The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia.

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.

International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors.

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARalpha is expressed at high levels in organs with significant catabolism of fatty acids. PPARbeta/delta has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARgamma is expressed as two isoforms, of which PPARgamma2 is found at high levels in the adipose tissues, whereas PPARgamma1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARalpha and PPARgamma agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

Genetic differentiation and zoogeography of Asian Suncus murinus (Mammalia: Soricidae)

Protein electrophoresis was used to assess the phylogenetic relationships of populations of the phenotypically variable Asian house shrew Suncus murinus. These populations represent a sample of both commensal and wild forms. They were compared to another taxon, S. montanus, which was formerly considered conspecific with S. murinus. Suncus dayi was used as an outgroup in all phylogenetic reconstructions. Within the S. murinus lineage, the allozyme data show very low levels of genetic differentiation among both wild and commensal Southeast Asian and Japanese samples when compared to the Indian populations. This pattern is consistent with the classical hypothesis of a recent introduction by man in Eastern Asia. The higher genetic diversity found within S. murinus from India, as well as previous mitochondrial and karyological results suggest that this area is the probable centre of origin for the species. Although the lack of gene flow between S. murinus and S. montanus is clearly established in an area of sympatry in Southern India, one Asian house shrew sampled in Nepal was more closely related to S. montanus. This could either reflect the retention of an ancestral polymorphism, or result from a hybridization episode between S. murinus and S. montanus. Similar conclusions were also suggested in mitochondrial DNA studies dealing with animals sampled in the Northern parts of the Indian subcontinent. Clearly, further data on Suncus from this area are needed in order to assess these hypotheses. (C) 1995 The Linnean Society of London

Leishmania major: histone H1 gene expression from the sw3 locus.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.

Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa.

The high-affinity siderophore salicylate is an intermediate in the biosynthetic pathway of pyochelin, another siderophore and chelator of transition metal ions, in Pseudomonas aeruginosa. The 2.5-kb region upstream of the salicylate biosynthetic genes pchBA was sequenced and found to contain two additional, contiguous genes, pchD and pchC, having the same orientation. The deduced amino acid sequence of the 60-kDa PchD protein was similar to those of the EntE protein (2,3-dihydroxybenzoate-AMP ligase) of Escherichia coli and other adenylate-forming enzymes, suggesting that salicylate might be adenylated at the carboxyl group by PchD. The 28-kDa PchC protein showed similarities to thioesterases of prokaryotic and eukaryotic origin and might participate in the release of the product(s) formed from activated salicylate. One potential product, dihydroaeruginoate (Dha), was identified in culture supernatants of iron-limited P. aeruginosa cells. The antifungal antibiotic Dha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine. Inactivation of the chromosomal pchD gene by insertion of the transcription and translation stop element omega Sm/Sp abolished the production of Dha and pyochelin, implying that PchD-mediated activation of salicylate may be a common first step in the synthesis of both metabolites. Furthermore, the pchD::omega Sm/Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA genes constitute a transcriptional unit. A full-length pchDCBA transcript of ca. 4.4 kb could be detected in iron-deprived, growing cells of P. aeruginosa. Transcription of pchD started at tandemly arranged promoters, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pchR gene encoding an activator of pyochelin biosynthesis. This promoter arrangement allows tight iron-mediated repression of the pchDCBA operon.

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma.

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.

Trichophyton rubrum secreted and membrane-associated carboxypeptidases

Dermatophytes are the most common agents of superficial mycoses, and exclusively infect stratum corneum, nails or hair. Therefore, secreted proteolytic activity is considered a virulence trait of these fungi. In a medium containing protein as a sole nitrogen and carbon source Trichophyton rubrum secretes a metallocarboxypeptidase (TruMcpA) of the M14 family according to the MEROPS proteolytic enzyme database. TruMcpA is homologous to human pancreatic carboxypeptidase A, and is synthesized as a precursor in a preproprotein form. The propeptide is removed to generate the mature active enzyme alternatively by either one of two subtilisins which are concomitantly secreted by the fungus. In addition, T. rubrum was shown to possess two genes (TruSCPA and TruSCPB) encoding serine carboxypeptidases of the S10 family which are homologues of the previously characterized Aspergillus and Penicillium secreted acid carboxypeptidases. However, in contrast to the Aspergillus and Penicillium homologues, TruScpA and TruScpB enzymes are not secreted into the environment, but are membrane-associated with a glycosylphosphatidylinositol (GPI) anchor. During infection, T. rubrum secreted and GPI-anchored carboxypeptidases may contribute to fungal virulence by cooperating with previously characterized endoproteases and aminopeptidases in the degradation of compact keratinized tissues into assimilable amino acids and short peptides.

GENCODE: producing a reference annotation for ENCODE.

