Pdx1-related homeodomain transcription factors are distinctly expressed in mouse adult pancreatic islets.

Complex gene networks are responsible for the proper operation of the endocrine pancreas. A central member of such networks, the homeodomain transcription factor Pdx1, belongs to the ParaHox gene cluster, an array of Hox-like homeobox genes. With a combination of mRNA in situ hybridisation and immunodetection, we have found that the rest of ParaHox cluster genes, Cdx1, Cdx2/3, and Cdx4, and Gsh1 and Gsh2, are all expressed in specific islet cell types of the endocrine pancreas. To our knowledge, this is the first report that locates ParaHox genes other than Pdx1 and Cdx2/3 in a place as to be involved in the pancreatic transcriptional regulatory networks, potentially regulating glucagon-insulin homeostasis.

GEM, a cluster of repetitive sequences in the Drosophila subobscura genome.

GEM is a new family of repetitive sequences detected in the D. subobscura genome. Two of the four described GEM elements encompass a heterogeneous central module, with no detectable ORF, flanked by two long inverted repeats. These elements are composed of a set of repetitive modules, which are inverted repeat (IR), direct repeat (DR), palindromic sequence (PS), long sequence (LS) and short sequence (SS). These five modules can be found either clustered or dispersed as single modules in the D. subobscura genome, in euchromatic and heterochromatic regions. In addition to the 3' region of Adh retrosequences, single IR and LS blocks were found associated with the promoter region of different genes, in particular, LS-like blocks have also been found associated with functional genes in D. melanogaster and D. virilis. Conversely, the DR block is highly similar to satellite DNAs from some other species of the obscura group. In addition, GEM elements share some structural features with IS elements described in different Drosophila species. It is likely that both GEM and IS sequences would be vestiges of an ancestral transposable element.

Homodimerization of presenilin N-terminal fragments is affected by mutations linked to Alzheimer's disease.

Mutations on human presenilins 1 and 2 cause dominant early-onset familial Alzheimer's disease (FAD). Presenilins are polytopic transmembrane proteins endoproteolytically processed in vivo to N- and C-terminal fragments (NTFs and CTFs). The functional presenilin unit consists of a high molecular weight complex that contains both fragments. Here we show NTF:NTF, CTF:CTF and NTF:CTF interactions by yeast two-hybrid and in vivo endoplasmic reticulum split-ubiquitin assays. Our results also highlight the involvement of HL1--the hydrophilic loop between TMI and TMII--in the NTF:NTF binding site. Besides, nine FAD-linked presenilin mutations substantially affected HL1:HL1 binding. From the evidence of NTF and CTF homodimerization, we propose the contribution of two NTFs and two CTFs, instead of a single NTF:CTF heterodimer, to the functional presenilin-gamma-secretase complex and that FAD mutations affect the assembly or stability of this complex.

Effect of site-directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase.

Tyr152 and Lys156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short-chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli. Mutation of Tyr152 to Glu or Gln, Lys156 to Ile, Gly184 to Leu, and the double mutant Gly130 to Cys and Gly133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr152 and Lys156 are involved in catalysis and that Gly130, Gly133 and Gly184 contribute substantially to the structure of the active form.

Novel splice donor site mutation in MERTK gene associated with retinitis pigmentosa.

BACKGROUND/AIM: Mutations in MERTK, a member of the MER/AXL/TYRO3 receptor kinase family, have been associated with disruption of the Retinal Pigment Epithelium (RPE) phagocytosis pathway and settling of autosomal recessive RP (arRP) in humans. This study reports a novel MERTK mutation (IVS16+1G>T) in a Spanish consanguineous family presenting arRP. METHODS: 21 genes were screened by high-throughput SNP multiplexing assay. Subsequent direct sequencing was performed in exons and intronic boundaries of the cosegregating gene. The effect of the mutation in mRNA splicing was confirmed by cDNA analysis. RESULTS: Haplotypic data revealed MERTK cosegregation with RP in affected individuals. MERTK sequencing showed a G-to-T substitution at the first nucleotide of intron 16. Finally, cDNA analysis confirmed the lack of exon 16 in the mRNA splicing process. CONCLUSIONS: IVS16+1G>T disrupts the splice donor site causing exon 16 skipping. Absence of exon 16 causes a frameshift and, subsequently, the introduction of a premature termination codon into exon 17 creating an altered mRNA transcript with a seriously affected tyrosine kinase domain.

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins.

The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate.

Characterization and evolution of the Adh genomic region in Drosophila guanche and Drosophila madeirensis.

We have sequenced the genomic region of the Adh (alcohol dehydrogenase) gene of Drosophila guanche and Drosophila madeirensis. Two genes, Adh and Adh-dup, whose main features are shared by other Drosophila species, are contained in this region. Interspecies comparisons of this genomic region of these two species with the region in D. subobscura have been made. Our data are in agreement with the phylogenetic relationship of the three species and particularly with the very close proximity of D. madeirensis and D. subobscura. The correct alignment of the noncoding as well as the coding sequences allows us to infer how this region has evolved. The degree of divergence of D. madeirensis and D. subobscura Adh sequences is low and replacement substitutions are as frequent as silent substitutions. In addition, heterogeneous divergence rates for coding and noncoding sequences have been observed. Our data also reveal that a recent evolutionary event is responsible for the branching of the three species.

