Histone H4 mRNA from the nematode Ascaris lumbricoides is cis-spliced and polyadenylated.

A histone H4 gene from Ascaris lumbricoides contains an intron of approx. 2040 bp. Transcripts of the gene are spliced and polyadenylated. This is the first intron-containing H4 gene described for a metazoan. Notably, H4 mRNA from another nematode, Caenorhabditis elegans, is intron-less and lacks poly A (Roberts, S.B., Emmons, S.W. and Childs, G. (1989) J. Mol. Biol. 206, 567-577).

From gene to protein: Investigating the disease etiology of retinitis pigmentosa

Retinitis pigmentosa (RP) is a name given to a group of inherited retinal dystrophies that lead to progressive photoreceptor degeneration, and thus, visual impairment. It is evident at both the clinical and the molecular level that these are heterogeneous disorders, with wide variation in severity, mode of inheritance, and phenotype. The genetics of RP are not simple; the disease can be inherited in dominant, recessive, X-linked, and digenic modes. Autosomal dominant RP (adRP) results from mutations in at least ten mapped loci, but there may be dozens of genetic loci where mutations can cause RP. To date, there are over a hundred genes known to cause retinal degenerative diseases, and less than half of these have been cloned (RetNet). Among the dozens of retinitis pigmentosa loci known to exist, only a few have been identified and the remainders are inferred from linkage studies. Today, the genes for seven of the twelve-adRP loci have been identified, and these are rhodopsin, peripherin/RDS, NRL, ROM1, CRX, RP13 and RP1. My research projects involved a combination of the continued search for genes involved in retinal dystrophies, as well the investigation into the role of peripherin/RDS and RP1 in the disease etiology of autosomal dominant RP. ^ Most of the mutations leading to inherited retinal disorders have been identified in predominately retina expressed genes like rhodopsin, peripherin/RDS, and RP1. Expressed sequence tags (ESTs) that were retina-specific were culled from sequence databases and, together with laboratory analysis, were analyzed as potential candidate genes for retinal dystrophies. Thirteen of the fifty-five identified retina-specific ESTs mapped to within candidate regions for inherited retinopathies. One of these is RP1L1, a homologue of RP1 and a potential cause of adRP. ^ Once a disease-associated gene has been identified, elucidating the role of that gene in the visual process is essential for understanding what happens when the process is defective as it is in adRP. My next projects involved investigating the role of a novel 5′ donor +3 splice site mutation on the mRNA of peripherin/RDS in adRP affected individuals, and comparative sequencing in RP1 to define conserved regions of the protein. Comparative sequencing is a powerful way to delineate critical regions of a sequence because different regions of a gene have different functions, and each region is subject to different levels of functional or structural constraints. Establishing a framework of conserved domains is beneficial not only for structural or functional studies, but can also aid in determining the potential effects of mutations. With the completion of sequencing of human genome, and other organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans , and Drosophila, the facility of comparative sequencing will only increase in the future. Comparative sequencing has already become an established procedure for pinpointing conserved regions of a protein, and is an efficient way to target regions of a protein for experimental and/or evolutionary analysis. ^

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. ^

Planktonic foraminifera in the latest Maastrichtian of Walvis Ridge, South Atlantic

An abrupt global warming of 3-4°C occurred near the end of the Maastrichtian at 65.45-65.10 Ma. The environmental effects of this warm event are here documented based on stable isotopes and quantitative analysis of planktonic foraminifera at the South Atlantic DSDP Site 525A. Stable isotopes of individual species mark a rapid increase in temperature and a reduction in the vertical water mass stratification that is accompanied by a decrease in niche habitats, reduced species diversity and/or abundance, smaller species morphologies or dwarfing, and reduced photosymbiotic activity. During the warm event, the relative abundance of a large number of species decreased, including tropical-subtropical affiliated species, whereas typical mid-latitude species retained high abundances. This indicates that climate warming did not create favorable conditions for all tropical-subtropical species at mid-latitudes and did not cause a massive retreat in the local mid-latitude population. A noticeable exception is the ecological generalist Heterohelix dentata Stenestad that dominated during the cool intervals, but significantly decreased during the warm event. However, dwarfing is the most striking response to the abrupt warming and occurred in various species of different morphologies and lineages (e.g. biserial, trochospiral, keeled globotruncanids). Dwarfing is a typical reaction to environmental stress conditions and was likely the result of increased reproduction rates. Similarly, photosymbiotic activity appears to have been reduced significantly during the maximum warming, as indicated by decreased delta13C values. The foraminiferal response to climate change is thus multifaceted resulting in decreased species diversity, decreased species populations, increased competition due to reduced niche habitats, dwarfing and reduced photosymbiotic activity.

