Methods Discovering functional transcription-factor combinations in the human cell cycle
Activation Tagging Identifies a Conserved MYB Regulator of
The Plant Cell, Vol. 12, 2383–2393, December 2000, © 2000 American Society of Plant Physiologists Activation Tagging Identifies a Conserved MYB Regulator of Phenylpropanoid BiosynthesisJustin O. Borevitz,a,1 Yiji Xia,a,b,1 Jack Blount,b Richard A. Dixon,b and Chris Lamb a,2a Plant Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037b Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures. Unfortu-nately, these complex natu ral produ cts u su ally occu r at very low abu ndance and with restricted tissu e distribu tion,thereby hindering their evaluation. Here, we report a novel approach for enhancing the accumulation of natural prod-ucts based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mo-saic virus 35S enhancer sequences at its right border. Among ف5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development. This upregulationof pigmentation reflected a dominant mu tation that resu lted in massive activation of phenylpropanoid biosyntheticgenes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocya-nins that were responsible for the purple color. These phenotypes, caused by insertion of the viral enhancer sequencesadjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic con-trols regulating the accumulation of specific natural products during plant development. Our findings suggest a func-tional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation ofmassively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes. INTRODUCTIONEthnobotany and limited screens of medicinal plants indi-cate that the huge repertoire of chemical diversity in plants contains many potentially useful bioactive structural princi-ples for developing novel drug s, flavors, frag rances, and other specialty chemicals. Unfortunately, these complex natural products usually occur in very low abundance and with a restricted tissue distribution. Moreover, almost all of this phytochemical biodiversity resides in exotic, unculti-vated species. Whereas drug s such as taxol, vinblastine, and vincristine illustrate the potential of plants as sources of new drugs, the development of rational approaches for the g eneration of useful amounts of complex natural products for industrial evaluation remains an unsolved problem. In particular, an intense 30-year effort using cell and tissue cul-tures from medicinal plants has failed to g enerate useful quantities of complex products for the commercial produc-tion of established drugs in vitro or for high-throughput, mul-tiplex screening of phytochemicals (Facchini and Deluca, 1995; McCaskill and Croteau, 1998). This failure probably reflects the string ent spatial and temporal transcriptional controls governing the biosynthesis of specific natural prod-ucts during plant development (Fowler, 1983; Robins, 1994; Facchini and Deluca, 1995). Transg enic manipulation to override these genetic controls thus may provide the key to enhancing natural product biosynthesis for industrial evalu-ation and exploitation.Activation tagging with the enhancer from the cauliflower mosaic virus 35S transcript promoter (35Se) is an emerging technology in plant functional genomics (Weigel et al., 2000). This approach, based on Agrobacterium-mediated transfor-mation, can create transgenic plants in which the T-DNA car-rying 35Se at its right border is spliced into the plant genome at random sites. In each independent transgenic line, 35Se strongly activates the plant gene to which, by chance, it lies adjacent. Activation of a gene in this fashion may lead to ob-servable effects on the modified plant, providing important clues about the function of the activated g ene. Screening larg e collections of independent, activation-tag g ed lines thus represents a powerful way of surveying the g enome and isolating genes that affect traits of interest.Using activation tagging, we have isolated a bright-purple mutant, pro ductio n o f antho cyanin pigment 1-Dominant (pap1-D), in which genes encoding enzymes involved in the biosynthesis of phenylpropanoid natural products exhibit massive and widespread activation throughout plant de-velopment. The pap1-D phenotype, which is caused by1These authors contributed equally to this work.2To whom correspondence should be addressed. E-mail mb @; fax 44-1603-456844.2384The Plant CellFigure 1.pap1-D Phenotypes.(A)pap1-D (left) and Col-0 (right) flowers.(B) Roots of pap1-D (left) and Col-0 (right) plants.(C) Six-week-old adult pap1-D (front) and Col-0 (back) plants.Activation Tagging of PAP12385 overexpression of a g ene encoding an MYB transcriptionfactor, indicates that activation tagging can be used to over-come the stringent genetic controls regulating the develop-mental accumulation of specific natural products. Thesefindings suggest a new approach for the systematic biotech-nological evaluation of phytochemical biodiversity through theg eneration of massively enriched tissue sources for drugscreening and for isolation of the underlying regulatory andbiosynthetic genes.RESULTSMutant CharacterizationApproximately 5000 activation-tagged primary lines of Ara-bidopsis ecotype Columbia (Col-0) were generated by usingpSKI015, which contains four copies of 35Se at the rig htborder of the T-DNA, pBluescript KSϩ for plasmid rescue,and the BAR g ene for Basta resistance as a selectablemarker (Kardailsky et al., 1999; Weigel et al., 2000). A singlebright-purple plant was observed in this collection, and itsseed was collected for prog eny analysis. T2 plants seg re-g ated for the purple coloration characteristic of anthocya-nins in a 3:1 ratio, which is consistent with this trait being determined by a single dominant allele, an allele we named pap1-D (see above). The whole plant, including the roots, stems, leaves, primary and secondary branches, and cauline leaves as well as sepals, anthers, and carpels, ex-hibited purple pigmentation (Figures 1B and 1C). The purple coloration was more pronounced when plants were grown under high-intensity light or other stress conditions, such as droug ht and pathog en infection (data not shown). Under these conditions, leaves and stems of wild-type plants also show a slig ht pig mentation, sug g esting that the pap1-D phenotype might in part reflect enhancement of an endoge-nous stress response. However, we never observed pig-mentation in the roots of wild-type plants—in marked contrast to the strong pigmentation at the base of pap1-D roots (Fig ure 1B). Except for very weak pig mentation in flower petals (Figure 1A), enhanced pigmentation in pap1-D was observed throughout development. No other morpho-logical phenotypes were observed.Because anthocyanins are a subclass of flavonoid natural products derived from the phenylpropanoid skeleton, we examined the expression of phenylpropanoid biosynthetic g enes and the accumulation of natural products. RNA g el blot analysis showed massive enhancement of the expres-sion of phenylpropanoid biosynthetic g enes in pap1-D plants (Figure 2). The amounts of transcripts encoding chal-cone synthase (CHS), the entry point enzyme into the fla-vonoid branch pathway, and dihydroflavonol reductase, an enzyme of flavonoid biosynthesis specific for anthocyanins, were greater in pap1-D plants than in wild-type Col-0 plants.Transcripts encoding g lutathione S-transferase, which has been implicated in the transport of anthocyanins into the vacuole (Alfenito et al., 1998), also were expressed in in-creased amounts. Moreover, the accumulation of tran-scripts that encode phenylalanine ammonia-lyase (PAL), the first enzyme of the overall phenylpropanoid biosynthetic pathway, also was markedly enhanced, indicating that tran-scriptional activation was not confined to the flavonoid branch.To determine the extent of changes in anthocyanins and other phenylpropanoid-derived compounds in pap1-D, we extracted and analyzed soluble and cell wall–bound phe-nolic compounds by HPLC. Analysis of the soluble fraction, which was obtained by extraction in acetone, revealed sev-eral quantitative differences between pap1-D and wild-type Col-0 plants—in particular, increased concentrations of cer-tain flavonol g lycosides, including Glc-rhamnose (Rha)-quercetin, Glc-Rha-kaempferol, and unidentified conjugates of kaempferol and quercetin (Figures 3A and 3B). After alka-line hydrolysis of the residue that was obtained after ace-tone extraction, one portion was freeze-dried for analysis of anthocyanidins (anthocyanin aglycones); the remainder was partitioned into ethyl acetate for determination of cell wall–bound hydroxycinnamic acids. Anthocyanidins were present in g reater concentrations in pap1-D than in Col-0 (Fig ures 3C and 3D), as were coumaric and sinapic acids measured in alkaline hydrolysates of the wall-bound phenolic fraction (Fig ures 3E and 3F). Thus, pap1-D is characterized by strongly increased concentrations of glycosylated anthocya-nins, flavonols, and cell wall–esterified hydroxycinnamic acidsin comparison with wild-type Col-0.Figu re 2.Enhanced Expression of Phenylpropanoid Biosynthetic Genes in pap1-D.RNA g el blot hybridization was conducted with total RNA isolated from 6-week-old pap1-D and Col-0 wild-type plants. DFR, dihydrofla-vonol reductase; GST, glutathione S-transferase; UBQ, ubiquitin.2386The Plant CellThe observation of increased wall-bound hydroxycin-namic acids in pap1-D prompted us to measure