Management and conservation can be greatly informed by considering explicitly how environmental factors influence population genetic structure. Using simulated larval dispersal estimates based on ocean current observations, we demonstrate how explicit consideration of frequency of exchange of larvae among sites via ocean advection can fundamentally change the interpretation of empirical population genetic structuring as compared with conventional spatial genetic analyses. Both frequency of larval exchange and empirical genetic difference were uncorrelated with Euclidean distance between sites. When transformed into relative oceanographic distances and integrated into a genetic isolation-by-distance framework, however, the frequency of larval exchange explained nearly 50 per cent of the variance in empirical genetic differences among sites over scales of tens of kilometres. Explanatory power was strongest when we considered effects of multiple generations of larval dispersal via intermediary locations on the long-term probability of exchange between sites. Our results uncover meaningful spatial patterning to population genetic structuring that corresponds with ocean circulation. This study advances our ability to interpret population structure from complex genetic data characteristic of high gene flow species, validates recent advances in oceanographic approaches for assessing larval dispersal and represents a novel approach to characterize population connectivity at small spatial scales germane to conservation and fisheries management. In many cases, however, no linear fit is found despite high variance in pairwise genetic difference values indicating diversity in population connectivity .In the marine environment, currents can be circuitous and oceanographic features like eddies and fronts can prevent mixing and diffusion of pelagic larvae, decoupling pelagic larval dispersal from Euclidean distance . Two adjper se), but only the knowledge of the (relative) distance between samples’. The challenge then is to use a model of oceanographic forces to map frequencies of dispersal between genetic sampling sites, and translate them into relative distances that can be tested for a linear fit with pairwise genetic difference. We estimated frequencies of larval exchange among populations of a marine species based on simulations of dispersal trajectories of the species' larval stage in a data-assimilated oceanographic circulation model produced from ocean temperature, salinity, current and wind observations in the study area. We focused our analysis on Kelletia kelletii, a subtidal whelk in US and Mexico Pacific waters that is a significant predator in kelp forest ecosystems (K. kelletii using the isolation-by-distance framework. Our approach enabled the construction of genetic isolation by ‘derived oceanographic distance’ (DOD) plots that were far more effective at organizing the pattern of population genetic structure than conventional isolation-by-distance methods based on Euclidian distance.This study represents an important advancement in the evaluation of spatial marine population genetic data (i.e. seascape genetics) by applying a new approach for incorporating ocean circulation observations directly into the isolation-by-distance analysis. As osystems . Recogni2.K. kelletii at nine microsatellite loci, K13, Kk2b, Kk7a, Kk28a, Kk33a, Kk34a, Kk41a, Kk48b and Kk52a, described and amplified with laboratory methods published previously . Particles were advected by simulated currents (e.g. as done by b) and were allowed to settle during K. kelletii's natural age-at-settlement competency window . Variability in the resulting connectivity matrices reached an asymptote at 25 particles per grid unit per day, prompting our use of this sample size here. To estimate potential larval connectivity, we assumed constant larval production for all release locations and no larval mortality (see §4).Simulations of larval trajectories for the Santa Barbara Channel, California, and the surrounding southern California area were used to estimate probability of larval dispersal and ultimately DOD among genetic sampling sites. Data-assimilated models of ocean currents for the study region were produced by combining available observations of ocean temperature, salinity, currents and winds in a numerical model to provide a dynamic interpolation of the near-surface ocean flow field . The observational period included the 1997/1998 El Niño event and thus represented both ‘normal’ and ‘exceptional’ oceanographic conditions. For each year, we recorded the proportion of larvae released from each grid cell that dispersed to a given grid cell. This procedure resulted in a 448 × 448 source–destination (row–column) matrix, K. kelletii larvae to grid cell i from grid cell j.Estimation of DOD for each pair of genetic sampling locations was done after subjecting the source–destination matrices to five transformations. First, we calculated a single, all-years 448 × 448 matrix as the element-by-element mean of the frequencies in the annual matrices. Second, we standardized the values in the all-years matrix so that for each source, destination probabilities sum to one . This procedure generated a forward transition matrix , which n2–104 generations, dependent on the demographics, marker mutation rate and other factors  intermediary sites. Thus, the Markov chain captures effects of both single- and multiple-generation dispersal processes on the long-term probability of dispersal among locations. Let M represent the original forward transition matrix that was used to set the initial condition. When n is sufficiently large, Mn = Mss, the Markov chain output of steady-state (long-term) dispersal probabilities between pairwise locations that is independent of the initial state of the population. We iterated the Markov chain until it reached numerically computed convergence, which took about 103 iterations.In the third transformation, we incorporated the effects of multi-generational gene flow on the long-term probability of dispersal between locations. Gene flow data from neutral markers represents a long-term average of dispersal integrated over 10 factors . In cont factors . The Mar factors . The Mar factors . We usedFST and Dest values represent genetic differences between subpopulations without indicating directionality of gene flow. In contrast, both M and Mss contain two values for each pair of locations, each representing a uni-directional probability of dispersal. To correspond with our empirical estimates of population genetic differences between each pair of sampling locations, in the fourth transformation step we averaged uni-directional dispersal probabilities to estimate mean probability of dispersal between pairwise locations. We performed this transformation step for both M and Mss in order to measure the effect of consideration of multi-generational dispersal events (transformation step 3) on our ability to explain the observed pattern of population genetic structure.Pairwise The isolation-by-distance analysis tests for a positive linear relationship between genetic differentiation and distance, requiring conversion of the matrix of mean dispersal probability into a distance matrix for the final step of the transformation process. Because each row in the matrix sums to one, the collection of dispersal probabilities for each source can be considered a probability density function (PDF). This type of PDF is termed a ‘larval dispersal kernel’ and provides a mathematical framework for transforming dispersal probabilities into relative dispersal distances . No two geo-referenced sites were closest to the same centroid; however, if this had been the case, we would have calculated DOD in relation to the mean probability of dispersal between that grid cell and itself. DODs could have been calculated for any number of pairs of grid cells, but was limited to only those pairs corresponding with the empirical genetic data.The dispersal kernel of FST and Dest) with Euclidean and derived oceanographic distance metrics. Correlations were tested using IBDWS Mantel tests with 10 000 permutations . Genetic differentiation between pairwise sites ranged from −0.0023 to 0.0068 (FST) and from −0.0048 to 0.023 (Dest) , compared with approximately 2 expected by chance. Results from pairwise FST and Dest were correlated , and the interpretation of the patterns was unchanged regardless of which measure of population structure was applied. For both FST and Dest, we did not detect a fit with a conventional isolation-by-distance model based on pairwise Euclidean distance  . Six of distance . In eachM, representing dispersal over a single generation), mean probabilities of dispersal among coastal grid units representing the 10 geo-referenced genetic sampling sites ranged from zero to 0.51 per cent (a). Using Siegel et al.'s dispersal kernel, we transformed the probabilities into DODs that ranged from ∞ − 47 km, respectively .In the original forward transition matrix . From these probabilities, we calculated DODs 171–82 km, respectively . Jackknifing the data found the observed pattern to be largely independent of any single locus or sampling location . Although convergence in Mss took many generations, corresponding with evolutionary time scales, similar regression correlation strengths were obtained when the Markov chain was run for fewer iterations . Setting negative FST and Dest values to zero had a minimal effect on the correlation with DOD, and did not improve the significance of the correlation with Euclidean distance .The steady-state transition matrix, per cent b. From tectively . Inclusi4.This study demonstrates the value of considering dispersal from the perspective of the organism for estimating population connectivity. Calculation of oceanographic distance in relation to the frequency of dispersal enabled us to construct genetic isolation-by-distance plots that were more effective at explaining population genetic structure than