The Adh genomic region of Drosophila ambigua: evolutionary trends in different species.

The study of individual genes is essential to a comprehensive understanding of genome evolution. The wealth of information on alcohol dehydrogenase (Adh) in Drosophila makes this gene particularly suitable for such analysis. We have characterized more than 4 kb of the genomic Adh region in Drosophila ambigua and compared this region to Drosophila mauritiana and Drosophila pseudoobscura. The presence of two genes, Adh and 3'ORF (open reading frame), has been confirmed and some of their essential features have been inferred from primary structural analysis. Inter- and intraspecific comparisons have led us to support that both genes may have diverged from an ancient precursor. They appear to be evolving independently, and show a species-specific pattern. The Adh in the obscura group species lacks amino acids three and four when compared to the species of the melanogaster group and has accumulated most of its amino acid replacements in the third exon. Neither characteristic is observed when any other group species are compared, which suggests that these may be particular features of the evolution of the obscura group. The 3'ORF is highly conserved among the three species analyzed, although variability in the length of the third exon and the nucleotide substitution rate, which is much higher than in Adh, are worth noting. According to our data, both mutation/fixation rates and the distribution of mutations vary over time, which makes it difficult to predict the evolutionary dynamics of specific genome regions.

Evidence for retrotranscription of protein-coding genes in the Drosophila subobscura genome.

Evidence is provided for the presence of retrosequences (also named retroposons) arising from Adh in the Drosophila subobscura genome. Restriction analysis and primary structure of two different retrosequence-containing clones, S812 and S135, are reported. The fact that these retrosequences lack introns and a recognizable promoter strongly supports their retrotranscriptional origin. Adjacent to the two retrosequences analyzed, a middle repetitive DNA element has been found which bears no clear similarity to any sequence reported to date in the GenBank/EMBL Data Library. A comparative analysis of these retrosequences with the functional Adh gene of D. subobscura is presented. In addition, a model concerning the origin, functionality, and propagation of these genome elements is discussed.

The Drosophila subobscura Adh genomic region contains valuable evolutionary markers.

We have sequenced 4 kb of the genomic region comprising the Adh (Alcohol dehydrogenase) gene of Drosophila subobscura. In agreement with other species which belong to the same subgenus, two structural genes, Adh and Adh-dup, are contained in this region. The main features of these two genes of D. subobscura have been inferred from the sequence data and compared with the homologous region of D. ambigua and D. pseudoobscura. Drosophila subobscura Adh and Adh-dup differ from those of D. ambigua at a corrected estimation of 10.1% and 12.5%, respectively, while from those of D. pseudoobscura they differ by 9.5% and 8.1%, respectively. Our data suggest that Adh and Adh-dup are evolving independently, showing a species-specific pattern. Moreover, particular features of some regions of these genes make them valuable evolutionary hallmarks. For instance, replacement substitutions in the third exon of Adh may indicate the branching of the melanogaster-obscura groups, whereas replacement substitutions in the third exon of the Adh-dup could be used to assess speciation within the obscura group.

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.

Characterization and molecular analysis of Adh retrosequences in species of the Drosophila obscura group.

Retrosequences, genes, and pseudogenes originated by retrotranscription are frequent components of vertebrate genomes, but they have only occasionally been described in invertebrates. In Drosophila, very few retrosequences have been reported, among them those of alcohol dehydrogenase (Adh) and phosphoglyceromutase (Pglym). Although 52 Adh gene sequences are available for comparison, Adh retrosequences have been described only in the sibling species D. teissieri and D. yakuba (melanogaster subgroup) and in D. subobscura (obscura subgroup). Here, we report the presence of Adh retrosequences in two closely related species of D. subobscura: D. madeirensis and D. guanche. Extensive sequence comparisons with their functional paralogs suggest separate retrotranscriptional events: one in the melanogaster subgroup in the ancestor of D. teissieri and D. yakuba, and the other in the obscura subgroup before the radiation of the lineages leading to D. subobscura, D. madeirensis, and D. guanche. In the former, the Adh retrotranscript originated a new expressed gene, named jingwei. However, in the obscura Adh retrosequences, retention of codon bias and higher Ks than Ka values, both distinctive evolutionary features supporting functionality, have to be considered together with a frameshift, premature stop codons, and other nucleotide substitutions, which, added to the lack of the original promoter elements, suggest that they are pseudogenes. At least two different Adh retrosequences have been characterized in each of the obscura species, and their phylogenetic analysis indicates that paralogs and their flanking genomic regions share a higher degree of similarity than orthologous sequences. Two alternative hypotheses could explain this current organization and structure: either a multiplication event occurred independently in each species, or gene conversion events should be invoked after a single duplication in the species ancestor. The significance of retrotranscriptional events in the evolution of invertebrate genomes is discussed.

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.

Identification of proteolytic cleavage sites within the gag-analogue protein of Ty1 virus-like particles.