the content and composition of lignin, which is derived from hydroxycin-namic acid precursors. Lig nin was analyzed by derivati-zation followed by reductive cleavag e, which helps to determine the absolute amounts of guaiacyl (G, monomethyl-ated) and syringyl (S, dimethylated) lignin monomers (Lu and Ralph, 1997). The results in Table 1 indicate increases in both total G and total S residues in the cell wall fraction of pap1-D compared with those in Col-0, but the S/G ratio var-ied little. The change in lignin monomers could reflect an in-crease in lignin content or a change in composition that led to more efficient monomer extractability.Changes in lignin content and composition have been en-g ineered in transg enic plants by downreg ulation of PAL,caffeic acid O -methyltransferase, and caffeoyl-CoA O -meth-yltransferase, enzymes of the lig nin branch of phenylpro-panoid biosynthesis (Atanassova et al., 1995; Zhong et al.,1998). PAL activity in stems of pap1-D plants was approxi-mately twice that found in stems of wild-type plants,whereas the activities of the two O -methyltransferases dif-fered little between the two (Table 1). Thus, the changes in lig nin composition and increased concentrations of wall-bound sinapic acid in pap1-D reflect the change in PAL ac-tivity but do not appear to be associated with increases in lignin O -methyltransferase activities.The Arabidopsis transparent testa glabra1-1 ( ttg1-1 ) mu-tation blocks anthocyanin accumulation and trichome for-mation (Koornneef, 1981). TTG1 encodes a WD40 repeat protein homolog ous with an AN11-encoded protein from petunia, which also controls anthocyanin production (deVetten et al., 1997; Walker et al., 1999). To test the geneticFigure 3.Effect of pap1-D Mutation on Accumulation of Phenylpropanoid Products.(A) to (F) HPLC profiles of phenylpropanoid metabolites in extracts from wild-type Col-0 ([A], [C], and [E]) and pap1-D ([B], [D], [F]) plants.(A) and (B) Soluble phenolics: peak 1, rhamnose (Rha)-Glc-Rha-quercetin; peak 2, quercetin conjugate; peak 3, Rha-Glc-Rha-kaempferol; peak 4, Glu-Rha-quercetin; peak 5, Rha-Rha-quercetin; peak 6, Glc-Rha-kaempferol; peaks 7 and 8, kaempferol conjugates; peak 9, sinapic acid;peak 10, Rha-Rha-kaempferol.(C) and (D) Anthocyanidins; the inset in (D) shows the UV light absorption spectrum of the major anthocyanidin eluting at 21.5 min.(E) and (F) Wall-bound phenolics: peak 1, trans -4-coumaric acid; peak 2, sinapic acid; peak 3, cis -4-coumaric acid.Activation Tagging of PAP12387relationship between TTG1 and PAP1, we crossed ttg1-1 with pap1-D. The pap1-D allele was tracked by Basta resis-tance, and the ttg1-1 mutation was scored visibly. The dou-ble mutant F2 plants failed to accumulate anthocyanins, indicating that TTG1 is required for the production of antho-cyanins mediated by PAP1 overexpression and acts either downstream from or at the same step as PAP1.Cloning of PAP1In a population of Ͼ100 seg reg ating T2 plants, each plant that had the pap1-D phenotype showed resistance to Basta, and all plants with a wild-type phenotype (i.e., lacking purple pig mentation) were sensitive to Basta, indicating that the mutation was tightly linked to the T-DNA insert. Moreover, hybridization of DNA gel blots of pap1-D genomic DNA that had been digested with EcoRI or KpnI showed that the 35Se sequences were localized to single fragments of 10 and 12 kb, respectively (data not shown), indicating that the mutant contained a single, simple insertion. The 12-kb KpnI and 10-kb EcoRI fragments were cloned by plasmid rescue (Weigel et al., 2000), g enerating pPAP1-K1 and pPAP1-E1, respec-tively. Nucleotide sequencing and restriction analysis showed that the 12-kb KpnI frag ment fully overlapped the smaller EcoRI frag ment. Probing the Arabidopsis CD4-7 cDNA li-brary at high stringency with pPAP1-K1 resulted in isolation of a single 956-bp cDNA, which defined three exons in the genomic DNA of pPAP1-K1 encoding an MYB transcription factor (Figures 4A and 4B). In the pap1-D line, the 35Se se-quences had inserted 5.1 kb 3Ј to the start of this gene, des-ignated PAP1, and RNA gel blot hybridization with the PAP1 cDNA revealed a sing le 1-kb transcript, which was mas-sively overexpressed in the pap1-D mutant (Figure 4C).To confirm that overexpression of PAP1 caused the pap1-D phenotype, we cloned a 3-kb g enomic frag ment spanning the three PAP1 exons and flanking sequences into pMN20-2, which contains two copies of 35Se (Weig el et al., 2000), thereby creating pMN-PAP1. Transformation of wild-type Col-0 with this construct, which mimics the g enomic con-text of the pap1-D allele, generated multiple transgenic lines with the characteristic purple phenotype (Fig ure 5B). As would be expected from position effects, these transgenic lines represented an allelic series ranging from the wild type to an even more intense phenotype than pap1-D, in some cases having strong purple pigmentation in the petals (Fig-ure 5E). In contrast, transformation with pMN20-2 as an empty vector control gave no enhanced pigmentation phe-notype (Figures 5A and 5D).Sequence alig nments with the Arabidopsis databases showed that PAP1 is a member of the R2, R3 MYB family, which is estimated to have Ͼ100 members in Arabidopsis (Kranz et al., 1998; Romero et al., 1998) and Ͼ80 membersin maize (Rabinowicz et al., 1999). PAP1 is identical to ATMYB75 (Kranz et al., 1998) except that ATMYB75 con-tains a sequencing error (1-bp deletion at position 695), cre-ating an early stop codon. The PAP1 protein shares hig h homology with other MYB-like transcription factors that reg-ulate anthocyanin production (Figures 4B and 4D). PAP1 is closely related to the product of the petunia AN2 g ene, showing 82% identity throug h the MYB reg ion and 50% identity overall. The products of the maize anthocyanin MYB genes C1 and pl are 64% identical through the MYB region, with 38% identity overall. The MYB transcription factor GLABAROUS1 from Arabidopsis and MIXTA from snap-dragon both control trichome development (Oppenheimer et al., 1991; Glover et al., 1998) and are each 58% identical to PAP1 in the MYB domain and 33% identical overall. A phy-log enetic tree constructed with these full-leng th MYB pro-teins shows that PAP1 belong s to a branch involved in anthocyanin biosynthesis (Figure 4D).The PAP1 gene was mapped to 0.2 centimorgan (cM) up from mi303 at 83.7 cM on chromosome 1 by using an Xba1 restriction frag ment leng th polymorphism and the Col/Ler recombinant inbred lines (Nottingham University Stock Cen-tre, Nottingham, UK). The sequencing project recently came to PAP1 on bacterial artificial chromosome F25P12 just be-low mi303 at 85 cM.PAP2Also discovered in the Arabidopsis database was 193M15, an expressed sequence tag with very hig h homolog y withTable 1.Enhanced PAL Activity and Lignin Levels in pap1-D PlantsPlant PAL Activity(mkat/g FW)aStemCOMT b Activity(kat/g FW)StemCCOMT c Activity(pkat/g FW)StemLignin CompostionTotal G(mol/g)Total S(mol/g)Total G and S(mol/g)S/GWild type30.544.250.421.6 2.524.10.12 pap1-D70.344.460.630.6 5.035.60.16a FW, fresh weight; kat, katal.b COMT, caffeic acid O-methyltransferase.c CCOMT, caffeoyl-CoA O-methyltransferase.2388The Plant CellFigure 4.Molecular Characterization of PAP1.Activation Tagging of PAP12389PAP1. 193M15 encodes a protein with 93% identity to PAP1 in the MYB domain and 77% identity overall. To test whether overexpression of 193M15 also could cause the production of anthocyanin pig ments, we made a 35S pro-moter::cDNA fusion construct, pCHF3-PAP2. Transgenic plants containing pCHF3-PAP2 had purple leaves and stems (Fig-ure 5C), althoug h the pig mentation was somewhat weaker than that of the pap1-D mutant or some pMN-PAP1 lines. In view of the sequence similarity between the PAP1 protein and that encoded by 193M15 (Figures 4B and 4D) and similar overexpression phenotypes (Fig ure 5C), we named the 193M15 cDNA PAP2. PAP2 is on bacterial artificial chromo-some T27F4 near mi424 on chromosome 1, ف9 cM below PAP1. PAP2 is also a member of the Arabidopsis MYB family reported as ATMYB90 (Kranz et al., 1998).Overexpression of PAP1 and PAP2 in TobaccoTo test whether PAP1 and PAP2 could enhance pigmenta-tion in other plants, we transformed tobacco cv xanthi with pCHF3-PAP1 and pCHFS-PAP2. Both constructs g ener-ated purple tobacco plants, indicating that the Arabidopsis PAP1 and PAP2 genes could activate production of antho-cyanin pig ments in another species (Fig ures 5G and 5H). These transgenic tobacco plants also produced flowers with much more pig mentation than did the pCHF3 transg enic control plants (Figures 5I and 5J). Tobacco transformed with pCHF3 as an empty vector control did not have an in-creased pigmentation phenotype (Figures 5F and 5I). DISCUSSIONAccumulation of phenylpropanoid products during develop-ment is under tight transcriptional regulation, and the pap1-D phenotype represents a striking override of these g enetic controls. Thus, specific tranches of the overall pathway ap-pear to be controlled by separate sets of transcription fac-tors. For example, the maize myb g enes C1 and pl are involved in the reg ulation of anthocyanin synthesis from CHS onward but do not regulate PAL and other genes of the upstream central pathway (Cone et al., 1993a, 1993b; Mol et al., 1996), whereas P independently controls the 3-deoxy flavonoid branch pathway (Grotewold et al., 1994). In con-trast, the pap1-D phenotype, which results from overex-pression