Werner Kunz

Struktur und Funktion der Oocytenchromosomen und Nukleolen sowie der Extra-DNS whrend der Oogenese panoistischer und meroistischer Insekten

In panoistic ovaries (without nurse cells) there are three predominating structures: lampbrush chromosomes, multiple nucleoli, and the hitherto undescribed endobody (Binnenkörper). Nucleoli are always multiple during the growth period of the oocyte of panoistic ovaries. This is true even in the case of Blattella which seems to possess only one big nucleolus, if examined in the light microscope (cf. Figs. 2 and 14b).—In the meroistic type of ovary (with nurse cells) the development of nucleoli and lampbrush chromosomes in the oocyte is very reduced. Only in the early growth stages of the oocyte the chromosomes despiralisize in a speciesspecific degree before they condense to a karyosphere (Pigs. 8, 9). On the other hand the endobody is bigger in the meroistic than in the panoistic ovary (Figs. 5, 8,14). — Lampbrush chromosomes and multiple nucleoli are sites of a very intensive RNA-synthesis (Fig. 1). The nucleoli are built up by granules measuring 125 Å in diameter (Figs. 15, 16). In the endobody, no RNA-metabolism could be demonstrated (Figs, 1a, b, 8c). The endobody is very homogeneous in electron microscope pictures and clearly distinct from the granular nucleoli (Fig. 17). The labelling pattern after incubation with 3H-amino acids suggests a permanent exchange of protein molecules between the karyoplasm and the endobody. — In the meroistic type of ovary the oocyte obtains RNA from the nurse cells, and RNA-synthesis in the oocyte nucleus is decreased in the same measure as its chromosomes are condensed. — The water-beetles Dytiscus and Acilius possess extra-DNA and deviate from the rule of restricted RNA-synthesis in the oocyte nucleus of the meroistic ovary albeit their chromosomes form a karyosphere too (Fig. 11) and RNA streams also from the nurse chamber into the ooplasm (Fig. 10). The extra-DNA resolves itselve into a network of fine fibrils no longer stainable by the Feulgen reaction. True multiple nucleoli develop on the fibrils suggesting the extra-DNA contains a huge mass of nucleolus organizers. The case of Dytiscus is very similar to the development of the multiple nucleoli in Gryllus.Zusammenfassung1.Die Oocytenkerne von Insekten mit panoistischem Ovar enthalten drei vorherrschende Strukturelemente: Lampenbürstenchromosomen, multiple Nukleolen und als bisher noch nicht beschriebenes Kernorganell den Binnenkörper. Auch der scheinbar einheitliche Nukleolus der Blattella-Oocyte erweist sich im elektronenmikroskopischen Bild als multipel. Im meroistischen Ovar ist die Ausbildung von Lampenbürstenchromosomen und multiplen Nukleolen stark reduziert. Lediglich in der prävitellogenetischen Frühphase durchlaufen die Chromosomen eine begrenzte Entspiralisierung, ehe sie zur Karyosphäre kondensieren. Der Binnenkörper ist im meroistischen Ovar stärker entwickelt als im panoistischen.2.Die Lampenbürstenchromosomen und die multiplen Nukleolen des panoistischen Ovars sind Orte kräftiger RNS-Synthese. Die Nukleolen setzen sich aus Granula von 120 Å Durchmesser zusammen und haben meist eine dichtere Rindenstruktur. Der Binnenkörper besitzt dagegen keinen autoradiographisch erfaßbaren RNS-Stoffwechsel. Weiterhin unterscheidet er sich von den Nukleolen durch eine sehr homogene, feinfibrilläre Ultrastruktur. Sein Markierungsverhalten nach Inkubation mit 3H-Aminosäuregemischen läßt auf einen ständigen Austausch von Proteinmolekülen zwischen dem Karyoplasma und dem Binnenkörper schließen.3.Im meroistischen Ovar erhält die Oocyte RNS aus dem Nährfach und die RNS-Synthese der Oocytenchromosomen wird im Maße ihrer Kondensierung zur Karyosphäre vermindert.4.Die Dytisciden weichen von der Regel ab, daß die RNS-Synthese im Oocytenkern bei Anwesenheit von Nährzellen gedrosselt ist, obwohl RNS aus dem Nährfach in die Oocyte strömt. Während sich die Oocytenchromosomen zur Karyosphäre kondensieren, lockert sich die fibrilläre Extra-DNS der Keimbahn zu einem feinfädigen Netzwerk auf, das den ganzen Oocytenkern erfüllt und nicht mehr mit der Feulgen-Reaktion dargestellt werden kann. Durch Behandlung mit hypertonischen Medien vor der Fixierung gelingt es jedoch wieder, ein grobes Feulgen-positives DNS-Gerüst im Kern zu erzeugen. Im Maße der Auflockerung des Extra-DNS-Körpers verstärken sich die RNS-Synthese und die Bildung von Nukleolenkügelchen. Die Nukleolen bestehen aus den typischen 120Å-Granula und erfüllen schließlich den Kernraum bis auf den Binnenkörper und die Karyosphäre. Alle Befunde sprechen dafür, daß die Extra-DNS im Dytiscidenkern die Matrizen für eine intensive nukleoläre RNS-Synthese liefert. Die übereinstimmende Bedeutung der Extra-DNS bei Gryllus und den Dytisciden für die Bildung multipler Nukleolen wird erörtert.

