Hartmut Voss

Identification of farnesoid X receptor beta as a novel mammalian nuclear receptor sensing lanosterol

ABSTRACT Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRβ as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRβ is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRα and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRβ. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRβ as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.

Simultaneous sequencing of nucleic acids

The invention concerns a method for the sequence-specific labelling of nucleic acids comprising the generation of labelled nucleic acid fragments by an enzymatic labelling reaction in which a labelled deoxyribonucleoside triphosphate is attached to a nucleic acid primer molecule and the nucleic acid sequence is determined by means of the label, wherein the labelling reaction is carried out in a single reaction vessel with the simultaneous presence of one or several nucleic acid primer molecules and at least two labelled deoxyribonucleoside triphosphates which each contain different labelling groups and different bases and under conditions in which only one single type of labelled deoxyribonucleoside triphosphate can be attached to a nucleic acid primer molecule.

A Chimeric Subunit of Yeast Transcription Factor IIIC Forms a Subcomplex with τ95

ABSTRACT The multisubunit yeast transcription factor IIIC (TFIIIC) is a multifunctional protein required for promoter recognition, transcription factor IIIB recruitment, and chromatin antirepression. We report the isolation and characterization of TFC7, an essential gene encoding the 55-kDa polypeptide, τ55, present in affinity-purified TFIIIC. τ55 is a chimeric protein generated by an ancient chromosomal rearrangement. Its C-terminal half is essential for cell viability and sufficient to ensure TFIIIC function in DNA binding and transcription assays. The N-terminal half is nonessential and highly similar to a putative yeast protein encoded on another chromosome and to a cyanobacterial protein of unknown function. Partial deletions of the N-terminal domain impaired τ55 function at a high temperature or in media containing glycerol or ethanol, suggesting a link between PolIII transcription and metabolic pathways. Interestingly, τ55 was found, together with TFIIIC subunit τ95, in a protein complex which was distinct from TFIIIC and which may play a role in the regulation of PolIII transcription, possibly in relation to cell metabolism.

Characterization of SRp46, a Novel Human SR Splicing Factor Encoded by a PR264/SC35 Retropseudogene

ABSTRACT The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with β-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.

DNA Sequencing and Analysis of 130 kb from Yeast Chromosome XV

We have determined the nucleotide sequence of 129 524 bases of yeast (Saccharomyces cerevisiae) chromosome XV. Sequence analysis revealed the presence of 59 non‐overlapping open reading frames (ORFs) of length >300 bp, three tRNA genes, four delta elements and one Ty‐element. Among the 21 previously known yeast genes (36% of all ORFs in this fragment) were nucleoporin (NUP1), ras protein (RAS1), RNA polymerase III (RPC1) and elongation factor 2 (EF2). Further, 31 ORFs (53% of the total) were found to be homologous to known protein or DNA sequences, or sequence patterns. For seven ORFs (11% of the total) no homology was found. Among the most interesting protein identifications in this DNA fragment are an inositol polyphosphatase, the second gene of this type found in yeast (homologous to the human OCRL gene involved in Lowes syndrome), a new ADP ribosylation factor of the arf6 subfamily, the first protein containing three C2 domains, and an ORF similar to a Bacillus subtilis cell‐cycle related protein. For each ORF detailed sequence analysis was carried out, with a full consideration of its biological function and pointing out key regions of interest for further functional analysis. The sequence has been submitted to the EMBL data library under Accession Number X94335.© 1997 John Wiley & Sons, Ltd.

Sequencing 39,350 bp of saccharomyces cerevisiae chromosome XII utilizing ordered shotgun libraries

A 39,350 bp cosmid containing DNA of Saccharomyces cerevisiae chromosome XII was sequenced by making use of ordered sub-clones of 1 kb insert-length selected from a physical clone map. In a first analysis, 96 clones were sequenced from both ends (10 gels) with two standard sequencing primers covering 91% of the total sequence (49% double-stranded). After selection of another eight clones six gaps of a total of 1.8 kb and several single-stranded stretches remained. These gaps were closed by 86 primer walks leading to an overall redundancy of 4.4 per base and a total of 292 sequencing reactions. The number of walking primers can be reduced significantly by more uniform clone lengths and longer sequencing reads, thus, the total amount of sequencing reactions can approach the minimum value achieved with primer walking strategies, with only very few walking primers needed for gap closure.

Sequencing and analysis of 51 kb on the right arm of chromosome XV from Saccharomyces cerevisiae reveals 30 open reading frames

We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.