of the PAP1 MYB transcription factor, reflects massively enhanced expression of PAL as well as CHS, the g ene encoding dihydroflavonol reductase, and the g lu-tathione S-transferase gene. This broad transcriptional acti-vation of the overall pathway is accompanied by a corresponding ly broad pattern of enhanced product accu-mulation with increases in lig nin, wall-bound hydroxycin-namic acid esters, flavonols, and anthocyanins. Moreover, pathway activation in pap1-D was observed in all vegetative organs and maintained throughout development, in marked contrast to activation in wild-type plants of individual branch pathways at defined developmental stages and with charac-teristic cell-type, tissue-type, and organ specificities (Graham, 1991; Grotewold et al., 1994). The relatively modest in-crease in lig nin content probably reflects a major control point at the polymerization stag e (Bate et al., 1994) with consequent spillover of lig nin monomers and their precur-sors, which contributes to the marked accumulation of wall-bound hydroxycinnamic acid esters in pap1-D.MYB genes contribute to the control of flavonoid biosyn-thesis in a wide range of plant species, often in combination with other regulatory genes. The extensive sequence simi-larity with AN2, c1, and pl, together with the overexpression phenotypes, suggests that PAP1 and PAP2 may be the Ara-bidopsis orthologs of these petunia and maize myb genes, with genetically defined functions in phenylpropanoid regu-lation. In maize, c1 and pl but not P require R and B, encod-ing basic helix-loop-helix proteins, to activate transcription of maize flavonoid biosynthetic genes (Mol et al., 1996). Ba-sic helix-loop-helix proteins and MYB proteins also function together in the control of flower pigmentation in snapdragon (Goodrich et al., 1992) and petunia (Quattrocchio et al., 1998). Moreover, the WD40 proteins TTG1 and AN11 are re-quired for MYB control of flavonoid biosynthesis in bothFigure 4.(continued).(A) Genomic context of T-DNA insertion in pap1-D and structure of pMN-PAP1. Basta r, Basta resistance; Kan r, Kanamycin resistance; LB, left border; pBS, pBluescript KSϩ plasmid; RB, right border; 4 ϫ 35S denotes four copies of 35Se.(B) Sequence homology of R2R3 MYB. Proteins were aligned using the ClustalW software program. Red shading denotes 100% conserved res-idues, and yellow shading denotes matching residues with PAP1. R2 and R3 MYB domains are shown. GenBank accession numbers PAP1 (AF325123), PAP2 (AF325124), AN2 (AAF66727), C1 (AAA33482), P1 (AAB67720), P (AAC49394), GL1 (P27900), Mixta (CAA55725), and c-myb (AAA52031).(C) Gel blot hybridization of total RNA from 4-week-old pap1-D and Col-0 plants.1.0 denotes transcript size in kilobases.(D) Neighbor-joining phylogenetic tree built by using full-length proteins, showing the anthocyanin MYB family branch. The human gene c-myb is used as an outgroup.2390The Plant CellArabidopsis and petunia (de Vetten et al., 1997; Larkin et al.,1999; Walker et al., 1999), and the pap1-D phenotypes re-quire the WD40 gene TTG1. Despite the stringent and often complex g enetic control of phenylpropanoid biosynthesis,strong overexpression of PAP1 in the pap1-D line was suffi-cient to hyperactivate the pathway, which is reminiscent of the enhancement of flavonoid biosynthesis by deliberate ectopic expression of P in suspension cultures of maize cells (Grotewold et al., 1999). The pap1-D phenotypes may reflect involvement of PAP1 as the limiting factor in a novel reg ulatory circuit with atypically broad control functions in phenylpropanoid biosynthesis or may indicate functional spillover from one regulatory circuit to related circuits when PAP1 is massively overexpressed.A recent report describes the use of activation tagging in Catharanthus cell cultures to isolate ORCA3, a jasmonate-responsive transcriptional regulator of primary and second-ary metabolism, the upregulation of which promotes biosyn-thesis of indole alkaloids (Van der Fits and Memelink, 2000).These data, along with the present finding s, indicate that activation tag g ing can be used to g enerate novel g ain-of-function mutations that affect complex biosynthetic path-ways under polygenic control; as such, this presents a po-tentially powerful new approach for isolating the genes that reg ulate biosynthesis of plant natural products. Loss-of-function screens for transparent testa have been saturated,and no mutations map to the PAP1 or PAP2 loci (Shirley et al., 1995). Moreover, examination of Ͼ100 PAP1 antisense lines showed no visible phenotype (data not shown). The similar overexpression phenotypes of PAP1 and PAP2 sug-gest that these genes may be functionally redundant, such that only activation tagging or some other gain-of-function screen could have readily revealed their key attributes.Activation tag g ing as a g ene discovery tool based on g ain-of-function is intrinsically oriented toward biotechno-logical utility, and the ability to activate a biosynthetic path-way that will lead to the enhanced accumulation of several distinct subclasses of natural products has several impor-tant potential applications. Thus, hyperactivated tissues or org ans provide massively enriched sources for passag e through multiplex drug screens with the potential for discov-ery of novel activities based on what combinatorial effectsFigure 5.Overexpression of PAP1 or PAP2 Enhances Pigmentation in Arabidopsis and Tobacco.(A) to (E) Arabidopsis plants transformed with pMN20-2 ([A] and [D]), pMN-PAP1 ([B] and [E]), and pCHF3:PAP2 (C). (A) to (C) show six-week-old plants. (D) and (E) show flowers on 12-week-old plants.(F) to (J) Tobacco plants transformed with pCHF3 ([F] and [I]), pCHF3:PAP1 (G), and pCHF3:PAP2 ([H] and [J]). Plantlets in (F) to (H) were pho-tographed at age 4 weeks, and flowers in (I) and (J) at 10 weeks after transfer to soil. pCHF3-PAP1 plants had brilliant flower pigmentation, iden-tical to that of pCHF3-PAP2 (data not shown).Activation Tagging of PAP12391might arise from complex mixtures as well as allowing con-venient isolation and characterization of individual bioactive components. This approach in principle could be aug-mented by feeding studies using pathway intermediates or synthetic derivatives. Moreover, activation of phenylpro-panoid biosynthesis in pap1-D reflects massively enhanced expression of g enes encoding pathway enzymes; hence, these tissues provide a correspondingly enriched source for isolating the cDNAs that encode key biosynthetic enzymes not readily identified by biochemical approaches.Although the plant kingdom has a remarkable diversity of natural products, the underlying pathway regulatory mecha-nisms appear to be at least partially conserved between species (Mol et al., 1996; Quattrocchio et al., 1998). For ex-ample, the maize anthocyanin reg ulatory g ene R functions appropriately when expressed in Arabidopsis or tobacco (Lloyd et al., 1992). Likewise, ectopic expression of PAP1 or PAP2 in transg enic tobacco caused phenotypes similar to those observed in Arabidopsis. Therefore, convenient, readily transformed genetic model species, such as Arabi-dopsis, can be used to isolate candidate regulatory genes for direct evaluation in medicinal plants and other exotic species or as a platform for the identification of ortholog s and potentially useful, related genes in target species.The serendipitous discovery of pap1-D among a larg e collection of activation-tag g ed lines was possible because activation of PAP1 enhanced the accumulation of anthocya-nin pigments, which was easily scored. Several other plant natural products, such as the isoprenoids lycopene and car-otene and the alkaloid sang uinarine, also are colored. Hence, genetic activation of these tranches of plant metab-olism also could be scored by visual inspection, but this is not a generally applicable approach. In principle, activation-tag g ed lines with enhanced accumulation of natural prod-ucts of interest could be identified by high-throughput meta-bolic profiling. However, a more promising general strategy may be to make transg enic plants that express easily screened marker genes under the control of promoters from g enes encoding enzymes involved in the biosynthesis of natural products of interest.METHODSPlant Growth and TransformationArabidopsis thaliana ecotype Columbia (Col-0) plants were grown in growth rooms at 22ЊC in long days (16 hr of light) or short days (9 hr of light) and received 250 E from three 35-W cool white bulbs and one 35-W Sylvania GrowLux bulb (Osram Sylvania, Danvers, MA). Nico tiana tabacum cv xanthi plants were reg enerated under 24-hr-light conditions at 25ЊC and then transferred to the greenhouse. Tobacco and Arabidopsis transformation was performed as previ-ously described (Neff et al., 1999; Weig el et al., 2000) except that 0.02% Silwet-L77 (Lehle Seeds, Round Rock, TX) was used for the latter. Basta was obtained from AgrEvo (Montvale, NJ).Gel Blot HybridizationDNA and RNA gel blot hybridizations were performed according to standard procedures (Sambrook et al., 1989). RNA samples used in the gel blot analysis shown in Figure 2 were from vegetative leaves of pap1-D and Col-0 plants g rown under short-day conditions for 4 weeks and long-day conditions for 2 weeks. Probes were full-length cDNA fragments of PAP1, the glutathione S-transferase gene, and the g ene encoding ubiquitin. The chalcone synthase (CHS) probe was a polymerase chain reaction product amplified