Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.Choanoflagellates have long fascinated evolutionary biologists for their marked similarity to the 'feeding cells' (choanocytes) of sponges and the possibility that they might represent the closest living relatives of metazoans 1,2 . Over the past decade or so, evidence supporting this relationship has accumulated from phylogenetic analyses of nuclear and mitochondrial genes [3][4][5][6] , comparative genomics between the mitochondrial genomes of choanoflagellates, sponges and other metazoans 7,8 , and the finding that choanoflagellates express homologues of metazoan signalling and adhesion genes 9-12 . Furthermore, species-rich phylogenetic analyses demonstrate that choanoflagellates are not derived from metazoans, but instead represent a distinct lineage that evolved before the origin and diversification of metazoans (Fig. 1a, Supplementary Fig. 1 and Supplementary Note 3.1) 8,13 . By virtue of their position on the tree of life, studies of choanoflagellates provide an unparallelled window into the nature of the unicellular and colonial progenitors of metazoans 14 .Choanoflagellates are abundant and globally distributed microbial eukaryotes found in marine and freshwater environments 15,16 . Like sponge choanocytes, each cell bears an apical flagellum surrounded by a distinctive collar of actin-filled microvilli, with which choanoflagellates trap bacteria and detritus (Fig. 1b). Using this highly effective means of prey capture, choanoflagellates link bacteria to higher trophic levels and thus have critical roles in oceanic carbon cycling and in the microbial food web 17,18 .More than 125 choanoflagellate species have been identified, and all species have a unicellular life-history stage. Some can also form simple colonies of equipotent cells, although these differ substantially from the obligate associations of differentiated cells in metazoans 19 . Studies of basal metazoans indicate that the ancestral metazoan was multicellular and had differentiated cell types, an epithelium, a body plan and regulated development including gastrulation. In contrast, the last common ancestor of choanoflagellates and metazoans was unicellular or possibly capable of forming simple colonies, underscoring the abundant biologi...

Tyrosine kinase signaling has long been considered a hallmark of intercellular communication, unique to multicellular animals. Our genomic analysis of the unicellular choanoflagellate Monosiga brevicollis discovers a remarkable count of 128 tyrosine kinases, 38 tyrosine phosphatases, and 123 phosphotyrosine (pTyr)-binding SH2 proteins, all higher counts than seen in any metazoan. This elaborate signaling network shows little orthology to metazoan counterparts yet displays many innovations reminiscent of metazoans. These include extracellular domains structurally related to those of metazoan receptor kinases, alternative methods for membrane anchoring and phosphotyrosine interaction in cytoplasmic kinases, and domain combinations that link kinases to small GTPase signaling and transcription. These proteins also display a wealth of combinations of known signaling domains. This uniquely divergent and elaborate signaling network illuminates the early evolution of pTyr signaling, explores innovative ways to traverse the cellular signaling circuitry, and shows extensive convergent evolution, highlighting pervasive constraints on pTyr signaling.choanoflagellate ͉ evolution ͉ genome ͉ kinome ͉ phosphotyrosine C hoanoflagellates such as Monosiga brevicollis are unicellular aquatic protists and the closest known relatives of multicellular animals (metazoans). The sequencing of the Monosiga genome now provides a key evolutionary node between metazoans and fungi, close to the origin of animal multicellularity (1). The role of the tyrosine-specific group of kinases (TKs) in intercellular signaling and their restriction to metazoans suggested that TKs were key to metazoan evolution (2). Plants and unicellular organisms lack TKs, although they have a small number of dual-specificity kinases and associated tyrosine phosphatases (PTPs) and SH2 phosphotyrosine-binding domains generally not involved in intercellular signaling. The surprising discovery of TKs in choanoflagellates (3)(4)(5) showed that invention of these key mediators of intercellular signaling preceded their expansion in metazoans. We show here that choanoflagellates have invested hugely in a largely independent pTyr signaling system, yet many of these genes suggest functional convergence between choanoflagellates and metazoans and new combinations of signaling modules, both of which hint at restricted pathways through the signaling network. ResultsDetermination and Classification of Monosiga Tyrosine Kinases. Our analysis of the draft Monosiga genome predicts 128 TKs within a total kinome of Ϸ380 protein kinases (http://kinase.com/kinbase). Extensive gene model curation and selected cDNA and genome resequencing allowed us to improve predictions for 102 of these sequences, although several fragments and likely imperfect predictions remain. These constitute the largest known tyrosine kinome and make up over twice the fraction of the proteome than that of any metazoan (6-9), a startling result for a unicellular organism. Sequence analysis of the kinase domain and oth...