Schistosome male–female interaction: induction of germ-cell differentiation

Male and female schistosomes are permanently paired while they live in the bloodstream of their vertebrate hosts. Female schistosomes produce eggs only when they are in intimate association with a male. Here, I combine classical cytological knowledge about the cellular processes in the female that are affected by the male with recent molecular results that are beginning to allow speculation about the signalling events involved.

HSP70-controlled GFP expression in transiently transformed schistosomes☆

Among the parasitic helminths schistosomes are of high medical and economic importance. Despite of the world-wide relevance of this parasite, very little is known about the cellular mechanisms controlling its development and concerning the host-parasite interaction. Within the last decade a great effort has been made in this blood fluke to identify genes which play important roles during these processes. However, molecular analysis was limited by the fact, that neither function nor regulation of candidate genes could be investigated in this organism due to the lack of transformation protocols. Here, we present the strategy of ballistic gene transfer to introduce and characterize transgenes in different schistosome life stages. As a transformation vector, the heat shock protein 70 (hsp70) gene promoter and terminator from Schistosoma mansoni were cloned and fused to the green fluorescent protein (GFP) reporter gene. In a first attempt, the hsp70--GFP vector was successfully tested in a eukaryotic cell line. Thereafter, adult male schistosomes and sporocysts were transformed with this vector, and GFP expression was demonstrated using molecular and microscopical methods. PCR, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses confirmed the presence, transcription and translation of the transgene in adults. Confocal laser scanning microscopy revealed GFP-activity at various sites along the surface of the worms after hs induction and within sporocysts. These results suggest diverse roles for hsp70 during the development of schistosomes. Furthermore, the results demonstrate the feasibility of this method and open the perspective to analyze a variety of molecular functions in schistosomes.

A multiple-staining procedure for the detection of different DNA fragments on a single blot.

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Female-specific gene expression in Schistosoma mansoni is regulated by pairing

Gene expression studies in adult females of Schistosoma mansoni cultured in vitro revealed that the transcription of female-specifically expressed genes is influenced by pairing. In contrast, the activity of genes that are expressed in both genders was not affected by contact with the male. The transcription of genes was monitored in paired, separated and remated females. The transcript level of female-specifically expressed genes decreases within a few days following separation from males. Remating of uncoupled females with males leads to the reinitiation of transcription. These results provide strong evidence for the influence of the male on gene transcription in the female and contribute a molecular basis for the classical histological observation that the maturation of females is male dependent. The data also show that the culture system is suitable to monitor gene expression and, furthermore, they indicate de novo RNA synthesis in vitro.

Ferritins of Schistosoma mansoni: sequence comparison and expression in female and male worms.