by using the primers 5Ј-TGGTCTCCGTCCTTCCGTCAA and 5Ј-CCCTCAAATGTCCGT-CTATGGAA. The phenylalanine ammonia-lyase (PAL) probe was am-plified by using the primers 5Ј-CTATACGCTTACCTACCAACAAAC and 5Ј-TCTCCGATGAGAAGTAGCACCAA, and the dihydroflavonol reductase probe was amplified with primers 5Ј-AAAAAGATGACA-GGATGGGT-3Ј and 5Ј-CCCCTGTTTCTGTCTTGTTA-3Ј.Enzyme AssaysPAL activity was measured by using a microcuvette spectrophoto-metric assay (Blount et al., 2000). Caffeic acid O-methyltransferase and caffeoyl-CoA O-methyltransferase activities were assayed by standard methods (Inoue et al., 1998). Protein concentrations were determined by the procedure of Bradford (1976).Phenylpropanoid AnalysisSoluble and wall-bound phenolics in whole-plant extracts as well as extracts of individual tissues were analyzed by HPLC (Blount et al., 2000). The aqueous phase, which remained after ethyl acetate ex-traction of the wall-bound phenolics, was lyophilized and resus-pended in 70% methanol for analysis. The HPLC eluates were monitored by absorbance at 270, 310, and 550 nm, and the peaks were identified by comparing their retention times and UV light spec-tra with those of known standards. Lignin was assayed by derivatiza-tion followed by reductive cleavage (Lu and Ralph, 1997).pMN-PAP1, pCHFS-PAP1, and pCHFS-PAP2 ConstructsA PAP1 genomic fragment was amplified by using 5Ј-AACTACTGC-AGCTAGAGCGTAGAGG-3Ј and 5Ј-TCAAACTGCAGAAACTAAGCC-CA-3Ј to construct 5Ј and 3Ј Pst sites. This fragment was cloned into pMN20-2 (Weigel et al., 2000), which contains two copies of 35Se to create pMN-PAP1. PCHF3-PAP1 was created by amplifying the PAP1 cDNA with primers 5Ј-ACTGGTACCTTTTACAATTTGTTTA-3Јand 3Ј-AAGGGATCCTATACACAAACGCA-5Ј and cloning it into the KpnI and BamHI sites of pCHF3, a pPZP211-based plant expression vector carrying the cauliflower mosaic virus 35S promoter and a pea ribulose 1,5-bisphosphate carboxylase/oxyg enase terminator (C. Fankhauser, K. Hanson, and J. Chory, unpublished data). The PAP2 cDNA was excised from expressed sequence tag clone 193M15 with KpnI and BamHI and was cloned into pCHF3 to create pCHF3-PAP2.ACKNOWLEDGMENTSWe thank the following (all at Salk Institute unless otherwise noted): Tseg aye Dabi for help with transg enic tobacco; Mary Anderson at。
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C.parvum全基因组序列
DOI: 10.1126/science.1094786, 441 (2004);304Science et al.Mitchell S. Abrahamsen,Cryptosporidium parvum Complete Genome Sequence of the Apicomplexan, (this information is current as of October 7, 2009 ):The following resources related to this article are available online at/cgi/content/full/304/5669/441version of this article at:including high-resolution figures, can be found in the online Updated information and services,/cgi/content/full/1094786/DC1 can be found at:Supporting Online Material/cgi/content/full/304/5669/441#otherarticles , 9 of which can be accessed for free: cites 25 articles This article 239 article(s) on the ISI Web of Science. cited by This article has been /cgi/content/full/304/5669/441#otherarticles 53 articles hosted by HighWire Press; see: cited by This article has been/cgi/collection/genetics Genetics: subject collections This article appears in the following/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprints Information about obtaining registered trademark of AAAS.is a Science 2004 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m3.R.Jackendoff,Foundations of Language:Brain,Gram-mar,Evolution(Oxford Univ.Press,Oxford,2003).4.Although for Frege(1),reference was established rela-tive to objects in the world,here we follow Jackendoff’s suggestion(3)that this is done relative to objects and the state of affairs as mentally represented.5.S.Zola-Morgan,L.R.Squire,in The Development andNeural Bases of Higher Cognitive Functions(New York Academy of Sciences,New York,1990),pp.434–456.6.N.Chomsky,Reflections on Language(Pantheon,New York,1975).7.J.Katz,Semantic Theory(Harper&Row,New York,1972).8.D.Sperber,D.Wilson,Relevance(Harvard Univ.Press,Cambridge,MA,1986).9.K.I.Forster,in Sentence Processing,W.E.Cooper,C.T.Walker,Eds.(Erlbaum,Hillsdale,NJ,1989),pp.27–85.10.H.H.Clark,Using Language(Cambridge Univ.Press,Cambridge,1996).11.Often word meanings can only be fully determined byinvokingworld knowledg e.For instance,the meaningof “flat”in a“flat road”implies the absence of holes.However,in the expression“aflat tire,”it indicates the presence of a hole.The meaningof“finish”in the phrase “Billfinished the book”implies that Bill completed readingthe book.However,the phrase“the g oatfin-ished the book”can only be interpreted as the goat eatingor destroyingthe book.The examples illustrate that word meaningis often underdetermined and nec-essarily intertwined with general world knowledge.In such cases,it is hard to see how the integration of lexical meaning and general world knowledge could be strictly separated(3,31).12.W.Marslen-Wilson,C.M.Brown,L.K.Tyler,Lang.Cognit.Process.3,1(1988).13.ERPs for30subjects were averaged time-locked to theonset of the critical words,with40items per condition.Sentences were presented word by word on the centerof a computer screen,with a stimulus onset asynchronyof600ms.While subjects were readingthe sentences,their EEG was recorded and amplified with a high-cut-off frequency of70Hz,a time constant of8s,and asamplingfrequency of200Hz.14.Materials and methods are available as supportingmaterial on Science Online.15.M.Kutas,S.A.Hillyard,Science207,203(1980).16.C.Brown,P.Hagoort,J.Cognit.Neurosci.5,34(1993).17.C.M.Brown,P.Hagoort,in Architectures and Mech-anisms for Language Processing,M.W.Crocker,M.Pickering,C.Clifton Jr.,Eds.(Cambridge Univ.Press,Cambridge,1999),pp.213–237.18.F.Varela et al.,Nature Rev.Neurosci.2,229(2001).19.We obtained TFRs of the single-trial EEG data by con-volvingcomplex Morlet wavelets with the EEG data andcomputingthe squared norm for the result of theconvolution.We used wavelets with a7-cycle width,with frequencies ranging from1to70Hz,in1-Hz steps.Power values thus obtained were expressed as a per-centage change relative to the power in a baselineinterval,which was taken from150to0ms before theonset of the critical word.This was done in order tonormalize for individual differences in EEG power anddifferences in baseline power between different fre-quency bands.Two relevant time-frequency compo-nents were identified:(i)a theta component,rangingfrom4to7Hz and from300to800ms after wordonset,and(ii)a gamma component,ranging from35to45Hz and from400to600ms after word onset.20.C.Tallon-Baudry,O.Bertrand,Trends Cognit.Sci.3,151(1999).tner et al.,Nature397,434(1999).22.M.Bastiaansen,P.Hagoort,Cortex39(2003).23.O.Jensen,C.D.Tesche,Eur.J.Neurosci.15,1395(2002).24.Whole brain T2*-weighted echo planar imaging bloodoxygen level–dependent(EPI-BOLD)fMRI data wereacquired with a Siemens Sonata1.5-T magnetic reso-nance scanner with interleaved slice ordering,a volumerepetition time of2.48s,an echo time of40ms,a90°flip angle,31horizontal slices,a64ϫ64slice matrix,and isotropic voxel size of3.5ϫ3.5ϫ3.5mm.For thestructural magnetic resonance image,we used a high-resolution(isotropic voxels of1mm3)T1-weightedmagnetization-prepared rapid gradient-echo pulse se-quence.The fMRI data were preprocessed and analyzedby statistical parametric mappingwith SPM99software(http://www.fi/spm99).25.S.E.Petersen et al.,Nature331,585(1988).26.B.T.Gold,R.L.Buckner,Neuron35,803(2002).27.E.Halgren et al.,J.Psychophysiol.88,1(1994).28.E.Halgren et al.,Neuroimage17,1101(2002).29.M.K.Tanenhaus et al.,Science268,1632(1995).30.J.J.A.van Berkum et al.,J.Cognit.Neurosci.11,657(1999).31.P.A.M.Seuren,Discourse Semantics(Basil Blackwell,Oxford,1985).32.We thank P.Indefrey,P.Fries,P.A.M.Seuren,and M.van Turennout for helpful discussions.Supported bythe Netherlands Organization for Scientific Research,grant no.400-56-384(P.H.).Supporting Online Material/cgi/content/full/1095455/DC1Materials and MethodsFig.S1References and Notes8January2004;accepted9March2004Published online18March2004;10.1126/science.1095455Include this information when citingthis paper.Complete Genome Sequence ofthe Apicomplexan,Cryptosporidium parvumMitchell S.Abrahamsen,1,2*†Thomas J.Templeton,3†Shinichiro Enomoto,1Juan E.Abrahante,1Guan Zhu,4 Cheryl ncto,1Mingqi Deng,1Chang Liu,1‡Giovanni Widmer,5Saul Tzipori,5GregoryA.Buck,6Ping Xu,6 Alan T.Bankier,7Paul H.Dear,7Bernard A.Konfortov,7 Helen F.Spriggs,7Lakshminarayan Iyer,8Vivek Anantharaman,8L.Aravind,8Vivek Kapur2,9The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals,and causes an unrelenting infection in immuno-compromised individuals such as AIDS patients.We report the complete ge-nome sequence of C.parvum,type II isolate.Genome analysis identifies ex-tremely streamlined metabolic pathways and a reliance on the host for nu-trients.In contrast to Plasmodium and Toxoplasma,the parasite lacks an api-coplast and its genome,and possesses a degenerate mitochondrion that has lost its genome.Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected.Elu-cidation of the core metabolism,including enzymes with high similarities to bacterial and plant counterparts,opens new avenues for drug development.Cryptosporidium parvum is a globally impor-tant intracellular pathogen of humans and animals.The duration of infection and patho-genesis of cryptosporidiosis depends on host immune status,ranging from a severe but self-limiting diarrhea in immunocompetent individuals to a life-threatening,prolonged infection in immunocompromised patients.Asubstantial degree of morbidity and mortalityis associated with infections in AIDS pa-tients.Despite intensive efforts over the past20years,there is currently no effective ther-apy for treating or preventing C.parvuminfection in humans.Cryptosporidium belongs to the phylumApicomplexa,whose members share a com-mon apical secretory apparatus mediating lo-comotion and tissue or cellular invasion.Many apicomplexans are of medical or vet-erinary importance,including Plasmodium,Babesia,Toxoplasma,Neosprora,Sarcocys-tis,Cyclospora,and Eimeria.The life cycle ofC.parvum is similar to that of other cyst-forming apicomplexans(e.g.,Eimeria and Tox-oplasma),resulting in the formation of oocysts1Department of Veterinary and Biomedical Science,College of Veterinary Medicine,2Biomedical Genom-ics Center,University of Minnesota,St.Paul,MN55108,USA.3Department of Microbiology and Immu-nology,Weill Medical College and Program in Immu-nology,Weill Graduate School of Medical Sciences ofCornell University,New York,NY10021,USA.4De-partment of Veterinary Pathobiology,College of Vet-erinary Medicine,Texas A&M University,College Sta-tion,TX77843,USA.5Division of Infectious Diseases,Tufts University School of Veterinary Medicine,NorthGrafton,MA01536,USA.6Center for the Study ofBiological Complexity and Department of Microbiol-ogy and Immunology,Virginia Commonwealth Uni-versity,Richmond,VA23198,USA.7MRC Laboratoryof Molecular Biology,Hills Road,Cambridge CB22QH,UK.8National Center for Biotechnology Infor-mation,National Library of Medicine,National Insti-tutes of Health,Bethesda,MD20894,USA.9Depart-ment of Microbiology,University of Minnesota,Min-neapolis,MN55455,USA.*To whom correspondence should be addressed.E-mail:abe@†These authors contributed equally to this work.‡Present address:Bioinformatics Division,Genetic Re-search,GlaxoSmithKline Pharmaceuticals,5MooreDrive,Research Triangle Park,NC27009,USA.R E P O R T S SCIENCE VOL30416APRIL2004441o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mthat are shed in the feces of infected hosts.C.parvum oocysts are highly resistant to environ-mental stresses,including chlorine treatment of community water supplies;hence,the parasite is an important water-and food-borne pathogen (1).The obligate intracellular nature of the par-asite ’s life cycle and the inability to culture the parasite continuously in vitro greatly impair researchers ’ability to obtain purified samples of the different developmental stages.The par-asite cannot be genetically manipulated,and transformation methodologies are currently un-available.To begin to address these limitations,we have obtained the complete C.parvum ge-nome sequence and its predicted protein com-plement.(This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the project accession AAEE00000000.The version described in this paper is the first version,AAEE01000000.)The random shotgun approach was used to obtain the complete DNA sequence (2)of the Iowa “type II ”isolate of C.parvum .This isolate readily transmits disease among numerous mammals,including humans.The resulting ge-nome sequence has roughly 13ϫgenome cov-erage containing five gaps and 9.1Mb of totalDNA sequence within eight chromosomes.The C.parvum genome is thus quite compact rela-tive to the 23-Mb,14-chromosome genome of Plasmodium falciparum (3);this size difference is predominantly the result of shorter intergenic regions,fewer introns,and a smaller number of genes (Table 1).Comparison of the assembled sequence of chromosome VI to that of the recently published sequence of chromosome VI (4)revealed that our assembly contains an ad-ditional 160kb of sequence and a single gap versus two,with the common sequences dis-playing a 99.993%sequence identity (2).The relative paucity of introns greatly simplified gene predictions and facilitated an-notation (2)of predicted open reading frames (ORFs).These analyses provided an estimate of 3807protein-encoding genes for the C.parvum genome,far fewer than the estimated 5300genes predicted for the Plasmodium genome (3).This difference is primarily due to the absence of an apicoplast and mitochondrial genome,as well as the pres-ence of fewer genes encoding metabolic functions and variant surface proteins,such as the P.falciparum var and rifin molecules (Table 2).An analysis of the encoded pro-tein sequences with the program SEG (5)shows that these protein-encoding genes are not enriched in low-complexity se-quences (34%)to the extent observed in the proteins from Plasmodium (70%).Our sequence analysis indicates that Cryptosporidium ,unlike Plasmodium and Toxoplasma ,lacks both mitochondrion and apicoplast genomes.The overall complete-ness of the genome sequence,together with the fact that similar DNA extraction proce-dures used to isolate total genomic DNA from C.parvum efficiently yielded mito-chondrion and apicoplast genomes from Ei-meria sp.and Toxoplasma (6,7),indicates that the absence of organellar genomes was unlikely to have been the result of method-ological error.These conclusions are con-sistent with the absence of nuclear genes for the DNA replication and translation machinery characteristic of mitochondria and apicoplasts,and with the lack of mito-chondrial or apicoplast targeting signals for tRNA synthetases.A number of putative mitochondrial pro-teins were identified,including components of a mitochondrial protein import apparatus,chaperones,uncoupling proteins,and solute translocators (table S1).However,the ge-nome does not encode any Krebs cycle en-zymes,nor the components constituting the mitochondrial complexes I to IV;this finding indicates that the parasite does not rely on complete oxidation and respiratory chains for synthesizing adenosine triphosphate (ATP).Similar to Plasmodium ,no orthologs for the ␥,␦,or εsubunits or the c subunit of the F 0proton channel were detected (whereas all subunits were found for a V-type ATPase).Cryptosporidium ,like Eimeria (8)and Plas-modium ,possesses a pyridine nucleotide tran-shydrogenase integral membrane protein that may couple reduced nicotinamide adenine dinucleotide (NADH)and reduced nico-tinamide adenine dinucleotide phosphate (NADPH)redox to proton translocation across the inner mitochondrial membrane.Unlike Plasmodium ,the parasite has two copies of the pyridine nucleotide transhydrogenase gene.Also present is a likely mitochondrial membrane –associated,cyanide-resistant alter-native oxidase (AOX )that catalyzes the reduction of molecular oxygen by ubiquinol to produce H 2O,but not superoxide or H 2O 2.Several genes were identified as involved in biogenesis of iron-sulfur [Fe-S]complexes with potential mitochondrial targeting signals (e.g.,nifS,nifU,frataxin,and ferredoxin),supporting the presence of a limited electron flux in the mitochondrial remnant (table S2).Our sequence analysis confirms the absence of a plastid genome (7)and,additionally,the loss of plastid-associated metabolic pathways including the type II fatty acid synthases (FASs)and isoprenoid synthetic enzymes thatTable 1.General features of the C.parvum genome and comparison with other single-celled eukaryotes.Values are derived from respective genome project summaries (3,26–28).ND,not determined.FeatureC.parvum P.falciparum S.pombe S.cerevisiae E.cuniculiSize (Mbp)9.122.912.512.5 2.5(G ϩC)content (%)3019.43638.347No.of genes 38075268492957701997Mean gene length (bp)excluding introns 1795228314261424ND Gene density (bp per gene)23824338252820881256Percent coding75.352.657.570.590Genes with introns (%)553.9435ND Intergenic regions (G ϩC)content %23.913.632.435.145Mean length (bp)5661694952515129RNAsNo.of tRNA genes 454317429944No.of 5S rRNA genes 6330100–2003No.of 5.8S ,18S ,and 28S rRNA units 57200–400100–20022Table parison between predicted C.parvum and P.falciparum proteins.FeatureC.parvum P.falciparum *Common †Total predicted proteins380752681883Mitochondrial targeted/encoded 17(0.45%)246(4.7%)15Apicoplast targeted/encoded 0581(11.0%)0var/rif/stevor ‡0236(4.5%)0Annotated as protease §50(1.3%)31(0.59%)27Annotated as transporter 69(1.8%)34(0.65%)34Assigned EC function ¶167(4.4%)389(7.4%)113Hypothetical proteins925(24.3%)3208(60.9%)126*Values indicated for P.falciparum are as reported (3)with the exception of those for proteins annotated as protease or transporter.†TBLASTN hits (e Ͻ–5)between C.parvum and P.falciparum .‡As reported in (3).§Pre-dicted proteins annotated as “protease or peptidase”for C.parvum (CryptoGenome database,)and P.falciparum (PlasmoDB database,).Predicted proteins annotated as “trans-porter,permease of P-type ATPase”for C.parvum (CryptoGenome)and P.falciparum (PlasmoDB).¶Bidirectional BLAST hit (e Ͻ–15)to orthologs with assigned Enzyme Commission (EC)numbers.Does not include EC assignment numbers for protein kinases or protein phosphatases (due to inconsistent annotation across genomes),or DNA polymerases or RNA polymerases,as a result of issues related to subunit inclusion.