Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.Although A. fumigatus using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2s-M2) are controlled by the A. nidulans gpdA promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into A. fumigatus by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the E. coli hygromycin resistance gene, hph, was placed under the control of seven copies of the TetR binding site (tetO7) in a plasmid vector and co-transfected into A. fumigatus protoplasts together with one of the two transactivator plasmids. Since the hph gene is essential to A. fumigatus in the presence of hygromycin, resistance to hygromycin was used as a marker of hph reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO7-hph reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2s-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO7-hph reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation.Here we describe the construction of plasmid vectors that can be used to regulate gene expression in A. fumigatusThe vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in  Aspergillus fumigatus is a saprophytic filamentous fungus that has become the leading mould pathogen in leukemia treatment centers and transplantation units in developed countries, second only to Candida spp. as a cause of systemic mycosis [Aspergillus case-fatality rate demonstrated that more than 50% of patients die with, or as a result of, aspergillosis, despite having received the reference standard of therapy [ mycosis . Despite therapy . The conA. fumigatus genome is expected to greatly facilitate efforts to determine the contribution of specific gene products to the virulence of this opportunistic pathogen. Unfortunately, the genetic tractability of A. fumigatus has lagged behind some other fungal systems, particularly in the area of conditional expression systems. Inducible promoter systems have proven to be instrumental for the elucidation of gene function in a number of species, most notably with essential genes. Experimental manipulation of gene expression in A. fumigatus is presently accomplished through the use of DNA cassettes that are introduced into the organism as transgenes [alcA promoter from A. nidulans has been successfully used in A. fumigatus [alcA promoter can have significant effects on the metabolism of the organism and thus remain a concern for many applications, particularly for in vivo studies.The completion of the annotated sequence of the ansgenes -5, inseransgenes ,6 or expansgenes . An induumigatus . HoweverE. coli tetracycline-resistance operon, a regulatory unit that detects minute concentrations of tetracycline and mounts an appropriate resistance response. Expression of the operon is controlled by a repressor protein, TetR that binds to operator sequences (tetO) in the promoter/enhancer region of the operon and prevents transcription. In the presence of tetracycline TetR is unable to bind tetO, which releases the repression and allows the operon to be expressed. This system has been adapted for experimental gene regulation in eukaryotes by fusing TetR to the VP16 transcriptional activating domain of herpes simplex virus VP16, thereby creating a synthetic tetracycline-regulatable transcriptional activator protein (tTA) that can be used to regulate a gene that is under the control of a tetracycline-responsive promoter  linked to a 175 bp minimal gpdA promoter from A. nidulans  and one showing moderate hygromycin sensitivity (tTA-1). Conidia from each of these transformants were spotted into the center of a plate of minimal medium containing both doxycycline and hygromycin and the radial growth of the colony was monitored with time. The pH of the medium in this experiment was adjusted to 8 in order to maximize the hygromycin toxicity. As shown in Fig. A. fumigatus .Inducible promoter systems are particularly useful for creating strains that can be inducibly depleted of an essential gene product . To modeulans p48, Fig. 2.tus Fig.  can be uhph RNA than tTA-2  and/or basal expression from one or more integrated copies of the tetO7-hph reporter gene (which would not be affected by doxycycline). Since there was a clear dose-response effect of doxycycline on hph expression and hygromycin resistant growth in this strain  that requires interaction of the transactivator with tetracyclines before 'tet-on' . Unfortunditions , we chosnditions . One paric cells .tetO7-hph reporter . Doxycycline was incorporated into the medium at 100 μg/ml to ensure that the tetO7-hph transgene would be expressed at sufficient levels to protect against hygromycin toxicity. Approximately 15% of 27 hygromycin resistant colonies showed reduced growth when shifted to hygromycin medium without doxycycline, one of which was selected for further analysis. As shown in Fig. tetO promoter in A. fumigatus. A further increase in hygromycin resistance was achieved at 15 μg/ml of doxycycline, but concentrations above 15 μg/ml had no additional effect. Northern blots analysis confirmed that the levels of hph RNA in the rtTA transformant were increased by the addition of doxycycline to the medium .A recent report has shown that iron blocks the accumulation and activity of tetracyclines in bacteria . Since iH20 Fig. . This inS. cerevisiae [Candida albicans and C. glabrata are the only pathogenic fungi in which the system has been successfully applied thus far, however neither of these studies used the tetR-VP16 fusions upon which the tTA and rtTA systems are based [The tetracycline-inducible method of gene regulation has become one of the most popular tools to manipulate gene expression in eukaryotes . The effrevisiae , the sysre based -17.A. fumigatus. Since the hph gene is essential in the presence of toxic levels of hygromycin, the ability to control hygromycin resistance by modulating the levels of hph transcription validates the system as a tool for analysis of essential genes in A. fumigatus. In the tTA system we found that individual transformants varied in the amount of doxycycline that was necessary to regulate expression of the tetO7-hph reporter gene. Since doxycycline prevents the tTA protein from binding to the tetO sequence, this is most likely due to variability in the amount of tTA protein that is expressed in each transformant. A limitation of the tTA approach described here is that the majority of the hygromycin-resistant transformants from the tTA/tetO7-hph co-transfection were not susceptible to regulation by doxycycline. This may be due in part to leaky expression of the tetO7-hph reporter, caused by enhancers in the proximity of the integration site [gpdA promoter used in this study were too high to be removed by non toxic concentrations of doxycycline. Since lower levels of tTA expression are more readily suppressed by doxycycline, it is conceivable that a weaker promoter used to drive tTA would increase the frequency with which doxycycline-regulatable transformants can be isolated. Lower levels of tTA expression could also be accomplished by using a shorter segment of the gpdA promoter used in this study.In this study we show that both the tet-off (tTA) and tet-on (rtTA) systems can be used to regulate the expression of a hygromycin resistance reporter gene in ion site ,25. A setetO7-hph reporter gene was also observed in a strain expressing the reverse transactivator, rtTA. Concentrations of doxycycline from 2 μg/ml to 15 μg/ml gave a graded response of hygromycin resistance, indicating that A. fumigatus is responsive to concentrations of doxycycline that are similarly effective in S. cerevisiae [C. albicans [A. fumigatus. Only 15% of the hygromycin-resistant colonies from an rtTA/tetO7-hph co-transfection showed doxycycline-dependent hygromycin resistance however, suggesting that some of the hygromycin resistance was due to leaky expression of the tetO7-hph gene. Leakage of tetO7-regulated genes has been described in other systems, and is attributed to enhancers located in the proximity of the integration site that increase expression of the tetO-linked gene [tetO7-controlled genes regardless of whether they are integrated randomly in the genome or targeted to specific loci.The ability to quantitatively control expression from the revisiae  and C. aalbicans , raisingked gene ,25. ThisA. fumigatus. A limitation of the system was that only 10–15% of the transformants could be regulated by doxycycline, either when tTA or rtTA were used, emphasizing the need to screen for regulatable transformants. A recent approach to limit the problem of leakiness of a tetO-driven gene is the use of trans-silencer proteins comprised of fusions between tetR and a transcriptional silencing domain [A. fumigatus-derived trans-silencer protein into the co-transfection approach described in this study would improve the efficiency of the system.This report establishes the utility of the tetracycline-regulated system as an approach to regulate gene expression in g domain ,27. It iAll vectors are based on the pBluescript plasmid (Stratagene) and were linearized prior to transfection. PCR amplification of components were performed using standard amplification protocols using PfuTurbo DNA polymerase (Stratagene).tet operator sequence (tetO7) was PCR amplified from pUHD10-3 [aagcttgcgtatcacgaggccctttc and the reverse primer 5'-aagcttctcgacccgggtaccgag (added HindIII cloning sites are underlined) and cloned into the HindIII site of pBluescript. A 1.6 kb fragment containing a minimal gpdA promoter from A. nidulans (-175 relative to the ATG of the hph open reading frame), the hph gene encoding resistance to hygromycin, and the trpC terminator from A. nidulans, was then PCR amplified from pAN7-1 [gagctccccatcttcagtatattcatc (added SstI cloning site underlined) and reverse primer 5'-tctagatcgcgtggagccaagagcgg (added XbaI cloning site underlined) and cloned downstream of tet07 into the SstI and XbaI sites of the plasmid, creating p482. To minimize read-through from flanking sequences into tet07, a 280 bp segment of the terminator region of A. fumigatus cgrA [tet07 PCR to create p500. The cgrA terminator was PCR amplified from genomic DNA of A. fumigatus isolate H237 using the forward primer 5'aagcttacagcagaagaatctctc (added HindIII cloning site underlined) and reverse primer 5'ctcgagatgattcatgacgtatattc (added XhoI cloning site underlined), cloned into pCR2.1-Topo (Invitrogen), excised with HindIII, and inserted upstream of tetO7 in p482 to create p500.A segment containing seven copies of the pUHD10-3  with them pAN7-1  with fortus cgrA  was inseA. nidulans gpdA promoter was amplified from pAN7-1 [hph open reading frame) using the forward primer 5'-aagcttcggagaatatggagctt (added HindIII cloning site underlined) and the reverse primer 5'-gaattcggtgatgtctgctcaag (added EcoRI cloning site underlined) and cloned into pBluescript at the same sites. The tTA gene was then PCR amplified from pUHD15-1 [gaattctggcaatgtctagattagataaaag (added EcoRI cloning site underlined) and reverse primer 5'-atcatgtctggatcctcgcg  and cloned into the EcoRI and BamHI sites downstream of the gpdA (-679) promoter. A 280 bp segment of the terminator region of A. fumigatus cgrA [actagtacagcagaagaatctctc (added SpeI site underlined) and reverse primer 5'-gcggccgcatgattcatgacgtatattc (added NotI site underlined) and inserted into the SpeI and NotI sites downstream of tTA. To introduce phleomycin selection into this construct, a phleomycin resistance cassette containing the A. nidulans gpdA promoter, the Streptoalloteichus hindustanus ble gene encoding resistance to phleomycin, and the S. cerevisiae CYC1 terminator was amplified from pBCphleo  using the forward primer 5'-cctcaggcggagaatatggagcttcatcg and the reverse primer 5'-cctcaggaattaaagccttcgagcgtccc. The PCR product was cloned into pCR-Blunt II-TOPO (Invitrogen), excised with KpnI and XhoI and inserted into the PgpdA-tTA construct to create p444. The phleomycin cassette was excised from p444 with HindIII and re-ligated to create p473. To introduce hygromycin selection into p444, the phleomycin cassette was excised with KpnI and HindIII and replaced with a hygromycin resistance cassette  that was amplified from pAN7-1 [ggtacccggagaatatggagcttc (added KpnI cloning site underlined) and reverse primer 