Hydrothermal liquefaction of sewage sludge: use of HCOOH and KOH to improve the slurry pumpability in a continuously operated plant.

We studied the hydrothermal liquefaction (HTL) of digested sewage sludge (DSS) as model of waste biomass in batch and continuous reactors. HCOOH and KOH were used to improve the slurry pumpability. HTL experiments were conducted at the same kinetic severity factor in a batch reactor of 25 mL of volume and in a continuously operated tubular reactor with 350 mL of volume. The observed outcomes suggested that it was not possible to achieve the pumpability of native DSS when a high concentrated stream of suspended solid particles has been fed to the HTL continuous plant. Using acidic or basic homogeneous additives, as potassium hydroxide or formic acid, it was possible to enhance the pumpability of a concentrated slurry of DSS in the continuous plant achieving yields of heavy oil (fraction of biocrude) similar to those obtained in the batch reactor and with higher H/C ratios. Hence, we found that HCOOH and KOH are promising additives for the practical implementation of a continuous HTL process.

Expression of the male determining gene dmrt1bY and its autosomal coorthologue dmrt1a in medaka.

Dmrt1bY (aka dmy) has been identified as the master male sex-determining gene of medaka by positional cloning and genetic evidence. How this gene on the molecular level determines the differentiation of the gonad primordium towards a testis, however, is unknown. To better understand the roles of dmrt1bY and its autosomal counterpart dmrt1a in the process of sex determination, a detailed expression analysis by quantitative real-time RT-PCR was performed showing non-organ-specific expression of dmrt1bY during embryogenesis and low expression in adult testes. Surprisingly a high expression of dmrt1bY was noted in the spleen of males. Dmrt1a is exclusively expressed in adult testes, 50 times higher than dmrt1bY. Dmrt1bY mRNA and protein have short half-lives and the GFP fusion protein products of both dmrt1 genes are localized to the nucleus. These features are consistent with the suggestion that Dmrt1 proteins might function as DNA-binding transcription factors.

Genomic organization and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1.

Genes related to the Drosophila melanogaster doublesex and Caenorhabditis elegans mab-3 genes are conserved in human. They are identified by a DNA-binding homology motif, the DM domain, and constitute a gene family (DMRTs). Unlike the invertebrate genes, whose role in the sex-determination process is essentially understood, the function of the different vertebrate DMRT genes is not as clear. Evidence has accumulated for the involvement of DMRT1 in male sex determination and differentiation. DMRT2 (known as terra in zebrafish) seems to be a critical factor for somitogenesis. To contribute to a better understanding of the function of this important gene family, we have analyzed DMRT1, DMRT2, and DMRT3 from the genome model organism Fugu rubripes and the medakafish, a complementary model organism for genetics and functional studies. We found conservation of synteny of human chromosome 9 in F. rubripes and an identical gene cluster organization of the DMRTs in both fish. Although expression analysis and gene linkage mapping in medaka exclude a function for any of the three genes in the primary step of male sex determination, comparison of F. rubripes and human sequences uncovered three putative regulatory regions that might have a role in more downstream events of sex determination and human XY sex reversal.

Fish retroposons related to the Penelope element of Drosophila virilis define a new group of retrotransposable elements.

Poseidon and Neptune are two ancient lineages of retroposons related to the Penelope element from Drosophila virilis. They have been identified in various teleost fish species, including the medakafish (Oryzias latipes), and the pufferfishes Fugu rubripes and Tetraodon nigroviridis, whose genomes are currently being sequenced. Some of these elements are highly reiterated in fish genomes. Penelope-related elements were also identified in blood fluke, shrimp, sea urchin, cichlid fish and frog, showing that they are widespread in animals. Penelope-related retroposons were not detected among sequences from the Drosophila melanogaster and human genome projects, suggesting that they have been lost from certain animal lineages. A sequence encoding a putative Uri (also called GIY-YIG) endonuclease domain was detected downstream from the gene for reverse transcriptase. To the best of our knowledge, this type of endonuclease sequence has previously been identified in group I introns and in genes for prokaryotic excinucleases but not in retrotransposable elements. Penelope-related elements are frequently truncated at their 5' ends and can also be flanked by long terminal repeat-like structures. Phylogenetic analysis of the reverse transcriptase domain failed to assign Penelope-related retroposons to one of the major groups of retroelements. Overall, therefore, the evidence strongly suggests that these sequences represent a new group of retrotransposable elements.

Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element.

The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus.

Triplet repeat variability in the signal peptide sequence of the Xmrk receptor tyrosine kinase gene in Xiphophorus fish.