those based on Euclidian distance. Euclidean distance was uncorrelated with the probability of dispersal or DOD . Thus, it is not surprising that Euclidean distance was a poor predictor of genetic structure because probabilities of larval exchange, and presumably gene flow, among sites have little to do with the physical distance between them. The decoupling of dispersal probability and DOD from Euclidean distance between sites is probably due to the complex geography and circulation of the region.post hoc analysis highlighted variation in how well the consideration of larval dispersal in the oceanographic model explained genetic structure among different groups of sites. For example, model-based probabilities of dispersal were high between several cross-channel pairs linked by a within-channel eddy . This nel eddy   distance was a poor explanatory variable compared with DOD. Given the relatively low and equivalent levels of gene diversity observed among the sampling sites  of Dest and FST with DOD based on M. Consideration of Anacapa North may compromise the strength of the isolation by oceanographic distance regression because the oceanographic model poorly characterizes currents flowing to and from this location. Thus, limitations in the oceanographic model in relation to the experimental design of study sites may have generated results that undervalue the potential for single-generation dispersal probabilities to contribute significantly to the interpretation of population genetic structure. Alternatively, oceanography may simply be less influential on Anacapa Island's genetic structure compared with at other sites. Use of an improved circulation model and consideration of additional environmental factors  and demonstrations of the effect of vertical migration on dispersal . In transformation step 4, we represented mean dispersal probability as an average of uni-directional dispersal probabilities. This necessary simplification of the output provided by the oceanographic model is accurate given the isolation-by-distance approach we have taken, but future improvements on this approach might consider asymmetrical uni-directional probabilities of dispersal among sites. Additional explanatory power may be gained using genetics models that estimate directionality in migration, because asymmetry in migration rate can influence genetic structure (Our simulation model makes several important simplifying assumptions. Larvae are assumed passive, despite laboratory observations of diel vertical migration in ispersal . Also, atructure . OverallIt is likely that there are many situations where a stepping-stone model is an inadequate framework for describing the gene flow of marine species. A conventional stepping-stone model assumes that diffusion dominates ocean circulation over equilibrium population genetic time scales. However, ocean flow simulations with even simple but real coastlines can generate complex spatial patterns of connectivity whose spatial and temporal average departs considerably from expected Gaussian dispersal kernels based on homogeneous flow conditions . In the FST ≈ Dest ≈ 0.001, there is significant patterning organized by ocean currents on scales less than 30 km. Furthermore, our results serve as validation for estimating population connectivity using an oceanographic approach, one of the few methods for simulating larval dispersal in marine systems (This study advances the lower bound of seascape genetics for interpreting fine-scale population structure from seemingly chaotic genetic patchiness characteristic of marine species ( systems . Finally systems . Our dem


BOULE homologs are present in the genomes of representative species of each of the major lineages of metazoans and exhibit reproductive-specific expression in all species examined, with a preponderance of male-biased expression. Examination of Boule evolution within insect and mammalian lineages revealed little evidence for accelerated evolution, unlike most reproductive genes. Instead, purifying selection was the major force behind Boule evolution. Furthermore, loss of function of mammalian Boule resulted in male-specific infertility and a global arrest of sperm development remarkably similar to the phenotype in an insect boule mutation. This work demonstrates the conservation of a reproductive protein throughout eumetazoa, its predominant testis-biased expression in diverse bilaterian species, and conservation of a male gametogenic requirement in mice. This shows an ancient gametogenesis requirement for Boule among Bilateria and supports a model of a common origin of spermatogenesis.Sex-specific traits that lead to the production of dimorphic gametes, sperm in males and eggs in females, are fundamental for sexual reproduction and accordingly widespread among animals. Yet the sex-biased genes that underlie these sex-specific traits are under strong selective pressure, and as a result of adaptive evolution they often become divergent. Indeed out of hundreds of male or female fertility genes identified in diverse organisms, only a very small number of them are implicated specifically in reproduction in more than one lineage. Few genes have exhibited a sex-biased, reproductive-specific requirement beyond a given phylum, raising the question of whether any sex-specific gametogenesis factors could be conserved and whether gametogenesis might have evolved multiple times. Here we describe a metazoan origin of a conserved human reproductive protein, BOULE, and its prevalence from primitive basal metazoans to chordates. We found that  Boule evolution in insect and mammalian lineages, representing the Protostome and Deuterostome clades of bilateral animals, failed to detect any evidence for accelerated evolution. Instead, purifying selection is the major force behind Boule evolution. Further investigation of Boule homologs among Deuterostome species revealed reproduction-specific expression, with a strong prevalence of testis-biased expression. We further determined the function of a deuterostomian Boule homolog by inactivating Boule in mice . Like its counterpart in Drosophila (a representative of the opposing Protostome clade), mouse Boule is also required only for male reproduction. Loss of mouse Boule prevents sperm production, resulting in a global arrest of spermatogenesis in remarkable similarity to that of Drosophila boule mutants. Our findings are consistent with a common origin for male gametogenesis among metazoans and reveal the high conservation of a reproduction-specific protein among bilaterian animals.While sexual reproduction is widespread among animals, it remains enigmatic to what extent sexual reproduction is conserved and when sex-specific gametogenesis (spermatogenesis and oogenesis) originated in animals. Here we demonstrate the presence of the reproductive-specific protein Boule throughout bilaterally-symmetric animals (Bilateria) and the conservation of its male reproductive function in mice. Examination of  Evolution of sexual reproduction, consisting of the origin and maintenance of sex, has been a central focus of evolutionary biology since the time of Darwin. The origin of sex has generally been simplified to the question of the origin of meiosis, which is known to have a single origin among all eukaryotes What remains unknown is to what extent features of sexual reproduction can be conserved. Most animals produce sex-specific gametes distinct in size, morphology, motility and development. Animal sperm are predominantly small and motile, with compact nuclei and often a beating flagellum, which are produced through a series of male-specific developmental steps called spermatogenesis. Eggs are usually large in size and immotile, and are produced through a distinct developmental process called oogenesis. The evolutionary origin of such dimorphic features of animal sexual reproduction is intriguing yet difficult to address experimentally since they left no trace in the fossil record. However, the identification of conserved male or female-specific gametogenic proteins across large evolutionary distances could uncover molecular traces of any ancient gametogenic machinery, providing evidence for a common origin of sexually dimorphic traits among animals. While reproductive proteins with conserved homologs in different phyla are not uncommon, most of them are involved in general cellular functions, and are hence also required for other non-reproductive processes Drosophila, C. elegans and mice, no conserved male- or female-specific gametogenic factors common to all lineages of animals have been clearly demonstrated Besides meiosis and the specification of germ cells, most of the other components of gametogenesis appear to be sex-biased or sex-specific. Despite a large number of sex-specific gametogenesis proteins uncovered in the model organisms BOULE, the ancestral member of the human DAZ gene family. The human DAZ gene family consists of a Y-linked DAZ gene and the autosomal DAZ-Like (DAZL) and BOULE genes, all of which share a conserved RNA recognition motif (RRM) and a more divergent DAZ repeat consisting of 24 amino acids rich in N, Y, and Q residues DAZ gene is commonly deleted in men with few or no sperm DAZL or BOULE have been shown to be responsible for infertility in men or women, homologs of DAZL and BOULE are required for fertility in other species, and over-expression of DAZ family proteins promote the differentiation of human embryonic stem cells towards the germ cell lineage BOULE transgene rescued partial testicular defects of fly boule mutants, suggesting functional similarity between these two distant homologs Identification of such conserved core components is the key to distinguishing between these two possibilities. Such ancient core reproductive components should fulfill the following criteria: present in most, if not all, of the major lineages of animals undergoing sexual reproduction; originated around the time when gametogenesis likely evolved; demonstrated