Choanoflagellates, unicellular organisms that are closely related to metazoans, possess cell adhesion and signaling proteins previously thought to be unique to animals, suggesting that these components may have played roles in the evolution of metazoan multicellularity. We have cloned, expressed, and purified the nonreceptor tyrosine kinase MbSrc1 from the choanoflagellate Monosiga brevicollis. The kinase has the same domain arrangement as mammalian Src kinases, and we find that the individual Src homology 3 (SH3), SH2, and catalytic domains have similar functions to their mammalian counterparts. In contrast to mammalian c-Src, the SH2 and catalytic domains of MbSrc1 do not appear to be functionally coupled. We cloned and expressed the M. brevicollis homolog of c-Src C-terminal kinase (MbCsk) and showed that it phosphorylates the C terminus of MbSrc1, yet this phosphorylation does not inhibit MbSrc to the same degree seen in the mammalian Src/ Csk pair. Thus, Src autoinhibition likely evolved more recently within the metazoan lineage, and it may have played a role in the establishment of intercellular signaling in metazoans.A defining feature of multicellular organisms is the cellular machinery that enables cell-cell interaction and communication. Recent analyses show that an unexpected diversity of cell signaling and adhesion components are present in choanoflagellates, a group of unicellular organisms that are closely related to metazoans (1, 2). The presence of these proteins in choanoflagellates demonstrates that they evolved before the origin of animals and suggests that they may have served as preadaptations for the evolution of multicellularity (3).The genome of the choanoflagellate Monosiga brevicollis contains 128 tyrosine kinase genes.3 Expressed sequence tags (ESTs) generated from an M. brevicollis cDNA library demonstrated the expression of members of the receptor-tyrosine kinase and nonreceptor-tyrosine kinase families. Anti-phosphotyrosine Western blotting of M. brevicollis cell lysates confirmed the phosphorylation of cellular proteins in response to treatment with nutrients (1). M. brevicollis contains four Src kinase homologs (designated MbSrc1-4) (1), each of which contains the diagnostic SH3 4 and SH2 domains of mammalian Src kinases. Src family nonreceptor-tyrosine kinases are widely expressed in animals and are involved in the regulation of many processes including cell growth and differentiation, adhesion, and motility (4 -6). Mutation of Src or elevated Src expression or activity can lead to cell transformation and metastasis (7, 8) (Src was initially identified as the cellular homolog of the transforming gene from Rous sarcoma virus). For this reason the normal activity of Src in animal cells is tightly regulated. Src kinases possess a conserved domain architecture that is critical for enzymatic regulation; they have an N-terminal myristoylation sequence followed by SH3, SH2, and kinase catalytic domains. The C terminus possesses a tyrosine (Tyr-530) that is phosphorylated by the tyrosine...

Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered G␤␥ freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic ␣-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388-518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains G␤␥ and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK).[Keywords: Pheromone response; plasma membrane; mutants; yeast; Saccharomyces cerevisiae; Ste20] Supplemental material is available at http://www.genesdev.org.

The origin of metazoans required the evolution of mechanisms for maintaining differentiated cell types within a multicellular individual, in part through spatially differentiated patterns of gene transcription. The unicellular ancestor of metazoans was presumably capable of regulating gene expression temporally in response to changing environmental conditions, and spatial cell differentiation in metazoans may represent a co-option of preexisting regulatory mechanisms. Myc is a critical regulator of cell growth, proliferation, and death that is found in all metazoans but absent in other multicellular lineages, including fungi and plants. Homologs of Myc and its binding partner, Max, exist in two of the closest living relatives of animals, the choanoflagellate Monosiga brevicollis (Mb) and Capsaspora owczarzaki, a unicellular opisthokont that is closely related to metazoans and choanoflagellates. We find that Myc and Max from M. brevicollis heterodimerize and bind to both canonical and noncanonical E-boxes, the DNA-binding sites through which metazoan Myc proteins act. Moreover, in M. brevicollis, MbMyc protein can be detected in nuclear and flagellar regions. Like metazoan Max proteins, MbMax can form homodimers that bind to E-boxes. However, cross-species dimerization between Mb and human Myc and Max proteins was not observed, suggesting that the binding interface has diverged. Our results reveal that the Myc/Max network arose before the divergence of the choanoflagellate and metazoan lineages. Furthermore, core features of metazoan Myc function, including heterodimerization with Max, binding to E-box sequences in DNA, and localization to the nucleus, predate the origin of metazoans.

High resolution mass spectrometry (HRMS) is a valuable tool for the analysis of chemical contaminants in food. Our laboratory has successfully developed methods to screen for veterinary drug residues using liquid chromatography quadrupole time-of-flight (Q-TOF). There have been, however, significant challenges as methods are transferred from the development stage to routine regulatory analysis. Having experimental retention time and product ion information for analytes greatly facilitates the ability to determine if residues found by the HRMS searching software are false detects. These data were collected for over 200 veterinary drug residues using LC Q-TOF MS. The screening levels of detection for over 150 veterinary drug residues in milk were determined, and over half of those tested can be detected at concentrations of 10 ng/mL or less; 72% can be found in milk when present at 100 ng/mL. Tentative identification of the product ions from these analytes is also presented.

INTRODUCTIONChoanoflagellates are the closest living relatives of the metazoa, and the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. These heterotrophic flagellates are a cosmopolitan group of small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Monosiga brevicollis, a marine species, has emerged as a representative of the group because it is easily grown and manipulated in the laboratory and was the subject of a recently completed genome project. Because of the similarity in morphology and ecology shown across the order Choanoflagellida, the protocols mentioned in this article should be transferable to most choanoflagellate species.

INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. Most, if not all, choanoflagellates are heterotrophs that prey on bacteria. Thus, all choanoflagellate media provide nutrition for bacteria, which are in turn consumed by the choanoflagellates. If a culture is axenic or has low bacterial content, it can be supplemented with a specific bacterial strain that has been grown separately. On the other hand, if a culture already contains a well-established flora of bacteria, it can be cocultured with the choanoflagellate isolate. In either case, it is common to enrich the medium with an organic extract, such as liver extract, cereal grass infusion, or a mixture of proteose peptone and yeast extract. This protocol describes how to start cultures of the marine choanoflagellate species, Monosiga brevicollis, from frozen stocks. These cultures can then be maintained and expanded in preparation for DNA or RNA isolation or cell biological assays.