Cloning and genetic characterization of Skb1: A novel regulator of Shk1 and mitosis in Schizosaccharomyces pombe

A partial skb1 gene was originally isolated in a yeast two-hybrid screen for Shk1-interacting polypeptides. Shk1 is one of two Schizosaccharomyces pombe p21Cdc42/Rac-activated kinases (PAKs) and is an essential component of the Ras1-dependent signal transduction pathways regulating cell morphology and mating responses in fission yeast. After cloning the skb1 gene we found the Skb1 gene product to be a novel, nonessential protein lacking homology to previously characterized proteins. However the identification of Skb1 homologs in C. elegans, S. cerevisiae, and H. sapiens reveals evolution has conserved the skb1 gene. Fission yeast cells carrying a deletion of skb1 exhibit a defect in cell size but not mating abilities. This defect is suppressed by high copy shk1. Fission yeast overexpressing skb1 were found to undergo cell division at a length 1.5X greater than normal. In the two-hybrid system, Skb1 interacts with a subdomain of the Shk1 regulatory region distinct from that with which Cdc42 interacts, and forms a ternary complex with Shk1 and Cdc42. By use of yeast genetics, we have established a role for Skb1 as a positive regulator of Shk1. Co-overexpression of shk1 with skb1 was found to suppress the morphology defect, but not the sterility, of ras1Δ fission yeast. Thus, the function of Skb1 is restricted to a morphology control pathway. We determined that Skb1 functions as a negative regulator of mitosis and does this through a Shk1-dependent mechanism. The mitotic regulatory function of Skb1 and Shk1 was also partially dependent upon Wee1, a direct negative regulator of the cyclin-dependent kinase Cdc2. The role for Skb1 and Shk1 as mitotic regulators is the first connection from a PAK protein to control of the cell cycle. Furthermore, Skb1 is the first non-Cdc42/Rac PAK modulator to be identified. ^

Trying to make sense in nonsense-mediated mRNA decay

Despite over 30 years of research, the molecular mechanisms of nonsense-mediated mRNA decay (NMD) are still not well understood. NMD appears to exist in most eukaryotes and is intensively studied in S. cerevisiae, C. elegans, D. melanogaster and in mammalian cells. Current evidence suggests that the core of NMD – involving UPF1, UPF2 and UPF3 – is evolutionarily conserved, but that different species may have evolved slightly different ways to identify target mRNAs for NMD and to degrade them. Our lab has shown that the exon junction complex (EJC) is not absolutely required for NMD in human cells (Bühler et al., NSMB 2006) and that it is neither restricted to CBP80-bound mRNAs as classical models claim (Rufener & Mühlemann, NSMB 2013). Together with the finding that long 3’ UTRs often are an NMD-inducing feature (Eberle et al, PLoS Biol 2008; Yepiskoposyan et al., RNA 2011), our data is consistent with much of the data from other species and hence has led to a “unified” working model for NMD (Stalder & Mühlemann, Trends Cell Biol 2008; Schweingruber et al., Biochim Biophys Acta 2013). Our recent iCLIP experiments with endogenous UPF1 indicate that UPF1 binds mRNAs indiscriminately with respect to being an NMD target or not before they engage with ribosomes (Zünd et al., NSMB 2013). After onset of translation, UPF1 is cleared from the coding region but remains bound to the 3’ UTR of mRNAs. Why this 3’ UTR-associated in some cases induces NMD and in others not is currently being investigated and not yet understood. Following assembly of a phospho-UPF1-containing NMD complex, decay adaptors (SMG5, SMG7, PNRC2) and/or the endonuclease SMG6 are recruited. While the latter cleaves the mRNA in the vicinity of the termination codon, the former proteins induce deadenylation, decapping and exonucleolytic degradation of the mRNA. In my talk, I will give an overview about the latest developments in NMD – with a focus on our own work – and try to integrate the bits and pieces into a somewhat coherent working model.

The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning.

Gene clustering of the small leucine -rich repeat gene family

The small leucine-rich repeat proteoglycans (or SLRPs) are a group of extracellular proteins (ECM) that belong to the leucine-rich repeat (LRR) superfamily of proteins. The LRR is a protein folding motif composed of 20–30 amino acids with leucines in conserved positions. LRR-containing proteins are present in a broad spectrum of organisms and possess diverse cellular functions and localization. In mammals, the SLRPs are abundant in connective tissues, such as bones, cartilage, tendons, skin, and blood vessels. We have discovered a new member of the class I small leucine rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N-terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9g21.3 where asporin is part of a SLRP gene cluster that includes ECM2, osteoadherin, and osteoglycin. This gene cluster of four LRR-encoding genes is embedded in a 238 kilobase intron of another novel gene named Tes9orf that is expressed primarily in the testes of the adult mouse. The SLRP genes are not present in Drosophila or C. elegans , but reside in three separate gene clusters in the puffer fish, mice and humans. Targeted disruption of individual mouse SLRP genes display minor connective tissue defects such as skin fragility, tendon laxity, minor growth plate defects, and mild osteoporosis. However, double and triple knockouts of SLRP genes exacerbate these phenotypes. Both the double epiphycan/biglycan and the triple PRELP/fibromodulin/biglycan knockout mice exhibit premature osteoarthritis. ^

