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.

---

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.

     

DNA sequence analysis of mitochondrial Cyt-b and the species status ofLaniarius dubiosus (Rchw. 1899)

In 1899Reichenow described an african bushshrike in juvenile plumage as a new species. He named itLaniarius dubiosus. DNA from type material (feathers and skin) was extracted and DNA sequences from the mitochondrial Cyt-b gene were analysed. Comparisons of DNA-sequences from other bushshrikes (including the type-specimen from Luehder's bushshrikeLaniarius lühderi) support the judgement that dubiosus does not represent a full species rather than a representative of the western subspecies of the Luehder's bushshrikeLaniarius luehderi.

---

Zusammenfassung Der vonReichenow (1899) als neue Art beschriebene, sich im Jugendkleid befindliche BuschwürgerLaniarius dubiosus ist nach DNA-Sequenzanalysen des mitochondrialen Cyt-b Genes mit hoher Wahrscheinlichkeit ein Jungvogel der westlichen Rasse des BraunscheitelwürgersLaniarius lühderi. Auf der Basis eines DNA-Stammbaumes von sieben verschiedenen Buschwürgern ist der 1991 neu beschriebeneLaniarius liberatus am wenigsten eng mitL. dubiosus verwandt.

     

Instantaneous force-velocity-length relationship in diaphragmatic sarcomere

Coirault, Catherine, Denis Chemla, Jean-Claude Pourny,Francine Lambert, and Yves Lecarpentier. Instantaneousforce-velocity-length relationship in diaphragmatic sarcomere.J. Appl. Physiol. 82(2): 404-412, 1997.The simultaneous analysis of muscle force, length, velocity, andtime has been shown to precisely characterize the mechanicalperformance of isolated striated muscle. We tested the hypothesis thatthe three-dimensional force-velocity-length relationship reflectsmechanical properties of sarcomeres. In hamster diaphragm strips,instantaneous sarcomere length (SL) and muscle length were simultaneously measured during afterloaded twitches. SL was measured by means of laser diffraction. Wealso studied the influence of initialSL, abrupt changes in total load, and2 × 10⁷ M dantrolene.Baseline resting SL at the apex of thelength-active tension curve was 2.2 ± 0.1 µm, whereasSL at peak shortening was 1.6 ± 0.1 µm in the preloaded twitch and 2.1 ± 0.1 µm in the "isometric" twitch. Over the whole load continuum and at anygiven level of isotonic load, there was a unique relationship between instantaneous sarcomere velocity and instantaneousSL. Part of this relationship was timeindependent and initial SL independent and was markedly downshifted after dantrolene. When five different muscle regions were considered, there were no significant variations ofSL and sarcomere kinetics along themuscle. These results indicate that the time- and initiallength-independent part of the instantaneous force-velocity-lengthrelationship previously described in muscle strips reflects intrinsicsarcomere mechanical properties.

     

Transformation of the wild tomatoLycopersicon chilense Dun. byAgrobacterium tumefaciens

Summary Leaf disc transformation-regeneration technique was applied to the drought tolerant wild relative of cultivated tomato,Lycopersicon chilense, using a plasmid construct which contained the coding sequences of neomycin phosphotransferase (NPTII) and chloramphenicol acetyltransferase (CAT) genes. The two genotypes used, LA2747 and LA1930, showed a distinct difference in their aptitude to transformation; a higher success rate was obtained for the first genotype in every stage of the process. Shoots were formed on the regeneration medium containing 100 g/ml kanamycin through direct or indirect organogenesis. Root formation became only possible when the concentration of kanamycin was reduced to 50 g/ml. Expression of chloramphenicol acetyltransferase gene was observed in all of the kanamycin-screened plants after they matured; the activity of the gene was absent or low in some of the young plants. The presence of the CAT gene in transgenic plants was further confirmed by Southern blot analysis. Although transgenic plants grew to maturity, they did not produce fruit, owing to the self incompatibility ofL. chilense. Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - LB Luria Broth - EDTA ethylenediamine-tetraacetic acid