The winged-helix transcription factor JUMU regulates development, nucleolus morphology and function, and chromatin organization of Drosophila melanogaster.

The PEV-modifying winged-helix/forkhead domain transcription factor JUMU of Drosophila is an essential protein of pleiotropic function. The correct gene dose of jumu is required for nucleolar integrity and correct nucleolus function. Overexpression of jumu results in bloating of euchromatic chromosome arms, displacement of the JUMU protein from the chromocenter and the nucleolus, fragile weak points, and disrupted chromocenter of polytene chromosomes. Overexpression of the acidic C terminus of JUMU alone causes nucleolus disorganization. In addition, euchromatic genes are overexpressed and HP1, which normally accumulates in the pericentric heterochromatin and spreads into euchromatic chromosome arms, although H3-K9 di-methylation remains restricted to the pericentric heterochromatin. The human winged-helix nude gene shows similarities to jumu and its overexpression in Drosophila causes bristle mutations.

The winged-helix transcription factor JUMU is a haplo-suppressor/triplo-enhancer of PEV in various tissues but exhibits reverse PEV effects in the brain of Drosophila melanogaster.

Gene expression goes along with changes in chromatin structure and is regulated by chromatin-modifying factors. If genes are transposed from their euchromatic position to the vicinity of heterochromatin, their expression can underly a position effect variegation (PEV). In Drosophila melanogaster a few genes are known that function in a gene dose-dependent manner as haplo-suppressors and triplo-enhancers of PEV or vice versa. The gene jumeaux (jumu) encodes a winged-helix transcription factor of multiple regulatory functions. A novel PEV test system for Drosophila melanogaster reveals that JUMU behaves as a haplo-suppressor/triplo-enhancer in different larval and adult tissues, but surprisingly behaves in the reverse manner as a haplo-enhancer/triplo-suppressor in larval and adult brains. Like jumu, the Su(var)3-9 gene also behaves as a haplo-suppressor/triplo-enhancer, but in our test system does not show any PEV effect in the brains.

Umbrea, a chromo shadow domain protein in Drosophila melanogaster heterochromatin, interacts with Hip, HP1 and HOAP.

Drosophila melanogaster HP1-interacting protein (Hip) is a partner of heterochromatin protein 1 (HP1) and is involved in transcriptional epigenetic gene silencing and the formation of heterochromatin. Recently, it has been shown that HP1 interacts with the telomere capping factor HP1/ORC (origin recognition complex)-associated protein (HOAP). Telomeres, complexes of DNA and proteins at the end of linear chromosomes, have been recognized to protect chromosome ends from degradation and fusion events. Both proteins are located at telomeres and prevent telomere fusions. Here, we report the identification and characterization of the Hip-interacting protein Umbrea. We found that Umbrea interacts directly with Hip, HP1 and HOAP in vitro. Umbrea, Hip and HP1 are partners in a protein complex in vivo and completely co-localize in the pericentric heterochromatin and at telomeres. Using a Gal4-induced RNA interference system, we found that after depletion of Umbrea in salivary gland polytene chromosomes, they exhibit multiple telomeric fusions. Taken together, these results suggest that Umbrea cooperates with Hip, HP1 and HOAP and plays a functional role in mediating normal telomere behaviour in Drosophila.

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR.

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 was performed using the SoilMaster() DNA Extraction Kit (Epicentre, Madison, Wisconsin) and hybridization probe based real-time PCR. The detection target was the alkane hydroxylase gene (alkM). Standard curve construction showed a linear relation between log values of cell concentrations and real-time PCR threshold cycles over five orders of magnitude between 5.4+/-3.0x10(6) and 5.4+/-3.0x10(2)CFUml(-1) cell suspension. The detection limit was about 540CFUml(-1), which was ten times more sensitive than conventional PCR. The quantification of Acinetobacter sp. strain MUB1 cells in soil samples resulted in 46.67%, 82.41%, and 87.59% DNA recovery with a detection limit of 5.4+/-3.0x10(4)CFUg(-1) dry soil. In this study, a method was developed for the specific, sensitive, and rapid quantification of the Acinetobacter sp. strain MUB1 in soil samples.

Hip, an HP1-interacting protein, is a haplo- and triplo-suppressor of position effect variegation.

The Drosophila heterochromatin protein 1 (HP1) regulates epigenetic gene silencing and heterochromatin formation by promoting and maintaining chromatin condensation. Here we report the identification and characterization of an HP1-interacting protein (Hip). Hip interacts with HP1 in vitro and is associated with HP1 in vivo. This interaction is mediated by at least three independent but similar HP1-binding modules of the Hip protein. Hip and HP1 completely colocalize in the pericentric heterochromatin, and both haplo- and triplo-dosage mutations act as dominant suppressors of position effect variegation. These findings identify a player in heterochromatinization and suggest that Hip cooperates with HP1 in chromatin remodeling and gene silencing.

Comparative molecular analysis of two asparaginyl endopeptidases and encoding genes from Fasciola gigantica.