(For consistency,46proteins were excluded from the reported P.falciparum values.)R E P O R T S16APRIL 2004VOL 304SCIENCE 442 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mare otherwise localized to the plastid in other apicomplexans.C.parvum fatty acid biosynthe-sis appears to be cytoplasmic,conducted by a large(8252amino acids)modular type I FAS (9)and possibly by another large enzyme that is related to the multidomain bacterial polyketide synthase(10).Comprehensive screening of the C.parvum genome sequence also did not detect orthologs of Plasmodium nuclear-encoded genes that contain apicoplast-targeting and transit sequences(11).C.parvum metabolism is greatly stream-lined relative to that of Plasmodium,and in certain ways it is reminiscent of that of another obligate eukaryotic parasite,the microsporidian Encephalitozoon.The degeneration of the mi-tochondrion and associated metabolic capabili-ties suggests that the parasite largely relies on glycolysis for energy production.The parasite is capable of uptake and catabolism of mono-sugars(e.g.,glucose and fructose)as well as synthesis,storage,and catabolism of polysac-charides such as trehalose and amylopectin. Like many anaerobic organisms,it economizes ATP through the use of pyrophosphate-dependent phosphofructokinases.The conver-sion of pyruvate to acetyl–coenzyme A(CoA) is catalyzed by an atypical pyruvate-NADPH oxidoreductase(Cp PNO)that contains an N-terminal pyruvate–ferredoxin oxidoreductase (PFO)domain fused with a C-terminal NADPH–cytochrome P450reductase domain (CPR).Such a PFO-CPR fusion has previously been observed only in the euglenozoan protist Euglena gracilis(12).Acetyl-CoA can be con-verted to malonyl-CoA,an important precursor for fatty acid and polyketide biosynthesis.Gly-colysis leads to several possible organic end products,including lactate,acetate,and ethanol. The production of acetate from acetyl-CoA may be economically beneficial to the parasite via coupling with ATP production.Ethanol is potentially produced via two in-dependent pathways:(i)from the combination of pyruvate decarboxylase and alcohol dehy-drogenase,or(ii)from acetyl-CoA by means of a bifunctional dehydrogenase(adhE)with ac-etaldehyde and alcohol dehydrogenase activi-ties;adhE first converts acetyl-CoA to acetal-dehyde and then reduces the latter to ethanol. AdhE predominantly occurs in bacteria but has recently been identified in several protozoans, including vertebrate gut parasites such as Enta-moeba and Giardia(13,14).Adjacent to the adhE gene resides a second gene encoding only the AdhE C-terminal Fe-dependent alcohol de-hydrogenase domain.This gene product may form a multisubunit complex with AdhE,or it may function as an alternative alcohol dehydro-genase that is specific to certain growth condi-tions.C.parvum has a glycerol3-phosphate dehydrogenase similar to those of plants,fungi, and the kinetoplastid Trypanosoma,but(unlike trypanosomes)the parasite lacks an ortholog of glycerol kinase and thus this pathway does not yield glycerol production.In addition to themodular fatty acid synthase(Cp FAS1)andpolyketide synthase homolog(Cp PKS1), C.parvum possesses several fatty acyl–CoA syn-thases and a fatty acyl elongase that may partici-pate in fatty acid metabolism.Further,enzymesfor the metabolism of complex lipids(e.g.,glyc-erolipid and inositol phosphate)were identified inthe genome.Fatty acids are apparently not anenergy source,because enzymes of the fatty acidoxidative pathway are absent,with the exceptionof a3-hydroxyacyl-CoA dehydrogenase.C.parvum purine metabolism is greatlysimplified,retaining only an adenosine ki-nase and enzymes catalyzing conversionsof adenosine5Ј-monophosphate(AMP)toinosine,xanthosine,and guanosine5Ј-monophosphates(IMP,XMP,and GMP).Among these enzymes,IMP dehydrogenase(IMPDH)is phylogenetically related toε-proteobacterial IMPDH and is strikinglydifferent from its counterparts in both thehost and other apicomplexans(15).In con-trast to other apicomplexans such as Toxo-plasma gondii and P.falciparum,no geneencoding hypoxanthine-xanthineguaninephosphoribosyltransferase(HXGPRT)is de-tected,in contrast to a previous report on theactivity of this enzyme in C.parvum sporo-zoites(16).The absence of HXGPRT sug-gests that the parasite may rely solely on asingle enzyme system including IMPDH toproduce GMP from AMP.In contrast to otherapicomplexans,the parasite appears to relyon adenosine for purine salvage,a modelsupported by the identification of an adeno-sine transporter.Unlike other apicomplexansand many parasitic protists that can synthe-size pyrimidines de novo,C.parvum relies onpyrimidine salvage and retains the ability forinterconversions among uridine and cytidine5Ј-monophosphates(UMP and CMP),theirdeoxy forms(dUMP and dCMP),and dAMP,as well as their corresponding di-and triphos-phonucleotides.The parasite has also largelyshed the ability to synthesize amino acids denovo,although it retains the ability to convertselect amino acids,and instead appears torely on amino acid uptake from the host bymeans of a set of at least11amino acidtransporters(table S2).Most of the Cryptosporidium core pro-cesses involved in DNA replication,repair,transcription,and translation conform to thebasic eukaryotic blueprint(2).The transcrip-tional apparatus resembles Plasmodium interms of basal transcription machinery.How-ever,a striking numerical difference is seenin the complements of two RNA bindingdomains,Sm and RRM,between P.falcipa-rum(17and71domains,respectively)and C.parvum(9and51domains).This reductionresults in part from the loss of conservedproteins belonging to the spliceosomal ma-chinery,including all genes encoding Smdomain proteins belonging to the U6spliceo-somal particle,which suggests that this par-ticle activity is degenerate or entirely lost.This reduction in spliceosomal machinery isconsistent with the reduced number of pre-dicted introns in Cryptosporidium(5%)rela-tive to Plasmodium(Ͼ50%).In addition,keycomponents of the small RNA–mediatedposttranscriptional gene silencing system aremissing,such as the RNA-dependent RNApolymerase,Argonaute,and Dicer orthologs;hence,RNA interference–related technolo-gies are unlikely to be of much value intargeted disruption of genes in C.parvum.Cryptosporidium invasion of columnarbrush border epithelial cells has been de-scribed as“intracellular,but extracytoplas-mic,”as the parasite resides on the surface ofthe intestinal epithelium but lies underneaththe host cell membrane.This niche may al-low the parasite to evade immune surveil-lance but take advantage of solute transportacross the host microvillus membrane or theextensively convoluted parasitophorous vac-uole.Indeed,Cryptosporidium has numerousgenes(table S2)encoding families of putativesugar transporters(up to9genes)and aminoacid transporters(11genes).This is in starkcontrast to Plasmodium,which has fewersugar transporters and only one putative ami-no acid transporter(GenBank identificationnumber23612372).As a first step toward identification ofmulti–drug-resistant pumps,the genome se-quence was analyzed for all occurrences ofgenes encoding multitransmembrane proteins.Notable are a set of four paralogous proteinsthat belong to the sbmA family(table S2)thatare involved in the transport of peptide antibi-otics in bacteria.A putative ortholog of thePlasmodium chloroquine resistance–linkedgene Pf CRT(17)was also identified,althoughthe parasite does not possess a food vacuole likethe one seen in Plasmodium.Unlike Plasmodium,C.parvum does notpossess extensive subtelomeric clusters of anti-genically variant proteins(exemplified by thelarge families of var and rif/stevor genes)thatare involved in immune evasion.In contrast,more than20genes were identified that encodemucin-like proteins(18,19)having hallmarksof extensive Thr or Ser stretches suggestive ofglycosylation and signal peptide sequences sug-gesting secretion(table S2).One notable exam-ple is an11,700–amino acid protein with anuninterrupted stretch of308Thr residues(cgd3_720).Although large families of secretedproteins analogous to the Plasmodium multi-gene families were not found,several smallermultigene clusters were observed that encodepredicted secreted proteins,with no detectablesimilarity to proteins from other organisms(Fig.1,A and B).Within this group,at leastfour distinct families appear to have emergedthrough gene expansions specific to the Cryp-R E P O R T S SCIENCE VOL30416APRIL2004443o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mtosporidium clade.These families —SKSR,MEDLE,WYLE,FGLN,and GGC —were named after well-conserved sequence motifs (table S2).Reverse transcription polymerase chain reaction (RT-PCR)expression analysis (20)of one cluster,a locus of seven adjacent CpLSP genes (Fig.1B),shows coexpression during the course of in vitro development (Fig.1C).An additional eight genes were identified that encode proteins having a periodic cysteine structure similar to the Cryptosporidium oocyst wall protein;these eight genes are similarly expressed during the onset of oocyst formation and likely participate in the formation of the coccidian rigid oocyst wall in both Cryptospo-ridium and Toxoplasma (21).Whereas the extracellular proteins described above are of apparent apicomplexan or lineage-specific in-vention,Cryptosporidium possesses many genesencodingsecretedproteinshavinglineage-specific multidomain architectures composed of animal-and bacterial-like extracellular adhe-sive domains (fig.S1).Lineage-specific expansions were ob-served for several proteases (table S2),in-cluding an aspartyl protease (six genes),a subtilisin-like protease,a cryptopain-like cys-teine protease (five genes),and a Plas-modium falcilysin-like (insulin degrading enzyme –like)protease (19genes).Nine of the Cryptosporidium falcilysin genes lack the Zn-chelating “HXXEH ”active site motif and are likely to be catalytically inactive copies that may have been reused for specific protein-protein interactions on the cell sur-face.In