5'-aagcttgcttgagagttcaaggaag (added HindIII cloning site underlined) to make p434.A segment of the m pAN7-1   to create p474. To introduce phleomycin resistance into p474, the phleomycin resistance cassette was excised from p444 with KpnI and HindIII and cloned into the same sites in p474 to create p480. To introduce hygromycin resistance into p474, the hygromycin resistance cassette described in p434 was excised from an unrelated plasmid as a HindIII fragment and cloned into the HindIII site of p474 to make p502.The tTA gene was excised from p473 with A. fumigatus strains used in this study are listed in Table-Aspergillus minimal medium plates [4·4H20. For low-iron minimal medium, the FePO4·4H20 concentration was reduced to 0.45 μM.The m plates . This miA. fumigatus protoplasts as previously described [tetO7-hph reporter construct was co-transfected with 5 μg of the linearized tTA plasmid (p444), or 50 μg of the linearized rtTA plasmid (p474).Plasmids were introduced into escribed . FollowiAspergillus minimal medium agar containing hygromycin and doxycycline at the concentrations specified in the Figure legends. The plates were then incubated at 37°C, and colony diameter was measured with time. Radial growth rates were calculated from the exponential part of the resulting growth curves.For experiments addressing the effects of doxycycline on hygromycin sensitivity, ten thousand conidia were spotted onto the surface of hph gene expression, RNA was isolated from overnight cultures in minimal medium supplemented with the indicated concentrations of doxycycline by crushing in liquid nitrogen and extracting RNA from the crushed mycelium with phenol/chloroform. Twenty micrograms of total RNA were fractionated by formaldehyde gel electrophoresis as previously described [32P-labeled hph DNA probe under stringent conditions in 50% (v/v) formamide/5XSSC /2X Denhardt's solution/10% (w/v) dextran sulfate/1% (w/v) sodium dodecyl sulfate (SDS). The hph probe was an 800 bp EcoRI-BamHI fragment from pAN7-1 [hph open reading frame. Hybridization intensity was quantified with a Phosphorimager (Molecular Dynamics) and normalized for differences in gel loading by quantitating the relative levels of SYBR-green II-stained rRNA .For analysis of escribed , transfem pAN7-1  containitTA tetracycline transactivatorrtTA reverse tetracycline transactivatorTetR tetracycline repressortetO TetR binding sequencehph hygromycin resistance geneble phleomycin resistance geneA. fumigatus, screening of transformants and drafting the manuscript. RB participated in plasmid construction. JCR contributed to the planning of the study. DSA conceived of the project and directed its design and execution. All authors have read and approved the final manuscript.KV participated in vector construction, gene transfer into


We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene.The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish  luciferase gene under a clock gene promoter enables the study of molecular circadian rhythms in vivoGeneration of zebrafish that express the firefly  The circadian clock controls biological processes such as behavior, gene expression, and physiology in diverse organisms, ensuring that these processes to take place at appropriate times of the day. This is crucial for many organisms, such as plants, which must synchronize photosynthesis with day-night cycles. Animals also synchronize to environmental cycles because of more subtle but nevertheless important needs such as predator avoidance, food availability, and optimal temperatures for various processes. Circadian clocks in all species share the following properties: They persist even in constant environmental conditions with periods near 24 h; they can be reset by environmental stimuli such as light and temperature; and their periods are relatively constant at different temperatures.Drosophila, has been extremely valuable in identifying important players of the clockworks and their roles ) were raised as transgenic F1 fish. Three of the five lines emitted bioluminescence above background. The level of bioluminescence varied depending on the line. The strongest-glowing line (#23) was used for this study unless otherwise stated. All the animals used in this study were the progeny of crosses between a transgenic line and the *AB wild-type strain. Therefore, these animals carried the transgene in hemizygous condition.per3-luc transgenic zebrafish is screening for mutations that affect bioluminescence rhythms. Since it is most convenient to screen the youngest possible animals, bioluminescence from transgenic embryos was monitored first. It was also expected that per3-luc-mediated bioluminescence in embryos should cycle from day 1 of development even in constant conditions, because per3 mRNA expression detected by in situ hybridization has been demonstrated to oscillate from day 1 postfertilization in constant conditions  in 2 l of ddH2O) aerated overnight and containing 0.5 mM D-luciferin potassium salt  and 0.013% Amquel Instant Water Detoxifier . Once loaded with animals, four such plates were subjected to automatic monitoring of bioluminescence every 30 min by the Topcount multiplate scintillation counter (Perkin-Elmer) equipped with six detectors and plate stackers. The room temperature was set at 21–22 °C, and the machine at 24 °C. However, due to the heat created by the machine, temperature at the bottom of the stacker was 1–2 °C higher than the room temperature. In order to minimize high background counts under lighted conditions, each plate was dark-adapted for approximately 5 min before being counted for bioluminescence. Each well was counted for 4.8 s every 30 min. The plates were illuminated with two white fluorescent lamps, each facing the left or right side of the stacker. The approximate intensity of the light that reached the plates was 17–35 lux, depending on the position of the plates within the stacker.Embryos or larval fish were placed individually in every other well of a white 96-well Optiplate  with 200 μl of Holtfreter solution . This burst of luminescence is typically followed by a low background level of luminescence (< 50 cps). Furthermore, intermediate levels of spikes were also found in many plots just before the burst of high bioluminescence. Therefore, in order to eliminate data from dead fish, data that exceeded 5,000 cps, or those that went down below 50 cps, at any point of the analyzed portion of the data were first discarded. Then an averaged plot of the remaining data from each clutch of embryos was examined, and the highest count on days 8–9 was determined for experiments presented in Bioluminescence data from the Topcount were imported into Microsoft Excel 2000 by the Import and Analysis macro . In many of the experiments performed on the Topcount, some plates were placed in the machine several days earlier than others in order to monitor fish that experienced different numbers of LD cycles . TherefoPeriod and rhythmicity for each animal were determined by a macro  based on12–18 (Invitrogen) as the primer in a 25–40 μl reaction volume. Real-time PCR was performed in a 25 μl reaction volume containing a probe, forward and reverse primers, and qPCR Mastermix according to the manufacturer's instruction . Each reaction was quadrupled in order to minimize pipetting errors. The primers and TaqMan MGB probes for per3 and luc were designed and synthesized by the Assays-by-Design Gene Expression service : per3 forward, 5′-GCCCTGGCAGCACCA-3′; per3 reverse 5′-GAAAGCTGGAGGACGAGGAA-3′; probe, 5′-6-FAM-CTAAGAGCTCAAAATCC-NFQ-3′; luc forward, 5′-GCAGGTGTCGCAGGTCTT-3′; luc reverse, 5′-GCGACGTAATCCACGATCTCTTTT-3′; probe, 5′-6-FAM-TCACCGGCGTCATCG-NFQ-3′. The ABI Sequence Detection System 7000 (Applied Biosystems) was programmed to perform the following protocol: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.Total RNA was extracted from 9–42 embryos or larval fish raised in petri dishes using TRIzol reagent . The number of animals used for each extraction was recorded. Once extracted, total nucleic acid concentration was determined by a spectrophotometer. In order to prevent genomic DNA contamination, RNA samples were treated with Turbo DNA-free , and the concentration determined again by a spectrophotometer. Total RNA (0.5–1 μg) was subjected to cDNA synthesis by Superscript II Reverse Transcriptase (Invitrogen) using Oligo (dT)per3 or luc cDNA per animal was calculated by the standard-curve method [per3 and luc were compared between two different developmental stages as well as among different times of the day. It was also important to calculate the amount of each mRNA species per animal in order to compare these data to bioluminescence data. The amount of a specific control RNA, as well as the total RNA, may differ among fish of different ages, in which case RNA per animal cannot be calculated by the relative quantification method using a constitutive control. As a concentration standard, a single-stranded DNA oligonucleotide of known concentration was used for each gene. These oligonucleotides span from the 5′ end of the forward primer to the 5′ end of the reverse primer, and including 75 bp for per3 and 110 bp for luc . The standard concentration was varied from 102 to 107 copies per reaction in 10-fold increments. For every qPCR experiment, reactions for standards were performed in four replicates along with reactions for cDNA samples.In this study, relative amount of e method  rather tt-test was performed. The nonparametric Wilcoxon/Kruskal-Wallis test was performed on data that were not normally distributed even after various transformations  were tried. Where multiple tests were performed on a set of data, the experimentwise error rate (α) was adjusted by the Dunn-Sˇidák method [To test whether percentages of rhythmic fish among different experimental groups were equal, the G-test was performed using Microsoft Excel 2000 according to Sokal and Rohlf . If multk method .Protocol S1(27 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession numbers of the sequences discussed in this paper are per 3 cDNA (NM_131584) and BAC clone CH211–138E4 (AL929204). The Ensembl (http://www.ensembl.org/Danio_rerio/) ID of the flanking gene of per 3 mentioned in The GenBank (


A genome-wide study of nucleosome occupancy at yeast promoters shows that promoters that regulate active genes, contain multiple conserved motifs, or contain Rap1 binding sites tend to be depleted of nucleosomes.  