer resistance to DNA damage-induced apoptosis. Results from this study demonstrate that RB can also inhibit apoptosis independent of growth suppression. RB1) encodes a nuclear phosphoprotein RB with tumor suppression function RB1 mutation causes retinoblastoma with 90% penetrance in children; the tumor cells exhibit loss of heterozygosity (LOH) at the RB1 locus with the invariable loss the normal RB1 allele RB1 gene has also been detected in sporadic human cancers of a variety of tissue origins at an average frequency of approximately 10% (COSMIC database at the Sanger Genome Center). The current knowledge suggests that RB suppresses tumor development by inhibiting cell proliferation and promoting terminal differentiation The retinoblastoma susceptibility gene  as per manufacturer's instructions. K530A and N757F mutations were introduced into human RB cDNA via PCR-mediated mutagenesis and the resulting mutation confirmed by DNA sequencing.c antibody was obtained from Pharmingen. For co-immunoprecipitation, cells were lysed in the binding buffer, the lysates clarified by centrifugation, and then incubated with anti-RB or anti-HA. The immune complexes were collected on protein G Sepharose, solubilized with SDS sample buffer for immunoblotting by standard methods.GST fusion proteins of RB , E2F1 , E2F3 , DP1  and E7  were expressed in bacteria and purified on Glutathione Sepharose (Pharmacia) by standard methods Recombinant adenovirus was constructed and amplified using the AdEasy system SAOS-2 cells were transfected with plasmids encoding RB, RB-K, RB-N or RB-KN and the neomycin resistance gene c release from mitochondria, cells were harvested and resuspended in Mitobuffer (20 mM HEPES (pH 7.5), 1 mM EGTA, 1 mM EDTA, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, 2 µg/ml Leupeptin, 2 µg/ml Pepstatin, 2 µg/ml Aprotinin) and then mechanically ruptured with 25 strokes of pestle in a glass homogenizer placed on ice. The lysates were spun free of nuclei and intact cells at a speed of 500×g. The supernatant was then subjected to centrifugation at 8,000×g to pellet mitochondria. To measure the sub-G1 DNA content, cells were fixed in ice-cold ethanol (70%), re-suspended in phosphate buffered saline containing 40 µg/ml RNase and 20 µg/ml propidium iodide and then subjected to FACS analyses.To measure the extent of cytochrome The structure of RB A/B domain in complex with the C-terminal E2F peptide (aa 409–426) has been determined The RB and mutant proteins were expressed in the RB-deficient breast cancer cell line MBA-MD468 through adenoviral-mediated gene transfer, and lysates from the adenovirus-infected cells were then applied to glutathione-agarose beads loaded with GST-E2F1 or GST-E7 recombinant proteins purified from bacteria . As we hThe defect of RB-K in binding E2F1 was further demonstrated by co-immunoprecipitation  and tranCyclin A promoter to contain E2F-binding site and the promoter activity is repressed by the RB-E2F complex Cyclin A promoter, it can be shown that RB, RB-K and RB-N are each capable of transcription repression (Cyclin A promoter abolished the RB-mediated transcription repression (not shown). These results suggest that RB-K can still interact with E2F, although not through binding to the E2F C-terminal region.It is well established that RB can inhibit E2F-dependent transcription through two mechanisms. RB binds the C-terminal trans-activation domain of E2F1 to inhibit transcription pression . The RB-It has been reported that the RB C-region contains a second binding site for the E2F/DP heterodimer We next measured the growth suppression activity of RB mutants by a short-term assay of BrdU-incorporation and a long-term assay of flat cell formation. In the short-term assay, MDA-MB468 cells were infected with adenovirus expressing GFP, RB, RB-K, RB-N or RB-KN and then labeled with BrdU . RelativCyclin A promoter  , cytochr release , and the release . Etoposi release . The inc release . This re release , despite release .c release in GFP-virus infected cells  to inhibit apoptosis. Alternatively, RB-KN may sequester pro-apoptotic factors other than E2F1, for example, the nuclear ABL tyrosine kinase that has been shown to promote DNA damage-induced apoptosis Previous studies have shown that RB-deficient cells are hypersensitive to DNA damage-induced apoptosis, and that expression of wild-type RB in RB-deficient cells can rescue this hypersensitivity


Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells.Native as well as recombinant bacterial cell surface layer (S-layer) protein of E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua)-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S.) cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1  promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM) studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE) promoter.Upon expression in Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of otherwise instable proteins in their native conformation. In addition the self assembly properties of the fusion proteins allow their simple purification. Moreover the binding properties of the S-layer part can be used to immobilize the fusion proteins to various surfaces. Arrays of highly ordered and densely structured proteins either immobilized on surfaces or within living cells may be advantageous over the respective soluble variants with respect to stability and their potential interference with cellular metabolism.The mSbsC-EGFP fusion protein is stably expressed both in the yeast,  Contrary to the situation in vivo, the in vitro self-assembly process of S-layer proteins in solution can also result in double layer sheets or in tube-like structures [Bacterial cell surface layers (S-layer) as the outermost cell envelope components are a common feature of many bacteria and archaea species . Withructures .E. coli, Bacillus subtilis, Lactobacillus casei [Lactococcus lactis [Geobacillus stearothermophilus ATCC 12980 has been reported in E. coli [E. coli, the mature S-layer protein mSbsC(31–1099) with an apparent molecular mass of 112 kDa forms monolayer cylinders and spirally wound sheets-like structures in the cytosol that can be recovered from the insoluble fraction upon cell lysis. Neither deletion of the C-terminal 179 aa (rSbsC(31–920)) [(31–920)-Betv1) interferes with the assembly process and the oblique lattice symmetry. Interestingly, the Bet v1 epitope was accessible to antibodies demonstrating that the fused portion is protruding from the respective assembly structure [B. sphaericus CCM 2177 and its derivatives fail to assemble in the E. coli cytosol, but form insoluble inclusion bodies [S-layers have been recognized as important structures for biotechnological applications . Howeverus casei , or Lacts lactis . For exatructure . Contrarn bodies .In the present study we addressed the question whether the cytosol of eukaryotic host cells can provide a suitable environment for the formation of S-layer self assembly products, despite the presence of numerous chaperons and proteases. Assembly of S-layer fusion proteins could provide a simple purification scheme for soluble proteins, especially if the formation of their native structures depends on the conditions of the eukaryotic cytosol. In addition, such fusion proteins could stabilize proteolytically sensitive proteins and thus increase their yield.Saccharomyces cerevisiae and the HeLa cells as eukaryotic model organisms for expression of a bifunctional S-layer fusion protein, consisting of mature SbsC (aa 31–1099) and the enhanced green fluorescent protein (EGFP).We used the yeast (31–1099) was fused with the 720 bp ORF of EGFP and cloned into pET17b as described in Methods. Both ORFs are separated by a two-aa-linker (Leu-Glu) that was introduced by the non-template-encoded XhoI site (CTCGAG). E. coli BL21(DE3), transformed with pET17b-mSbsC-EGFP, were cultivated at 30°C and 37°C and analysed by fluorescence microscopy. Expression at 30°C resulted in an enhanced fluorescence emission, indicating a higher yield of correctly folded EGFP [The 3207 bp open reading frame (ORF) encoding mSbsCded EGFP . 