Marine diatoms in deep sea sediments

Long-term evolution is thought to take opportunities that arise as a consequence of mass extinction (as argued, for example, by Gould, 2002) and the following biotic recovery, but there is absolutely no evidence for this being the case. However, our study shows that eutrophication by oceanic mixing also played a part in the enhancement of several evolutionary events amongst marine organisms, and these results could indicate that the rates of oceanic biodiversification may be slowed if upwelling becomes weakened by future global warming. This paper defines three distinct evolutionary events of resting spores of the marine diatom genus Chaetoceros, to reconstruct past upwelling through the analysis of several DSDP, ODP and land-based successions from the North, South and equatorial Pacific as well as the Atlantic Ocean during the past 40 million years. The Atlantic Chaetoceros Explosion (ACE) event occurred across the E/O boundary in the North Atlantic, and is characterized by resting spore diversification that occurred as a consequence of the onset of upwelling following changes in thermohaline circulation through global cooling in the early Oligocene. Pacific Chaetoceros Explosion events-1 and -2 (PACE-1 and PACE-2) are characterized by relatively higher occurrences of iron input following the Himalayan uplift and aridification at 8.5 Ma and ca. 2.5 Ma in the North Pacific region. These events not only enhanced the diversification and increased abundance of primary producers, including that of Chaetoceros, other diatoms and seaweeds, but also stimulated the evolution of zooplankton and larger predators, such as copepods and marine mammals, which ate these phytoplankton and plants. Current thinking suggests new evolutionary niches open up after a mass extinction, but our study finds that eutrophication can also stimulate evolutionary diversification. Moreover, in the opposite fashion, our results show that as thermohaline circulation abates, global warming progresses and the ocean surface becomes warmer, many marine organisms will be affected by the environmental degradation.

Neogene diatoms of the Japan Sea

A series of excellent upper Miocene through Quaternary diatomaceous sequences recovered at four sites during Leg 127 was examined for diatoms. The diagenetic transition from opal-A to opal-CT is a diachronic horizon from the uppermost part of the Denticulopsis katayamae Zone (8.5 Ma) at Hole 797B to the uppermost part of the Neodenticula kamtschatica Zone (5.73 Ma) at Hole 795A. The diatom zonation of Koizumi (1985) best divides the upper Miocene to Quaternary sequences above the opal-A/opal-CT boundary and also is useful to date carbonate concretions including diatoms below the boundary. Forty diatom datum levels were evaluated biostratigraphically based on the sediment accumulation rate curve, and several isochronous datum levels are newly proposed for the Japan Sea area. A warm-water current did not penetrated into the Japan Sea through the Tsushima strait during the late Miocene and Pliocene time, because subtropical warm-water diatoms are essentially not present in such sediment samples. The occurrences of diatom are cyclic throughout the Quaternary sediments and are affected by eustatic sea level changes.

Distribution of diatoms in Oligocene to Pleistocene sediments of the tropical Pacific

According to the drilling probes of the Deep Waier Drilling Project, Neogene sediments in a tropical area of the Pacific Ocean are divided into 15 zones based on diatoms. The author shows that a unique zonation may be applied for the entire region. Identification of diatoms zones boundaries was conducted through their direct correlation with nannoplancton, radiolarian and foraminiferal zonal sceals. Their ultra-structure and morphological relationship are being analysed. The mode of siliceous accumulation within the equatorial belt differed through the western central and eastern region since the early Miocene and the difference become more evident from the end of Middle Miocene. The distribution of Neogene diatomaceous silt in the tropical area is controlled by the character of gyre-water circulation and agrees with the modern geographical zonation.