conservation at the sequence, expression and functional levels in species from diverse animal phyla. The most stringent criteria for a conserved male or female gametogenesis factor requires a clear demonstration that these conserved components are only required for gametogenesis in one sex among animals, and not for any other processes, thus excluding any possibility that such factors are conserved due to essential functions outside of gametogenesis. A strong candidate for a conserved male gametogenic factor in animals appears to be Boule homologs whose function has been characterized in depth revealed opposite gametogenic requirements Boule homologs in Drosophila and Caenorhabditis elegans reveal their divergent roles in reproduction, with boule required for male reproduction in flies and for oogenesis in the worm Boule homologs among other metazoan phyla remains unexplored, raising the possibility that Boule may have undergone adaptive evolution like many other reproductive genes and subsequently diverged at the functional level among different metazoan branches. While partial rescue of the fly boule mutant defect by a human BOULE transgene suggests functional conservation, this may only reflect similar biochemical properties of all DAZ family members Dazl is able to partially rescue the Drosophila boule mutant, despite the fact that frog Dazl performs a reproductive function distinct from fly bouleBoule and to determine if it has a general conserved reproductive function, or also a conserved sex-specific function. We systematically examined the prevalence of Boule homologs in major animal phyla and also the molecular evolution of Boule in two distant bilaterian classes. To understand the functional evolution of bilaterian Boule, we surveyed the expression of Boule homologs in representative bilaterian species and determined the functional conservation of deuterostomian Boule through expression and genetic analyses of the mouse Boule homolog.However, the only two metazoan Boule homologs besides insects, mammals, and nematodes. In order to distinguish Boule from homologs of other DAZ family members as well as other general RNA binding proteins, we established the signature features of Boule that would allow us to identify Boule homologs in distant lineages with confidence. We separately aligned the protein sequences of known Boule homologs among two distant metazoan groups, mammals and insects, and established a consensus sequence for the RNA recognition motif (RRM) in each group , Tunicata , Cephalochordata , Echinodermata , and Hemichordata . Boule homologs were present in many protostomian species of the Ecdysozoa and Lophotrochozoa superphyla (D. melanogaster), mosquitoes (Anopheles gambiae), lobster (Homarus americanus), green shore crab (Carcinus Maenas), wasp (Nasonia vitripennis) and nematodes (C. elegans), representing the Arthropoda and Nematoda phyla (Schistosoma japonicum), Annelida , and Mollusca   , LTZ. ThBoule arose during evolution by determining whether Boule homologs are present in basal, non-bilaterian metazoans or beyond the animal kingdom in plants or fungi.Next, we asked when Boule features, we determined that Boule homologs are absent in fungi and plants, suggesting that Boule is restricted to the animal lineage  reveals two proteins with high similarity Drosophila boule genomic structure, the sea anemone boule gene has separate exons 3 and 4 as in humans, suggesting that the ancestral Boule gene contained separate exons 2, 3, 4 and 5 that encoded the RRM. This gene (XM_001637198) is predicted to encode a protein around 22 kDa, close to the typical size of Boule proteins. Hence, a Boule homolog is present in the sea anemone, a representative of Cnidaria , sea urchin from Echinodermata (Strongylocentrotus purpuratus), lancelet from Cephalochordata (Branchiostoma floridae), and sea squirt from Tunicata . We could not detect any canonical Dazl homologs  and could not identify a Dazl homolog (http://genome.wustl.edu/genomes/view/petromyzon_marinus/)  and found no evidence of a Dazl homolog, though a shark Boule homolog is present Dazl originated around the time of vertebrate radiation, likely in the ancestral lineage of bony fish (homologs . The higarinus/) . Given tony fish .Boule homologs, we performed phylogenetic analysis of Boule homologs from the major animal branches, together with homologs of the other members of the DAZ family, Dazl and DAZ  as well as eight representative mammalian species The ancient origin and widespread presence of such a reproductive gene is in stark contrast with the pervasive rapid evolution usually associated with reproductive genes, especially male reproductive genes Boule evolution, we compared the ratio of the rate of nucleotide changes that result in a non-synonymous amino acid substitution (Ka) to the rate of nucleotide changes that cause a synonymous amino acid substitution (Ks). Positive selection is a process that favors the retention of mutations that are beneficial to the reproductive success of an individual. Neutral theory predicts that the rate of non-synonymous substitutions (that by definition affect protein sequence) is equal to the rate of synonymous substitutions. If a protein has evolved under positive selection, there are more non-synonymous substitutions (Ka) than synonymous substitutions (Ks), and an accordingly high Ka/Ks ratio. If the protein evolved under purifying selection or negative selection, a process that removes deleterious alleles, there is a decrease or absence of non-synonymous substitutions, and therefore Ka/Ks is much smaller than that expected under neutral theory. A Ka/Ks greater than 1 is a strong indication of positive selection whereas only a Ka/Ks smaller than 0.1 usually suggests a role of purifying selection.To determine if positive selection has played a role in Drosophila species, we found that non-synonymous substitutions (Ka) were not in excess of synonymous substitution (Ks). Instead, Ka/Ks ratios for all pairwise comparisons were below 0.1 , we reasoned that by determining Boule expression patterns in deuterostomes we could ascertain whether or not the expression or function of Boule is conserved among bilaterians.The prevalence and strong functional constraint of Boule homologs are preferentially expressed in the testis or ovary. Like Drosophila and C. elegans, the sea urchin is also an invertebrate and has only the Boule gene, whereas chicken is a vertebrate with both Boule and Dazl. We identified homologs of Boule in chicken  and purple sea urchin (S. purpuratus)  from separate phyla and asked if puratus)  [51], . Wh compare , similarBoule mutant epididymides lacked mature sperm  and a deuterostome (mouse).In DAZ family of reproductive genes, with homologs in diverse species, many of which are specifically expressed in reproductive tissues, are ideal candidates for the study of reproduction-specific gene evolution Boule, the ancestral gene member, is reproductive specific in flies and worms.Evolution of reproductive traits and genes is of the utmost interest to our understanding of the central questions in evolutionary biology such as speciation. However, the relatively rapid divergence of sex-biased reproductive genes in comparison with somatic cell proteins or non sex-biased reproductive proteins during evolution has made it difficult to study the evolution of sex-specific reproductive systems across extended evolutionary distances. Even though some reproductive genes are conserved beyond a given phyla, they are often also involved in other developmental processes. Such broad functionality compounds studies of their reproductive evolution because the selective pressures driving their evolution may be due to critical somatic functions, and not a reproduction-related function. The human Boule in the major phyla of metazoans, reconstructed the evolutionary history of Boule, and began to determine its functional divergence. We found that Boule, unlike other reproductive proteins, has been maintained in all major phyla of bilaterian animals as well as in Cnidarians, but are absent in the most primitive animals (the placozoan Trichoplax), fungi and plants , but were present in bony fish and tetrapod animals  as well as eumetazoans  argue against such a possibility. Furthermore, the predominance of testis-biased expression of Boule homologs among distinct bilaterian species  in the sea anemone, an outgroup to the bilaterian lineage, could provide further insights into the ancestral roles of Boule.It is important to note, however, that this model does not exclude the possibility of an additional ancestral ovarian function of Urbilaterian Boule is also conserved, our discovery that mammalian Boule is required only for sperm development like its fly counterpart is the first such demonstration of a conserved spermatogenesis-specific function in both lineages. While spermatogenesis occurs in the testes of different animal lineages, it is not known if either spermatogenesis or the testis itself is evolutionarily related between vertebrates and invertebrates. The fly testis, which is a single tube with a linear progression of spermatogenesis, appears different from the mouse testis, which is composed of many seminiferous tubules with a concentric progression of spermatogenesis from the periphery of the seminiferous tubules towards the lumen. However, if we focus on a single cycle of spermatogenesis within a segment of a mouse seminiferous tubule and compare it with a fly testis tubule, we see similar spermatogenic cell types present inside the fly testis tubule and the mouse seminiferous tubule segment Whether or not an ovarian function of Drosophila, fish, rodents and primates Protamine-1 or Protamine-2 leads to male sterility, but in contrast, fly sperm carrying a deletion of both protamine-like homologs appears to be functional Furthermore, male reproductive traits and genes undergo rapid adaptive evolution in diverse lineages such as Boule homologs have been maintained throughout