contrast to the Plasmodium falcilysin,the Cryptosporidium genes possess signal peptide sequences and are likely trafficked to a secretory pathway.The expansion of this family suggests either that the proteins have distinct cleavage specificities or that their diversity may be related to evasion of a host immune response.Completion of the C.parvum genome se-quence has highlighted the lack of conven-tional drug targets currently pursued for the control and treatment of other parasitic protists.On the basis of molecular and bio-chemical studies and drug screening of other apicomplexans,several putative Cryptospo-ridium metabolic pathways or enzymes have been erroneously proposed to be potential drug targets (22),including the apicoplast and its associated metabolic pathways,the shikimate pathway,the mannitol cycle,the electron transport chain,and HXGPRT.Nonetheless,complete genome sequence analysis identifies a number of classic and novel molecular candidates for drug explora-tion,including numerous plant-like and bacterial-like enzymes (tables S3and S4).Although the C.parvum genome lacks HXGPRT,a potent drug target in other api-complexans,it has only the single pathway dependent on IMPDH to convert AMP to GMP.The bacterial-type IMPDH may be a promising target because it differs substan-tially from that of eukaryotic enzymes (15).Because of the lack of de novo biosynthetic capacity for purines,pyrimidines,and amino acids,C.parvum relies solely on scavenge from the host via a series of transporters,which may be exploited for chemotherapy.C.parvum possesses a bacterial-type thymidine kinase,and the role of this enzyme in pyrim-idine metabolism and its drug target candida-cy should be pursued.The presence of an alternative oxidase,likely targeted to the remnant mitochondrion,gives promise to the study of salicylhydroxamic acid (SHAM),as-cofuranone,and their analogs as inhibitors of energy metabolism in the parasite (23).Cryptosporidium possesses at least 15“plant-like ”enzymes that are either absent in or highly divergent from those typically found in mammals (table S3).Within the glycolytic pathway,the plant-like PPi-PFK has been shown to be a potential target in other parasites including T.gondii ,and PEPCL and PGI ap-pear to be plant-type enzymes in C.parvum .Another example is a trehalose-6-phosphate synthase/phosphatase catalyzing trehalose bio-synthesis from glucose-6-phosphate and uridine diphosphate –glucose.Trehalose may serve as a sugar storage source or may function as an antidesiccant,antioxidant,or protein stability agent in oocysts,playing a role similar to that of mannitol in Eimeria oocysts (24).Orthologs of putative Eimeria mannitol synthesis enzymes were not found.However,two oxidoreductases (table S2)were identified in C.parvum ,one of which belongs to the same families as the plant mannose dehydrogenases (25)and the other to the plant cinnamyl alcohol dehydrogenases.In principle,these enzymes could synthesize protective polyol compounds,and the former enzyme could use host-derived mannose to syn-thesize mannitol.References and Notes1.D.G.Korich et al .,Appl.Environ.Microbiol.56,1423(1990).2.See supportingdata on Science Online.3.M.J.Gardner et al .,Nature 419,498(2002).4.A.T.Bankier et al .,Genome Res.13,1787(2003).5.J.C.Wootton,Comput.Chem.18,269(1994).Fig.1.(A )Schematic showing the chromosomal locations of clusters of potentially secreted proteins.Numbers of adjacent genes are indicated in paren-theses.Arrows indicate direc-tion of clusters containinguni-directional genes (encoded on the same strand);squares indi-cate clusters containingg enes encoded on both strands.Non-paralogous genes are indicated by solid gray squares or direc-tional triangles;SKSR (green triangles),FGLN (red trian-gles),and MEDLE (blue trian-gles)indicate three C.parvum –specific families of paralogous genes predominantly located at telomeres.Insl (yellow tri-angles)indicates an insulinase/falcilysin-like paralogous gene family.Cp LSP (white square)indicates the location of a clus-ter of adjacent large secreted proteins (table S2)that are cotranscriptionally regulated.Identified anchored telomeric repeat sequences are indicated by circles.(B )Schematic show-inga select locus containinga cluster of coexpressed large secreted proteins (Cp LSP).Genes and intergenic regions (regions between identified genes)are drawn to scale at the nucleotide level.The length of the intergenic re-gions is indicated above or be-low the locus.(C )Relative ex-pression levels of CpLSP (red lines)and,as a control,C.parvum Hedgehog-type HINT domain gene (blue line)duringin vitro development,as determined by semiquantitative RT-PCR usingg ene-specific primers correspondingto the seven adjacent g enes within the CpLSP locus as shown in (B).Expression levels from three independent time-course experiments are represented as the ratio of the expression of each gene to that of C.parvum 18S rRNA present in each of the infected samples (20).R E P O R T S16APRIL 2004VOL 304SCIENCE 444 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。
胡壮麟语言学教程笔记、重点
《语言学教程》重难点学习提示第一章语言的性质语言的定义：语言的基本特征（任意性、二重性、多产性、移位、文化传递和互换性）；语言的功能（寒暄、指令、提供信息、询问、表达主观感情、唤起对方的感情和言语行为）；语言的起源（神授说，人造说，进化说）等。
第二章语言学语言学定义；研究语言的四大原则（穷尽、一致、简洁、客观）；语言学的基本概念（口语与书面语、共时与历时、语言与言学、语言能力与言行运用、语言潜势与语言行为）；普通语言学的分支（语音、音位、语法、句法、语义）；；语言学的应用（语言学与语言教学、语言与社会、语言与文字、语言与心理学、人类语言学、神经语言学、数理语言学、计算语言学）等。
第三章语音学发音器官的英文名称；英语辅音的发音部位和发音方法；语音学的定义；发音语音学；听觉语音学；声学语音学；元音及辅音的分类；严式与宽式标音等。
第四章音位学音位理论；最小对立体；自由变异；互补分布；语音的相似性；区别性特征；超语段音位学；音节；重音（词重音、句子重音、音高和语调）等。
第五章词法学词法的定义；曲折词与派生词；构词法（合成与派生）；词素的定义；词素变体；自由词素；粘着词素（词根，词缀和词干）等。
第六章词汇学词的定义；语法词与词汇词；变词与不变词；封闭词与开放词；词的辨认；习语与搭配。
第七章句法句法的定义；句法关系；结构；成分；直接成分分析法；并列结构与从属结构；句子成分；范畴（性，数，格）；一致；短语，从句，句子扩展等。
第八章语义学语义的定义；语义的有关理论；意义种类（传统、功能、语用）；里奇的语义分类；词汇意义关系（同义、反义、下义）；句子语义关系。
第九章语言变化语言的发展变化（词汇变化、语音书写文字、语法变化、语义变化）；第十章语言、思维与文化语言与文化的定义；萨丕尔-沃夫假说；语言与思维的关系；语言与文化的关系；中西文化的异同。
第十一章语用学语用学的定义；语义学与语用学的区别；语境与意义；言语行为理论（言内行为、言外行为和言后行为）；合作原则。
MSP与BSP
BSP和MSP区别：1）检测的内容不完全相同BSP引物不包括CpG位点，在CpG位点的上下游，扩增后通过测序检测改位置的甲基化状态(测序如果CG不变则为甲基化，如果CG变为TG则为非甲基化）MSP/USP是根据修饰后甲基化和非甲基化的引物不同扩增出不同的条带而判断是否为甲基化(只扩增出MSP条带表明为甲基化，如果扩增出USP则表明为非甲基化）2）MSP原理：甲基化特异性PCR(MSP)其原理为：双链DNA变性解链后，在HSO3-作用下发生C→U转化，C若已有甲基化则无此改变；甲基化修饰只发生于5′→3′方向C-G相联结构的C上，因此在HSO3-作用后，DNA CpG岛若无甲基化，则序列中的改变为C→U，CG→UG，若有甲基化则为C→U，CG→CG，用不同的引物做PCR，即可检测出这种差异，从而确定基因有无CpG岛甲基化。
从理论上说，DNA发生甲基化的MSP产物的序列与原序列比较，变化为C→T，CG→CG，相应其互补链的改变为G→A，CG→CG。
然后根据目的基因修饰前后的改变，相应设计M和U引物，有时我们需要设计两轮引物！BSP法原理：用亚硫酸氢钠修饰处理基因组DNA，所有未发生甲基化的胞嘧啶（C）被转化为尿嘧啶（U）而甲基化的胞嘧啶则不变。
基因组DNA经亚硫酸盐处理后，设计BSP引物扩增目的片段，此时尿嘧啶（U）全部转化为胸腺嘧啶（T），最后对PCR产物进行测序就可以判断CpG位点是否发生甲基化。
3)一般用BSP找到甲基化位点，然后根据甲基化位点设计MSP引物，摸索条件以得到最优检测甲基化的方法。
BSP因为涉及测序，其结果准确但要求克隆时所挑克隆较多等原因，操作繁琐，不易大批量操作。
MSP则可以检测大量标本并用于检测。
详细参见/forums/topic/5371-differences-between-msp-and-bsp/引物和探针你可以查文献看别人设计好的，然后再找公司合成，也可以自己设计1、ABI Methyl primer Express DNA甲基化引物设计软件使用ABI Methyl primer Express DNA甲基化引物设计软件使用- 丁香园论坛=甲基化引物设计#141597232、甲基化的一些旧贴总结整理较全的甲基化（MSP)原理及问题总结，希望对大家有帮助！- 丁香园论坛=荧光定量方法测甲基化#91849273、荧光探针设计软件操作说明荧光探针设计软件操作说明- 丁香园论坛=甲基化探针设计#6763407DNA甲基化PCR引物的设计.pdf(931.16k) 在线查看精品：荧光定量PCR技术原理与结果分析..pdf(2157.35k) 在线查看关于DNA甲基化引物设计除了常用的在线设计工具MethPrimer : /methprimer/index1.htmlBiSearch Web Server: http://bisearch.enzim.hu/ABI公司还给我们提供了另外一个选择：Methyl primer Express，并且这个工具还是免费的。
EMP-1 基因对人食管癌细胞系生长的影响
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Nevertheless, the importance of CRYs in determining the effects of HMF has not been examined in animal cells. Thus, although the biological effects of the HMF are becoming more well-documented, the cellular and molecular mechanisms underlying the effect of the HMF remain unclear. Quantitative transcriptomic methods are widely used for understanding and assessing changes in cellular and molecular activity in response to changes in the environment [27,28]. Massively parallel sequencing technology, namely deep sequencing, is a powerful method for investigating DNA expression patterns. This technology has most recently been used to evaluate transcriptomic changes in mosquitoes under different environmental conditions, screen for the pathways involved in neurodevelopment and neurodegeneration, predict drug resistance genes for hepatitis, and understand the cellular mechanisms involved in irradiationinduced tumorigenesis [2932]. Deciphering the transcriptomic responses to HMF exposure would allow for the identification of key genes involved in the processes of HMF-response/GMF-adaptation, and may ultimately lead to the development of effective counter-measures to prevent the adverse effects of the HMF. We previously found that 2 d of exposure to the HMF accelerates the proliferation of human neuroblastoma cells (SH-SY5Y cell line), and predicted that the G1/S transition plays an important role in the bio-HMF response [18]. In the present study, to identify the HMF responding genes in neuronal cells, we collected RNA samples from SH-SY5Y cells after 2 d of culturing in a permalloy magnetic shielding system, where they were exposed to either the HMF (<200 nT) or GMF (~15 μT), after which the RNA was sent for massively parallel sequencing based on Illumina/Solexa sequencing [33]. Compared to controls, 2464 differentially expressed genes (DEGs) were identified, of which 216 were up-regulated and 2248 were down-regulated. Real-time quantitative PCR (RT-qPCR) was used to identify and verify the DEGs. Also, the dynamic expression changes of selected DEGs during the incubation period were measured. Our results provide some insight into the mechanisms underlying the effects of the HMF.
