Charakterisierung spezieller pflanzlicher Gamma-Tubulin-Sequenzbereiche und Detektion potentieller Interaktionspartner mittels Yeast-two-Hybrid-System

Mitotische und postmitotische Vorgänge pflanzlicher Zellen basieren auf der Funktion von Mikrotubuli. Es liegen nur wenige gesicherte Erkenntnisse zur Organisation dieser Multifunktionalität vor. Eine zentrale Bedeutung wird bei der Nukleation der Mikrotubuli an MTOCs durch γ-Tubulin zugeschrieben. Deren Zusammenlagerung an MTOCs ist jedoch noch nicht richtig verstanden. Domänen, die an der Proteinoberfläche exponiert werden, könnten in Interaktionen involviert sein. Hier werden im Besonderen der γ-A und γ-B-Peptivmotiv diskutiert. Es wurde das γ-A- und γ-B-Peptidmotiv des γ-Tubulins hinsichtlich einer Konservierung innerhalb des Pflanzenreiches untersucht. Die beiden Bereiche sind bei den grünen Landpflanzen stark konserviert. Sie divergieren stark zu den einzelligen Grünalgen Chlamydomonas reinhardtii und Chlorella spec. Es wurden daher in der bestehenden phylogentischen Lücke weitere Organismen hinsichtlich des γ-A und γ-B Peptidmotivs untersucht. Auswahlkriterien der Organismen waren Ein-/Mehrzelligkeit, Besitz/Abwesenheit von Centriolen und Besitz/Abwesenheit von Geißeln. Des weiteren wurde mit verschiedenen γ-Tubulin-Konstrukten um das γ-A- und γ-B-Peptidmotiv, gewonnen aus Nicotiana tabacum (BY2) mittels Y2H-System nach Interaktionspartnern gesucht. Bei den Sequenzuntersuchungen des γ-A- und γ-B-Peptidmotivs konnte festgestellt werden, dass die Konservierung innerhalb der Streptophytenlinie erfolgt. Interessant erweist sich die Tatsache, dass dieses Motiv bei den Jochalgen, welche ebenfalls den Streptophyten angehören, nur im γ-A-Peptidmotiv auftritt. Es besteht die Möglichkeit, dass die beiden potentiellen Interaktionspartner verschiedene Proteine als Interaktions-partner besitzen. Durch eine Anwendung eines auf dem GAL4-Protein basierenden Y2H-Systems mit vier unterschiedlichen Konstrukten des γ-Tubulin-A/B-Peptidbereichs als Köder-konstrukt und einer cDNA-Bibliothek als Beutekonstrukt, wurden diverse Sequenzen identifiziert. Identifiziert wurden das Poly(A)-Bindeprotein, Glycerin-aldehyd-3-phosphatdehydrogenase, die S-adenosyl-L-methionine-Synthetase, diverse Proteasom-Untereinheiten, eine sekretorische Peroxidase, eine Ascorbat-Peroxidase, die NtPOX1-Peroxidase und verschiedene Peroxidasen aus Nicotiana tabacum, Sequenzen des Chloroplastengenoms, ein Myosin-ähnliches Protein und eine Sequenz auf dem 5. Chromosom des Medicago truncatula-Klons mth2-16f8 und diverse humane Sequenzen der Proteine DKFZp68 und DKFZp77. Die Ergebnisse weisen auf eine komplexe Funktionsweise der unterschiedlichen Komponenten des pflanzlichen Cytoskeletts und des γ-Tubulins hin. Zur Aufklärung müsste dies in Zukunft mittels anderer genetischer, biochemischer oder funktioneller Methoden untersucht werden. Hypothesen über Interaktionen der Cytoskelettkomponenten können wahrscheinlich nicht allein durch die Anwendung des Y2H-Systems aufgeklärt werden.