all major lineages of animals from a common eumetazoan ancestral gene and are required only for sperm development in both Drosophila and mice. We have shown that Boule proteins have resisted sexual selective pressure, and instead evolved under purifying selection. Though ancestral Boule may have also functioned in oogenesis, our findings that bilaterian Boule homologs tend toward male-biased expression, taken together with the similar spermatogenesis arrest phenotypes in both Drosophila and mouse mutants, supports the model of a common origin of bilaterian spermatogenesis.However, Boule homologs are restricted only to spermatogenesis or also function in the ovary, we have shown a clear case of conservation of a reproduction-specific gene across Bilateria. We found that among a broad representation of bilaterian animals, Boule expression was restricted to the gonads , samples were stored at 4°C overnight, solution was removed, and the tissues were stored at −80°C for later RNA extraction. A minimum of two pairs of primers spanning the RRM region and other regions were used to confirm the expression of the Boule gene (Boule amplicons were confirmed by sequencing. Dmrt1 (Doublesex and mab-3 related transcription factor 1) was a testis-specific positive control in chicken Bnd (Bindin) was a testis-specific positive control in the sea urchin RNA was extracted by Trizol from tissues and reverse transcribed for amplification of ule gene . All theMus musculus) were housed and bred in a barrier facility according to the guidelines approved by the ACUC committee at Northwestern University. Boule mutant mice were created in the mixed background of C57B6 and 129svj. Ripe purple sea urchins (Strongylocentrotus purpuratus) in spawning season  were collected from Pacific Ocean off Carslad, California  and shipped overnight on ice to Chicago. The sex of sea urchins was determined by the presence of eggs  and by the presence of distinct gametes in the gonad tissue biopsies. Gonads, guts and ampullae from at least three male and three female purple sea urchins were collected and either stored in Trizol for immediate RNA extraction or snap-frozen in liquid nitrogen and stored at −80°C. Chicken gonadal and other tissues were collected from euthanized White leghorn chickens at the completion of a research project approved by UIUC animal committee at the University of Illinois at Urbana-Champaign Veterinary School. Tissues from two four-year old roosters and two three-year old hens were snap-frozen in liquid nitrogen or stored directly in RNA later.Mice . We designed primers with built-in SalI sites to amplify a 2-kb left arm next to exon 3, and primers with built-in XhoI and NotI sites to amplify a 5.9-kb right arm from 129svj mouse genomic DNA. High fidelity platinum PCR kits (Invitrogen) were used to amplify the fragment with minimal PCR error. The amplified fragments were cloned into Topo vectors and later released with appropriate enzymes for subcloning into the NZTK2 vector. The clones with correct orientation of left and right arm insertions were chosen for sequencing. Sequencing of the genomic arms in the selected clones indicated that both arms had greater than 99% sequence identity with the genomic sequence. Gene targeting was performed on 200 ES cell clones (129svj E14 feeder cell-less ES cells) and four positive ES clones  were identified by the presence of both the 2-kb and 6-kb arms using primers outside each arm and on the vector. The 1D5 clone was used to inject blastocysts at the Northwestern University Transgenic Core Facility. Four chimerical mice produced lacZ-positive progeny and mice from two independent founders were used to generate mutant mice for the analysis. The phenotypes were identical among the mutant mice from two independent lines and we did not distinguish our analyses between the two lines.Figure S1Boule homologs. The seven Drosophila species used are D. melanogaster, D. sechellia, D. yakuba, D. virilis, D. erecta, D. willistoni and D. ananassae. Mosquito (Aedes aegypti), Honeybee (Apis mellifera), Beetle (Tribolium castaneum), Wasp (Nasonia vitripennis), SMonkey , Marmoset , Tamarin (Saquinus Oedipus), mouse (Mus musculus), Rat (Rattus norvegicus), Lemur (Microcebus murinus), Human (Homo sapiens) Frog (Xenopus laevis), Chimp (Pan troglodytes) PChimp , RMonkey , Hedgehog (Erinaceus europaeus), Microbat (Myotis lucifugus), GuineaPig (Cavia porcellus), Dog (Canis familiaris), Cow (Bos Taurus), Horse , Bushbaby (Otolemur garnettii), Playtypus (Ornithorhynchus anatinus). Opossum (Monodelphis domestica), Chicken . Color scheme: blue– A,I,L,M,F,W,V; red—R,K; green—N,Q,S,T; pink—C; Magenta—E,D; orange—G; cyan—H,Y; yellow—P Alignments for RNA binding domains (RRM) from insects (A) and mammals (B) reveal strong conservation among homologs. Chicken Boule RRM was added as an outgroup to mammalian domain alignment for comparison. (C) Based on the sequences from mammals and insects, the consensus RRM sequences (RNP1 and RNP 2) for Boule are likely representative of bilaterian Boule. The site of exon/intron junction is marked by the arrows and is also conserved among (5.46 MB TIF)Click here for additional data file.Figure S2Dazl homologs from diverse vertebrate species. The Dazl RRM is two amino acids shorter than the Boule RRM because of a two amino acid deletion. The consensus sequence for Dazl RRM was generated and is distinct from Boule RRM (lower panel). XL Frog (Xenopus laevis), XT Frog , Cow (Bos taurus), Human (Homo sapiens), PP Chimpanzee (Pan paniscus), PT Chimpanzee (Pan troglodytes), SS Monkey , RhesusMonkey (Macaca mulatta), Rat (Rattus norvegicus), mouse (Mus musculus), chick , DR Zebrafish (Danio rerio), OL Killifish (Oryzias latipes), MM Lemur (Microcebus murinus), SO tamarin (Saguinus Oedipus), LS Seabass , RP Frog (Rana pipiens), CP Newt (Cynops pyrrhogaster), CF Dog (Canis familiaris), EE Hedgehog (Erinaceus europaeus), Opossum (Monodelphis domestica), Platypus (Ornithorhynchus Anatinus), Bushbaby (Otolemur garnettii), SA Shrew (Sorex araneus), TB Shrew (Tupaia Belangeri), FuguFish (Takifugu rubripes). (B). Alignment of Boule and Dazl RRM consensus sequences.Dazl homologs share similar signature RRM motifs but distinct from that of Boule. (A). RRM sequence alignment of (5.91 MB TIF)Click here for additional data file.Figure S3Boule homolog. Sequence alignment of Boule and Hrp1 is shown. The Hrp1 RRM does not contain the characteristic amino acids of the Boule RRM. When the yeast Hrp1 sequence is compared to that of the fly or human genome, the fly gene with the greatest similarity is not boule but hRNP. Although yeast Hrp1 has a sequence similarity to Boule in the RRM and probably represents the closest RRM protein to Boule, Hrp1 is unlikely to be a Boule ortholog. Boule homologs are also absent in other single-cell eukaryotes and in plants, including Schizosaccharomyces pombe, Dictyostelium discoideum and Arabidopsis thaliana. We thus concluded that Boule homologs are restricted to animals. (B) In Trichoplax, the protein with highest sequence similarity to the Boule consensus sequence does not contain the key signature amino acids of Boule and is not a Boule homolog. Furthermore, the RNA binding domains of these proteins do not contain any introns, whereas Boule genes contain a conserved genomic structure with at least two introns separating the RRM-encoding exons at conserved junctions. Hence, a Boule homolog appears to be absent in the TrichoplaxBoule, whereas sea anemone Boule2 only shares two exon-intron junctions. For phylogenetic tree construction (Boule1 was used as the Boule homolog sequence. The arrows mark the sites of exon-intron junctions based on genomic sequences. RNP2 and RNP1 are underlined.Absence in yeast and Trichoplax but presence in sea anemone of Boule homologs. (A) The yeast protein with highest similarity to the Boule consensus sequence is Hrp1, but it is not a truction , sea ane(1.84 MB TIF)Click here for additional data file.Figure S4Dmrt1 is testis-specific control in the chicken. In the chicken, Boule is highly enriched in the testis, and Dazl is 10-fold more abundant in the testis than the ovary. Dmrt1 RNA was not detectable in muscle, kidney or ovary, while Dazl RNA was not detectable in muscle or kidney.Real-time PCR analysis of chicken gene expression. (0.53 MB TIF)Click here for additional data file.Table S1boule homologs. We performed pair-wise comparison of Ka and Ks for the entire boule coding sequences among seven Drosophila species . All Ka/Ks ratios were significantly below 1.Ka/Ks ratio analysis revealed no evidence for accelerated evolution among Drosophila (0.05 MB DOC)Click here for additional data file.Table S2Boule homologs. Molecular evolutionary analysis of the entire coding sequence in the eight representative mammalian species: Monotremes (platypus), Marsupials (opossum) and Eutherians , revealed no excessive non-synonymous nucleotide changes in comparison with synonymous changes. A total of 477 nucleotides were used from each homolog. Ka/Ks ratios were all below 1, with the highest value less than 0.1. These data suggest that not only was the RRM conserved through mammalian evolution, but the entire Boule gene, including less conserved regions, did not undergo rapid evolution.Ka/Ks ratio analysis also failed to identify any evidence for accelerated evolution among mammalian (0.08 MB DOC)Click here for additional data file.Table S3Boule, Dazl and DAZ used in this analysis are listed with species names and their sequence ID from genbank other databases.Summary table listing information for all genes used in the analysis. Homologs of (0.09 MB DOC)Click here for additional data file.Table S4Boule homologs.List of primers used for RT-PCR and qRT-PCR analyses of Chicken, Sea urchin and mouse (0.06 MB DOC)Click here for additional data file.Table S5Color code table for amino acid residues in (0.04 MB DOC)Click here for additional data file.Text S1Supplemental data.(0.04 MB DOC)Click here for additional data file.


