Conditional gene targeting in macrophages and granulocytes using LysMcre mice

Conditional mutagenesis in mice has recently been made possible through the combination of gene targeting techniques and site–directed mutagenesis, using the bacteriophage P1–derived Cre/loxP recombination system. The versatility of this approach depends on the availability of mouse mutants in which the recombinase Cre is expressed in the appropriate cell lineages or tissues. Here we report the generation of mice that express Cre in myeloid cells due to targeted insertion of the cre cDNA into their endogenous M lysozyme locus. In double mutant mice harboring both the LysMcre allele and one of two different loxP–flanked target genes tested, a deletion efficiency of 83–98 was determined in mature macrophages and near 100 in granulocytes. Partial deletion (16) could be detected in CD11c⁺ splenic dendritic cells which are closely related to the monocyte/macrophage lineage. In contrast, no significant deletion was observed in tail DNA or purified T and B cells. Taken together, LysMcre mice allow for both specific and highly efficient Cre–mediated deletion of loxP–flanked target genes in myeloid cells.     

Rhythmische Erregungen in den optischen Zentren von Calliphora erythrocephala

Zusammenfassung Die rhythmischen Aktionspotentiale in den optischen Ganglien der Schmeißfliege (Calliphora erythrocephala) werden untersucht.Wird das Komplexauge von Calliphora belichtet, so können vom Ganglion opticum II schnelle, rhythmische Aktionspotentiale, 'Belichtungsrhythme , abgegriffen werden (Abb. 1). Sie treten im Bereich physiologischer Temperaturen und Lichtintensitäten stets und unabhängig von Schädigungen auf. Sie sind die einzige Form von Erregung, die zwischen dem retinalen Bereich und dem Cerebralganglion nachgewiesen werden kann. Die Belichtungsrhythmen zeigen gesetzmäßige Abhängigkeiten von den Reizgrößen. Es ist daher wahrscheinlich, daß sie in die Kausalkette der bei Belichtung des Auges ablaufenden zentralen Vorgänge eingeschaltet sind.Die optischen Ganglien werden mit einer Doppelmikroelektrode abgetastet. Da die Spannung zwischen zwei eng benachbarten Elektroden in der Nähe der Spannungsquelle am größten sein muß, kann gezeigt werden, daß die Belichtungsrhythmen wahrscheinlich in der äußeren Körnerschicht des Ganglion opticum II entstehen (Abb. 14 und 15).Als Maß für die Größe der Belichtungsrhythmen wird die größte während einer Belichtung auftretende Amplitude gewählt, die 'Maximalamplitud ; sie hängt stetig und reproduzierbar von der Zahl belichteter Ommatidien, von der Lichtintensität und vom Adaptationszustand des Auges ab (Abb. 5, 6, 7, 8, 10, 11 und 12).Die Amplituden der Belichtungsrhythmen klingen bei längerer Belichtung allmählich ab (Helladaptation), (Abb. 1C, Abb. 5). Die Heiladaptationszeit ist der Maximalamplitude proportional (Abb. 6, 8, 9 und 10). Wird die Belichtung vor dem völligen Abklingen der Rhythmen unterbrochen, so werden sie durch den Aus-Effekt des Retinogramms gehemmt und brechen sofort und vollkommen ab (Abb. 1 D). Die Dunkeladaptation ist selbst nach vorangegangener Belichtung mit sehr hohen Lichtintensitäten nach spätestens einer Minute abgeschlossen (Abb. 6 und 7).Die Frequenz der Belichtungsrhythmen liegt zwischen 100 sec^–1 und 250 sec^–1, sie nimmt mit steigender Temperatur zu (Tabelle 1). Die Frequenz ist unabhängig von der Lichtintensität, vom Adaptationszustand d von der Zahl belichteter Ommatidien.Während der einzelnen Belichtung zeigen die Rhythmen ein verschieden starkes Schwanken der Amplitude, eine Amplitudenmodulation. Die Modulation hängt vom Präparat und vom Präparationszustand ab.Durch den Vergleich der verschiedenen Modulationstypen und durch gleichzeitige Ableitung an mehreren Stellen des Ganglions können die physikalischen Überlagerungsvorgänge untersucht werden. Die Einzelschwingungen physiologischer Einheiten überlagern sich am gemeinsamen Ableitwiderstand zwischen den Elektroden. Durch die Art der Überlagerung wird die Modulationsform bestimmt. Sie hängt im besonderen von der Frequenz und der Phasenlage der Einzelrhythmen und von physiologischen Synchronisationsvorgängen ab (Abb. 1, 2 und 16).Auch wenn ein Bereich der Retina gereizt wird, der nur wenige Sinneszellen umfaßt, treten Belichtungsrhythmen wie bei großen Reizflächen auf (Abb. 12). Deshalb wird die Möglichkeit diskutiert, daß bereits die kleinste physiologische Einheit im Ganglion mit rhythmischer Erregung antwortet, die in ihrer Amplitude, nicht aber in ihrer Frequenz vom Reiz abhängt.Herrn Prof. Dr. H. Autrum danke ich für das stete Interesse, das er den Untersuchungen entgegengebracht hat. Die Untersuchungen wurden zum Teil mit Apparaten durchgeführt, die die Deutsche Forschungsgemeinschaft Herrn Prof. Autrum zur Verfügung stellte.     

Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy

The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mM], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i. e. in the P/P state, under the polyamine buffer conditions.     

The Sp8 zinc-finger transcription factor is involved in allometric growth of the limbs in the beetle Tribolium castaneum

Members of the Sp gene family are involved in a variety of developmental processes in both vertebrates and invertebrates. We identified the ortholog of the Drosophila Sp-1 gene in the red flour beetle Tribolium castaneum, termed T-Sp8 because of its close phylogenetic relationship to the vertebrate Sp8 genes. During early embryogenesis, T-Sp8 is seen in segmental stripes. During later stages, TSp8 is dynamically expressed in the limb buds of the Tribolium embryo. At the beginning of bud formation, TSp8 is uniformly expressed in all body appendages. As the limbs elongate, a ring pattern develops sequentially and the expression profile at the end of embryogenesis correlates with the final length of the appendage. In limbs that do not grow out like the labrum and the labium, T-Sp8 expression remains uniform, whereas a two-ring pattern develops in the longer antennae and the maxillae. In the legs that elongate even further, four rings of T-Sp8 expression can be seen at the end of leg development. The role of T-Sp8 for appendage development was tested using RNAi. Upon injection of double stranded T-Sp8 RNA, larvae develop with dwarfed appendages. Affected T-Sp8(RNAi) legs were tested for the presence of medial and distal positional values using the expression marker genes dachshund and Distal-less, respectively. The results show that a dwarfed TSp8(RNAi) leg consists of proximal, medial and distal parts and argues against T-Sp8 being a leg gap gene. Based on the differential expression pattern of T-Sp8 in the appendages of the head and the thorax and the RNAi phenotype, we hypothesise that T-Sp8 is involved in the regulation of limb-length in relation to body size - a process called allometric growth.     

Nicotine's Defensive Function in Nature

Plants produce metabolites that directly decrease herbivore performance, and as a consequence, herbivores are selected for resistance to these metabolites. To determine whether these metabolites actually function as defenses requires measuring the performance of plants that are altered only in the production of a certain metabolite. To date, the defensive value of most plant resistance traits has not been demonstrated in nature. We transformed native tobacco(Nicotiana attenuata) with a consensus fragment of its two putrescine N-methyl transferase (pmt) genes in either antisense or inverted-repeat (IRpmt) orientations. Only the latter reduced (by greater than 95%) constitutive and inducible nicotine. With D₄-nicotinic acid (NA), we demonstrate that silencing pmt inhibits nicotine production, while the excess NA dimerizes to form anatabine. Larvae of the nicotine-adapted herbivore Manduca sexta (tobacco hornworm) grew faster and, like the beetle Diabrotica undecimpunctata, preferred IRpmt plants in choice tests. When planted in their native habitat, IRpmt plants were attacked more frequently and, compared to wild-type plants, lost 3-fold more leaf area from a variety of native herbivores, of which the beet armyworm, Spodoptera exigua, and Trimerotropis spp. grasshoppers caused the most damage. These results provide strong evidence that nicotine functions as an efficient defense in nature and highlights the value of transgenic techniques for ecological research.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Variation in number and differentiation of the abdominal infrared receptors in the Australian 'fire-beetle' Merimna atrata (Coleoptera, Buprestidae)

Most individuals of the Australian ‘fire-beetle’ Merimna atrata have two pairs of IR receptors which are located ventrolaterally on the second and third abdominal sternite. An IR receptor consists of a specialized IR absorbing area, which is innervated by a neural complex. This complex contains one thermoreceptive multipolar neuron with a unique terminal dendritic mass (TDM) and two scolopidia and was termed ‘sensory complex’. However, also individuals with one pair of IR receptors on the second sternite and beetles with three pairs on the second, third, and fourth sternites were found. Additionally, beetles having one or two pairs of IR receptors may have preliminary stages of IR receptors on the third and fourth sternite, respectively. We found two kinds of preliminary stages, both of which are characterized by a much less pronounced absorbing area. In all five abdominal sternites segmental nerves are attached to the cuticle with a neural complex. Investigation of complexes of non-IR sternites suggests that the sensory cells inside the sensory complex of an IR receptor have developed from common internal stretch receptors. From our results it can be hypothesized that the IR sensory system in Merimna atrata has not yet reached a stage, which can be regarded as evolutionary stable.     

Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts

In higher plants, photorespiratory Gly oxidation in leaf mitochondria yields ammonium in large amounts. Mitochondrial ammonium must somehow be recovered as glutamate in chloroplasts. As the first step in that recovery, we report glutamine synthetase (GS) activity in highly purified Arabidopsis thaliana mitochondria isolated from light-adapted leaf tissue. Leaf mitochondrial GS activity is further induced in response to either physiological CO(2) limitation or transient darkness. Historically, whether mitochondria are fully competent for oxidative phosphorylation in actively photorespiring leaves has remained uncertain. Here, we report that light-adapted, intact, leaf mitochondria supplied with Gly as sole energy source are fully competent for oxidative phosphorylation. Purified intact mitochondria efficiently use Gly oxidation (as sole energy, NH(3), and CO(2) source) to drive conversion of l-Orn to l-citrulline, an ATP-dependent process. An A. thaliana genome-wide search for nuclear gene(s) encoding mitochondrial GS activity yielded a single candidate, GLN2. Stably transgenic A. thaliana ecotype Columbia plants expressing a p35S::GLN2::green fluorescent protein (GFP) chimeric reporter were constructed. When observed by laser scanning confocal microscopy, leaf mesophyll and epidermal tissue of transgenic plants showed punctate GFP fluorescence that colocalized with mitochondria. In immunoblot experiments, a 41-kD chimeric GLN2::GFP protein was present in both leaf mitochondria and chloroplasts of these stably transgenic plants. Therefore, the GLN2 gene product, heretofore labeled plastidic GS-2, functions in both leaf mitochondria and chloroplasts to faciliate ammonium recovery during photorespiration.