Localization and characterization of the RNA binding protein TLS in skin and stratified mucosa.

Translocated in liposarcoma (TLS), a member of the Ewing's sarcoma family of RNA binding proteins, is targeted to the product of RNA POL II and functions in nuclear events as well as in nuclear-cytoplasmic transport of mRNA. It has been most extensively studied in cell lines, but was identified in several rat tissues by northern blot analysis, with adipose tissue showing the highest expression followed by whole skin. This paper describes a protein with amino acid sequence homology to TLS that was isolated from bovine tongue epithelium using an affinity column made with an antibody to the cornified envelope precursor sciellin. Using reverse transcriptase polymerase chain reaction technology and total RNA isolated from bovine tongue epithelium, a cDNA was obtained whose nucleotide sequence coded for a protein homologous to human TLS. Nuclear staining in all layers of human epidermis and bovine stratified epithelium was observed with an antibody to TLS, whereas peripheral staining of the upper layers of these tissues was observed with the antibody to sciellin. Cultured cells gave similar results; however, adult tissue required boiling in citrate buffer to unmask antigenic sites before reacting with the TLS antibody. Western blots of extracts of human and bovine keratinocytes using TLS and sciellin antibodies showed that the two proteins shared at least one epitope, but that they were different. TLS was lost from the nucleus following inhibition of RNA POL II activity and the protein was identified in CNBr extracts of purified keratinocytes cornified envelopes by western blot. These results clearly indicate that TLS functions as an RNA binding protein in keratinocytes in vivo and in vitro. Furthermore the sequestration of TLS to the cell envelope may play a role in regulating its nuclear-cytoplasmic transport and effect its role in transcription.

Adh and Adh-dup sequences of Drosophila lebanonensis and D. immigrans: interspecies comparisons.

We have cloned and sequenced the Adh genomic region of Drosophila lebanonensis (subgenus Scaptodrosophila) and D. immigrans (subgenus Drosophila). This region, which contains Adh, encoding the alcohol dehydrogenase enzyme, and Adh-dup (duplicate of Adh), has been compared with the same fragment from D. subobscura (subgenus Sophophora). Even though the flanking regions and introns of both genes have been affected by high substitution rates, the consensus sequences have been clearly identified. Although the overall homology of the coding regions was 76-78% among the species compared, there were differences in the exon distribution of the nucleotide substitutions when Adh or Adh-dup were compared, thus showing that these two genes differ in their evolutionary pattern.

Characterization of the amphioxus presenilin gene in a high gene-density genomic region illustrates duplication during the vertebrate lineage.

Molecular studies on the pathology of Alzheimer's disease (AD) have revealed that mutations in presenilin genes (PS) account for some familial cases. Although the contribution of these genes to the etiology is clear, their biological function remains obscure. Approaches using model organisms have been hampered by the fact that rodents contain two PS copies in the genome and do not develop the hallmark features associated with AD upon aging. To understand PS function and evolution, we have searched for PS homologous sequences in the genome of a lower chordate, Branchiostoma floridae. We report the structure of a single copy Branchiostoma floridae PS gene, named BfPS, and describe new features at the molecular level. Moreover, molecular phylogenetic analysis suggests that BfPS is orthologous to the vertebrate PS-1 and PS-2 forms. Finally, the analysis of more than 16 kb of genomic DNA encompassing BfPS identified three novel genes, which cluster with BfPS in a high gene-density region.

Protein engineering of Drosophila alcohol dehydrogenase. The hydroxyl group of Tyr152 is involved in the active site of the enzyme.

Drosophila alcohol dehydrogenase is the most studied member of the family of short-chain alcohol dehydrogenases, although its tridimensional structure still remains unknown. We have engineered a Drosophila alcohol dehydrogenase in which tyrosine-152, an invariant residue in all members of the family, has been substituted by phenylalanine. The mutated gene has been expressed in yeast and pure mutant enzyme has been prepared by a one-step FPLC chromatographic procedure. Drosophila alcohol dehydrogenase-phenylalanine-152 shows no enzymatic activity. This result suggests not only that tyrosine-152 could constitute an essential building block of the active site but also that its hydroxyl group is directly involved in the redox reaction catalyzed by the enzyme.

Drosophila lebanonensis ADH: analysis of recombinant wild-type enzyme and site-directed mutants. The effect of restoring the consensus sequence in two positions.

Unique amino acid substitutions occur in D. lebanonensis ADH. They are found within the putative NAD(+)-binding domain and affect residues that are otherwise highly conserved in all other species of the genus. To restore the consensus amino acids, we have constructed an expression system for this enzyme in E. coli, and engineered two mutants, Ala13Gly and Asn56Thr. The biochemical and kinetic features of these retromutants are consistent with increased catalytic efficiency and thermal stability. Thus, results show that wild-type D. lebanonensis ADH can be improved by site-directed mutagenesis.

A statistical analysis of nucleotide substitutions in the Drosophila Adh region reflects irregularities in molecular clocks.