Gefitinib is a new molecular-targeted agent for the treatment of patients with advanced non-small cell lung cancer that fail to respond to conventional chemotherapy. Gefitinib is considered to be well tolerated and less toxic compared with conventional cytotoxic drugs. However, interstitial lung disease (ILD) has been reported as a serious adverse effect. The precise management of a gefitinib responder having severe adverse events remains unknown.EGFR) gene, L858R in exon 21, which was identified in the primary tumor.We report the case of gefitinib readministration in a patient with lung adenocarcinoma who had once responded but in whom treatment had to be discontinued owing to gefinitib-related ILD. A dramatic response was achieved both at the time of initial treatment (250 mg/day) and at readministration of gefitinib (125 mg/day). The effectiveness of gefitinib therapy in our patient could be explained in part by the presence of an activating mutation of epidermal growth factor receptor (EGFR gene mutations, and that the currently approved dose may be excessively potent in some of these patients, thus resulting in the onset of adverse events.A reduced dose of gefitinib might be sufficient for patients having tumors with  Gefitinib is a new molecular-targeted agent for the treatment of patients with advanced non-small cell lung cancer that fail to respond to conventional chemotherapy. Early clinical trial data demonstrated that gefitinib was well tolerated and was less toxic compared with conventional cytotoxic drugs,2. The mWe herein report a thought-provoking case of readministration of gefitinib in a patient with lung adenocarcinoma who had previously responded and developed gefinitib-related ILD. Gefitinib readministration with 50% dose was successful in managing disease progression.A 56-year-old male was referred to our hospital because of a lung nodule detected on population-based radiological screening. He was a current smoker with a smoking index of 15 pack-years. A chest computed tomography (CT) scan demonstrated a15 mm solid-density nodule with pleural indentation in the left lower lobe. Mediastinal lymph nodes were not swollen in the mediastinal setting images . He underwent left lower lobectomy and systematic lymphadenectomy. He was diagnosed as having lung adenocarcinoma with multiple mediastinal lymph node metastases and solitary pulmonary metastasis . He received two courses of postoperative adjuvant chemotherapy (carboplatin/paclitaxel).Multiple bone metastases developed 10 months after the operation. He received two courses of chemotherapy (cisplatin/docetaxel) and palliative irradiation therapy. Subsequently, at 17 months after the operation, a follow-up CT scan indicated miliary pulmonary metastases with lymphangitis carcinomatosa throughout both lungs and mediastinal lymphadenopathy Figure . ILD wasHowever, the patient developed progressive general fatigue and shortness of breath 45 days after the initiation of gefitinib therapy. A chest CT scan demonstrated new areas of patchy ground glass opacity (GGO) accompanied by interstitial markings bilaterally, without evidence of tumor growth Figure . The serIn the five months following the withdrawal of gefitinib, miliary pulmonary metastases with lymphangitis carcinomatosa gradually progressed Figure . The patEGFR) gene was conducted at Mitsubishi Kagaku Bio-Clinical Laboratories, Inc. Genomic DNA was prepared from a paraffin-embedded section using macrodissection in a surgically resected primary tumor specimen. The peptide nucleic acid-locked nucleic acid PCR clamp protocol [Because he gave his written informed consent to let us use surgically resected specimens for genetic analyses before operation, a mutation analysis of the epidermal growth factor receptor . Similar efficacy but higher toxicity was observed with the 500 mg dose. Consequently, 250 mg has been the recommended dose ,10. Howe and 2. SEGFR gene mutations [EGFR-mutated tumors respond dramatically to lower dose of EGFR-TKIs such as gefitinib in experimental studies [Numerous studies suggest that gefitinib has fairly effective anti-tumor activity, especially for tumors with utations ,12. It i studies ,14. ThesThe prognosis of patients with lymphangitis carcinomatosa is extremely poor, with approximately 50% of the patients dying within three months of their first respiratory symptoms . FortunaWe herein presented the case of gefitinib readministration in a patient with lung adenocarcinoma who had once responded but in whom treatment had to be discontinued owing to gefinitib-related ILD. Gefitinib readministration with 50% dose was found to successfully control disease progression.We believe clinical trials are warranted to evaluate gefitinib readministration with a dose reduction for patients who have once responded but later discontinued this treatment owing to severe adverse events including ILD. The appropriate dose and schedule of gefitinib readministration also should be determined in these trials.CT: computed tomography; EGFR: epidermal growth factor receptorGGO: ground glass opacity;ILD: interstitial lung disease;WJTOG: West Japan Thoracic Oncology Group;   The author(s) declare that they have no competing interests.KT reviewed the medical records and imaging findings, drafted the manuscript and coordinated the submission. KF ans KS participated in care of the patient, and helped draft the manuscript. AHB critically revised the manuscript. KY, TM, and TI participated in reviewing the manuscript. All authors read and approved the final manuscript.Written informed consent was given for publication from the patient's next-of-kin.


Sir,Elevated expression of carcinoembryonic antigen  has been implicated in various biological aspects of neoplasia. Carcinoembryonic antigen has been shown to be involved in both homophilic and heterophilic binding , demonstrating its ability to react with a panel of CEA-expressing colorectal cancer cell lines and induce ADCC activity. The authors state that ‘there are so far no unconjugated, or ‘naked’ antibodies to CEA being used for treatment of colorectal cancer’  in vitro against CEA-positive human colonic tumour cells (in vitro (Labetuzumab, as an IgGin vitro .et al in March 2008 demonstrates some promising in vitro activity, the experience with such humanised anti-CEA MAbs goes well beyond these observations, as described by our own in vitro and preclinical studies demonstrating direct, specific, anti-tumour effects without conjugation to a cytotoxic agent, in both colorectal and medullary thryroid cancer xenografts, providing evidence for the superiority of the combined modality naked anti-CEA MAb immunotherapy and chemotherapy treatments. The results support further studies focused on the integration of anti-CEA MAb therapy into chemotherapeutic regimens for the improved management CEA-expressing neoplasms.Although the PR1A3 antibody described by Conaghan


Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable.Halobacterium salinarum NRC-1 that batch culturing in complex medium stimulates meaningful changes in the expression of approximately two thirds of all genes. While the majority of these changes occur during transition from rapid exponential growth to the stationary phase, several transient physiological states were detected beyond what has been previously observed. In sum, integrated analysis of transcript and metabolite changes has helped uncover growth phase-associated physiologies, operational interrelationships among two thirds of all genes, specialized functions for gene family members, waves of transcription factor activities, and growth phase associated cell morphology control.We have discovered in the model organism Simple laboratory culturing in complex medium can be enormously informative regarding the activities of and interrelationships among a large fraction of all genes in an organism. This also yields important baseline physiological context for designing specific perturbation experiments at different phases of growth. The integration of such growth and perturbation studies with measurements of associated environmental factor changes is a practical and economical route for the elucidation of comprehensive systems-level models of biological systems. One of the main goals of current molecular systems biology is to generate quantitative models for the structure and behavior of cellular, metabolic, signal transducing and gene regulatory networks in a complete organism. Such knowledge may enable us to ultimately predict global changes in gene expression of an organism in response to cellular stimuli and enable the predictable reengineering of cells for therapeutic or industrial purposes . The relIn this light, it is worthwhile to recall that for nearly a century the microbial growth curve has been instrumental in shaping our investigation and understanding of cell function and evolution -8. The pH. salinarum NRC-1 is one of few that is easily cultured and manipulated in the laboratory. For this reason it has become an important model system for characterizing archaea with respect to their global cellular responses to diverse stresses [H. salinarum NRC-1 have only received basic characterization on a global scale. The work by Lange et al. [Among the archaea, stresses -15, archstresses , their pstresses , transcrstresses  and aspestresses , archaeastresses , archaeastresses -23, archstresses -26, archstresses  and numee et al. , comes cH. salinarum NRC-1 during growth in batch cultures. Our analysis demonstrates the scale at which growth in simple, reproducible batch cultures can induce major physiological changes encoded by 1,518, or 63% of the approximately 2400 putatively identified non-redundant genes in H. salinarum NRC-1. Transcriptional changes are interpreted in the context of biological functions. In