Megabenthic communities in the waters around Svalbard during POLARSTERN cruise ARK-VIII/2 (EPOS II)

Composition and distribution of megabenthic communities around Svalbard were investigated in June/July 1991 with 20 Agassiz trawl and 5 bottom trawl hauls in depths between 100 and 2100 m. About 370 species, ranging from sponges to fish, were identified in the catches. Species numbers per station ranged from 21 to 86. Brittle stars, such as Ophiacantha bidentata, Ophiura sarsi and Ophiocten sericeum, were most important in terms of constancy and relative abundance in the catches. Other prominent faunal elements were eunephthyid alcyonarians, bivalves, shrimps, sea stars and fish (Gadidae, Zoarcidae, Cottidae). Multivariate analyses of the species and environmental data sets showed that the spatial distribution of the megabenthos was characterized by a pronounced depth zonation: abyssal, bathyal, off-shore shelf and fjordic communities were discriminated. However, a gradient in sediment properties, especially the organic carbon content, seemed to superimpose on the bathymetric pattern. Both main factors are interpreted as proxies of the average food availability, which is, hence, suggested to have the strongest influence in structuring megabenthic communities off Svalbard.

(Table 2) Radiolaria species in nodule samples from the Clarion-Clipperton province, Pacific Ocean

Radiolaria were studied in 19 manganese nodules raised from the bottom. The nodules occurred mainly on the surface of thin Quaternary sediments covering Tertiary deposits of various ages (Middle Eocene to Early Miocene). Radiolaria in nodule cores and in inner and surface layers were studied. We found 85 radiolaria species and groups of species. Usually 1-4 to 6-19 radiolaria species were detected in each of the samples. Species belonging to Middle Eocene, Late Miocene to Early Oligocene, and Oligocene to Early Miocene were found. Rare Neogene species were revealed only in fractured surface layers. Age of the nodules is mainly Oligocene. Seismic waves cause sediment vibration, loosening disintegration, and removal of suspension by bottom currents. The vibration effect causes ancient nodules to float up to the surface of Quaternary sediment. This hypothesis suggests the reason for characteristics of the Clarion-Clipperton zone: regional stratigraphic hiatus, accumulation of residual fields of nodules, and the ''floating up'' of nodules to the surface of the Quaternary sediments.

Lower Cretaceous nannofossils of the northwestern Australian margin

Moderately to sparsely nannofossiliferous Neocomian siliciclastics and rich Aptian-Albian nannofossil chalks were cored at two Leg 123 sites on the abyssal plains off northwestern Australia. At Site 765, the basal 70 m of cored section yields questionable Tithonian and Berriasian to early Hauterivian assemblages of moderate diversity containing Cruelellipsis cuvillieri, Tegumentum striatum, Speetonia colligata, and Crucibiscutum salebrosum. The overlying Hauterivianlower Aptian is represented by 140 m of sediments barren of nannofossils. Above this, the remaining 80 m of the Lower Cretaceous section has been assigned to the Rhagodiscus angustus Zone (late Aptian-early Albian in age) and the Prediscosphaera columnata Zone (middle-late Albian in age). Common species include Rhagodiscus angustus, Prediscosphaera columnata, Eprolithus floralis, Eprolithus sp., Chiastozygus litterarius, Rucinolithus irregularis, and Flabellites biforaminis. At Site 766, the Neocomian, represented by 200 m of sediment, yields C. cuvillieri, T. striatum, S. colligata, and C. salebrosum. Within the overlying Aptian-Albian sequence of 80 m, the Rhagodiscus angustus, and P. columnata zones were recognized. The paleobiogeographic patterns and implications are discussed, with special emphasis paid to the bipolar high-latitude distribution pattern of C. salebrosum in the Valanginian-Hauterivian. Biostratigraphically important species are discussed and their occurrence in the Indian Ocean is compared with one from the Tethys and Boreal realms. Two new species, Serbiscutum gaultensis and Eprolithus bettenstaedtii, are described.