Substitutions rates are expected to be rather constant when a gene is compared between species. To analyze this feature, Ka/Ks ratios have been studied for Alcohol dehydrogenase (Adh) and Alcohol dehydrogenase duplication (Adh-dup) genes in Drosophila species. Adh Ka/Ks values are lower in intrasubgenus comparisons involving species of the Sophophora group than when these species are compared to the D. immigrans and S. lebanonensis, and this difference does not occur in the Adh-dup comparisons.

Characterization of a microsomal retinol dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates.

Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems - the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) - have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identified the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phylogenetic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms.

Travelling waves in two-dimensional smectic-C domains.

Continued irradiation of smectic-C-like domains of photosensitive Langmuir monolayers from azobenzene derivatives induces the nucleation and propagation of orientational travelling waves as observed with Brewster angle microscopy (BAM). BAM image analysis has allowed to identify different dynamical behaviors involving the generation and propagation of such waves. A model based on the coupling between an orientational and a composition field proposes a scenario for dynamic self-assembly that accounts for most of the observed phenomena, and allows to pinpoint the relevance of boundary defects in wave-emitting structures.-1.

Merging protein, gene and genomic data: the evolution of the MDR-ADH family.

Multiple members of the MDR-ADH (MDR: Medium-chain dehydrogenases/reductases; ADH: alcohol dehydrogenase) family are found in vertebrates, although the enzymes that belong to this family have also been isolated from bacteria, yeast, plant and animal sources. Initial understanding of the physiological roles and evolution of the family relied on biochemical studies, protein alignments and protein structure comparisons. Subsequently, studies at the genetic level yielded new information: the expression pattern, exon-intron distribution, in silico-derived protein sequences and murine knockout phenotypes. More recently, genomic and EST databases have revealed new family members and the chromosomal location and position in the cluster of both the first and new forms. The data now available provide a comprehensive scenario, from which a reliable picture of the evolutionary history of this family can be made.

A novel effector domain from the RNA-binding protein TLS or EWS is required for oncogenic transformation by CHOP.

In human myxoid liposarcoma, a chromosomal rearrangement leads to fusion of the growth-arresting and DNA-damage-inducible transcription factor CHOP (GADD153) to a peptide fragment encoded by the TLS gene. We have found that wild-type TLS and a closely related sarcoma-associated protein, EWS, are both abundant nuclear proteins that associate in vivo with products of RNA polymerase II transcription. This association leads to the formation of a ternary complex with other heterogeneous RNA-binding proteins (hnRNPs), such as A1 and C1/C2. An NIH-3T3-based transformation assay was used to study the oncogenic role of the sarcoma-associated domain of these RNA-binding proteins. Transduction of the TLS-CHOP oncogene into cells by means of a retroviral expression vector leads to loss of contact inhibition, acquisition of the ability to grow as colonies in soft agar, and tumor formation in nude mice. Mutations that interfere with the function of the leucine zipper dimerization domain or the adjacent basic region of CHOP abolish transformation. The essential role of the TLS component was revealed by the inability of truncated forms to fully transform cells. Domain swap between TLS- and EWS-associated oncogenes demonstrated that the component contributed by the RNA-binding proteins are functionally interchangeable, whereas the transcription factor component specifies tumor phenotype. The sarcoma-associated component of TLS and EWS contribute a strong transcriptional activation domain to the fusion proteins; however, transforming activity cannot be fully substituted by fusion of CHOP to other strong trans-activators. The juxtaposition of a novel effector domain from sarcoma-associated RNA-binding proteins to the targeting domain of transcription factors such as CHOP leads to the creation of a potent oncogene.

Endogenous beta-galactosidase activity in amphioxus: a useful histochemical marker for the digestive system.

Endogenous beta-galactosidase activity has been shown in the digestive tract of amphioxus from the larval to the adult stage and it can be easily followed as a histochemical marker. Enzymatic activity first appeared in 30-h larvae, became evident in 36-h larvae and remained in adults. In situ detection of beta-galactosidase activity was used to monitor morphological and functional differentiation of the digestive system and the posteriorization of the endodermal structures in retinoic-acid treated embryos. The endogenous beta-galactosidase activity was distinguished from the bacterial lacZ reporter by incubation at low pH.

Analysis of nucleotide substitutions and amino acid conservation in the Drosophila Adh genomic region.

The homologous genomic region that contains two paralogous genes, Adh and Adh-dup, was compared in several Drosophila species. Sequences were analyzed as follows: a) At the nucleotide level, Ka and Ks values were determined for each pair of species. Ka-Adh and Ka-Adh-dup are not significantly different. However, Ks-Adh values are significantly lower than Ks-Adh-dup, which are more variable. In agreement with other reports, lower Ks values for Adh correlate with a high level of gene expression and relatively high percentage of G+C content in the third codon position, while the opposite applies to Adh-dup. b) At the protein level, amino acid comparisons reveal conserved regions shared by ADH and ADH-DUP, which have been assigned to known functional domains. Key residues for dehydrogenasic function are also found in ADH-DUP, thus pointing to a dehydrogenase activity for ADH-DUP, albeit very different from that of ADH.