addition this manuscripts demonstrates how the analysis of transcriptome growth data in the context of other data types, including metabolomic data and gene perturbation can lead to novel biological insight.This manuscript reports a systems-level interrogation of physiological changes that occur in H. salinarum NRC-1 in rich complete medium (CM) . A typical growth curve is shown in Figure We first characterized growth of pyrF uracil auxotroph mutant of H. salinarum NRC-1, supplemented with uracil  at 7 different points along the growth curve for two cultures, the wild type strain in standard growth medium with or without uracil supplementation and 6 different points for strain MPK407, a l Figure . We noteH. salinarum NRC-1 showed significant changes in gene expression over the growth curve. We applied K-means clustering to this filtered data set to identify dominant patterns of gene expression changes. The two patterns with the largest gene memberships were those typified by (a) elevated gene expression during exponential growth followed by severe decreased transcript abundance in stationary phase . Moreover, numerous genes encoded on the replicon pNRC200 appear to be differentially regulated in the MPK407 strain. Given that many of the genes encode products of unknown function, we cannot offer a functional rationale for these observations other than to suggest caution when interpreting expression changes for these genes in responses of cells with the ΔpyrF genotype. A similar analysis did not reveal any alterations in gene expression changes due to the addition of uracil to the media.Using these criteria, we found 31 genes  of all plasmid-encoded genes increased in abundance during logarithmic phase, 28% (151 genes) increased in abundance at stationary phase. These observations show that functions of genes encoded on the plasmids are disproportionally associated with stationary phase physiology (p = 0.004).H. salinarum NRC-1 by searching the 772 genes against KEGG's pathway database  and biosynthesis (from precursors chorismate and PRPP) is worth noting. Since serine is required as one of the final building blocks of tryptophan biosynthesis, one possibility is that this pathway could be operated in conjunction with a serine deaminase (vng2100G) to maximize the breakdown of serine into pyruvate.Finally, the simultaneous increase in transcript abundance during stationary phase of genes encoding tryptophan breakdown , vng6315G (arcB), vng6316G (arcC) and vng6317G (arcA) decrease in abundance transiently during exponential phase, relative to levels in early growth and stationary phases. These genes are involved in the fermentation of arginine as an alternate form of energy production under anaerobic environmental conditions. Therefore, while the increased transcript abundance of these genes during stationary phase was to be expected, their relatively high transcript levels in early exponential growth was surprising. However, experiments tracking amino acid consumption in defined media during growth of Halobacterium salinarum sp. R1 have shown cells apparently consume available arginine very quickly during early phases of growth [vng2436G (argH) and vng2437G (argG) in the stationary phase might feed additional fumarate into the reductive carboxylate cycle.Transcripts for arginine import and fermentation genes, f growth . An intevng0414G, vng0864G, vng0876G, vng1395G) encode enzymes for the conversion of 5-phosphoribosylamine to 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate, a direct precursor to thiamin phosphate, and as such, link purine metabolism with thiamin metabolism. Notwithstanding the caveat that some enzymes of thiamin biosynthesis in H. salinarum NRC-1 are yet to be discovered, this may provide thiamine based cofactors for enzymes.Transcripts for ten genes are transiently elevated during exponential growth relative to stationary phase and early exponential Table . Among tvng2196G, vng1188G), one putative quinole oxidase (vng2413H), a putative Cu containing reductase (vng1187G) and two alternative cytochrome C oxidase subunits . This suggests that the organism may be retooling some of its capacity for redox chemistry at the end of exponential growth. It is known that other organisms will exchange cytochrome C oxidase subunits to remain more metabolically versatile in response to changing redox state [vng0602C, vng1182H, vng1486H, vng2197H, vng2199H and vng2412H), a putative putative haloacid dehalogenase-like hydrolase (vng0719G), a putative iron-sulfur cluster containing protein (vng1184Gm), a putative universal stress protein (vng1898C), a putative phosphoribosyl transferase (vng1912G), a heavy-metal transporting CPx-type ATPase (vng2201G) and an putative aminopeptidase (vng2546G).The transcript for eighteen genes show transient increased in abundance during the transition from exponential growth into the initial stages of stationary phase Table . The funox state . The othH. salinarum NRC-1 [TFBf and TBPe, were significantly higher during exponential growth. Together with analysis of genome-wide distribution of their transcription factor binding sites (TFBSs) generated in a previous study [TFBf and TBPe during active growth. The primary role of TBPe in exponential growth was also recently supported by qRT-PCR experiments that quantified the expression of TBPs A, B, D and E in exponential phase under both anaerobic and aerobic conditions [TBPe transcript is nearly 8-fold more abundant in exponentially growing cells than any other detectable TBP transcript.The scale of the physiological shift during transition of actively dividing cells to stationary phase suggests that this global phenomenon must to some degree be mediated by the activity of general transcription factors (GTFs), which are known to transcriptionally segregate large groups of functionally-related genes in um NRC-1 . This vius study , their sus study ,34, and nditions . These eTFBa, TFBb, TFBd, TFBe, TFBg, TBPf) showed increased abundance during stationary phase of growth. This suggested that the GTF regulatory network of stationary phase might be more complex than the network operating during exponential growth. It is tempting to speculate that gene regulation is of greater importance to optimize energy utilization when resources are limited and that this might consequently require a more complex regulatory network to regulate appropriately. If this is indeed the case, though, one might also suspect that deletion or over-expression of these GTFs would also result in significant perturbations to growth behavior. However, results from a previous study [Transcripts for seven of the 13 GTFs  in Using three observations we illustrate how the study of growth can provide evidence and generate testable hypotheses for a variety of research areas in cellular physiology. Specifically we highlight (a) the integration of metabolomic data with gene expression data to generate hypotheses regarding growth-associated nutrient fluxes (b) use of growth-associated transcriptional patterns for distinguishing among paralogous functions and (c) analysis of perturbed culture characteristics in a GTF mutant to explain transcriptional control of cellular morphology during growth.Metabolite profiling in halophilic archaea is complicated by the incompatibility of residual salt in the extraction buffer that interferes with most MS-based profiling technologies. We attempted some preliminary intracellular metabolite extraction and analysis over different growth phases to see whether or not growth studies could justify the effort and resources required for streamlining and integrating metabolomics-based approaches with gene expression profiling for reconstructing cellular responses to environmental changes.Cells were processed and metabolites were extracted and analyzed as described in Methods. Among the final set of identifiable metabolites whose abundance changed significantly during growth, we noticed patterns of changes in metabolite abundance that mirrored the patterns of gene expression we described earlier  show a similar pattern of relative abundance in across growth  show steady increase in abundance over growth , vng0628G (gdhA1) and vng1204G (gdhA2) are shown in Figure gdhB and gdhA1 have anti-correlated expression patterns upon transition to stationary phase. Whereas transcript levels for gdhB increase upon entering stationary phase, the transcript level for gdhA1 decreases. The transcript levels for gdhA2 do not show significant change in this experimentAn interesting example of how this simple study has led to a potential hypothesis for functional annotation involves the analysis of three genes annotated as glutamate dehydrogenases (GDH). Expression profiles for each of the GDH homologs, GdhA1 has been experimentally identified as an NADPH dependent enzyme while the cofactor preference of the other two enzymes remains unknown [+ and NADPH-associated GDH enzymes [+ and NADPH-associated GDH enzymes are reciprocally regulated as a function of changes in various factors including ammonium ion levels [H. salinarum NRC-1 encodes, like yeast, multiple genes that are true GDHases, a direct comparison to yeast would suggest that, the expression anti-correlated gdhB and gdhA1 genes could, respectively, encode an NAD+ dependent GDH that would drive the oxidation of glutamate to oxoglutarate and an NADPH dependent enzyme catalyzing the reverse reaction. Meanwhile, the role of the third annotated GDH gene, gdhA2, remains less clear. This hypothesis presumes that each gene annotated as a GDH actually function as GDHs in vivo. Further biochemical characterisation will be necessary to confirm or refute this hypothesis. Our interpretation of the data nevertheless represents a reasonable and testable hypothesis that was generated simply by inspection