《细胞生物学》学习题解答
第一章：绪论1、填空题：1、细胞生物学是细胞整体、超微结构和分子水平上研究及其规律的科学。
、2、名词解释：1、细胞学说（cell theory）3、选择题：1、现今世界上最有影响的学术期刊是。
a：Natune b: Cell c: PNAS d: Science2、自然界最小的细胞是（a）病毒（b）支原体（c）血小板（d）细菌4、是非题：1、现代细胞生物学的基本特征是把细胞的生命活动和亚细胞的分子结构变化联系起来。
…………………………………（）5、问答题：1. 当前细胞生物学研究的热点课题哪些？2. 细胞学说的基本要点是什么？细胞学说在细胞学发展中有什么重大意义？3. 细胞生物学的发展可划分为哪几个阶段？各阶段的主要特点是什么？第二章：细胞基本知识概要1、名词解释：1. 血影（Ghost）2. 通道形成蛋白（Porin）3. 纤维冠（fibrous corona）2、选择题：1、立克次氏体是（a）一类病毒（b）一种细胞器（c）原核生物（d）真核生物2、原核细胞的呼吸酶定位在（a）细胞质中（b）质膜上（c）线粒体内膜上（d）类核区内3、最小的细胞是（a）细菌（b）类病毒（c）支原体（d）病毒4、在英国引起疯牛病的病原体是：（a）朊病毒（prion）（b）病毒（Virus）（c）立克次体（rickettsia）（d）支原体（mycoplast）5、逆转病毒（retro virus）是一种（a）双链DNA病毒（b）单链DNA病毒（c）双链RNA病毒（d）单链RNA病毒6、英国疯牛病病原体是（a） DNA病毒（b） RNA病毒（c）类病毒（d）朊病毒7、线虫基因组的全序列测定目前已接近尾声，发现其一共约有（）种的编码基因（a） 6000（b） 10000（c） 20000（d） 500008、原核细胞与真核细胞虽有许多不同，但都是（a）核仁（b）核糖体（c）线粒体（d）内质网9、前病毒是（a） RNA病毒（b）逆转录RNA病毒RNA病毒（c）整合到宿主DNA中的逆转录DNA（d）整合到宿主DNA中的DNA病毒3、是非题：1.类病毒仅由裸露的DNA所构成，不能制造衣壳蛋白。
宁波大学基因工程考研题库-精
第一章绪论1.分子生物学要研究的主要内容是（）（）（）参考答案是：基因工程；基因表达调控研究；结构分子生物学第二章DNA结构一、填空题(6分)1.天然存在的DNA分子形式为右手（）型螺旋参考答案是：B2.Cot曲线方程为：（）参考答案是：C/C0=1/（1+K2C0t）3.DNA复性必须满足两个条件：（）（）参考答案是：盐浓度必须高；温度必须适当高4.DNA 携带有两类不同的遗传信息，即（）信息和（）信息。
参考答案是：基因编码；基因选择性表达二、判断题(12分)1.拓扑异构酶I解旋需要ATP酶。
参考答案是：不正确2.假基因通常与它们相似的基因位于相同的染色体上。
参考答案是：不正确3.水蜥的基因组比人的基因组大。
参考答案是：正确4.一段长度100bp的DNA，具有4100种可能的序列组合形式。
参考答案是：正确5.单个核苷酸通过磷酸二酯键连接到DNA骨架上。
参考答案是：正确6.在高盐和低温条件下由DNA单链杂交形成的双螺旋表现出几乎完全的互补性，这一过程可看作是一个复性（退火）反应。
参考答案是：不正确7.生物的遗传密码只存在于细胞核中参考答案是：不正确8.Top I解旋需要ATP参考答案是：不正确9.琼脂糖凝胶电泳—EBr电泳法分离纯化超螺旋DNA原理是根据EBr可以较多地插入到超螺旋DNA 中，因而迁移速度较快参考答案是：不正确10.高等真核生物的大部分DNA是不编码蛋白质的参考答案是：正确11.Cot1/2与基因组复杂性有关参考答案是：正确12.Cot1/2与基因组大小有关参考答案是：正确三、单选题(4分)1.DNA变性是由于（D）A 磷酸二酯键断裂；B 多核苷酸解离；C 碱基的甲基化修饰；D 互补碱基之间氢键断裂；E 糖苷键断裂；2.1953年，Watson 和Crick提出（A）A 多核苷酸DNA链通过氢键连接成一个双螺旋；B DNA的复制是半保留的，常常形成亲本-子代双螺旋杂合链；C 三个连续的核苷酸代表一个遗传密码；D 遗传物质通常是DNA而非RNA。
植物干细胞调控研究新进展
植物干细胞调控研究新进展中国细胞生物学学报Chinese Journal of Cell Biology 2015, 37(7): 1021–1028DOI: 10.11844/cjcb.2015.07.0024收稿日期: 2015-01-15 接受日期: 2015-04-07973计划前期研究专项(批准号: 2014CB160306)、重庆市教委创新团队建设基金(批准号: KJTD201307)和重庆师范大学引进人才启动基金项目(批准号: 12XLR36)资助的课题*通讯作者。
Tel: 023-********, E-mail: hanmazhang@/doc/f017810486.html, Received: January 15, 2015 Accepted: April 7, 2015This work was supported by the National Grand Fundamental Research Pre-973 Program of China (Grant No.2014CB160306), the Innovation T eam Fund of the Education Department of Chongqing Municipality (Grant No.KJTD201307) and a Start-Up Fund from Chongqing Normal University (Grant No.12XLR36)*Corresponding author. Tel: +86-23-65912976, E-mail: hanmazhang@/doc/f017810486.html, 网络出版时间: 2015-07-01 16:53 URL: /doc/f017810486.html,/kcms/detail/31.2035.Q. 20150701.1653.001.html植物干细胞调控研究新进展赵中华南文斌梁永书张汉马*(植物环境适应分子生物学重庆市重点实验室, 重庆师范大学生命科学学院, 重庆 401331)摘要植物干细胞是植物胚后发育形成各种组织和器官的细胞来源和信号调控中心, 其调控机理是植物学研究的重要内容。
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Discoveringfunctionaltranscription-factorcombinationsinthehumancellcycle
ZhouZhu,1JayShendure,andGeorgeM.Church1DepartmentofGenetics,HarvardMedicalSchool,Boston,Massachusetts02115,USAWiththecompletionoffullgenomesequencesandadvancementinhigh-throughputtechnologies,insilicomethodshavebeensuccessfullyusedtointegratediversedatasourcestowardunravelingthecombinatorialnatureoftranscriptionalregulation.Sofar,almostallofthesestudiesarerestrictedtolowereukaryotessuchasbuddingyeast.Wedescribehereacomputationalsearchforfunctionaltranscription-factor(TF)combinationsusingphylogeneticallyconservedsequencesandmicroarray-basedexpressiondata.Takingintoaccountbothorientationalandpositionalconstraints,weinvestigatedtheoverrepresentationofbindingsitesinthevicinityofoneanotherandwhetherthesecombinationsresultinmorecoherentexpressionprofiles.Withoutanypriorbiologicalknowledge,thesearchledtothediscoveryofseveralexperimentallyestablishedTFassociations,aswellassomenovelones.Inparticular,weidentifiedaregulatorymodulecontrollingcellcycle-dependenttranscriptionofG2-Mgenesandexpandedits
functionalgenerality.Wealsodetectedmanyhomotypiccombinations,supportingtheimportanceofbinding-sitedensityintranscriptionalregulationofhighereukaryotes.
[Supplementalmaterialisavailableonlineatwww.genome.organdhttp://genetics.med.harvard.edu/∼zzhu/combination.html.]
Cis-regulationofgeneexpressionbythebindingoftranscriptionfactors(TFs)isacriticalcomponentofcellularphysiology.Ineukaryotes,abatteryofTFsoftenworktogetherinacombinato-rialfashiontoenablecellstorespondtoawidespectrumofenvironmentalanddevelopmentalsignals.Integrationofge-nomesequencesand/orChIP–chipdatawithgene-expressiondatahasfacilitatedinsilicodiscoveryofhowthecombinatoricsandpositioningofTF-bindingsitesunderliegeneactivationinavarietyofcellularprocessesforrelativelysimpleorganismssuchasSaccharomycescerevisiaeandCaenorhabditiselegans(Bussemakeretal.2001;Pilpeletal.2001;BanerjeeandZhang2003;BeerandTavazoie2004;Katoetal.2004;TeraiandTakagi2004).Appli-cationofthesemethodstothehumangenome,however,iscom-plicatedbyitssignificantlylargersizeandsubstantialrepetitivecontent,aswellasthegreatercomplexityofthetranscriptionalnetwork.Earlystudiessuggestthatphylogeneticfootprinting(WassermanandFickett1998;Wassermanetal.2000;Levyetal.2001;BlanchetteandTompa2002;Liuetal.2004)andafocusonmotifcombinations(Frechetal.1998;Keletal.1999;KrivanandWasserman2001;Aertsetal.2003)mayproveessentialtocom-putationalanalysesofhumantranscription-factorbindingsites.AsthefunctionalinteractionsbetweenTFsoftenrequirethemtobeinphysicalproximity,theirbindingsitesarelikelytobeoverrepresentedinthevicinityofeachother.Exploitingsuchproperty,wedevisedatwo-stepstrategy(Fig.1)torevealknownornoveltranscriptionfactorsthatworkinconcert.StartingfromaTF-bindingsiteofinterest,ouralgorithmfirstdiscoverssignifi-cantlyenrichedneighboringmotif(s)usinghuman–mousecon-servedsequences,andthenexaminesthefunctionalsignificanceoftheirphysicalproximitythroughtheassessmentofsimilarityinexpressionprofiles.Applyingthismethodologytohumanpro-motersequencesandacellcycleexpressiondataset,wefoundanumberofmotifpairsthatnotonlypreferentiallyco-occurnearby,butalsoappeartoacttogetherindetermininggeneex-pressionpattern,includingaG2-Mregulatorymodule.Inaddi-tiontoheterotypicinteractions,weobservedahomotypicdistri-butionoftranscription-factorbindingsites,asmanyofthemarespecificallyenrichedaroundthemselves.
ResultsExtractionofhumanpromotersequenceandphylogeneticfootprinting
WehadpreviouslymappedUniGeneclustersontothehumangenomeaswellasgenerateda“mousenized”versionofthege-nome(http://club.med.harvard.edu/hummus/hummus.html).Tobuildapromotersequencesset,weextractedthesequence1kbupstreamof11,436curatedRefSeqmRNAsasputativepro-moterregions.Whilesomeregulatoryelementscanactoververylargedistances,uptoseveralkilobasesfromtranscriptionalstartsites(TSS),wefocusedonsequencesintherelativeproximityofTSS,astheyaremostlikelytocontainregulatoryinformationforevolutionarilyconservedbiologicalprocessessuchasthecellcycle.
AccuracyofidentifyingbindingsitesbyweightmatrixForanchormotifs,weutilized134experimentallyderivedposi-tionweightmatricesfromtheTRANSFACdatabase.Theycorre-spondto∼70distinctmotifs,asestimatedbyCompareACE(Hughesetal.2000)withacut-offof0.85.PutativebindingsiteswereidentifiedbyscanningpromotersequenceswiththeanchormotifsusingPATSER(HertzandStormo1999).Toestimatetheaccuracyofourinsilicopredictions,wecomparedthelistofE2Fsite(M00516)-containinggeneswiththoseidentifiedasE2F4tar-getsinprimaryfibroblastsusingChIP–chiptechnology(Renetal.2002).Ourpromotersetincludes96oftheexperimentallydeterminedtargetgenes,and56ofthemwerepredictedcompu-tationallybasedon1-kbpromotersequences(P=1.1E-11).Phylo-geneticfootprintinghasbeendemonstratedtobeapowerfulstrat-egyforfilteringoutfalse-positiveresultsofmotifdiscoveryalgo-