In this study we describe the first cysteine proteinases of the MEROPS Clan CD family C13 in Fasciola gigantica. Family C13 contains asparaginyl endopeptidases and glycosylphosphatidylinositol-anchor transamidases and is also called the legumain family due to the discovery of the first asparaginyl endopeptidase in a legume. The cDNAs encoding two asparaginyl endopeptidases, FgLGMN-1 and FgLGMN-2, were cloned and used for the analysis of nucleic acid and protein properties. The deduced amino acid sequences showed 47.4% identity to each other and from 42.2 to 51.1% identity to homologs of other trematode species. The catalytic site residues histidine, cysteine and preceding hydrophobic residues, characteristic for the cysteine proteinase families C11, C13, C14, and C25, were found conserved. Northern and reverse transcription PCR analyses demonstrated that the transcriptional products are present in metacercariae, juveniles and adults. RNA in situ hybridization and immunohistochemistry revealed that RNA and protein products of the two genes are specifically expressed in the intestinal epithelium of juveniles and adults. Immune sera of mice infected with F. gigantica reacted with immunoblotted, bacterially expressed recombinant proteins starting 4 weeks after infection. Polyclonal antisera raised against the recombinant proteins detected 40 and 30 kDa antigens, respectively in crude worm protein extracts but not in the excretion-secretion products of adult parasites. Likewise, legumain-specific activity was found in crude worm protein extracts but not in excretion-secretion products. This study elucidates the molecular characteristics of these proteins in F. gigantica and demonstrates differences in the biology between Fasciola and Schistosoma which may prove useful for the development of vaccines against fasciolosis in domestic livestock.

Opisthorchis viverrini: identification of a glycine-tyrosine rich eggshell protein and its potential as a diagnostic tool for human opisthorchiasis.

A cDNA encoding a novel eggshell protein (OvESP) with high-glycine (49.2%) and -tyrosine (27.8%) content was cloned from the human liver fluke Opisthorchis viverrini. In the adult parasite, the RNA products of the OvESP gene are limited to the vitelline follicles. They have a size of 800 nucleotides and are already present in 2-week-old juveniles. Immune sera of hamsters, experimentally infected, and humans, naturally infected with O. viverrini, detect bacterially expressed recombinant OvESP (rOvESP). A rabbit anti-rOvESP antiserum only reacts with the shells of intrauterine eggs in tissue sections of the parasite. Comparison of rOvESP with the parasite's excretion/secretion products as diagnostic tools for human opisthorchiasis shows a higher sensitivity (0.82-0.48) and specificity (0.97-0.91) of the recombinant protein in the ELISA technique. But the observed weak cross-reactivity of immune sera from mice infected with Schistosoma mansoni, Schistosoma mekongi, and Fasciola gigantica in Western blots of rOvESP indicates that the diagnostic quality of this protein might be compromised if infections by other trematodes are present.

Molecular cloning and analysis of stage and tissue-specific expression of cathepsin B encoding genes from Fasciola gigantica.

The transcriptional products of Fasciola gigantica genes encoding cathepsin B proteases were cloned from adult, newly excysted juvenile (NEJ), and metacercarial stages. The obtained cDNAs were named FG cat-B1, FG cat-B2, and FG cat-B3. The deduced amino acid sequences of the encoded proteases have identities ranging from 64 to 79%. Sequence comparison with homologous proteins showed that all functional important residues formerly described for cathepsin B are conserved. Southern analysis confirmed the presence of a family of related cathepsin B genes in the genome of F. gigantica. Northern analysis revealed a common transcript size of 1400 nucleotides with abundant cathepsin B transcripts detected in metacercarial and NEJ stages. Cathepsin B transcripts were located by RNA in situ hybridization in the caecal epithelial cells, in cells underlining the proximal part of the digestive tract, and in the tegumental cells underlining the surface tegument. Furthermore, transcripts were detected in the tissues of the reproductive system including cells of prostate, Mehlis, and vitelline glands, testis, and eggs. Stage-specific gene expression was investigated by RT-PCR using gene-specific primers and hybridization with a labeled cathepsin B probe. FG cat-B1 transcripts were detected in all stages, whereas FG cat-B2 and FG cat-B3 transcripts were expressed in metacercariae, NEJ, and juvenile parasites only. The switching off of the cat-B2 and cat-B3 genes during the maturation of the parasites implicates that these enzymes may be involved in digesting host tissues during penetration and migration to the liver, whereas cat-B1 present in all stages may perform general digestive function.

Molecular cloning and characterization of a glutathione S-transferase encoding gene from Opisthorchis viverrini.

An adult stage Opisthorchis viverrini cDNA library was constructed and screened for abundant transcripts. One of the isolated cDNAs was found by sequence comparison to encode a glutathione S-transferase (GST) and was further analyzed for RNA expression, encoded protein function, tissue distribution and cross-reactivity of the encoded protein with other trematode protein counterparts. The cDNA has a size of 893 bp and encodes a GST of 213 amino acids length (OV28GST). The most closely-related GST of OV28GST among those published for trematodes is a 28 kDa GST of Clonorchis sinensis as shown by multiple sequence alignment and phylogenetic analysis. Northern analysis of total RNA with a gene-specific probe revealed a 900 nucleotide OV28GST transcriptional product in the adult parasite. Through RNA in situ hybridization OV28GST RNA was detected in the parenchymal cells of adult parasites. This result was confirmed by immunolocalization of OV28GST with an antiserum generated in a mouse against bacterially-produced recombinant OV28GST. Both, purified recombinant and purified native OV28GST were resolved as 28 kDa proteins by SDS-PAGE. Using the anti-recOV28GST antiserum, no or only weak cross-reactivity was observed in an immunoblot of crude worm extracts against the GSTs of Schistosoma mansoni, S. japonicum, S. mekongi, Eurytrema spp. and Fasciola gigantica. The enzyme activity of the purified recombinant OV28GST was verified by a standard 1-chloro-2, 4-dinitrobenzene (CDNB) based activity assay. The present results of our molecular analysis of OV28GST should be helpful in the ongoing development of diagnostic applications for opisthorchiasis viverrini.