Male Dominance in the New Zealand Longfin Eel Population of a New Zealand River: Probable Causes and Implications for Management

The endemic New Zealand longfin eel Anguilla dieffenbachi (hereafter, longfin eel), is overfished, and in southern South Island, New Zealand, rivers have recently become predominated by males. This study examined length and age at sexual differentiation in male eels in the Aparima River catchment (area, 1,375 km(2); mean flow, 20 m(3.)s(-1)) and the sex ratio and distribution of eels throughout the catchment. Longfin eels differentiated into males mostly at lengths from 300 to 460 mm and ages from 10 to 25+ years. Females were rare: Of 738 eels examined for sexual differentiation, 466 were males and 5 were females, and a few others, not examined, were large enough to be female. These counts suggest a male : female ratio among differentiated longfin eels of 68:1. Of 31 differentiated shortfin eels A. australis, less common in the Aparima River, 26 were females. Male longfin eels were distributed throughout the main stern and tributaries; undifferentiated eels were more prevalent in lower and middle reaches and in the main stem than in upper reaches and tributaries. In other studies, male longfin eels predominated commercial catches in the Aparima and four other southernmost rivers, by 2.4:1 to 13.6:1 males to females. The Aparima River had the most skewed sex ratio. Longfin eel catches from the Aparima River will become more male predominated because few sublegal-size females were present. The length-frequency distributions of eels in the present samples and in the commercial catches were truncated just above minimum legal size (about 460 mm), showing that few females escape the fishery. Historically, females predominated these rivers. The recent change in sex ratio is attributable partly to selective harvest of females, and partly to changes in the structure of the population from fishing, such that differentiation into males has been favored. Longevity, delayed sexual maturity, semel-parity, and endemism with restricted range make the longfin eel particularly vulnerable to overfishing.

Simulation of the Impact of Dams and Fishing Weirs on Reproductive Potential of Silver-Phase American Eels in the Kennebec River Basin, Maine

I modeled the cumulative impact of hydroelectric projects with and without commercial fishing weirs and water-control dams on the production, survival to the sea, and potential fecundity of migrating female silver-phase American eels, Anguilla rostrata in the Kennebec River basin, Maine, This river basin has 22 hydroelectric projects, 73 water-control dams, and 15 commercial fishing weir sites. The modeled area included an 8,324 km(2) segment of the drainage area between Merrymeeting Bay and the upper limit of American eel distribution in the basin. One set of input,, (assumed or real values) concerned population structure (Le., population density and sex ratio changes throughout the basin, female length-class distribution, and drainage area between dams), Another set concerned factors influencing survival and potential fecundity of migrating American eels (i.e., pathway sequences through projects, survival rate per project by length-class. and length-fecundity relationship). Under baseline conditions about 402,400 simulated silver female American eels would be produced annually reductions in their numbers due to dams and weirs would reduce the realized fecundity (i.e., the number of eggs produced by all females that survived the migration). Without weirs or water-control dams, about 63% of the simulated silverphase American eels survived their freshwater spawning migration run to the sea when the survival rate at each hydroelectric dam was 9017, 40% survived at 80% survival per dam, and 18% survived at 60% survival per dam. Removing the lowermost hydroelectric dam on the Kennebec River increased survival by 6.0-7.6% for the basin. The efficient commercial weirs reduced survival to the sea to 69-76%( of what it would have been without weirs', regardless of survival rates at hydroelectric dams. Water-control dams had little impact on production in this basin because most were located in the upper reaches of tributaries. Sensitivity analysis led to the conclusion that small changes in population density and female length distribution had greater effects on survival and realized fecundity than similar changes in turbine survival rate. The latter became more important as turbine survival rate decreased. Therefore, it might be more fruitful to determine population distribution in basins of interest than to determine mortality rate at each hydroelectric project.