PHO5 promoter in yeast is largely devoid of nucleosomes. We systematically evaluated nucleosome occupancy in yeast promoters by immunoprecipitating nucleosomal DNA and quantifying enrichment by microarrays.Although eukaryotic genomes are generally thought to be entirely chromatin-associated, the activated RPS11B promoter, by removing the Rap1 consensus sites.Nucleosome depletion is observed in promoters that regulate active genes and/or contain multiple evolutionarily conserved motifs that recruit transcription factors. The Rap1 consensus was the only binding motif identified in a completely unbiased search of nucleosome-depleted promoters. Nucleosome depletion in the vicinity of Rap1 consensus sites in ribosomal protein gene promoters was also observed by real-time PCR and micrococcal nuclease digestion. Nucleosome occupancy in these regions was increased by the small molecule rapamycin or, in the case of the The presence of transcription factor-binding motifs is an important determinant of nucleosome depletion. Most motifs are associated with marked depletion only when they appear in combination, consistent with a model in which transcription factors act collaboratively to exclude nucleosomes and gain access to target sites in the DNA. In contrast, Rap1-binding sites cause marked depletion under steady-state conditions. We speculate that nucleosome depletion enables Rap1 to define chromatin domains and alter them in response to environmental cues. Global gene-expression patterns are established and maintained by the concerted actions of transcription factors and the proteins that constitute chromatin. The global network of interactions between transcription factors and promoters in yeast is increasingly being characterized . The rolPHO5 promoter in yeast, this remodeling proceeds until essentially no nucleosomes are detected across a region of several hundred base-pairs [The nucleosome consists of approximately 146 base-pairs (bp) of DNA wrapped around an octamer of histone proteins - two each of histones H2A, H2B, H3 and H4. Eukaryotic genomes are packaged into repeating units of nucleosomes separated by around 10-80 bp of linker DNA. High occupancy by nucleosomes is thought to be generally repressive [Transcription factors and chromatin proteins each form complex regulatory networks that interact in a variety of ways ,7. TransWe used chromatin immunoprecipitation (ChIP) and DNA microarrays to evaluate nucleosome occupancy levels for essentially all promoters in yeast. Promoters that regulate active genes, contain multiple conserved motifs or recruit Rap1 tend to be relatively nucleosome-depleted. We also used real-time PCR and micrococcal nuclease digestion to show that nucleosomes are depleted in the vicinity of Rap1 consensus sites. This depletion can be partially reversed by the actions of the small molecule rapamycin or by removing Rap1-binding sites. We suggest that other transcription factors have less robust nucleosome-depleting activities than Rap1 and must therefore act collaboratively to gain access to their cognate sites in the DNA.PHO5 promoter [in vivo with histone H3 was isolated by ChIP using antibody against the carboxy terminus of histone H3 . ChIP DNA and unenriched control DNA were amplified by in vitro transcription and evaluated using microarrays. DNA associated with histone H2B was evaluated in a similar fashion using anti-FLAG antibody and a FLAG-H2B strain. H3 and H2B datasets were compiled by averaging four and three independent biological experiments, respectively. These datasets are remarkably similar as shown by a genome-wide correlation of 0.83 . dA.dT stretches destabilize nucleosome formation in vitro and in vivo [in vitro studies show that a 16-bp insertion leads to a 1.7-fold increase in accessibility of nucleosomal target sites [To identify additional determinants of occupancy, we sought sequence elements associated with nucleosome depletion. Specifically, we carried out an unbiased search for elements up to 10 bp in length that occur with higher frequency in nucleosome-depleted promoters. Two distinct categories of sequences emerged Figure . The fir in vivo ,18. The in vitro and in vivo [p < 10-5). Furthermore, multiple Rap1 sites are found in 19% of nucleosome-depleted promoters with Rap1 sites, compared to 8% of promoters with Rap1 sites overall (hypergeometric p < 10-3). These data suggest that Rap1 recruitment may lead to nucleosome loss.The second sequence element enriched in nucleosome-depleted promoters corresponds to the consensus motif for the Rap1 transcription factor. This motif commonly occurs in the promoters of ribosomal proteins genes and is required for Rap1 binding  in vivo ,20. Somep < 0.001. However, many of the implicated motifs appear in the same promoters. For example, nine of the over-represented motifs are associated with filamentation gene promoters [p <10-5; Figure p < 0.001; Figure Because only the Rap1 consensus site was identified in an unbiased search, we sought to identify additional sequence motifs by incorporating species conservation data. Specifically, we evaluated a set of 71 conserved motifs identified by Kellis and colleagues, a majority of which function in transcription factor recruitment . Nearly romoters . We therp < 0.001. Rap1 has the most significant association . Furthermore, an evaluation of nucleosome occupancy at promoters bound by both members of a cooperative pair revealed a significant association with nucleosome-depletion for 18 of the 31 pairs (hypergeometric p < 0.01). Together, these findings suggest that binding motifs and transcription factors act in combination to deplete nucleosomes and suggest a role for nucleosomes in transcription factor cooperativity [We sought an underlying binding mechanism or function common to the transcription factors we had identified. However, these factors utilize a variety of binding domains, regulate different pathways, and only a minority have significant associations with promoters of highly active genes. Nonetheless, a commonality does emerge when transcription factor cooperativity is considered. A recent informatics study by Banerjee and Zhang identified 31 functionally cooperative transcription factor pairs  on the basis of comprehensive binding and expression data . Only a rativity -25.in vivo [TUB2) with average occupancy by global analysis  growing in yeast extract/peptone/dextrose (YPD) were cross-linked in 1% formaldehyde for 15 min, washed twice in PBS, resuspended in 400 μl lysis buffer  and lysed with glass beads. The resulting extract was sonicated to fragment chromatin  and centrifuged for 15 min. Solubilized chromatin was then immunoprecipitated with polyclonal antibodies against the carboxy terminus of histone H3 . A unenriched whole-cell extract sample (WCE) was also retained as a control. After enrichment, cross-links were reversed by incubating samples in 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1.0% SDS, 150 mM NaCl at 65°C overnight. DNA was purified from ChIP and WCE samples by proteinase K treatment, phenol/chloroform extraction, ethanol precipitation, and incubation with RNAse. DNA associated with histone H2B in vivo was isolated in a similar manner from yeast containing epitope-tagged H2B [DNA associated with histone H3 eviously ,48,49. B8 cells) and whole-cell extract DNA (unenriched control) were amplified in a linear fashion as described [in vitro transcription reaction carried out with the T7 Megascript Kit (Ambion) and RNA samples were purified using an RNeasy Mini Kit (Qiagen). Amplified RNA was reverse-transcribed, incorporating amino-allyl dUTP, and the resulting DNA was fluorescently labeled by incubation with monofunctional reactive Cy5 (enriched sample) or Cy3 (unenriched control) dye as described [To obtain sufficient quantities for hybridization, immunoprecipitated DNA . The FLAG-H2B ChIP dataset was determined from three independent immunoprecipitations and hybridizations. The mRNA dataset was determined from three independent extractions and hybridizations of mRNA against genomic DNA. Relative transcription rates were determined by dividing transcript levels by half-life data collected by Wang and colleagues [Cy5 and Cy3 fluorescence were integrated for each feature using GenePix Pro Software (Axon). Data were processed and composite Cy5:Cy3 ratios determined according to protocols at the Stanford Microarray Database . Correlalleagues . A set olleagues .p < 0.001), consistent with the premise that these promoters are relatively free of nucleosomes.Z-scores were assigned to each intergenic that reflect depletion across the four H3 and three H2B ChIP experiments, using the formula Z = (x - μ)/σ where x is the average of the replicate measurements, μ is the average of all intergenics and σ is the standard error of the replicate measurements. We defined as nucleosome-depleted the 410 features with the highest Z-scores. This set, which includes 308 promoters, contains nucleosome-depleted outliers and is not inclusive of all promoters that immunoprecipitate with average or lower efficiency. The average aqueous enrichment ratio  for thesSaccharomyces Genome Database [P-values calculated by a hypergeometric probability model. The P-values reflect the extent to which observed overlaps exceed that expected under the null hypothesis that there is no relationship between the sets [p < 0.001 [Sequence elements common to nucleosome-depleted promoters were identified by searching between 10 and 500 bp upstream of gene start sites for over-represented sequences up to 10 bp in length using the GeneSpring program suite (Silicon Genetics). Enrichment was confirmed by evaluating the significance of overlap between the set of nucleosome-depleted promoters and the set of promoters containing Rap1 consensus motifs (ACACCCATACAT with up to two mismatches) or poly dA.dT stretches at least 10 bp in length . Statisthe sets . Where sthe sets  were colthe sets . Lists o < 0.001 .-ΔΔCT method described in the Applied Biosystems User Bulletin. For each region examined, the TUB2 promoter was used as the normalizer , and the unenriched control sample was used as the calibrator. Each reported ratio represents the average of three independent ChIP experiments analyzed in duplicate by real-time PCR. The following primer pairs were used:Regions approximately 200 bp in size that span one or more Rap1 consensus sites in ribosomal protein gene promoters were amplified from ChIP and unenriched control samples using SYBR green PCR mix (Qiagen) in an MJ Research real-time PCR machine according to the manufacturers' instructions. Fold-ratios that reflect relative enrichment or depletion of a given region in the H3 or FLAG-H2B ChIP assays were determined using the 2RPS22A promoter: 5'-GCCTAAAACGCCCATAAGTT-3' and 5'-ACTGCAAACCCATATTCAAGA-3'RPS15 promoter: 5'-TACACCGCGCGTATAAATCA-3' and 5'-CCCAGCAAGGAGTTTCTCAG-3'RPS11B promoter: 5'-GAAGAAATATTTCCTTGCTGCACC-3' and 5'-AAGGGAAACGTAAAGCTATTGGAC-3'RPL23A promoter: 5'-ATTAACATCTGTACACCCCCAACT-3' and 5'-TACAGTTCGTTTCCTGCC ATATTA-3'TUB2 promoter: 5'-GGCCTAACAGTAAAGATATCCTCC-3' and 5'-GTTGTAGTAGCTGCTATGT CACTC-3'RPS11B promoter lacking the two Rap1 consensus motifs [RPS11B promoter: 5'-CTGGAAGAAATATTTCCTT GCTCTAG-3' and 5'-AAGGGAAACGTAAAGCTATTGGAC-3'.Centromeric vectors containing either a mutant s motifs  or an es600 of 1.0 were spheroplasted with zymolase , at 30°C for 38 min shaking at 300 rpm), divided into five aliquots, and digested with increasing concentrations (20 U to 320 U) of micrococcal nuclease (Worthington Biochem) in 600 μl 0.5 mM spermidine, 1 mM β-ME, 0.075% NP-40. DNA from digested samples was extracted with phenol twice and chloroform once and precipitated in ethanol. Samples were washed, resuspended in 10 mM Tris pH 7.5, subjected to RNAse treatment, cleaned up with the MinElute kit (Qiagen) and run out in a 1% agarose gel. Following depurination, denaturation and neutralization of the gel, DNA was transferred onto nylon membranes by capillary action and covalently linked to the membranes by UV irradiation. Southern blotting was carried out using a DIG Luminescent Detection Kit (Roche) and DIG-labeled probe generated by PCR using the TUB2, RPS11B and RPS15 primers described above.Log-phase cultures of W303a yeast grown in 450 ml YPD to OD


Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. We have used the chromatin profiling method DamID to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. We also adapted DamID profiling such that we could use tightly gated collections of embryos (2–6 h) and found 20 Hairy targets related to early embryogenesis. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box–containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. We have identified 59 putative transcriptional targets of Hairy. In addition to finding putative targets for Hairy in segmentation, we find groups of targets suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairy's three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, we find here that it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development.Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper  A new chromatin profiling technique identifies targets of the transcription factor Hairy -- as well as targets of three co- repressors -- which implicate Hairy in several developmental processes Drosophila embryogenesis is marked by the subdivision of the embryo into progressively more precise spatial domains, achieved through the coordinated functions of both transcriptional activators and repressors  pair-rule gene, fushi tarazu  subclass of repressor bHLH proteins . These pac has been shown to be a direct transcriptional target of Hairy during peripheral nervous system development .HES proteins have been shown to bind preferentially to Class C sites  as homodimers in vitro . The preelopment . HoweverDrosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2)  . None ofhairy mutant phenotype (Drosophila histone deacetylase 1 homolog), but the evidence for the significance of this interaction is somewhat mixed  acid dehydrogenases  family members are an interesting new class of transcriptional coregulators that encode nicotinamide adenine dinucleotidedSir2 encodes NAD+-dependent histone deacetylase activity that is required for heterochromatic silencing  tethered to a chromatin binding protein leads to specific methylation of DNA adjacent to the protein binding/recruitment sites (Drosophila Kc cells (see Drosophila microarray chip containing approximately 6200 full-length Drosophila Gene Collection (DGC) cDNAs and ESTs , those affecting cell cycle or cell growth , ImpL2, Idgf2), and those with unknown/unlinked functions. Unfortunately, the two previously identified Hairy targets, ftz and ac, are not present in the DGC Release 1 cDNA set used to generate our microarray chips.We identified 40 statistically significant putative direct Hairy transcriptional targets in Kc cells . For jusDrosophila Myc/Max/Mad-Mnt network of bHLH leucine zipper proteins , or because the mechanism by which Hairy acts during segmentation is different than expected. To begin distinguishing among these possibilities, we used the DamID approach to identify Hairy targets in Drosophila embryos during segmentation.Since Hairy is part of the segmentation gene transcriptional regulatory cascade, we expected to find segmentation-related transcription factors as downstream targets of Hairy. The putative Hairy targets we identified in Kc cells do not fulfill this expectation, but rather suggest roles for Hairy in cell cycle, cell growth, and morphogenesis; these putative targets are likely targets for Hairy during its other developmental roles. This could be because of cellular context , or because only half of the Towards this aim, we generated functional transgenic flies carrying a UAS–Dam or UAS–Dam–Hairy fusion gene construct see . As withegh, overlapped between the datasets , is a homeobox-encoding transcription factor known to function in segmentation  in a hairy mutant background  or its target gene  alone were viable. The reduction of Hairy in this target-gene-sensitized background allows inappropriate target gene regulation .If Hairy is a direct regulator of a particular target gene, genetic interaction might be expected between th hairy . In all stg, a series of transgenic lines have been generated in which lacZ expression is driven from different promoter fragments , but not the other two (pstg β-E2.2 and pstg β-E6.7). Consistent with the presence of Hairy binding sites, the lacZ expression from pstg β-E4.9 and pstg β-E6.4, but not from pstg β-E2.2 or pstg β-E6.7, was derepressed (expanded) in a hairy mutant background compared to wild-type (TCGCA) to generate pstg β-E4.9Δhairy. This mutation abolishes Hairy binding in vitro . Wild-type flies carrying this pstg β-E4.9Δhairy reporter exhibit the same lacZ derepression as observed for the original pstg β-E4.9 reporter when in a hairy mutant background, indicating that the derepression is due to Hairy binding  that contain the canonical core Hairy site (CACGCG). We searched for this consensus site within the promoter and transcribed regions of three Hairy targets: stg, egh, and prd. We identified one site in prd, three in egh, and four in the stg genomic region (hairy mutant background (see above). We tested whether the identified sites are direct Hairy binding sites in electromobility shift assays (EMSAs), utilizing bacterially purified full-length Hairy protein and 32P-labeled oligos containing the appropriate Hairy binding sites , a zinc-finger-encoding transcription factor  reporter at cytological location 1F, the transgene insertion site .If particular Hairy targets require specific cofactors to be appropriately regulated, we would expect their expression to be altered (deregulated) in a cofactor mutant background. We performed RNA in situ hybridization for two Hairy Kc cell targets that differentially recruit Groucho, dCtBP, and dSir2. We chose to examine the expression of two ally cf. . Consistkgrounds A–8D. Simkgrounds E–8H. Whikgrounds I–8L, sugWhen DamID data for the three Hairy cofactors and Hairy itself are graphically projected onto chromosomes, several interesting features come to light A. For exWe have known for almost two decades that Hairy plays a pivotal role in the segmentation hierarchy, as well as other developmental processes, but the details of Hairy action have not been easy to tease apart. An important step in understanding the molecular mechanisms surrounding Hairy-mediated repression was made with the identification of Groucho as a Hairy binding protein . One of Drosophila cDNAs, we estimate the actual number of Hairy targets to be approximately twice that number . This predicted number of Hairy targets is close to the approximately 120 strongly staining sites we observe on polytene chromosomes. Of the 59 putative Hairy targets we identified in both the Kc cell and embryo DamID experiments, 58 correspond to bands of Hairy staining on the polytene chromosomes, suggesting that polytene chromosome staining is representing Hairy binding sites without regard to tissue specificity. It is not yet clear what is limiting Hairy accessibility in different tissues or why Hairy's access does not appear to be limited in salivary glands. It may be that polytene chromosome organization necessitates a looser chromatin structure or that the large number of factors that seem to be endogenously expressed in salivary glands affects accessibility. Ultimately, additional confirmation of the DamID and polytene staining correspondence will require microarray tiling chips containing overlapping genomic DNA fragments; however, such genomic DNA tiling chips are currently unavailable.The 59 putative Hairy targets we identified in the embryo and Kc cell DamID experiments correspond to bands of Hairy immunostaining on polytene chromosomes, suggesting that the polytene chromosome staining faithfully represents Hairy binding. Polytene chromosomes are functionally similar in transcriptional activity and display factor/cofactor binding properties similar to chromatin of diploid interphase cells, despite their DNA endoreplication , 2002. SDrosophila microarray chip we used was generated using full-length cDNAs. Indeed, as Hairy has been described as a long-range repressor  have been shown to function as either positive or negative regulators of transcription depending on promoter context and cofactor recruitment  was generated by standard PCR using primers containing a BamHI 5′ site and a NotI 3′ site, cut with BamHI and NotI, and subcloned into the BglII and NotI sites of pCMycDam plasmid, as described previously . As in Kc cells, induced expression of these Dam fusion constructs leads to high levels of nonspecific methylation. Therefore we utilized low-level leaky expression from the minimal promoter of the pUASp vector for these experiments. 2–6-h embryos were collected and dechorionated with 100% bleach. Approximately 500 μl of embryos were crushed in 1 ml of lysis buffer . SDS (to 0.5%) and proteinase K (to 100 μg/ml) were added immediately after homogenization, followed by incubation at 55 °C for 2 h. SDS was increased to 1.5%, followed by incubation for an additional 2–3 h. The genomic DNA was isolated and processed for microarray hybridizations essentially as described previously  for the Northwest Fly Consortium and contain approximately 6200 full-length DGC cDNAs . We employed the default window size of 101 and used a confidence value of ten in our CyberT analysis. The null hypothesis was rejected and a spot ratio was called significantly changed if pBon ≤ 0.05, where pBon is the Bayesian p-value adjusted for multiple hypothesis tests using the conservative Bonferroni correction. Based on prior “self versus self” DamID comparisons, we empirically determined a lower-bound ln(ratio) threshold = |0.405| as an additional significance criterion to discriminate spot intensity signals from the inherent noise in the hybridization process. For each protein analyzed, a fluor-reversed array comparison was performed and used to screen all significant calls for fluor-specific artifacts. For our analyses, we treated the small subset of replicated spots on the array independently. For those cases, both spots were required to be called significant. Reported ratio values were retransformed to log2 as a matter of convention. The complete raw and processed datasets can be accessed at http://www.fhcrc.org/labs/parkhurst/supplementary-data/.Arrays were scanned using a GenePix 4000 scanner , and image analysis was performed using GenePix Pro 3.0. For each array, spot intensity signals were filtered and removed if the values did not exceed 3 standard deviations above the background signal in at least one channel or if the spot was flagged as questionable by the GenePix Pro software. For each spot, background-corrected ratios were natural log transformed and a median-centered normalization strategy was applied across each array. Dam–protein and Dam transfections were independently replicated three times, and the subsequent array comparisons  were analyzed using CyberT , a Bayes7H rucucah/ TM3, 12C st eh/ TM3, Df(3 l)i22 Ki roe pph/TM3, and 2.45.17prd/CyO (D. Ish-Horowicz); FRT82B- P{ry+t7.2=PZ}03463 ry506CtBP/TM3 (N. Perrimon); FRT 82B- E47gro/TM3 , KG02223ImpL2 (#14083), k06602mae/CyO (#10633), Δ88pnt/TM3 (#861), and KG03110rgr (#13770) . Details of these strains are found on FlyBase (http://flybase.bio.indiana.edu:82/). AR2stg and the stg-lacZ reporter lines  were described previously (dCtBP and groucho were generated as previously described (Δhairy transgenic flies were generated by injecting vector (500 μg/ml) along with the pTURBO helper plasmid (100 μg/ml)   into isoLarval cuticle preparations were prepared and analyzed as described by Immunohistochemical whole mount RNA in situ hybridization was performed according to the protocol of 32P-end-labeled probe of each oligo was incubated with either GST– or GST–Hairy–purified proteins (200 ng each), in a 25-μl reaction supplemented with binding buffer  at room temperature. Where indicated, the binding was preformed in the presence of 15-fold excess of unlabeled wild-type or mutated ac competitor oligos. Following incubation, the complexes were resolved using 0.5% TBE-PAGE gels and visualized by autoradiograms. The following oligos were used (forward primers are shown): ac 5′-TAAACCGGTTGGCAGCCGGCACGCGACAGGGCCAGGTTTT-3′; egh egh1 5′-TGCGCGTCACGCGCCGTTC-3′, egh2 5′-TCATTCGCACGCGGAATCT-3′, and egh 3 5′-GCCGGACACGCGATGATGG-3′; mutated ac oligo 5′-TAAACCGGTTGGCAGCCGGGACGCGACAGGGCCAGGTTTT-3′; mutated stg oligo 5′-TCTACCACACACAAACACTCGCACGCGAAAACTGGG -3′; prd 5′-AAGTGACACGCGCTCCGCT-3′; and stg 5′-AAACACACGCGCGCGAAAA-3′.EMSA was carried out using either bacterially expressed GST or GST–Hairy ( full-length) proteins, similar to the procedure described by Drosophila promoter sequences were captured using Apollo Genome Sequence and Annotation Tool . Using the Hairy site closest to transcription start site, the composition of transcription factor binding sites adjacent to (within 500 bp of) the Hairy site was assessed. This was also performed for non-Hairy targets selected because they contained one or more core C-box sequences. Matrices were compared that matched percentages of known Hairy targets  to C-box–containing nontargets.Several bioinformatics approaches were employed to analyze Hairy target gene promoters. In particular, ion Tool . Match vWild-type or pstg βE4.9 third instar larval salivary gland polytene chromosomes were prepared and stained for endogenous proteins essentially as described by The insertion site for the pstg β-E4.9 reporter line was performed as described by Dataset S1(2.9 MB XLS).Click here for additional data file.Dataset S2(11.8 MB XLS).Click here for additional data file.Dataset S3(11.8 MB XLS).Click here for additional data file.Dataset S4(11.8 MB XLS).Click here for additional data file.Dataset S5(11.8 MB XLS)Click here for additional data file.Dataset S6(11.8 MB XLS)Click here for additional data file.Dataset S7(230 KB XLS).Click here for additional data file.Dataset S8(276 KB XLS).Click here for additional data file.Dataset S9(44 KB XLS).Click here for additional data file.