of transcriptional growth data. unknown . A compl enzymes . Howevern levels . If the tbpD. Along with a slow doubling time, this strain was defective in gas vesicle production . The gas vesicle minus phenotype observed during imaging is thus corroborated by the perturbed expression of gas vesicle genes, specifically gas vesicle cluster 1. The perturbed expression of aconitase may partially explain both the noted slow growth and the circular morphology. While it is reasonable to assume that reducing the expression of a TCA cycle intermediate could slow metabolic activity sufficiently to reduce growth rate, citrate accumulation is also thought to be a principle culprit for growth retardation in aconitase deficient mutants [Streptomyces coelicolor [Streptomyces viridochromogenes [Bacillus subtilis [Staphylococcus aureus [To interrogate this morphological phenotype we analyzed gene expression of patterns through growth in the  mutants -43. Meanelicolor , Streptoomogenes , Bacillusubtilis  and Staps aureus  suggestiH. salinarum NRC-1 is that of Lange et al. [One of the most surprising element of this study is the degree of changes in transcript abundance detected across the growth curve . The closest comparable study in e et al.  who repoe et al. ,28. We ntfbE , while transcriptionally increased in stationary phase may nevertheless have significant function during exponential growth.A critical component of Lange et al's. study is that in addition to changes in transcript abundance they also measured translational efficiencies between exponential and stationary growth. This provides the opportunity for some interesting comparisons. For instance, one notable difference between Lange et al. and this study is the near absence of differentially abundant transcription factors, particularly general transcription factors which contrasts with this study. Interestingly, however, Lange et al. do note significant translational regulation of numerous general transcription factors in exponential growth. This suggests that some of the transcription factors, like Escherichia. coli reported 27% of all transcripts showing differential abundance between stationary and exponential growth phases [It is, finally, worth comparing growth or growth-related studies in other organisms to the currently presented data. One early study in h phases . In methh phases -51.H. salinarum NRC-1 [H. salinarum have evolved. Therefore, it is reasonable to consider the proposition that other experiments conducted in this growth regime might be most informative regarding the physiology of the organism.The set of experiments presented herein, and the demonstrated extent to which transcriptional changes across a complete genome can be observed, serve to highlight the often forgotten potential for information inherent in relatively simple growth studies. A general approach to the systems-level study of any newly or poorly characterized organism could begin by characterizing changes in gene expression over a relatively simple growth experiment. For example, we have already applied this principle to capture the structure of the transcriptome (transcription start sites (TSSs), transcription termination sites (TTSs), non-coding RNAs (ncRNAs) etc.). By sampling at 13 distinct points covering the various phases of growth we were able to reveal fine mechanistic features of transcriptional control  associated with most genes of um NRC-1 . In yet um NRC-1 . We ratiIn the context of systems-level reconstructions of physiology and gene regulation, a simple experiment that can stimulate the changes in expression for over 60-80% of all genes without any externally administered perturbation(s) Tables  and 5 caAdditionally, one of the overarching goals in systems biology is not only to reconstruct a detailed understanding of cellular physiology/networks but to also place this understanding into an ecological context . Cells hTherefore, in the context of the systems-level study, it would also be highly appropriate to (a) provide mechanisms to control environmental factors in the experiment (e.g. by using bioreactors rather than flasks for culturing) and (b) make quantitative measurements on the levels of as many environmental factors as possible during the experiment. The latter could then be queried for their ability to predictably inform on the noted physiological changes. The critical, and yet undetermined, element in this scenario would be to use the bioreactor (or similar device) in a manner that would only control environmental factors sufficiently to allow for reproducibility in the aforementioned measurements while still allowing numerous environmental factors to change, thereby inducing differential gene expression.This last point is admittedly controversial because it challenges what have been very informative protocols established during years of single-perturbation studies. These single perturbation experiments typically compare the results from two experiments, control and perturbation, in which all environmental factors, except the perturbation, are held constant. What we are proposing above is effectively a multi-parameter perturbation experiment in which numerous environmental factors are simultaneously perturbed. Given both our ever-improving ability to make low cost measurements of numerous metabolites, likely the primary environmental factors changing during growth, and our improving ability to learn functional relationships from multi-parameter biological data sets, our proposed approach seems like it might be a reasonable approach. The potential for information rich data sets illustrated in this manuscript do suggest that it is worthwhile to pursue.H. salinarum NRC-1 during growth in simple batch culture, show the physiological changes encoded by 1,518, or 63% of the approximately 2400 putatively identified non-redundant genes. The exponential phase is characterized by genes associated with transcription, translation, and numerous genes associated with oxidative phosphorylation including the TCA cycle. Meanwhile, stationary phase seems associated with increased abundance of transcripts for genes encoding protein complexes involved in phosphate transport, oligo and dipeptide transport, glycerol-3-phosphate transport, manganese transport, branched chain amino acid transport and those encoding alternate oxygen-independent energy transduction processes including phototrophy, DMSO/TMAO respiration and arginine fermentation. The changes in transcript abundance noted in the stationary phase are also disproportionately associated with genes encoded on the two plasmids, pNRC100 and pNRC200, for whom functions are not known. In addition, while the majority of changes in transcript abundance are associated with classic exponential and stationary phases, the sampling frequency of this experiment has demonstrated that transient changes in transcript abundance occur during exponential growth and in the transitions between exponential and stationary phases which include thiamine biosynthesis and redox enzymes.Our analysis of global transcriptional changes that occur in This study also demonstrates how the analysis of transcriptome growth data in the context of other data types, including metabolomic data and gene perturbation can lead to novel biological insight. Finally, we suggest that one of the potential significant implications of this study is that the integration of growth as a parameter in traditional perturbation studies may prove to be useful in minimizing the number of experiments necessary to observe a near comprehensive set of functional association patterns between different genomically-encoded elements. This could help to drive further development of systems-level analysis in numerous non-model organisms whose genomes are being sequenced at an ever increasing rate.H. salinarum sp. NRC-1 is the wild-type strain [TBPd-cmyc non-native expression strain was constructed as previously described [TBPd has been cloned. The construction of the MPK407 strain  was described previously [4•7H2O, 3 g/L Sodium Citrate, 2 g/L KCl and 10 g/L Peptone  made with distilled water. Cultures were inoculated to a starting optical density at 600 nm of 0.02 with starter culture of optical density at 600 nm of 0.5 which was derived from a single colony. Cultures were grown in unbaffled flasks in which 40% of the flask volume is occupied by the culture. Cultures were shaken at 220 RPM illuminated at ~20 μmol/m2/sec (standard illumination in Innova9400 incubators (New Brunswick)). Duplicate cultures were grown and samples were harvested for RNA extraction at points indicated in Figure e strain . The TBPescribed  by transeviously . CulturiH. salinarum NRC-1 genome were bought from QIAGEN . Microarray fabrication, RNA preparation, labeling with Alexa 594, and Alexa 660 dyes , hybridization, and washing have been described previously [Unique 70-mer oligonucleotides spanning 2400 of the putatively identified non-redundant genes in eviously . Each coeviously .http://www.R-project.org). Data were row normalized and filtered according to the following criteria: genes must have lambda >15 for 4 consecutive conditions to be considered significant. A total of 1518 genes met this criteria. Fold change was calculated by taking the ratio between the average non-logged ratio for the last four samples (replicates included) taken in the growth curve to the average of the first four samples taken in the growth curve. A t-test, computed on logged data, was also used on the same selected sets to (first and last four data points for each strain) to ask whether the changes in expression were statistically significant given an overall p-value threshold = 0.05 and enforcing a false discovery rate of 0.05 or less.Microarray data was analyzed using Gaggle , TM4 3131, and ttbpDcmyc-expressing cells were grown as indicated above. Cell aliquots were harvested at each data point in Figure http://rsb.info.nih.gov/ij/) to extract individual cells and to quantify cellular length, width, circularity and average pixel intensity. Average values from a minimum of 50 individual cells were used to quantify each datapoint. Matlab  scripts were used to process single frame ImageJ output into single files. R scripts were written to generate mixture models for cellular morphology using the mixdist R package . Plots were generated with R.Wild type and 8 cells were harvested by centrifugation during early growth, exponential growth and stationary phase and washed with 2 mL basal salts to remove contamination from the media. Metabolites were extracted from cell pellets with 100 μl of 100% cold methanol. Samples were centrifuged at top speed in a microfuge to remove unsolubilized material. The supernatant was dried by SpeedVac at cold temperature. 