0, 1, 2Pure preparations of mSbsC-EGFP were obtained by FPLC as described in Methods. REM analysis of dialysed FPLC fractions of the purified full-size mSbsC-EGFP revealed predominantly tube-like structures. According to TEM analysis these tubes exhibit a regular symmetry Fig. . The cylG. stearothermophilus wild type strain ATCC 12980 devoid of its native S-layer due to Gua-HCl treatment, cells were covered with a green fluorescent layer indicating binding to the SCWP (data not shown). The same result was obtained with other G. stearothermophilus strains , but not with the Gua-HCl treated yeast strain BY4741.When recrystallization was performed in the presence of S. cerevisiae strain BY4741. As a control BY4741 was transformed with p426-GPD-EGFP. Expression of the S-layer fusion protein has only a very moderate effect on growth of the transformants , and the fractions analysed in a Western blot using anti-GFP antibodies. As shown in Figure Lactobacillus casei, B. subtilis, E. coli  and Lacs lactis  were useE. coli [S. cerevisiae, one of the best-studied eukaryotic organism. We observed no alterations of the cell shape and only a slightly reduced vegetative growth, possibly reflecting the high energy demand due to the constitutive high level synthesis of 139 kDa mSbsC-EGFP. Western blot analysis of insoluble material with anti-GFP antibody gave no indication for N-terminal proteolytic degradation of the recombinant protein as observed in E. coli. Possibly the organization of mSbsC-EGFP in assembled structures confers protection from ubiquitin-mediated degradation in yeast. In respect to protein stability S. cerevisiae seems to be a superior expression system. Fluorescence microscopy revealed that yeast transformants exhibited an intense green fluorescence indicating proper folding of the EGFP part within mSbsC-EGFP. In line with the formation of self-assembly structures in the cytosol, the fluorescence pattern differs significantly from that of EGFP-expressing yeast cells. TEM studies revealed the presence of closely packed cylindrical structures with regular symmetry comparable with those obtained after in vitro recrystallization. Our study demonstrates that the assembly process of SbsC-EGFP is not affected by the presence of eukaryote-specific cytosolic chaperons, like members of the CCT family [Our report describes for the first time the expression of a S-layer fusion protein in eukaryotic host cells. Because of the ability of mSbsC to form sheet-like and cylindrical structures in the cytosol of E. coli , we deciT family .G. stearothermophilus PV72/p2 and NRS 2004/3a form protein monolayers on substrates like silicon wafers with a native oxide layer [Recently it was reported that some S-layer monomers assemble on hydrophobic surfaces. S-layer proteins of the strains de layer . The selAs in yeast cells, mSbsC-EGFP monomers self assemble into filamentous structures within the cytosol of HeLa cells that are neither associated with subcellular structures nor affect the cell shape. We did not observe necrotic or apoptotic events in the transfected cells that expressed mSbsC-EGFP (data not shown). The detection of a single band of the expected molecular weight in Western blot analysis with anti-GFP antibodies indicates that the recombinant protein is stable and not subjected to N-terminal degradation.G. stearothermophilus wild-type strains [A positively charged N-terminal domain of S-layer proteins directly interacts via electrostatic forces with the negatively charged SCWP whose chemical composition is identical in  strains . In our in vitro and in vivo folding of both protein portions within the fusion protein is independent, and not affected by each other. This observation opens the possibility to fuse other proteins, e.g. enzymes, to the S-layer part in order to obtain functional fusion constructs in a densely packed and stable structure.Our results show that the mSbsC-EGFP fusion protein is efficiently synthesized, and self-assembles under the physiological conditions of the cytosol of eukaryotic cells. Both in yeast and human cells cytosolic accumulation of S-layer self assembly products is not accompanied by proteolytic degradation. The green fluorescence of mSbsC-EGFP suggests that both Fusion of otherwise instable proteins to S-layer proteins may be a useful tool for their high level expression in eukaryotic cells. The self assembly properties of the fusion proteins can be exploited to purify them by simple centrifugation steps from cell lysate. By engineering a suitable protease cleavage site between the S-layer and the fused protein part, incubation of the assembly products with the respective proteases could allow separation