Caenorhabditis elegans, combining genetics, gas chromatography, and DNA microarray analysis. We show that C. elegans synthesizes mmBCFAs de novo and utilizes the long-chain fatty acid elongation enzymes ELO-5 and ELO-6 to produce two mmBCFAs, C15ISO and C17ISO. These mmBCFAs are essential for C. elegans growth and development, as suppression of their biosynthesis results in a growth arrest at the first larval stage. The arrest is reversible and can be overcome by feeding the arrested animals with mmBCFA supplements. We show not only that the levels of C15ISO and C17ISO affect the expression of several genes, but also that the activities of some of these genes affect biosynthesis of mmBCFAs, suggesting a potential feedback regulation. One of the genes, lpd-1, encodes a homolog of a mammalian sterol regulatory element-binding protein (SREBP 1c). We present results suggesting that elo-5 and elo-6 may be transcriptional targets of LPD-1. This study exposes unexpected and crucial physiological functions of C15ISO and C17ISO in C. elegans and suggests a potentially important role for mmBCFAs in other eukaryotes.Monomethyl branched-chain fatty acids (mmBCFAs) are commonly found in many organisms from bacteria to mammals. In humans, they have been detected in skin, brain, blood, and cancer cells. Despite a broad distribution, mmBCFAs remain exotic in eukaryotes, where their origin and physiological roles are not understood. Here we report our study of the function and regulation of mmBCFAs in  C. elegans produces two mmBCFAs that are essential for growth, suggesting an important role in other eukaryotesMonomethyl branched chain fatty acids (mmBCFAs) are found in bacteria and up through mammals.  Fatty acids (FAs) belong to a physiologically important class of molecules involved in energy storage, membrane structure, and various signaling pathways. Different FAs have different physical properties that determine their unique functions. Among the most abundant in animal cells as well as the most studied are those of long-chain even-numbered saturated and unsaturated FAs.C15ISO and C17ISO are saturated tetradecanoic and hexadecanoic FAs with a single methyl group appended on the carbon next to the terminal carbon . MonometVernix caseosa , elo-4 (C40H1.4), elo-7 (F56H11.3), and elo-8 (Y47D3A.30)—did not produce any notable phenotypes, whereas suppression of elo-1 (F56H11.4) and elo-2 (F11E6.5) affected the elongation of straight long-chain saturated and polyunsaturated FAs (In characterizing FA elongation in  enzymes . To testated FAs .elo-5 (F41H10.7) and elo-6 (F41H10.8), affected the levels of branched-chain FA. Transcriptional reporter constructs (elo-5Prom::GFP and elo-6Prom::GFP) indicated that both genes are expressed in the gut . RNAi of Y39E4A.3 led to a significant decrease in C15ISO and C17ISO production  to wild-type animals did not cause obvious morphological or growth defects, the suppression of elo-5 resulted in more pronounced phenotypes (elo-5(RNAi) plates displayed no obvious growth or morphological abnormality until the second day of adulthood, when they developed an egg-laying defect , their progeny did not arrest in L1 but continued to develop into adulthood. These animals had no obvious defects in locomotion, pharyngeal pumping, intestinal contractions, chemotaxis response, touch sensitivity, or general anatomy (unpublished data). However, the growing worms became progressively sick , which has a 245-bp deletion eliminating the predicted first exon . This allele phenocopies the L1 arrest phenotype of the elo-5(RNAi) animals.We also obtained a likely null mutant of the elo-5(RNAi) animals resulted directly from the deficiency of C15ISO and C17ISO, then feeding these worms with C15ISO and C17ISO should mask a shortage of endogenous C15ISO and C17ISO and permit the animals to grow normally. As predicted, the C17ISO and C17anteISO supplements rescued the elo-5 RNAi defects . A partial rescue was observed on the plates supplemented with C15ISO and C15anteISO . Corroborating results were obtained when homozygous elo-5(gk208) animals supplied with C17ISO grew normally. In sharp contrast, neither saturated or mono- or polyunsaturated FA molecules , mmBCFAs with shorter or longer backbones , nor polymethyl branched phytanic acid were able to rescue or reduce defects (0 of 30 plates in each experiment). Therefore, we have determined that only dietary 17-carbon mmBCFAs are competent to bypass the biochemical defect caused by loss of ELO-5 function.We reasoned that if the defects observed in the elo-5(RNAi) worms grown on supplemented plates revealed that only C17ISO and C17anteISO are significantly incorporated into lipids (elo-5(RNAi) feeding Escherichia coli strain (HT115), which lacks mmBCFAs. This mmBCFA-producing strain was identified by chance; we noticed that in the presence of a certain bacterial contaminant the animals could overcome the elo-5(RNAi) effects. Using a rapid bacterial identification method  animals fed with S. maltophilia indicated that they not only accumulated bacterial C15ISO and C15anteISO but also efficiently elongated these FA species to C17ISO and C17anteISO, which are absent in S. maltophilia (elo-5(RNAi) animals. Therefore, ELO-6 function remains intact in elo-5(RNAi). Apparently, C15ISO added to the plates could not be utilized by ELO-6 whereas C15ISO-CoA and/or C15anteISO-CoA originating from the bacterial food could, suggesting that free and esterified mmBCFAs were likely to enter alternative pathways.GC analysis of FA composition in o lipids A–6C. Beco lipids A, we wann method , we dete mmBCFAs D. GC anatophilia D and 6E.elo-5(gk208) deletion mutant. The homozygous mutants grew without any obvious morphological defects when maintained on the plates supplemented with C17ISO or seeded with S. maltophilia. However, removal of the FA supplements or S. maltophilia by bleaching resulted in the same L1 arrest phenotype seen for the elo-5(RNAi) worms.The essential roles of C15ISO and C17ISO were also supported through an examination of the elo-5(RNAi) animals arrested at the L1 stage could be recovered by adding the 17-carbon mmBCFA supplements. Indeed, C17ISO and C17anteISO could effectively release L1 larvae from the developmental arrest; about 50% of 2-d-arrested and 1% of 4-d-old L1 were rescued to full growth and proliferation. Since C17anteISO could not be detected in the laboratory animals under normal conditions of culturing, C17ISO appeared to be the principal molecule conveying the ELO-5 function. Therefore, the L1 arrest of the C17ISO-depleted worms is both completely penetrant and reversible, indicating that C17ISO plays a critical role in growth and development at the L1 stage.We then asked if elo-5 nor elo-6 is significantly expressed in eggs or L1. Therefore, C17ISO likely accumulates in embryos during oogenesis. It may be directly transported from gut to gonads, since both ELO-5 and ELO-6 were expressed mainly in the gut and since feeding C17ISO rescued the elo-5 mutant phenotypes. When RNAi-mediated disruption of elo-5 occurs at the L1 stage of a parent and consequently blocks C17ISO synthesis from that stage on, the eggs and L1 animals of the next generation are expected to contain a critically low concentration of C17ISO, halting further development. Because the arrested L1 can be rescued by a dietary supply of the mmBCFA, the deficiency is not likely to cause critical defects during the embryonic and early postembryonic periods.The analysis of the FA levels of staged worms revealed that the C17ISO level increases gradually from a relatively low level at L1 to its peak in gravid adults containing eggs A. Based elo-5 RNAi is applied to the parent worms at or after the L2 larval stage, when the amount of C17ISO has already been elevated and/or the RNAi effect is less penetrant, the progeny may receive sufficient C17ISO to pass the L1 arrest stage. The resulting animals, however, become visibly unhealthy at the L4 and adult stages as mentioned earlier, suggesting that C17ISO also plays a role in late developmental stages.If Based on these results, we propose a relationship between the amounts of C17ISO and developmental stages B. In thin = 50) obtained from mixed populations of wild-type animals where the fractions of eggs, larvae, and adults randomly varied. We also included GC data separately obtained from staged worms: eggs, L1, L2, L3, L4, and gravid adults. We found that the amounts of C17ISO significantly correlated with only two other FA molecules: linoleic acid (C18:2 n6) and vaccenic acid (C18:1 n7)  . A potenStreptomyces avermitilis -treated worms, a substantial loss of C15ISO and C17ISO is also accompanied by a change in the FA composition, most noticeably by the elevation in C18:1 n7 (see elo-5(RNAi) worm compared to wild type . Therefore, elo-5(RNAi) may not cause massive cell membrane dysfunction.In the 1 n7 see C, a resuA positive correlation between the amounts of C17ISO and C18:2 n6 may suggest a potential common function during development. In addition to the importance of linoleic acid as a substrate for polyunsaturated FA biosynthesis, its hydroxylated fatty acid derivative (HODEs) is known as a signaling molecule affecting chemotaxis , cell prelo-5(RNAi) animals fed with C15ISO or C15anteISO containing bacterial supplement , the FA composition was significantly altered  , FAS, and glycerol 3-phosphate acyltransferase (G3PA) , which promotes the expression of FA metabolic enzymes . There ieted 1”) . McKay ae (G3PA) . Thus, slpd-1 also plays a role in mmBCFA metabolism. We first applied RNAi to lpd-1 and determined the FA composition of the mutant worms. As expected, the FA content of treated animals was significantly changed, but surprisingly the most reduced were the levels of C15ISO and C17ISO . McKay and coauthors have shown that the lpd-2(RNAi) and lpd-1(RNAi) phenotypes are quite similar; affected worms are defective in growth, pale and scrawny in appearance, and lacking in fat content (lpd-1(RNAi), the FA composition in lpd-2(RNAi) worms was not significantly different from that of wild-type animals even though these animals had a noticeably sick appearance (unpublished data). This result suggested that, in addition to having some common targets, LPD-1 and LPD-2 have distinct functions. LPD-1 is important for production of mmBCFAs as well as other very-long-chain