5 μL of sample reconstituted in 0.1% formic acid was injected for each run using the microwell autosampler of an Agilent 1200 HPLC system . Reverse phase chromatography was performed using a Waters Symmetry C18  column  at a flow rate of 250 μL/min. Buffer A water with 0.1% formic acid (v/v) and buffer B was acetonitrile with 0.1% formic acid. The column was equilibrated at 100% A, then ramped to 5% B over 5 minutes. Then the gradient elution was used over the next 30 minutes to 95% B and maintained for 5 minutes. A m/z scan range of 85-2000 was used on an Agilent ESI-q-TOF mass spectrometer in positive ion mode. The capillary was kept at 3500 V and the fragmentor was set to 175 V for the analysis. Collision energy for each metabolite was optimized for maximum fragmentation efficiency, and identical collision energies were used for each chemical standard and the endogenous metabolite for identification.Approximately 10http://www.genome.jp/kegg/, METLIN http://metlin.scripps.edu/, Lipidmaps http://www.lipidmaps.org/ and HMP http://www.hmdb.ca/ to look for known metabolites which correspond to the ions of interest. High purity chemical standards of hypothesized structures of ions of interest were bought  and identification was performed by a precise match of MS/MS pattern and chromatographic retention time under identical experimental conditions on an Agilent ESI-q-TOF mass spectrometer.The LC-MS runs performed on the Agilent ESI-TOF mass spectrometer were used for a comparative analysis using XCMS  to identhttp://metlin.scripps.edu/.Individual metabolites were identified with high statistical significance  and were further filtered based on a minimum 3-fold change between early and stationary phase abundance. Finally, manual inspection to eliminate noisy profiles reduced the final set of metabolites to nearly 150 that were searched against the METLIN database of characterized metabolites http://www.ncbi.nlm.nih.gov/projects/geo with accession numbers: GSE14832, GSE14835 and GSE14836. Metabolomic data can be accessed via http://baliga.systemsbiology.net/.Microarray data for this study can be accessed from the Gene Expression Omnibus MTF conceived of the study, participated in the design, collected data and drafted the manuscript. WLP collected growth curve data, wrote image analysis scripts and helped draft the manuscript. FYL conducted growth studies on GTF perturbation strains. KW, TK and KM conducted growth experiments and helped draft the manuscript. MP and AK conducted gene expression analysis. DL conducted experiments characterizing the morphology of TBPd mutants. DJR wrote scripts to analyze functional overrepresentation in gene sets. LH, EK, TN, SAT and GS designed and conducted the metabolite analysis. NSB participated in the design of the study and in its coordination and helped draft the manuscript. All authors have read and approve of the final manuscript.H. salinarum NRC-1 in rich mediaAdditional figure S1. Interrogation of the apparent second "growth" phase of . (A) Growth of H. salinarum NRC-1 was tracked by optical density measurements at both 600 nm and 700 nm to determine the contribution of bacteriorhodopsin (a protein whose broad absorption peak centered at 568 nm can contribute to absorption at 600 nm but not at 700 nm) accumulation. The growth curve generated at 700 nm displays similar behavior to the curve generated from measurements taken at 600 nm. In particular, the apparent doubling that occurs during what appears to be stationary phase is apparent in both curves suggesting that this is not due to bacteriorhodopsin accumulation. This increase in optical density is most likely due to an increase in light scattering gas vesicles that are visibly released during this late phase of the growth experiment. (B) Phase-contrast visible light microscopy image of H. salinarum NRC-1 near the end of data collection for the data presented in Figure Click here for fileH. salinarum NRC-1.Potential causes for the apparent second growth phase in Click here for fileAdditional figure S2. Heat maps of genes with significant up or down regulation during growth. Samples from each individual experiments  are organized by increasing optical density moving from left to right. The black volume bar indicates increasing optical density. Genes have been hierarchically clustered to show potential subpatterns of expression. (A) A heatmap showing the changes in expression of 451 genes whose transcript abundance is decreased upon entry into stationary phase. (B) A heatmap showing the changes in expression of 772 genes whose transcript abundance is increased upon entry into stationary phase.Click here for fileAdditional table S1 - Genes whose transcript abundance is decreased during the transition to stationary phase. This table lists ORF name, gene symbol, an estimate fold change in expression between pre-stationary and stationary phase expression, an indicator of significance of change between pre-stationary and stationary phase expression values and the putative gene function (if known). Fold change was calculated by taking the ratio between the average non-logged ratio for the last four samples (replicates included) taken in the growth curve to the average of the first four samples taken in the growth curve. A t-test, computed on logged data, was also used on the same selected sets to (first and last four data points for each strain) to ask whether the changes in expression were statistically significant given an overall p-value threshold = 0.05 and enforcing a false discovery rate of 0.05 or less. 418 genes of the 451 in this clustering derived set were deemed to have significantly different expression levels using this criteria while the remaining 33 genes did not. Genes meeting this criteria are marked with a number one while those not meeting the criteria are marked with a zero. Manual inspection of expression profiles of genes not meeting the above criteria suggest that the t-test in this instance may be too conservative as many gene expression profiles deemed not significant show what seems to be clear decrease in signal between pre-stationary and stationary phases.Click here for fileAdditional table S2 - Genes whose transcript abundance is increased during the transition to stationary phase. This table lists ORF name, gene symbol, an estimate fold change in expression between pre-stationary and stationary phase expression, an indicator of significance of change between pre-stationary and stationary phase expression values and the putative gene function (if known). Fold change was calculated by taking the ratio between the average non-logged ratio for the last four samples (replicates included) taken in the growth curve to the average of the first four samples taken in the growth curve. A t-test, computed on logged data, was also used on the same selected sets to (first and last four data points for each strain) to ask whether the changes in expression were statistically significant given an overall p-value threshold = 0.05 and enforcing a false discovery rate of 0.05 or less. 713 genes of the 772 in this clustering derived set were deemed to have significantly different expression levels using this criteria while the remaining 59 genes did not. Genes meeting this criteria are marked with a number one while those not meeting the criteria are marked with a zero. Manual inspection of expression profiles of genes not meeting the above criteria suggest that the t-test in this instance may be too conservative as many gene expression profiles deemed not significant show what seems to be clear increase in signal between pre-stationary and stationary phases.Click here for fileFunctional KEGG pathway and ontology annotations for those genes reported in Additional tables S1 and S2. Functional assignments are as reported by KEGG, unmodified.Click here for fileAdditional figure S3. Metabolites showing significant changes in abundance during growth. Each panel show a bar chart illustrating the changes in abundance for 51 detected metabolites whose levels change significantly during growth. The numbering scheme at the top of each panel does not have any practical significance and simply serves to identify each MS peak in the experiment. Histogram heights correspond to average counts for each metabolite in the triplicates. Measured levels are not indicative of absolute cellular concentrations. However, relative changes in concentrations between samples derived from different growth states may be inferred the histograms.Click here for fileAdditional figure S4. Spectra of citrulline, phenylalanine, riboflavin and 5-deoxyadenosine. Paired presentation of ion spectra measured from both H. salinarum NRC-1 and from purified metabolite standard for citrulline, phenylalanine, riboflavin and 5-deoxyadenosine. Relative abundance levels are also shown as a histogram.Click here for file