Insertional mutagenesis to isolate acetate-requiring mutants in Chlamydomonas reinhardtii

Abstract An arg 7 mutant of the green alga Chlamydomonas reinhardtii was transformed with pARG7.8, a plasmid bearing the wild-type ARG 7 gene. Out of 4100 arg⁺ transformants selected on an arginine-free medium supplemented with acetate, nine failed to grow on acetate-free medium (ac⁻ mutants). The results of the genetic and molecular analysis of several ac⁻ mutants are in agreement with the hypothesis that they originated from insertion of the incoming plasmid into the nuclear genome. These mutants should constitute valuable tools for isolating the corresponding wild-type genes after plasmid rescue into Escherichia coli .     

Detection of renal cell carcinoma-associated markers via proteome- and other 'ome'-based analyses.

Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the complementary technology for genetic profiling. It has been shown to be a powerful tool for studying human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. This review focuses on the identification of new biomarkers and therapeutic targets for renal cell carcinoma using different 'ome'-based technologies.     

Comparative chromosome painting between two marsupials: origins of an XX/XY1Y2 sex chromosome system

Cross-species chromosome painting was used to investigate genome rearrangements between tammar wallaby Macropus eugenii (2n = 16) and the swamp wallaby Wallabia bicolor (2n = 10♀/11♂), which diverged about 6 million years ago. The swamp wallaby has an XX female:XY₁Y₂ male sex chromosome system thought to have resulted from a fusion between an autosome and the small original X, not involving the Y. Thus, the small Y₁ should represent the original Y and the large Y₂ the original autosome. DNA paints were prepared from flow-sorted and microdissected chromosomes from the tammar wallaby. Painting swamp wallaby spreads with each tammar chromosome-specific probe gave extremely strong and clear signals in single-, two-, and three-color FISH. These showed that two tammar wallaby autosomes are represented unchanged in the swamp wallaby, two are represented by different centric fusions, and one by a tandem fusion to make the very long arms of swamp wallaby Chromosome (Chr) 1. The large swamp wallaby X comprises the tammar X as its short arm, and a tandemly fused 7 and 2 as the long arm. The acrocentric swamp wallaby Y₂ is a 2/7 fusion, homologous with the long arm of the X. The small swamp wallaby Y₁ is confirmed as the original Y by its painting with the tammar Y. However, the presence of sequences shared between the microdissected tammar Xp and Y on the swamp wallaby Y₂ implies that the formation of the compound sex chromosomes involved addition of autosome(s) to both the original X and Y. We propose that this involved fusion with an ancient pseudoautosomal region followed by fission proximal to this shared region. Received: 16 October 1996/Accepted: 30 January 1997     

Auswirkungen von Hochspannungstrassen auf die Fl?chennutzung überwinternder Bl??- und Saatg?nseAnser albifrons, A. fabalis

Zusammenfassung  Während das Mortalitätsrisiko für Vögel durch Drahtanflug und Stromschlag an Hochspannungsleitungen vielfach untersucht wurde, könnte auch ein Einfluß von Hochspannungstrassen (110 kV) auf Vögel bestehen, die sich am Boden aufhalten. Dieser wurde 1994/95 am Beispiel der Flächennutzung überwinternder Bläß- und Saatgänse (Anser albifrons, A. fabalis) am Unteren Niederrhein untersucht. Da Gänse offene ungestörte Äsungsflächen beanspruchen, wurde der Arbeitshypothese nachgegangen, daß trassennahe Bereiche von Gänsen gemieden bzw. vermindert genutzt werden. Die Besuchsfrequenz von vier häufig genutzten trassenüberspannten Äsungsflächen wurde ermittelt. Im Anschluß an die einzelnen mehrtägigen Beäsungsphasen wurde die Losungsdichte (LD) als Maß der Flächennutzung senkrecht zum Trassenverlauf bestimmt. An 16–20 m hohen Trassen stieg die LD mit zunehmender Entfernung zur Trasse. Eine signifikante Abnahme der LD trat unterhalb von 40–80 m Abstand zur Trasse ein. Kleine Teilflächen, die durch die Trassen von größeren Bereichen abgetrennt sind, wurden weniger genutzt. An einer 60 m hohen, den Rhein Überquerenden Trasse, konnte kein trassenbedingter Unterschied der LD festgestellt werden. Mögliche Ursachen für die beschriebenen Effekte werden diskutiert.

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The impact of power lines on field selection and grazing intensity of wintering White-fronted- and Bean GeeseAnser albifrons, A. fabalis

In addition to studies about the mortality risk by collision and electrocution power lines may have an impact on birds during foraging behaviour on the ground. We examined this question in geese (Anser albfrons, A. fabalis) wintering in the lower Rhine area of North Rhine Westfalia (FRG) 1994/95. Because geese prefer large areas with low levels of disturbance by human activities or urbanic structures like buildings or roads, we hypothesized that they might be disturbed by the presence of power lines in their foraging sites and thus avoid areas next to the lines. To quantify the grazing intensity in relation to the distance from the power lines (110 kV), dropping densities have been repeatedly counted after independant grazing intervals in four different feeding sites. Our data show that: 1. Grazing intensity increases with the distance to power lines of low height. Significantly reduced grazing levels have been found in distances less than 80 to 40 m to the power lines. 2. No effects have been observed for a power line in an area next to the river rhine where the wires crossed the ground in a height of about 60 metres. 3. In small pasture or field areas, cut off by power lines from large ones, a generally reduced grazing amount is evident. Several conceivable reasons for the described effects are discussed.

     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20