FAs, whereas LPD-2 has no specificity for any particular type of FA.LPD-2 (C48E7.3) is another  content . They halpd-1(RNAi) would be consistent with downregulation of elo-5, elo-6 (decrease in mmBCFA), elo-2 (increase in C16:0) (9- and/or Δ12-desaturase genes (decrease in C18:2 n6). The genes encoding mammalian orthologs of the C. elegans elo-2 and Δ9-desaturase genes are known targets of SREBP-1c  , and Δ9-SREBP-1c . To examlpd-1Prom::GFP fusion construct (a gift of J. Graff) in transgenic animals revealed that, in addition to the previously reported expression in intestinal cells  plates, the expression was noticeably weakened, suggesting a downregulation of the promoter activities .If LPD-1 promotes tivities A–11D. Nolpd-1(RNAi), we analyzed FA composition in FAS(RNAi) strains. There is one predicted FAS gene, F32H2.5, and its shorter homolog, F32H2.6, in the C. elegans genome. The latter can only encode the N-terminal portion of the protein. These genes share extended nucleotide identity, and RNAi of one could thus possibly affect the other. Consistent with a critical role for FAS in the first steps of FA biosynthesis, the RNAi-mediated disruption of F32H2.5 and F32H2.6 resulted in multiple defects and a lethal growth arrest (unpublished data). The FA composition (the content and relative amounts of various FA species) of the affected animals remained, however, unchanged. This suggests that disruption of FAS does not selectively alter FA biosynthesis and that neither FAS protein is specific for mmBCFA. Therefore, downregulation of FAS by loss of lpd-1 cannot account for the severe deficiency of mmBCFA in lpd-1(RNAi).To test if the disruption of FAS, a target of LPD-1 , could clpd-1 affects C15ISO and C17ISO biosynthesis. The fact that lpd-1, elo-5, and elo-6 are expressed in the same cells concurrently and that the GFP reporter analysis indicated that elo-5 and elo-6 transcription is downregulated in the absence of lpd-1 suggests that elo-5 and elo-6 are likely to be the targets of lpd-1.Thus, we have shown that disruption of C. elegans.Since ACC and FAS catalyze the first steps in the biosynthesis of straight-chain FAs while ELO-5 and ELO-6 extend mmBCFA molecules, LPD-1 appears to integrate conventional and “unusual” FA biosyntheses. It seems reasonable to predict that in order to differentiate between these metabolic pathways and mediate compensatory or adaptive changes in FA composition, LPD-1 must interact with other factors such as nuclear receptors activated by specific FA ligands. It is thus important to screen for such interactions to better understand FA homeostasis in lpd-1 could be regulated by mmBCFAs at the transcriptional level. Our microarray data (discussed below) indicated a 1.68-fold upregulation of lpd-1 in the elo-5(RNAi) animals, while no changes were detected in its levels between samples from wild-type animals at different developmental stages  and control plates to compare GFP fluorescence. No obvious difference in the GFP expression driven by the lpd-1 promoter in intestinal cells was detected on the elo-5(RNAi) plates versus the control plates. A modest change in the transcription level (1.68-fold) could be masked by a variability of the expression between individual animals and even between individual cells (unpublished data). In contrast to the observation for the intestinal cells, a strong induction of GFP was detected in amphid neurons of lpd-1Prom::GFP;elo-5(RNAi) animals (elo-5(RNAi) animals may transcriptionally stimulate LPD-1 production, at least in neuronal cells.To examine the influence of the mmBCFA deficiency on  animals E–11H. Thlpd-1 and C15ISO/C17ISO is reciprocal; while downregulation of lpd-1 transcription results in the C17ISO deficiency, the C15ISO and C17ISO deficiency upregulates lpd-1 transcription at least in a subset of cells. Therefore, the worm SREBP homolog, LPD-1, may play an important role in mmBCFA homeostasis.Collectively, our results suggest that the relationship between Because C15ISO and C17ISO play critical roles in animal development and growth, we suspected mechanisms might exist to respond to and regulate their levels. Regulation of mmBCFA homeostasis may involve transcription factors, metabolic enzymes, and transport and binding proteins. It is reasonable to suggest that a deficiency of mmBCFA triggers a compensatory alteration in the expression of these genes. It is also possible that a comparative analysis of global gene expressions between wild-type and mmBCFA-deficient animals may reveal these potential changes and the changes underlying developmental and growth functions of mmBCFA.elo-5(RNAi) and wild-type animals. To select candidate genes, we applied restrictive criteria and excluded genes of which the expression was also changed in the spt-1(RNAi) strain (spt-1 (C23H3.4) gene encodes a predicted C. elegans homolog of serine-palmitoyl transferase subunit 1. RNAi of spt-1 strongly affects the FA composition without reducing the C15ISO or C17ISO levels (unpublished data). The F1 generation of spt-1(RNAi) animals developed gonadal and egg-laying defects that were similar to the phenotype of F1 animals from parents treated with elo-5(RNAi) at a late larval stage , we would be able to eliminate variations in gene expressions unrelated to the mmBCFA deficiency. Such variations might emerge from altered straight-chain FA metabolism and from general sickness. Here, we discuss the analysis of the first set of candidate genes that are differentially expressed in elo-5(RNAi) and may relate to the C15ISO and C17ISO homeostasis.We used DNA microarray analysis to compare the total gene expression in ) strain . The sptpnk-1 [C10G11.5], nhr-49 [K10C3.6], acs-1 [F46E10.1], and C27H6.2) significantly affected the FA composition .Twenty-five genes were selected in the screen  and eachposition . All foupnk-1, nhr-49, and acs-1 were upregulated in C17ISO-deficient elo-5(RNAi). Lastly, a functional analysis shows that these three candidate genes are required for the normal level of mmBCFA production (RNAi of the genes affects mmBCFA production). The fourth candidate gene, C27H6.2, affects the level of vaccenic acid (C18:1 n7), which is related to the levels of mmBCFA  plates indicated a significantly brighter fluorescence in the RNAi worms   see L and 11Npnk-1 and acs-1 genes were previously selected in two different screens as potential targets of the daf-2/daf-16  pathway. pnk-1 had been identified in a screen for genes affecting the life span and metabolism of C. elegans through analysis of promoter regions, and it was confirmed as a direct target of DAF-16, a forkhead transcriptional factor  animals showed that reduction of nhr-49 activity results in upregulation of saturated FA biosynthesis that may contribute to fat accumulation. Although the regulatory path for this process remains unknown, the involvement of daf-2 has not been ruled out.It was interesting to note that the l factor . acs-1 hife span . A thirdry genes . It was C. elegans insulin-signaling pathway is involved in sensing nutritional state and metabolic conditions as well as controlling growth and diapause  animals . Second, worms may obtain mmBCFAs from their diet (bacteria). Therefore, C. elegans is able to produce, activate, transport, and utilize mmBCFAs and is vitally dependent on this system.Two mmBCFAs are normally detected in The level of C15ISO and C17ISO in eggs appears to be critical for growth and development, as animals depleted of C15ISO or C17ISO completely arrest at the L1 stage. The uniformity and reversibility of the arrest would be consistent with a regulatory role in growth and development for these mmBCFAs or for more complex lipid molecules containing them. However, it cannot be ruled out that the arrest is due to the failure of a metabolic or structural function that is essential for growth and development at the first larval stage. In addition, C15ISO and C17ISO may directly or indirectly regulate genes involved in FA homeostasis. Consistent with this, their deficiency triggers a large alteration in gene expression that may reflect a complex feedback mechanism. Among the potentially responsive genes are transcription factors and metabolic genes.Ubiquitous and unattended mmBCFAs come forth as physiologically important molecules that regulate essential functions in eukaryotes. Many interesting questions regarding mmBCFAs remain to be addressed. What are the other components of the mmBCFA biosynthetic machinery? What are the components of their transport system? Does an organism have a mechanism by which the mmBCFA level is measured? What are the signaling pathways involved in the mmBCFA responses? How do mmBCFAs exert their physiological function? Do mmBCFAs act alone or as parts of more complex lipids? How are mmBCFAs synthesized in mammals? Lastly, what are the specific physiological functions of mmBCFAs in mammals? Both genetic and biochemical approaches will be taken to address these questions.C. elegans whole genome RNAi feeding library .The RNAi feeding vectors were either made in our laboratory using Taq PCR and cloning genomic fragments into a double T7 vector, pPD129.36 (gift of A. Fire), or obtained from the E. coli HT115 transformed with either empty pPD129.36 vector (controls) or with dsRNA-producing constructs. Plates were prepared as described in The RNAi feeding strain was A mixed population of well-fed worms were washed off the plates with water, rinsed 3–4 times, and, after aspirating away water, frozen at −80 °C. FA methyl esters and lipid extraction were performed as described in After bleaching gravid adults, an aliquot of the eggs was set apart, and the rest was incubated overnight in M9 at room temperature. On the next day, an aliquot of L1 was frozen for GC analysis. The rest of L1 was plated on agar plates. Subsequently, L2, L3, L4, young adults, and adults along with hatched L1 were collected as the separate samples. Mixed populations of worms starved for 24–100 h were included in the experiment to monitor a possible effect of the starvation.elo-5 dsRNA-producing plasmid or the control HT115 vector. Two synchronized young adults were plated and their progeny was scored 4 and 5 d later. Each experiment was performed in at least 30 replicates. For recovering elo-5(RNAi) worms from L1 arrest, wild-type adults were placed on the elo-5(RNAi) plates. Four days later, their progeny was removed and eggs of the next generation were left on the plates. Hatched L1 were kept for 2 or 4 d before transferring as agar chunks to new elo-5(RNAi) plates. FA supplements were added to spots next to the chunks. Ten plates were prepared for each FA supplement. Control plates contained no supplements. To verify that an addition of supplements did not affect RNA interference per se, we used let-418(RNAi) animals, which have a sterile phenotype, as a control. Neither C15 nor C17 mmBCFA added to let-418(RNAi) plates modified the expected phenotype.Ninety microliters of the 4 mM solution of FA  in 1% NP40 was dropped on the side of the bacterial lawn that contained either To prepare the GFP fusion constructs, genomic fragments were PCR amplified and cloned in frame into one of the GFP fusion vectors (gift of A. Fire). The locations of the genomic fragments and PCR primers used are listed below:elo-5Prom::GFP, starting at 3.894 kb genomic upstream of the first codon and ending 4 bp into the first exon; primers, F-BamHI-TTTAGGTCATTTTTTGAGTCGCCA and R-BamHI-TAGTCTGGAATTTTGAAATTGAACGG; vector, pPD95.69;(1) elo-6Prom::GFP, a 4.764-kb fragment covering 3,104 bp upstream and 1,660 bp downstream of the predicted start codon and ending 14 bp into the third exon; primers, F-Sph1-GCCCTTGGAAACCATCTACGACGAATC and R-Sma1-TCCGAACAGAACGACATAAGAGATTTCC; vector, pPD95.77;(2) acs-1Prom::GFP, a 3.142-kb genomic fragment containing 3,048 kb upstream of the first predicted ATG and ending 24 bp into the second predicted exon; primers, F-SphI-CATAATTACTATTGCGTCACATG and R-SphI-CTCTTCCAAACTGGCGATGTCGA; vector, pPD95.69;(3) pnk-1Prom::GFP, a 1.14-kb fragment that includes 937 bp upstream of the first predicted codon of the C10G11.5 and 203 bp downstream, ending 24 bp into the second exon; primers, F-SphI-TCGTACGATCGGACCATAGGCTAA and R-SphI-CTGATCCTCTGTAGCAGCGGCCCT; vector, pPD95.69.(4) C. elegans at 10–50 ng/μl to form extrachromosomal arrays. In the case of acs-1, the extrachromosomal array had been integrated into the C. elegans genome.These constructs were injected into Worms were soaked in a 5-μg/ml solution of DiI  in M9 buffer for 1 h. They were then rinsed three times with M9 and visualized by fluorescence using the Texas Red filter.t test (two-tailed distribution) and correlation analysis were performed using the Microsoft Excel program.The FA quantities obtained by GC were expressed as a percentage of the total. Synchronized adults were placed on control  and RNAi  plates. Several worms of the next generation were picked from the control and RNAi plates and mounted on the same microscopic slide. GFP images were obtained with the fixed settings and exposure.E. coli HT115 strain transformed with empty pPD129.36 vector. Experimental RNAi plates contained E. coli HT115 transformed with corresponding dsRNA constructs. The growth conditions, RNA preparations, and data analyses are described in detail in One young adult of the N2 Bristol strain was plated on each control and RNAi feeding plate. Control plates contained the Dataset S1(1.8 MB TXT).Click here for additional data file.Figure S1elo-5(RNAi) phenotype have a lower number of the changed collagen genes, suggesting that populations on these experimental and control plates are similar.Samples I, II, and III represent mixed populations of wild-type animals started simultaneously from one young adult. Each was harvested at three time points, when mostly adults represented the F1 generation and the embryos and larvae in different proportions represented the F2 generation see . Sample (24 KB PPT).Click here for additional data file.Protocol S1(37 KB DOC).Click here for additional data file.Table S1(28 KB DOC).Click here for additional data file.


Comparative genome sequence analysis is powerful, but sequencing genomes is expensive. It is desirable to be able to predict how many genomes are needed for comparative genomics, and at what evolutionary distances. Here I describe a simple mathematical model for the common problem of identifying conserved sequences. The model leads to some useful rules of thumb. For a given evolutionary distance, the number of comparative genomes needed for a constant level of statistical stringency in identifying conserved regions scales inversely with the size of the conserved feature to be detected. At short evolutionary distances, the number of comparative genomes required also scales inversely with distance. These scaling behaviors provide some intuition for future comparative genome sequencing needs, such as the proposed use of “phylogenetic shadowing” methods using closely related comparative genomes, and the feasibility of high-resolution detection of small conserved features. The mathematical model presented in this work will help to inform comparative genomics strategies for identifying conserved DNA sequences Comparative genome sequence analysis is a powerful means of identifying functional DNA sequences by their evolutionary conservation ,2,3. It Saccharomyces and Drosophila species were done to choose appropriate species for comparative genome sequencing  points where the model predicts needing N = 183, 89, and 40 for the different values of FP and FN, the HKY/LLR simulation predicts needing N = 210, 80, and 35; for the  points, the model predicts N = 23, 12, and 5, and the simulation predicts N = 24, 11, and 5; and for the  points the model and simulation both predict N = 4, 2, and 1.This analysis showed that the predictions of the simple model's equations and the results of the HKY/LLR simulations are in close agreement. The maximum deviation was 15%. For example, for the  features, to achieve FP < 0.0001 and FN < 0.01 for distances of D = 1, 2, 3, 4, 5, and 10, the simple model predicts needing N = 8, 8, 11, 17, 26, and 335 genomes, respectively, whereas HKY/LLR simulations predict needing N = 8, 9, 9, 13, 17, and 82 genomes. Thus, the simple model's approximation breaks down somewhat at larger distances, beyond the D < 1 range that is considered here to be reasonable for comparative genomics.More significant discrepancies appear at larger distances. A simple Jukes-Cantor model has only one substitution rate, so all types of substitutions saturate equally fast. In an HKY model, some substitution rates are faster than others. Intuitively, one expects an HKY model to be able to extract information from slower, less quickly saturated substitutions at longer distances, resulting in more discrimination at large N that is not predicted by the simple model. For instance, GC-rich features are more easily detected than AT-rich features when substitution rates are biased towards high AT composition. Additional HKY/LLR simulations, using the same HKY matrices as above but specifically looking at poly-A features versus poly-C target features, show this effect; for instance, for  features at D = 0.19, to achieve FP < 0.0001 and FN < 0.01, we need at least N = 24 genomes to detect features on average, but specifically we need N = 19 for poly-C/G features and N = 29 for poly-A/T features.Additionally, nonuniform base composition causes some composition-dependent spreading around the mean One also wants to see that the model's predictions do not disagree with published results, at least to the extent that it is possible to crudely compare real phylogenies to the abstracted uniform star topology of the model. Three examples follow.D. However, using either N = 1 and D = 0.6 (pairwise comparison to one rodent using one full pairwise distance for D), or N =2 and D = 0.23 (approximating D as an average of three independent branch lengths), or N = 2 and D = 0.35 , the model predicts that 90% of 50-nt features with ω = 0.2 can be detected with a reasonable FP of between 0.0003 and 10−5; but for features just half that size (L = 25), FP collapses to between 0.02 and 0.006 .Cooper et al. estimated that the mouse and rat genomes suffice for about 50-nt resolution of human conserved features . The indN = 15 and D = 0.04 (average independent branch length of 0.6/15), one can detect 90% of 50-nt, ω = 0.2 features with a FP of 10−5; but for 25-nt features, FP collapses to 0.003 .Boffelli et al., in introducing “phylogenetic shadowing,” used 13–17 primate sequences with a total independent branch length of about 0.6 neutral substitutions/site to analyze conserved sequences smoothed in 50-nt windows, and conserved regions down to 40–70 nt were detected effectively . The modSaccharomyces cerevisiae using alignments of intergenic regions to three comparative Saccharomyces genomes, with a total independent branch length of about 0.8–0.9 [N = 3 and D = 0.27–0.30 (average independent branch length of 0.8/3 to 0.9/3), the model predicts that binding site–like features  would be detected with a FP of 0.001–0.002  and a sensitivity of about 25%, suggesting that these data are barely sufficient to identify individual short conserved features. Indeed, though both research groups showed examples of highly conserved individual sites, both groups analyzed their data primarily at the level of detecting motif consenses, rather than attempting to detect individual features genome-wide. That is, they required that the same motif be found conserved in multiple places upstream of multiple genes. This is a data aggregation strategy, multiplying the effective L by the number of copies of the feature. In this way, even when only a fraction of individual features are identified, the existence of a conserved consensus motif may be inferred from the average conservation of the aggregated data.Kellis et al. and Cliften et al. reported comparative analyses to identify transcription factor binding sites in  0.8–0.9 ,12. For The model assumes a pure, brute force detection of individual conserved features by comparative analysis. For many particular problems, one can leverage additional information and reduce the number of comparative genomes needed. Data aggregation strategies are one example . AnotheSome features are not just conserved, but also show informative patterns of substitution, insertion, and deletion, so we can gain power by using feature-specific evolutionary models instead of a general conservation screen. For instance, coding regions predominately show substitutions in wobble positions and strong selection against insertions/deletions, and those insertions/deletions that remain will generally preserve frame . ConservFinally, there are other important uses of comparative genomics in addition to DNA sequence analysis of conserved elements. For example, evolutionary/developmental studies choose species based on phylogenetic position, and population genetics studies choose multiple individuals within the same species.The principal results here are two inverse scaling behaviors that provide useful intuitions for planning comparative genome sequencing. All other things being constant, the required number of comparative genomes is inversely proportional to detectable feature size, and at small evolutionary distances, required genome number becomes inversely proportional to the neutral distance to the comparative genomes.L and N. And it is already common to use total independent neutral branch length as a measure of the strength of a comparative dataset [ND.Neither behavior is entirely surprising; the contribution of an abstract model is to see them more clearly. Obviously, it takes more comparative genomes to recognize smaller features, though one may not have predicted a simple inverse relationship between  dataset ,8,9,30, The model also shows clearly that for two analysis scenarios—identification of small conserved features and the identification of lineage-specific conserved features in closely related genomes—it will be useful to obtain large numbers of comparative genome sequences. Since a small number of comparative genome sequences are already enabling powerful analyses, this may be surprising. Even for simple conservation analyses, we have not begun to exhaust the power of comparative genome analysis.http://www.genetics.wustl.edu/eddy/publications/Eddy05.The model was implemented in several ANSI C programs, which can be downloaded at


Fcγ receptor (FcγR)–mediated phagocytosis of IgG-coated particles is regulated by 3′-phosphoinositides (3′PIs) and several classes of small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity of phagocytosis to brefeldin A (BFA), an inhibitor of certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show that ARF1 was activated during FcγR-mediated phagocytosis and that blocking normal ARF1 cycling inhibited phagosome closure. We examined the distributions and activation patterns of ARF6 and ARF1 during FcγR-mediated phagocytosis using fluorescence resonance energy transfer (FRET) stoichiometric microscopy of macrophages expressing CFP- or YFP-chimeras of ARF1, ARF6, and a GTP-ARF-binding protein domain. Both GTPases were activated by BFA-insensitive factors at sites of phagocytosis. ARF6 activation was restricted to the leading edge of the phagocytic cup, while ARF1 activation was delayed and delocalized over the phagosome. Phagocytic cups formed after inhibition of PI 3-kinase (PI-3K) contained persistently activated ARF6 and minimally activated ARF1. This indicates that a PI-3K-dependent signal transition defines the sequence of ARF GTPase activation during phagocytosis and that ARF6 and ARF1 coordinate different functions at the forming phagosome. The authors use fluorescence resonance energy transfer (FRET) to track the localization of activated Arf1 and Arf6 GTPases, and show that both proteins are involved in Fcγ receptor-mediated phagocytosis. Fcγ receptor (FcγR)-mediated phagocytosis by professional phagocytes requires the extension of a tightly apposed, actin- and membrane-rich pseudopod over the target particle. In spite of this commitment of plasma membrane, macrophages are able to engulf many particles without a concomitant loss in surface area . The obs2], can facilitate actin polymerization through recruitment of WASP/N-WASP proteins , blocking phagocytosis at a stage distal to receptor ligation and phagocytic cup formation  for 30 min; fixation and all subsequent steps were performed at room temperature. Following three rinses with PBS, cells were treated with sodium borohydride  to reduce primary fluorescence induced by glutaraldehyde. Coverslips were incubated with a 1 mg/mL solution of borohydride in PBS for 5 min. After four incubations in the borohydride solution, cells were rinsed six times with PBS and twice with PBS supplemented with 2% goat serum (PBS-GS); coverslips were left in the second PBS-GS rinse for 15 min to block nonspecific binding sites.To quantify the effects of the ARF GTPase mutants on phagocytosis, phagocytic efficiencies were measured for cells expressing the GTPases as CFP fusions. ˜5 × 10To mark uninternalized erythrocytes, coverslips were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG  at a concentration of 1 μg/mL in PBS-GS for 30 min. Cells were rinsed with PBS-GS followed by PBS and mounted on slides using Prolong Gold antifade reagent .−) and bound, uninternalized erythrocytes (Alexa488+) counted. The percent phagocytosis was calculated from at least 25 CFP-positive macrophages on each coverslip as:Visualization of CFP and Alexa Fluor 488 was accomplished on the inverted Nikon Eclipse TE300 FRET microscope using the JP4v2 filter set. Alexa Fluor 488 was imaged through the YFP channel of this filter set. Cells transfected with the CFP expression construct were located on coverslips, and then the internalized erythrocytes .Figure S1A, ED, and EAVG for each cell were then plotted against the corresponding measured RM values. For RM values below 1, the CFP donor is in excess, while for RM values above 1, the YFP acceptor is the excess binding partner. For low RM values, more acceptors are able to form complex with the excess donors, leading to relatively higher EA values; however, the lack of sufficient acceptors reduces the fraction of donors that can enter into FRET complexes and reduces ED. In the case of excess acceptors, the impact on EA and ED values is reversed, with EA being suppressed. EAVG, though, does not demonstrate this sharp, asymmetric dependence on RM. When graphed as a function of RM, EAVG is essentially independent of RM, particularly in the range RM = 0.1 – 10. Because EA and ED are distinctly dependent on RM, EAVG is preferred for comparing FRET values for multiple samples where average RM values differ between some samples.The average FRET values were calculated from ˜40 cells expressing ARF1-CFP and YFP-NGAT (A) or ˜125 cells expressing YFP-Rac2V12 and PBD-CFP (B). The construction of the Rac-PBD FRET pair is described elsewhere . The val(204 KB PDF)Click here for additional data file.Protocol S1(25 KB DOC)Click here for additional data file.Table S1(40 KB DOC)Click here for additional data file.Video S1A, ID, and IF images were used to calculate the time-lapse RM images (center panel), ED images (right-hand panel) and EA images (unpublished data). Activated ARF6 can be seen at a site of ruffling . Phagocytosis of an erythrocyte, initiated below the ruffle, induced the activation of ARF6 at the leading edge of the phagosome pseudopod during the extension phase. ARF6 was deactivated as the phagosome sealed. All videos seen here play at 6 frames/s. Frames were collected at 30-s intervals.A phase contrast movie of an RAW264.7 macrophage engulfing an IgG-coated RBC is shown in the panel to the left. I(2.9 MB MOV)Click here for additional data file.Video S2M and ED time-lapse images of a macrophage expressing ARF1-CFP and YFP-NGAT. The activation of ARF1-CFP at the phagosome is revealed in the ED image. ARF1 activation at the phagosome increased during the extension phase and was resolved during closure. The juxtanuclear Golgi compartment is visible in the ED image as well, to the lower right of the forming phagosome. Levels of activated ARF1 at the Golgi did not vary during phagocytosis.Images shown left to right: phase contrast, R(2.2 MB MOV)Click here for additional data file.Video S3M and ED images from a macrophage expressing ARF1-CFP and YFP-NGAT, and treated with 5 μM BFA for 30 min prior to microscopic observation. Exposure to BFA prevented the activation of ARF1 at the Golgi—no regions of high FRET are visible near the nucleus in the ED image at the start of the movie. However, the two phagocytic events  led to the activation of ARF1, indicating that ARF1 activation at the phagosome is mediated by a BFA-resistant ARNO/cytohesin-family GEF.Panels in the video, left to right: phase contrast, R(2.7 MB MOV)Click here for additional data file.Video S4M and ED time-lapse images. Following binding of a particle at the right, ARF6-CFP was activated and pseudopod extension commenced. GTP-bound ARF6-CFP continued to accumulate in the phagocytic cup and was not deactivated during observation. The accumulation of ARF6-CFP at the arrested phagocytic cup can be seen in the RM image series (the blue band at the phagocytic cup represents a molar excess of ARF6-CFP relative to YFP-NGAT). The generation of ARF6-GTP in the absence of PI-3K function indicates ARF6 is activated at the phagosome by a PI-3K-independent GEF activity.RAW264.7 macrophages co-transfected with ARF6-CFP and YFP-NGAT were incubated in 50 μM LY294002 for 30 min before the addition of IgG-opsonized erythrocytes. The presented images, left to right, are: phase contrast, R(2.8 MB MOV)Click here for additional data file.Video S5D (far-right panel) corresponds to the Golgi apparatus. The Golgi is also visible in the RM image (center panel) where ARF1-CFP is present in excess over YFP-NGAT. Cells treated with LY294002 could bind opsonized particles and form phagocytic cups but could not complete phagocytosis. At the upper left and right of the macrophage, binding of opsonized erythrocytes was not accompanied by the activation of ARF1-CFP. This result indicates that amplification of FcγR-signaling by PI-3K is required for the activation of ARF1 at the phagosome.RAW264.7 macrophages co-transfected with ARF1-CFP and YFP-NGAT demonstrated high FRET signals in the area of their Golgi network in the presence of LY294002: the nucleus is visible in the phase contrast image shown at the left; next to the nucleus, the crescent-shaped region of high E(2.9 MB MOV)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers are: ARF1 cDNA used to produce ARF1-CFP (AF493881), ARF6 source cDNA (AF493885), GGA1 transcript variant used to create NGAT (NM_013365). All plasmids used to produce the data for this manuscript can be obtained from the Addgene plasmid repository (http://www.addgene.org/Joel_Swanson, and the Addgene plasmid ID numbers are 11381–11390.Genbank (