Recombinant clones of Schistosoma mansoni cDNA libraries containing the complete coding regions of 2 different ferritin subunits have been isolated and sequenced. This allows for the first time a comparison of ferritin sequences from an invertebrate with those of vertebrates. The deduced amino acid sequences of both Schistosoma ferritin subunit clones show significant homology to vertebrate ferritin H chains. Similarity exceeds 50% identity and includes the recently identified ferroxidase center which is present only in H chains. However, non-conservative substitutions of amino acid residues lining the 3-fold symmetry channel were found, and a gap of 3 successive amino acids unique to the 2 Schistosoma ferritin sequences was identified. Remarkably, for each of the 2 genes, we found a conspicuous difference in the amount of ferritin transcripts between females and males: one of the genes is preferentially expressed in females, the other in males.

When is a parasite species a species

Regrettably, 140 years after the publication of Darwins Origin of Species, we face the grotesque situation that we still do not know what is a species whose origin Darwin wanted to explain. A generally applicable species definition is not available. Is there a basic unit of biodiversity above the level of individuals? Do we try to define something that does not exist in reality? The strong potential for the evolution of genetic variability in parasites together with the importance of species diagnosis for applied fields of parasite research make biodiversity research a key role in parasitology. Frequent occurrence of sympatric speciation, clonal reproduction, selfing, sib mating or parthenogenesis imply exceptional conditions for the evolution of gene pool diversities in parasites.

Characterisation of the cysteine protease ER60 in transgenic Schistosoma mansoni larvae.

Proteinases have been found to play important roles in parasites. They are involved in developmental processes and facilitate invasion of host tissues as well as the digestion of host molecules for nutrition. The cysteine protease ER60 from Schistosoma mansoni, originally characterised in adults to be expressed in excretory organs, was analysed in larval stages. Transcripts were found in miracidia, in vitro generated mother sporocysts and cercariae. After cloning the promoter and terminator of the ER60 gene, a transformation vector was constructed containing the green fluorescent protein reporter gene flanked by the regulatory elements. The ER60-green fluorescent protein vector was used for transfection experiments of COS-7 cells demonstrating the functionality of the promoter in the heterologous system. To analyse the expression pattern of ER60-green fluorescent protein in larval S. mansoni, in vitro generated mother sporocysts were transformed by particle bombardment, a method which allows gene transfer into schistosomes. Molecular analyses demonstrated transcription and translation of the transgene. Furthermore, confocal laser scanning microscopy revealed ER60-induced green fluorescent protein fluorescence within the larvae. Inside primary sporocysts, tissue-specific activity was localised in the gland cells, protonephridia and several cytons. These results suggest that ER60 is expressed in the ES system of larvae and, amongst other functions, may play a role in penetration and migration processes.

Identification of Ras, MAP kinases, and a GAP protein in Schistosoma mansoni by immunoblotting and their putative involvement in male-female interaction

The maturation of female Schistosoma mansoni depends on pairing with a male which induces mitotic activities in the reproductive organs of the female worm. Since in other organisms cell proliferation is regulated by well-conserved signal transducing molecules, we looked for such molecules on immunoblots of schistosomes, using antibodies against conserved epitopes of Ras, GAP and MAP kinases. We identified all 3 molecules in schistosomes and found that they are developmentally regulated. Furthermore, there is evidence for their involvement in the male-directed maturation of the female.

Sequence, characterization and localization of a cysteine proteinase cathepsin L in Schistosoma mansoni☆

A cDNA encoding Schistosoma mansoni cathepsin L was isolated from a cDNA library and sequenced. Alignment of the proposed amino-acid sequence with known members of cathepsin L shows highest homologies with sequences from mouse and rat. An expression plasmid was constructed in Escherichia coli to produce recombinant schistosome cathepsin L with an extension of six histidines at its N terminus. Using antibodies raised against the purified fusion protein, two polypeptide bands with approx. molecular masses of 38 and 31 kDa were identified in a schistosome extract. By use of specific radioiodinated inhibitors, a radioactively labeled protein could be detected at 31 kDa, suggesting that this is the active mature enzyme. The larger protein of 38 kDa did not react with the inhibitor, indicating that it represents the inactive precursor molecule. Immunohistological experiments revealed that the proteinase is localized in structures associated with the reproductive system of females and with the subtegumental region of the gynecophoric canal of males. However, Northern blot hybridization demonstrates that more transcripts are present in female parasites than in males. Genomic Southern blotting suggests that schistosome cathepsin L is expressed from a single-copy gene.