Like retroviruses, the yeast retrotransposon Ty1 produces its proteins as precursors that are subsequently cleaved by a protease encoded by the element. These cleavage events are essential for transposition as they release the active reverse transcriptase and integrase and they modify the structure of the virus-like particles in a way that is analogous to the morphological changes that occur during retrovirus core maturation. Using a combination of epitope tagging, amino acid analysis and mutagenesis, we have identified the major cleavage sites for the Ty1 protease within the particle-forming protein, p1, at 407S/408N. In addition, we present evidence indicating that the Ty1 protease may be a 17 kDa protein.

Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, is under-expressed in Down syndrome fetal brains.

Suppression subtractive hybridization performed on Down syndrome (DS) versus control fetal brains revealed differential expression of peroxiredoxin 2 (PRDX2), mapped at 13q12. Peroxiredoxins are antioxidant enzymes involved in protein and lipid protection against oxidative injury and in cellular signalling pathways regulating apoptosis. The under-expression of PRDX2 observed in DS samples was confirmed by real-time PCR (0.73-fold). To test whether decreased expression is associated with enhanced sensitivity of DS neurons to reactive oxygen species, we down-regulated PRDX2 through stable transfections of SH-SY5Y neuroblastoma cells with antisense contructs of the complete PRDX2 coding sequence. In addition, we over-expressed SOD1 and compared the effects of the two genes on cell viability. Cells transfected with either construct showed similar sensitivity to oxidative stress in addition to increased apoptosis under basal conditions and after treatment with oxidative cytotoxic agents. This suggests that the decreased expression of PRDX2 may contribute to the altered redox state in DS at levels comparable to that of the increased expression of SOD1.

Structural determinants within the subunit protein of Ty1 virus-like particles.

The Ty virus-like particles (VLPs) are functionally analogous to retroviral particles. They package the enzymes and the RNA necessary for retrotransposition, and mediate the integration of the reverse-transcription product into the genome of the host cell. Here we map three structural determinants of particle assembly in the subunit protein. We have also identified key residues in these regions that seem to be involved in subunit interaction and particle morphology. In particular, two point mutations in putative amphipathic helices have remarkable effects on VLP morphology, increasing the diameter as much as eightfold.

Sorbitol dehydrogenase of Drosophila. Gene, protein, and expression data show a two-gene system.

The Drosophila melanogaster sorbitol dehydrogenase (SDH) is characterized as a two-enzyme system of the medium chain dehydrogenase/reductase family (MDR). The SDH-1 enzyme has an enzymology with Km and kcat values an order of magnitude higher than those for the human enzyme but with a similar kcat/Km ratio. It is a tetramer with identical subunits of approximately 38 kDa. At the genomic level, two genes, Sdh-1 and Sdh-2, have a single transcriptional start site and no functional TATA box. Expression is greater in larvae and adults than in pupae, where it is very low. At all three stages, Sdh-1 constitutes the major transcript. Sdh-1 and Sdh-2 genes were located at positions 84E-F and 86D in polytene chromosomes. The deduced amino acid sequences of the two genes show 90% residue identity. Evaluation of the sequence and modeling of the structure toward that of class I alcohol dehydrogenase (ADH) show altered loop and gap arrangements as in mammalian SDH and establishes that SDH, despite gene multiplicity and larger variability than the "constant" ADH of class III, is an enzyme conserved over wide ranges.

USP25, a novel gene encoding a deubiquitinating enzyme, is located in the gene-poor region 21q11.2.

We have identified a new gene, USP25, spanning over 150 kb at 21q11. 2, one of the lowest gene-density regions of the human genome. USP25 is made up of 25 exons and encodes a 1087-aa protein. Database comparisons reveal high homology with members of the ubiquitin protease family (UBP). Basal expression was observed in all human tissues tested, and two main transcripts were identified. The homologous murine gene has also been characterized. In situ hybridization in mouse embryonic brains showed a clear correlation of expression with proliferative neuroepithelial cells and postmitotic neurons. Moreover, high expression was observed in adult mouse testis. UBPs belong to a complex family of deubiquitinating enzymes that specifically cleave ubiquitin conjugates on a great variety of substrates. These enzymes have an essential role in protein degradation via the 26S proteasome and thus regulate many cellular pathways. An increase in USP25 gene dosage in Down syndrome patients could seriously disturb the balance between ubiquitinated and deubiquitinated substrates.

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25.

BACKGROUND: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. RESULTS: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. CONCLUSIONS: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.

Frequency of the arylsulphatase A pseudodeficiency allele in the Spanish population.

Pseudodeficiency in arylsulphatase A (ASA) is a relatively frequent condition in healthy individuals. It produces a reduction in enzyme activity similar to that found in metachromatic leukodystrophy (MLD). Unambiguous discrimination between the two conditions cannot be achieved through conventional enzyme activity assays. A PCR method has been developed which detects the pseudodeficiency (pd) allele using a single pair of primers encompassing the mutation site and hair follicles as the source of DNA. The frequency of this allele in the Spanish population has been evaluated and correlations between different genotypes and ASA activity levels have been established. Ten out of 55 individuals were heterozygous for the ASA pd allele, while two were homozygous. The allele frequency was thus 12.7%.