Occurrence and abundance of diatoms at DSDP Holes 72-515B and 72-516F

Samples from the upper Oligocene and lower Miocene of Holes 515B (Brazil Basin) and 516F (Rio Grande Rise) were examined for fossil marine diatom content. The preservation of the diatoms was poor and the species diversity low in both holes. However, it was possible to zone portions of the intervals studied using the zonation proposed by Gombos and Ciesielski (1983), which is based, as far as possible, on common and robust species. Thus, the interval in Hole 515B represented by Cores 515B-15 and 515B-16 is assigned to the Coscinodiscus rhombicus Zone and the interval represented by Cores 515B-17 through 515B-44 is assigned to the Rocella gelida Zone. The C. rhombicus Zone is early Miocene in age and the R. gelida Zone is late Oligocene to early Miocene in age. In Hole 516F the interval represented by Cores 516F-6 through 516F-10 is assigned to the R. gelida Zone Gate Oligocene to early Miocene), and the interval represented by Cores 516F-11 through 516F-15 is assigned to the Triceratium groningensis Zone (late Oligocene). Two new fossil diatom taxa are defined herein: Coscinodiscus lewisianus Greville f. concavus n. f. and Rocella semigelida n. sp.

Oligocene and lowermost Miocene diatom stratigraphy and datums of ODP Site 199-1220 sediments

Completion of studies on material collected during Ocean Drilling Program Leg 199 at Site 1220 in the equatorial Pacific allows calibration of the ranges of >35 stratigraphically important diatoms to paleomagnetic stratigraphy for the Oligocene and earliest Miocene (~33.5-21.5 Ma). The taxonomy of these taxa is reviewed, and age estimates of their first and last occurrences are compiled. The diatom zonation for the Oligocene and earliest Miocene of the equatorial Pacific is revised and correlated with paleomagnetic stratigraphy. This biostratigraphy is likely to be applicable throughout the low-latitude regions of the world's oceans.

Presence-absence species/sample matrix for the measured sections at the area of Maghara, North Sinai, Egypt

As age-diagnostic fossils are rare in the Middle to Upper Jurassic sedimentary succession of Gebel Maghara, North Sinai, Egypt, and in order to ensure maximal stratigraphic resolution, chronostratigraphic boundaries were determined based on quantitative biostratigraphy. A data matrix comprising 231 macrofaunal taxa in 93 samples from four sections has been processed with the Unitary Association (UA) Method. This led to construction of a sequence of 29 UAs (maximal sets of actually or virtually coexisting taxa), which have been grouped into 14 laterally reproducible association zones. The UA method allowed an in-depth analysis of the stratigraphically conflicting taxa, enabled the biostratigraphic subdivision of the studied interval, and also provided stratigraphic correlation among the measured sections and with the Tethyan ammonite zones.

Latest Oligocene through early middle Miocene diatom biostratigraphy of the eastern tropical Pacific

Study of DSDP Sites 71, 77, and 495 has allowed the development of a refined diatom biostratigraphy for the latest Oligocene through early middle Miocene of the eastern tropical Pacific which is well correlated to the low-latitude zonations for planktonic foraminifers, coccoliths, and radiolarians. Six zones and 7 subzones are proposed, and correlation with high-latitude diatoms zonations for the North Pacific, the Norwegian Sea, and the Southern Ocean is suggested by the discovery of selected diatoms in these tropical sediments which were previously thought to be restricted to high latitudes. Six new species and one new variety of diatoms which are stratigraphically useful are proposed : Actinocyclus hajosiae, n. sp., A. radionovae, n. sp., Coscinodiscus blysmos, n. sp., C. praenodulifer, n. sp., Craspedodiscus rydei, n. sp., Thalassiosira bukryi, n. sp., and Coscinodiscus lewisianus var. robustus n. var.

The Global Plankton Database: an inventory and data from the Former Soviet Union expeditions

At present time, there is a lack of knowledge on the interannual climate-related variability of zooplankton communities of the tropical Atlantic, central Mediterranean Sea, Caspian Sea, and Aral Sea, due to the absence of appropriate databases. In the mid latitudes, the North Atlantic Oscillation (NAO) is the dominant mode of atmospheric fluctuations over eastern North America, the northern Atlantic Ocean and Europe. Therefore, one of the issues that need to be addressed through data synthesis is the evaluation of interannual patterns in species abundance and species diversity over these regions in regard to the NAO. The database has been used to investigate the ecological role of the NAO in interannual variations of mesozooplankton abundance and biomass along the zonal array of the NAO influence. Basic approach to the proposed research involved: (1) development of co-operation between experts and data holders in Ukraine, Russia, Kazakhstan, Azerbaijan, UK, and USA to rescue and compile the oceanographic data sets and release them on CD-ROM, (2) organization and compilation of a database based on FSU cruises to the above regions, (3) analysis of the basin-scale interannual variability of the zooplankton species abundance, biomass, and species diversity.

Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.

soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein–protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans.