Differential degradation of a recombinant albumin-binding receptor in Escherichia coli.

The degradation in Escherichia coli of the recombinant serum-albumin-binding receptor derived from streptococcal protein G was investigated using a dual-affinity fusion approach. The proteolytic degradation of the receptor was characterized when fused to human proinsulin and human secretin. Several cleavages occurred at sequences not normally regarded as proteolytically sensitive, such as the dipeptide sequences Ile-Gly, Val-Ser and Ser-Ala. Depending on the fusion partner, large differences in the degradation of the albumin-binding domain were observed. Thus, susceptibility to proteolysis of a recombinant protein can be affected by a neighbouring domain.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.     

Steuerung und potentielle Evolutionsgeschwindigkeit des obligaten Teilzieherverhaltens: Ergebnisse eines Zweiweg-Selektionsexperiments mit der M?nchsgrasmücke (Sylvia atricapilla)

Zusammenfassung 1. In Südfrankreich lebt eine obligat teilziehende Population der Mönchsgrasmücke (Sylvia atricapilla) mit rund drei Vierteln Zugvögeln und einem Viertel Standvögeln. Das Teilzugverhalten dieser Population wird in beträchtlichem Umfang von genetischen Faktoren gesteuert (Berthold &Querner 1982). Der starke Selektionserfolg eines Zweiweg-Selektionsexperiments bis zur F₁-Generation ließ eine hohe potentielle Evolutionsgeschwindigkeit des obligaten Teilzieherverhaltens vermuten (2). Wir haben das Evolutionspotential in einem Langzeit-Selektionsversuch experimentell untersucht.2. Wir zogen insgesamt 267 Individuen der freilebenden Mönchsgrasmückenpopulation in fünf Gruppen von Hand auf, und alle fünf Stichproben zeigten entsprechende Verhältnisse von Ziehern und Nichtziehern. Wir konnten das Langzeit-Zweiweg-Selektionsexperiment bis zu einer F₆-Generation durchführen und konnten dabei insgesamt 455 Mönchsgrasmücken in Volieren züchten.3. Das Zweiweg-Selektionsexperiment zeigt, daß die untersuchte teilziehende Mönchsgrasmückenpopulation bereits nach drei Generationen eine ausschließlich ziehende und nach vier bis sechs Generationen eine fast nicht mehr ziehende Population werden kann.4. Das Zweiweg-Selektionsexperiment zeigt starken Selektionserfolg und hohe Heritabilitätswerte (ca. 0,6–1), die deutlich höher liegen als bisher für das Verhalten von (meist domestizierten) Wirbeltieren angegeben. Demnach sind genetische Faktoren für die Steuerung dieses obligaten Teilzugs sehr bedeutsam und wahrscheinlich dominant über Umwelteinflüsse.5. Die Versuchsergebnisse lassen im Falle von schnellen Umweltveränderungen und der Wirkung von starken Selektionsfaktoren ein enorm großes Evolutionspotential des obligaten Teilzieherverhaltens annehmen und damit allgemein eine schnelle Mikroevolution der Ortsbewegung von Vögeln. Sollten die von Meteorologen prophezeiten Klimaänderungen in Bälde eintreten (Erwärmung aufgrund von Verunreinigungen der Atmosphäre durch den Menschen, Treibhauseffekt), dann könnten viele obligate Teilzieher auf genetischer Basis rasch zu Standvögeln werden und wenig ausgeprägte Zieher rasch zu Teilziehern. Unter diesem allgemeinen Anwachsen von Standvögeln könnten ausgeprägte Zugvögel mehr und mehr zu leiden haben.6. Eine genetische Kontrolle obligaten Teilzugs scheint weit verbreitet zu sein, und sie stellt wohl auch für alle Individuen den zuverlässigsten Steuerungsmechanismus dar. Beim fakultativen Teilzug hingegen dürften Umweltfaktoren mehr unmittelbar steuernd wirken.

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Control and evolutionary potential of obligate partial migration: results of a two-way selective breeding experiment with the Blackcap (Sylvia atricapilla)

Summary 1. In southern France breeds an obligate partially migratory population of the Blackcap with about three quarters of migrants and one quarter of residents. Partial migration of this population is considerably controlled by genetic factors (Berthold &Querner 1982). The large selection response of a two-way selective breeding experiment up to F₁-generations suggested a high evolutionary potential of obligate partial migration (2). We have this evolutionary potential experimentally investigated in a long-term selective breeding study.2. We raised by hand a total of 267 individuals of the free-living Blackcap population in five groups, and all five samples showed corresponding ratios of migrants and nonmigrants. We were able to conduct the long-term selective breeding experiment up to a F₆-generation, and thereby a total of 455 Blackcaps could be bred in aviaries.3. The two-way selective breeding experiment shows, that the investigated partially migratory Blackcap population can become completely migratory in just three generations and almost exclusively sedentary in just four to six generations.4. The two-way selective breeding experiment yielded large selection responses and high heritability values (about 0.6–1) which are distinctly higher than those previously reported for behavioural traits of (mostly domesticated) vertebrates. Hence, genetic factors are very important for the control of this obligate partial migration and are presumedly dominant over environmental influences.5. In case of rapid environmental changes and the effect of strong selection factors the experimental results suggest a strikingly high evolutionary potential of obligate partial migration and thus in general a rapid microevolution of bird movements. If climatic changes would occur in the near future as they are predicted by meteorologists (rise in temperature due to man-made pollution of the atmosphere, greenhouse-effect) then many obligate partial migrants could rapidly become sedentary on a genetic basis and many less typical migrants could then considerably suffer from the general increase of residents.6. A genetic control of obligate partial migration appears to be widespread and it also appears to be the most reliable control mechanism for all individuals of such a partially migratory population. Facultative partial migration, however, appears to be controlled more directly by environmental factors.

     

A simple and rapid procedure for the isolation of intact bovine rod outer segments (ROS)

A method is described which allows the rapid isolation and purification of intact rod outer segments (ROS) from cattle eyes. It requires very fresh retinal material and can be completed within less than 2 h of the death of the animals. Cattle eyes are dissected in the usual manner, the retinae are isolated and the ROS are separated from the rest of the retina by gentle vortexing and filtration through a nylon mesh. The resulting crude ROS suspension is purified on a discontinuous sucrose density gradient. Two fractions are obtained, the major one consisting of mostly intact ROS, the minor one of RIS-ROS, i.e. of ROS which are still connected to part of their inner segment. The ROS are washed once and can be stored on ice for several days without loosing their intact plasma membrane. They can be transformed to leaky ROS by a quick freeze/thawing cycle or, if one wants unobstructed access to the interdiskal space, they can be subjected to a mild lysis treatment. The resulting ROS material is characterised using light microscopy, electron microscopy, light scattering, gel electrophoresis and absorption spectroscopy. It contains unusually low levels of 48k-protein and very high levels of G-protein. The latter cannot be washed out in the presence of GTP-gamma-S, even in the case of leaky ROS.     

Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines

Six new non-small-cell lung cancer (NSCLC) cell lines were established directly from human tissue or indirectly via nude mouse xenografts in serum-supplemented media with success rates of 8% and 13%, respectively. They comprised one adenocarcinoma (ADLC-5M2), two squamous cell carcinomas (EPLC-32M1, EPLC-65H), two large cell carcinomas (LCLC-97TM1, LCLC-103H), and one malignant biphasic mesothelioma (MSTO-211H). All cell lines grew adherent to culture vessels with population doubling times (PDT) of 16-40 h, formed colonies in soft agarose with efficiencies of 0.1%-5.1%, and all grew in athymic nude mice. Xenograft histologies appeared as follows: (a) undifferentiated carcinomas with feeble resemblance to the original tumors in the case of adenocarcinomas and squamous cell carcinomas; (b) large cell carcinoma with high resemblance to the original tumor; (c) an undifferentiated tumor with predominance of large epithelial cells and few fibrous cells in the case of mesothelioma. Human chorionic gonadotropin (HCG) was found by radioimmunoassay and high-affinity binding sites for epidermal growth factor (EGF) by radio-receptor assay in 4/4 cell lines. A very low activity of L-DOPA decarboxylase (DDC) was detectable only in the adenocarcinoma cell line. All cell lines overexpressed the c-myc protooncogene, and no gene rearrangement or amplification was observed. Chromosome analysis revealed modal chromosome numbers of 70-73 in ADLC-5M2, EPLC-32M1, EPLC-65H, and MSTO-211H. Cell lines derived from large cell carcinoma had modal values of 65 and 170 and a wider chromosome distribution than all other cell lines. A NSCLC specific chromosomal aberration has been undetectable until now. These cell lines may aid in elucidating the biology of NSCLC and its interrelationship to other lung tumors.     

Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G

Chimeric Fc receptors, consisting of the IgG-binding domains of both staphylococcal protein A and streptococcal protein G, were constructed. An efficient bacterial expression system was used to produce the recombinant proteins, which vary in size and number of IgG-binding domains. The purified receptors were analyzed by immunodiffusion and a competitive enzyme-linked immunosorbent assay to establish the relative binding strength to various polyclonal and monoclonal immunoglobulins from different species. The results demonstrate that protein A and protein G have complementary binding patterns and that the chimeric receptors retain the binding capacities of both the parental constituents. This suggests that these novel chimeric receptors might be versatile reagents for immunochemical assays.     

Leaf lifespan as a determinant of leaf structure and function among 23 amazonian tree species

Summary The relationships between resource availability, plant succession, and species' life history traits are often considered key to understanding variation among species and communities. Leaf lifespan is one trait important in this regard. We observed that leaf lifespan varies 30-fold among 23 species from natural and disturbed communities within a 1-km radius in the northern Amazon basin, near San Carlos de Rio Negro, Venezuela. Moreover, leaf lifespan was highly correlated with a number of important leaf structural and functional characterisues. Stomatal conductance to water vapor (g) and both mass and area-based net photosynthesis decreased with increasing leaf lifespan (r²=0.74, 0.91 and 0.75, respectively). Specific leaf area (SLA) also decreased with increasing leaf lifespan (r²=0.78), while leaf toughness increased (r²=0.62). Correlations between leaf lifespan and leaf nitrogen and phosphorus concentrations were moderate on a weight basis and not significant on an area basis. On an absolute basis, changes in SLA, net photosynthesis and leaf chemistry were large as leaf lifespan varied from 1.5 to 12 months, but such changes were small as leaf lifespan increased from 1 to 5 years. Mass-based net photosynthesis (A/mass) was highly correlated with SLA (r²=0.90) and mass-based leaf nitrogen (N/mass) (r²=0.85), but area-based net photosynthesis (A/area) was not well correlated with any index of leaf structure or chemistry including N/area. Overall, these results indicate that species allocate resources towards a high photosynthetic assimilation rate for a brief time, or provide resistant physical structure that results in a lower rate of carbon assimilation over a longer time, but not both.     

A single fixation protocol for proteome-wide immunofluorescence localization studies

Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.