Susan L. Young

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans

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.

The protist, Monosiga brevicollis, has a tyrosine kinase signaling network more elaborate and diverse than found in any known metazoan

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.

Signaling Properties of a Non-metazoan Src Kinase and the Evolutionary History of Src Negative Regulation

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.

Premetazoan Ancestry of the Myc-Max Network

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.

Starting and Maintaining Monosiga brevicollis Cultures

Cold Spring Harb Protoc; Nicole King, Susan L. Young, Monika Abedin, Martin Carr and Barry S.C. Leadbeater Cultures Monosiga brevicollis Starting and Maintaining Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (882 articles) Laboratory Organisms, general (326 articles) Genetics, general (291 articles) Emerging Model Organisms (243 articles) Cell Culture (1068 articles) Cell Biology, general

Visualizing the Subcellular Localization of Actin, β-Tubulin, and DNA in Monosiga brevicollis

Cold Spring Harb Protoc; Nicole King, Susan L. Young, Monika Abedin, Martin Carr and Barry S.C. Leadbeater brevicollis Monosiga -Tubulin, and DNA in β Visualizing the Subcellular Localization of Actin, Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (339 articles) Visualization, general (90 articles) Visualization of Proteins (70 articles) Visualization of Organelles (459 articles) Visualization (876 articles) Laboratory Organisms, general (293 articles) Labeling for Imaging (25 articles) Immunostaining Cells (91 articles) Immunostaining (72 articles) Immunohistochemistry (32 articles) Immunofluorescence (531 articles) Imaging/Microscopy, general (320 articles) Genetics, general (394 articles) Fluorescence (284 articles) Emerging Model Organisms (454 articles) Cell Imaging (1020 articles) Cell Biology, general (214 articles) Antibodies, general

Preparation of Total RNA from Monosiga brevicollis and Other Choanoflagellates

Cold Spring Harb Protoc; Nicole King, Susan L. Young, Monika Abedin, Martin Carr and Barry S.C. Leadbeater and Other Choanoflagellates Monosiga brevicollis Preparation of Total RNA from Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (62 articles) RNA Purification (212 articles) RNA (1009 articles) Molecular Biology, general (880 articles) Laboratory Organisms, general (322 articles) Genetics, general (284 articles) Emerging Model Organisms (1037 articles) Cell Biology, general

Preparation of high-molecular-weight genomic DNA from Monosiga brevicollis and other choanoflagellates.

Cold Spring Harb Protoc; Nicole King, Susan L. Young, Monika Abedin, Martin Carr and Barry S.C. Leadbeater and Other Choanoflagellates Monosiga brevicollis Preparation of High-Molecular-Weight Genomic DNA from Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (1011 articles) Molecular Biology, general (880 articles) Laboratory Organisms, general (71 articles) Genomic DNA (323 articles) Genetics, general (284 articles) Emerging Model Organisms (113 articles) DNA Purification (1049 articles) Cell Biology, general

Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm

Although delaying fatherhood has become somewhat more popular, the heritable sequelae of this practice are not well understood. Advancing paternal age has, however, been implicated in numerous abnormal reproductive and genetic outcomes, including poorer semen quality, reduced fertility, and more frequent spontaneous abortions, as well as some 20 autosomal-dominant diseases such as Apert syndrome and achondroplasia. The investigators examined the effects of advancing male age on multiple genomic defects in sperm (reflected in the DNA fragmentation index [DFI]), chromatin integrity, gene mutations, and numeric chromosomal abnormalities. Participants were 97 men ranging in age from 22 to 80 years who were in good to excellent health and did not smoke. They were predominantly a white and highly educated population. Semen specimens were obtained after an average of 5 days without sexual activity. Age correlated positively with all 5 DFI end points analyzed. Thirty men, nearly one third of those studied, had percent DFI values at or above those previously associated with an increased risk of male infertility. After adjusting for age and abstinence, the frequency of sperm with high DNA stainability did not correlate with DFI end points. Age did correlate with fibroblast growth factor receptor 3 gene (FGFR3) mutations associated with achondroplasia. No associations were noted between age and the frequency of sperm with immature chromatin, aneuploidies or diploidies, or FGFR2 mutations (as in Apert syndrome). There also were no consistent correlations among genomic and semen quality end points except for an association between DFI and sperm motility. Male age did not correlate with the sperm sex ratio. Men who choose to delay fatherhood may be less likely to experience a successful pregnancy. Unlike older women, however, older men do not seem to be at increased risk of trisomic or triploid pregnancies. Semen quality does not reflect the presence of genomic damage to sperm. A small number of older men do appear to be at increased risk of transmitting multiple genetic and chromosomal defects.