The Adh in Drosophila: chromosomal location and restriction analysis in species with different phylogenetic relationships.

Restriction analysis of the genomic region containing the Adh gene and in situ hybridization assays were performed in six Drosophila species belonging to three different subgenera: D. ambigua, D. subobscura, D. madeirensis and D. guanche (sg. Sophophora); D. immigrans (sg. Drosophila); and D. lebanonensis (sg. Pholadoris). In agreement with previous observations, comparison of restriction maps of the Adh region shows that D. subobscura and D. madeirensis are very closely related. Partial homology is also observed with the rest of the obscura group species. Nevertheless, no resemblance at the restriction map level is detected when more distantly related species are compared. In D. ambigua, D. immigrans and D. lebanonensis in situ hybridization assays reveal a single chromosomal location for Adh, which in D. lebanonensis appears to be sex linked. In contrast, in D. subobscura, D. madeirensis and D. guanche multiple sites of hybridization with homologous and heterologous probes are observed. For example, in D. subobscura and D. madeirensis the functional Adh gene is located on the U chromosome and additional homologous retrosequences are found on the E chromosome.

Involvement of the C-terminal tail in the activity of Drosophila alcohol dehydrogenase. Evaluation of truncated proteins constructed by site-directed mutagenesis.

Drosophila alcohol dehydrogenase belongs to the heterogeneous family of short-chain dehydrogenases/reductases, which does not include the well characterized mammalian alcohol dehydrogenases. Although it is clear that the main biological role of this enzyme is in alcohol oxidation, in the absence of the three-dimensional conformation only partial information on the protein regions involved in the active site, and the coenzyme and substrate interacting cavities is available. Two segments have already been identified, a coenzyme-binding segment at the N-terminus, and the reactive Tyr152 and Lys156 residues. Limited proteolytic assays had suggested the involvement of the 13 C-terminal amino acids in the function of the enzyme. By site-directed mutagenesis, we have constructed eight different truncated mutant enzymes and expressed them in Escherichia coli. The purified mutant enzymes have been recovered and characterized using monoclonal antibodies. Kinetic analysis and stability assays have been performed, and clearly demonstrate the contribution of the last 13 amino acids to the activity. We hypothesize that the C-terminal tail constitutes an essential region for maintaining the hydrophobicity of the catalytic pocket needed for binding of the substrate.

Amphioxus alcohol dehydrogenase is a class 3 form of single type and of structural conservation but with unique developmental expression.

The coding region of amphioxus alcohol dehydrogenase class 3 (ADH3) has been characterized from two species, Branchiostoma lanceolatum and Branchiostoma floridae. The species variants have residue differences at positions that result in only marginal functional distinctions. Activity measurements show a class 3 glutathione-dependent formaldehyde dehydrogenase, with kcat/Km values about threefold those of the human class 3 ADH enzyme. Only a single ADH3 form is identified in each of the two amphioxus species, and no ethanol activity ascribed to other classes is detectable, supporting the conclusion that evolution of ethanol-active ADH classes by gene duplications occurred at early vertebrate radiation after the formation of the amphioxus lineage. Similarly, Southern blot analysis indicated that amphioxus ADH3 is encoded by a single gene present in the methylated fraction of the amphioxus genome and northern blots revealed a single 1.4-kb transcript. In situ experiments showed that amphioxus Adh3 expression is restricted to particular cell types in the embryos. Transcripts were first evident at the neurula stage and then located at the larval ventral region, in the intestinal epithelium. This tissue-specific pattern contrasts with the ubiquitous Adh3 expression in mammals.

The first non-LTR retrotransposon characterised in the cephalochordate amphioxus, BfCR1, shows similarities to CR1-like elements.

BfCR1 is the first non-long terminal repeat retrotransposon to be characterised in the amphioxus genome. Sequence alignment of the predicted translation product reveals that BfCR1 belongs to the CR1-like retroposon class, a family widely distributed in vertebrate and invertebrate lineages. Structural analysis shows conservation of the specific motifs of the ORF2-CR1 elements: the N-terminal endonuclease, the reverse transcriptase and the C-terminal domains. The BfCR1 element possesses an atypical 3' terminus consisting of the tandem repeat (AAG)6. We gathered evidence supporting the mobility of this element and report an estimated 15 copies of BfCR1, mostly truncated, per haploid genome, a remarkably low number when compared to that of vertebrates. Phylogenetic analysis, including the amphioxus element, seems to indicate that (i) CR1-like retroposons cluster in a monophyletic group and (ii) the CR1-like family was already present in the chordate ancestor. Our data provide further support for the horizontal transmission of CR1-like elements during early vertebrate evolution.