and subsequent purification of the protein of interest. Arrays of highly ordered and densely structured S-layer fusion proteins can efficiently be immobilized on a variety of surfaces, either directly or upon treating with SCWP.In vivo affinity chromatography" may be another potential field for applying S-layer fusion proteins. Enrichment of soluble biomolecules within living cells may be achieved by assembly structures consisting of fusion proteins between the respective receptor and a suitable S-layer protein.Besides technical applications, an ordered aggregation of S-layer fusion proteins within living cells may offer novel strategies for cell manipulation. For example, if expression of proteins would interfere with cellular functions, e.g. as a result of intracellular transport, fusion to an S-layer protein permits their retention in the cytosol. "Geobacillus (G.) stearothermophilus ATCC 12980G. stearothermophilus DSM 1550 (German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Braunschweig, Germany)G. stearothermophilus DSM 13240 (DSMZ)G. stearothermophilus DSM 297 (DSMZ)Escherichia (E.) coli DH5á (BRL)E. coli BL21(DE3) (Invitrogen)Saccharomyces (S.) cerevisiae BY4741 (EUROSCARF)HeLa cells #1: 5' TATATATACATATGGCAACGGACGTGGCGACGGTC 3'#2: 5' TATATATACTCGAGCGATGCTGATTTTGTACCAATTTG 3'#3: 5' TATATATACTCGAGATGGTGAGCAAGGGCGAGGAG 3'#4: 5' TATATATAGCTCAGCTTACTTGTACAGCTCGTCCATGC 3'#5: 5' TATATATAGGATCCATGGCAACGGACGTGGCGACGGTC 3'#6: 5' TATATATAGGTACCTCACTATTACTTGTACAGCTCGTCCATGC 3'#7: 5' TATATATACTCGAGATGGCAACGGACGTGGCG 3'#8: 5' TATATATACCGCGGCGATGCTGATTTTGTACCAATTTG 3'G. stearothermophilus strains were grown in tryptone-enriched LB-medium at 55°C (strain DSM 13240 at 65°C) to an OD600 of 0,6. Surface proteins attached to the cell wall layer via noncovalent forces were removed by Guanidine-HCl (Gua-HCl) treatment of cells from a 500 ml culture. Cells were washed 3 times in distilled water, suspended in 10 ml of 5 M Gua-HCl and incubated for 30 min on ice with occasional mixing. Cells devoid of their S-layers were recovered by centrifugation at 8,000 × g and washed twice with 5 M Gua-HCl. For preparation of S-layer proteins the supernatant obtained after centrifugation at 40,000 × g was dialyzed against distilled water for 18 h.Batch cultures of the ® SV Gel and PCR Claen-up system kit" (Promega).Genomic DNA was isolated with the QIAGEN Genomic-tip 100 kit according to the manufacturer's instruction. Plasmid DNA was prepared with the "Wizard(31–1099) was PCR-amplified with primers #1 and #2, using total DNA of G. stearothermophilus ATCC 12980 as a template. An ATG initiation codon was introduced 5'to the open reading frame by primer #1. The PCR-product was cut with NdeI and XhoI and ligated with plasmid pET17b (Novagen) to yield plasmid pET17b-mSbsC_oT.DNA encoding mSbsCXhoI and Bpu1102I sites of pET17b-mSbsC_oT were used to insert the EGFP reading frame, which was PCR-amplified from vector EGFP-N1 (Clontech) with primers #3 and #4. Primer #3 introduced a 5'-flanking XhoI-site in the PCR product, while primer #4 generated a 3'-flanking Bpu1102I-site and introduced a TAA termination codon. The PCR-product was cut with XhoI and Bpu1102I and ligated with pET17b-mSbsC_oT to yield plasmid pET17b-mSbsC_oT-EGFP.The BamHI and XhoI, and ligated with the S. cerevisiae expression vector p426-GPD, which bears the strong GPD promoter [in frame fusion the EGFP orf, which was amplified with primers #3 and #6. The respective PCR product harbours a 5' XhoI site and at the 3'-end three consecutive termination codons followed by a KpnI restriction site. Thereby plasmid p426-GPD-mSbsC_oT-EGFP was created.A DNA fragment encoding mSbsC was PCR-amplified as described above with primers #5 and #2, cut with promoter . The resvia the flanking XhoI and KpnI sites. Plasmids p426-GPD-mSbsC_oT-EGFP and p426-GPD-EGFP were propagated in E. coli DH5α and transformed into the S. cerevisiae wild type strain BY4741.Plasmid p426-GPD-EGFP for expression of EGFP in yeast was created by ligation of the PCR-fragment encoding the EGFP orf into p426-GPD XhoI and SacII restriction sites in the vector pEGFP-N1 (Clontech) as an in frame fusion with the EGFP-ORF.The reading frame coding for mSbsC was amplified with primers #7 and #8 and subsequently integrated within the