Sexually Different Growth Histories of the American Eel in Four Rivers in Maine

Growth histories of yellow-phase American eels Anguilla rostrata collected in four rivers in Maine, were back-calculated from sagittal otolith increments. Our objectives were to first determine whether sexually dimorphic growth rates exist and then compare the growth histories of American eels from four rivers within a geographic region. For female eels, the maximum growth rate was 31.9 +/- 1.7 mm/year at age 8, decreasing to 25.1 +/- 2.9 mm/year at age 14. Males attained a maximum of 29.8 +/- 1.6 min/year at age 3, decreasing to a minimum of 17.9 +/- 1.3 mm/year at age 11. Females grew faster than males after age 4 and had a slower reduction in growth rate with age. These faster growth rates among females were similar in all four rivers. The observed growth rates are not consistent with current life history hypotheses and may indicate an alternative life history strategy. Because female eels benefit from a larger size (i.e., size refuge, increased fecundity, and greater niche breadth), they would benefit from a higher-risk growth strategy that increases growth rate during their earlier years and reduces the amount of time spent in an unfavorable size-class. The tradeoffs (i.e., mortality, developmental rate, pathogen resistance, and longevity) associated with this faster growth rate may not favor the males' life history requirements. Male eels do not achieve the size of females and therefore are not subject to the advantages associated with being larger. Therefore, they may use a risk-averse strategy that maintains submaximum growth rates to obtain the minimum size necessary to mature and complete the spawning migration while reducing the adverse affects of faster growth rates. We postulate that, in eels, intrinsic growth rates should be considered a life history trait that has evolved to meet the life history requirements of each sex.

Homology Cloning, Heterologous Expression and Characterization of a New Channelrhodopsin

Channelrhodopsins are phototaxis receptors in the plasma membranes of motile unicellular algae. They function as light-gated cation channels and this channel activity has been exploited to trigger action potentials in neurons with light to control neural circuits (“optogenetics"). Four channelrhodopsins were identified in two algal species, Chlamydomonas reinhardtii and Volvox carteri, with known genome sequences; each species contains 2 channelrhodopsins, one absorbing at longer wavelengths and one at shorter wavelengths, named CrChR1 and CrChR2, respectively. Our goals are to expand knowledge of channelrhodopsin mechanisms and also to identify new channelrhodopsins from various algal species with improved properties for optogenetic use. For these aims we are targeting algae from extreme environments to establish the natural diversity of their properties. We cloned a new channelrhodopsin from the psychrophilic (cold-loving) alga, Chlamydomonas augustae, with degenerate primers based on the 4 known homologs. The new protein is 48% and 52% identical to CrChR1 and CrChR2, respectively. We expressed the channelrhodopsin in HEK293 cells and measured light-induced currents to assess their kinetics and action spectrum. Based on the primary structure, kinetics of light-induced photocurrents in HEK293 cells, and action spectrum maximum of 520 nm near that of the two previously found CrChR1, we named the new channelrhodopsin CaChR1. The properties of robust channel activity at physiological pH, fast on-and-off kinetics, and greatly red-shifted action spectrum maximum from that of CrChR2, make CaChR1 advantageous as an optogenetic tool. To know this new channelrhodopsin better, we expressed His-tagged CaChR1 in Pichia pastoris and the yield is about 6 mg/L. The purified His-tagged CaChR1 exhibited an absorption spectrum identical to the action spectrum of CaChR1-generated photocurrents. The future work will be measurement of the photocycles of CaChR1 by flash photolysis, crystallization of CaChR1 for the structure and mutagenesis of CaChR1 to find the critical amino acids accounting for red-shifted spectra, slow inactivation and rapid on-and-off kinetics. Seven new channelrhodopsins including CaChR1 from different algal species have been cloned in our lab at this time, bringing the total known to 13. The work of cloning of these new channelrhodopsins along with the expression of CaChR1 was published in Photochemistry and Photobiology in January 2012

Cocolos, emigración y narrativa dominicana

El desarrollo de la industria azucarera en la República Dominicana requirió mano de obra para el corte de caña y el trabajo en el ingenio. La dificultad en atraer trabajadores locales resultó en la contratación de obreros de las islas inglesas Anguila, St. Kitts, Antigua, Nevis, Tórtola, etc., donde el masivo desempleo provocado por la industrialización facilitaba el traslado de los isleños hacia las zafras dominicanas. En San Pedro de Macorís, lugar de su asentamiento, se los llamaba despectivamente “cocolos". Esta ponencia intenta estudiar la historia y delineación de estos obreros inmigrantes, su conducta, su idiosincrasia y modo de vida en el ingenio desde la narrativa dominicana del siglo XX. Las obras seleccionadas son un espejo del racismo, del rechazo cultural y de la explotación de los cocolos en el cañaveral. Cabe destacar que las novelas recientes asumen tardíamente una actitud reivindicatoria ante la ausencia de estudios críticos al respecto.