Trinucleotide repeats in several human genes have been found to undergo spontaneous variation in repeat numbers in succeeding generations. Expansion of the repeat beyond a certain length causes specific pathological disorders. So far, a naturally occurring triplet repeat instability of transcribed sequences has been reported only from humans. However, the signal peptide encoding region of the receptor tyrosine kinase gene Xmrk from fish of the genus Xiphophorus contains a CTG repeat that differs in length even between closely related individuals. The consequence of this variability is signal peptides with shorter or longer hydrophobic core regions reaching, in some individuals, the critical maximum length for functional protein export or even exceeding it. In one stock, animals that are homozygous for such an allele were extremely rare, indicating that the triplet repeat length variability of the Xmrk gene of Xiphophorus may indeed have an influence on the function of the gene product and, under certain conditions, may affect the fitness of the individual.

Susceptibility to the development of pigment cell tumors in a clone of the Amazon molly, Poecilia formosa, introduced through a microchromosome.

The Amazon molly Poecilia formosa is a gynogenetic fish that reproduces through the development of ameiotic diploid eggs triggered by insemination by males of related species without following karyogamie. This leads to clonal offspring. In rare cases, however, this gynogenesis is leaky, and paternal DNA in the form of small supernumerary chromosomes is included into the maternal genome. We have obtained a clone where one such microchromosome contains a pigmentary locus, resulting in macromelanophore pigmentation of the carrier. Approximately 5% of these fish spontaneously develop exophytic nodular or papillomatous pigment cell tumors. The tumors display considerable differences with respect to growth characteristics and invasiveness, despite the genetic uniformity of the affected animals. Following transplantation to syngeneic hosts, a remarkable clonal variability was observed. Oncogenes that are involved in tumorigenesis in hereditary melanoma of the closely related fish Xiphophorus appear not to be instrumental for induction of the P. formosa pigment cell tumors. Moreover, a new genetic locus is defined that mediates susceptibility to pigment cell tumor development and leads to transformation of chromatoblasts.

The activation domain of simian immunodeficiency virus SIVmac239 Rev protein is structurally and functionally analogous to the HIV-1 Rev activation domain.

The Rev proteins of primate immunodeficiency viruses are essential transactivators for the switch from early to late phase in the viral replication cycle. By mutational analysis, a putative activation domain (AD) has been assigned to the carboxy-terminus. This leucine-rich stretch of amino acids proved to be essential for the transactivating properties of HIV-1 Rev. Some mutants in the AD transdominantly inhibit the function of wild-type Rev protein very efficiently. We identified a similar domain structure for SIVmac239 Rev by sequence comparison and in vitro mutagenesis. The leucine/isoleucine residues of the SIVmac239 Rev activation domain appeared to be of similar importance for function. The mutants of these residues in the SIV AD displayed a dominant negative phenotype on both HIV-1 and SIVmac 239 rev-responsive elements (RRE). The prokaryotically expressed wild-type and mutant proteins were analyzed for RNA-binding properties in a gel-shift assay in vitro. This assay revealed a similar binding pattern of wild-type and transdominant proteins on either RRE.

Exchange of functional domains between Rev proteins of HIV-1 and SIVmac239 results in a dominant negative phenotype.

The Rev proteins of primate immunodeficiency viruses are essential transactivators to switch from early to late phase in the viral replication cycle. Surprisingly, the Rev protein of HIV-1 is able to substitute those of HIV-2 and, as shown in here, of SIVmac239, but not vice versa. To understand the underlying mechanism of this incomplete functional reciprocity, we constructed a series of chimeric HIV-1/SIVmac239 Rev proteins and tested for transcomplementation efficacy on Rev-dependent indicator plasmids. In addition, we analyzed the prokaryotically expressed wild type and chimeric proteins for RNA-binding properties in a gel-shift assay in vitro. The functional defect of SIVmac239 on the HIV-1 Rev response element (RRE) is not due to a lack of binding or multimerization. In cotransfection experiments, SIVmac239 Rev and the chimeric proteins were tested for potential inhibitory effects on HIV-1 Rev function using the HIV-1 based indicator plasmid. Some of these proteins turned out to be transdominant inhibitors almost as potent as the HIV-1 Rev mutant M10 which is localized in the activation domain and is one of the strongest transdominant inhibitors. Surprisingly, M10 was not able to inhibit the function of either Rev protein on SIVmac239 RRE, whereas a corresponding SIVmac239 Rev mutant (SIV M10) was a transdominant inhibitor of SIVmac239 Rev function on its homologous RRE.