E. coli DH5α and transfected into HeLa cells.The resulting plasmid pmSbsC-EGFP-N1 was propagated in 5 cells were plated on chamber slides (Nunc) and incubated after one day with TFX™-20 liposome reagent (Promega) and 3 μg DNA for 1 h. Subsequently DMEM with high glucose and 10 % (v/v) fetal calf serum (PAA laboratories) was supplemented. After over night cultivation transfected cells were analysed by Western blot and fluorescence microscopy.For transient transfection with plasmid pmSbsC-EGFP-N1, 2,5 × 10E. coli BL21(DE3) was used as a prokaryotic host for the expression of mSbsC-EGFP. E. coli transformants bearing pET17b-mSbsC-EGFP were cultivated in LB medium containing 100 mg/l ampicillin at 30°C to an OD600 of 0,4. S-layer synthesis was initiated by adding IPTG to a final concentration of 0,5 mM.8–16 h after induction cells obtained from a 500 ml culture were harvested by centrifugation at 10,000 × g for 10 min; 4°C. The cell pellet was washed 3 times with ice-cold distilled water and resuspended in 10 ml Tris-buffer ) containing 1% (v/v) Triton X-100. After disruption of cells by French press , soluble and insoluble fractions were separated by centrifugation at 20,000 × g for 30 min at 4°C. Nucleic acids were removed from the pellet fraction by treatment with DNase I (1 mg/ml) and RNase A (1 mg/ml) in Tris-buffer. After three washing steps with Tris-buffer the insoluble material was denatured in 5 ml 5 M Gua-HCl . The suspension was stirred at room temperature (RT) for 30 min and centrifuged for 30 min at 40,000 × g (10°C). Renatured S-layer proteins were obtained by dialyzing the supernatant twice against 5 l of distilled water or Tris-buffer over night at 4–8°C using dialysis tubes with an exclusion size of 14 kDa.2O over night and used for further analysis.For further purification the suspension containing renatured S-layer proteins was centrifuged at 12,000 × g for 30 min, the pellet washed 3 times with distilled water or Tris-buffer and dissolved in 3 M Gua-HCl in Tris-buffer. After a final centrifugation step  the clear supernatant was subjected to FPLC using a Sephacryl™ S-300 column (Pharmacia Biotech). Size exclusion chromatography was performed under denaturing conditions (3 M Gua-HCl in Tris-buffer) at RT with a flow rate of 0,5 ml/min. Fractions containing mSbsC-EGFP were dialyzed against dHS. cerevisiae strain BY4741 bearing plasmid p426-GPD-mSbsC-EGFP in YNB medium (Invitrogen) supplemented with Leu, His, Met and glucose as carbon source. 500 ml of an exponential culture (OD600 = 1) were centrifuged at 5,000 × g for 3 min and 20°C. Cells were washed 3 times with Tris-buffer and subsequently lysed by osmotic swelling of protoplasts obtained by treatment with zymolyase-20T (ICN) as described [Constitutive expression of S-layer protein coding genes was obtained by growing transformants of escribed . S-layer6 HeLa cells were transiently transfected with pmSbsC-EGFP-N1. After cultivation for 16 h cells were harvested by trypsin treatment followed by three washing steps with 1 × PBS w/o Ca2+, Mg2+ (PAA-Laboratories). Cell lysis was performed by 30 strokes in a dounce homogenizer in the presence of 0,1 % (v/v) Triton X-100, 1 mM AEBSF and Pi-cocktail . The cell lysate was centrifuged at 20,000 × g, and the resulting supernatant and pellet fractions were analysed in a Western blot.4 × 10S. cerevisiae transformants was used (Fig. Sample preparation and SDS-polyacrylamide gelelectrophoresis were carried out as described by Laemmli . Unless sed Fig. .et al. [Whole yeast cells and spheroplasts were fixed and prepared for electron microscopy as described by Waterham et al. . ImmunolStandard DNA techniques were as described . Yeast cAB carried out the molecular genetic studies, performed protein purification and drafted the manuscript.KZ performed part of the DNA cloning and sequencing and carried out the transfection studies of HeLa cells.KAS performed the immunogold labeling of yeast protoplasts and the TEM analysis. He provided the TEM images for publication.MV contributed to the design of the study and conducted the electron microscopy analysis.GR conceived of the study, participated in its design and coordination, and participated in drafting of the manuscript.All authors read and approved the final manuscript.