BACKGROUND: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manual annotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results. RESULTS: The GENCODE gene features are divided into eight different categories of which only the first two (known and novel coding sequence) are confidently predicted to be protein-coding genes. 5' rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentally verify the initial annotation. Of the 420 coding loci tested, 229 RACE products have been sequenced. They supported 5' extensions of 30 loci and new splice variants in 50 loci. In addition, 46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15 putative transcripts. We assessed the comprehensiveness of the GENCODE annotation by attempting to validate all the predicted exon boundaries outside the GENCODE annotation. Out of 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only two of them in intergenic regions. CONCLUSION: In total, 487 loci, of which 434 are coding, have been annotated as part of the GENCODE reference set available from the UCSC browser. Comparison of GENCODE annotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained within the two sets, which is a reflection of the high number of alternative splice forms with unique exons annotated. Over 50% of coding loci have been experimentally verified by 5' RACE for EGASP and the GENCODE collaboration is continuing to refine its annotation of 1% human genome with the aid of experimental validation.

How a haemosporidian parasite of bats gets around: the genetic structure of a parasite, vector and host compared.

Parasite population structure is often thought to be largely shaped by that of its host. In the case of a parasite with a complex life cycle, two host species, each with their own patterns of demography and migration, spread the parasite. However, the population structure of the parasite is predicted to resemble only that of the most vagile host species. In this study, we tested this prediction in the context of a vector-transmitted parasite. We sampled the haemosporidian parasite Polychromophilus melanipherus across its European range, together with its bat fly vector Nycteribia schmidlii and its host, the bent-winged bat Miniopterus schreibersii. Based on microsatellite analyses, the wingless vector, and not the bat host, was identified as the least structured population and should therefore be considered the most vagile host. Genetic distance matrices were compared for all three species based on a mitochondrial DNA fragment. Both host and vector populations followed an isolation-by-distance pattern across the Mediterranean, but not the parasite. Mantel tests found no correlation between the parasite and either the host or vector populations. We therefore found no support for our hypothesis; the parasite population structure matched neither vector nor host. Instead, we propose a model where the parasite's gene flow is represented by the added effects of host and vector dispersal patterns.

Localization and fine structure of a vaccinia virus gene encoding an envelope antigen.

The major antigen on the envelope of extracellular vaccinia virus particles is a polypeptide with an apparent molecular weight of 37,000 (p37K; G. Hiller and K. Weber, J. Virol. 55:651-659, 1985). The gene encoding p37K was mapped in the vaccinia virus genome by hybrid selection of RNA followed by in vitro translation. p37K was then identified among the in vitro translation products by immunoprecipitation with a monoclonal antibody. The gene is located close to the right-hand end of the HindIII F fragment. The corresponding region of the DNA was sequenced, and an open reading frame encoding a polypeptide of 41,748 daltons was observed. The 5' end of the mRNA, as defined by nuclease S1 analysis, maps within only a few nucleotides of the translation initiation codon. Examination of the DNA sequence around the putative initiation site of transcription revealed a characteristic sequence, TAAATG, which includes the ATG translation initiation codon and which is conserved in all but one late gene so far analyzed. It is therefore likely that this sequence is an important regulatory signal for late gene expression in vaccinia virus.

Population structure of three Psammodromus species in the Iberian Peninsula.

The knowledge of a species' population structure is essential for the development of adequate conservation actions as well as for the understanding of its evolution. The population structure is unknown in all species of the Genus Psammodromus, including the Western Sand Racer (Psammodromus occidentalis; a recently described species), the Edward's Sand Racer (P. edwardsianus) and the Spanish Sand Racer (P. hispanicus). In this article, the genetic variability and population structure of Psammodromus edwardsianus, P. hispanicus, and P. occidentalis were studied in the Iberian Peninsula covering their natural geographic distribution. Mitochondrial DNA showed genetically different units in all species with higher genetic variability in their southern populations (latitudinal variation). Genetic differentiation was different among species and contrasted to those of species with similar characteristics. Our results therefore highlight the importance of species-specific studies analysing population structure.

Fetal programming and epigenetic mechanisms in arterial hypertension.

PURPOSE OF REVIEW: To provide an overview of available evidence of the potential role of epigenetics in the pathogenesis of hypertension and vascular dysfunction. RECENT FINDINGS: Arterial hypertension is a highly heritable condition. Surprisingly, however, genetic variants only explain a tiny fraction of the phenotypic variation and the term 'missing heritability' has been coined to describe this phenomenon. Recent evidence suggests that phenotypic alteration that is unrelated to changes in DNA sequence (thereby escaping detection by classic genetic methodology) offers a potential explanation. Here, we present some basic information on epigenetics and review recent work consistent with the hypothesis of epigenetically induced arterial hypertension. SUMMARY: New technologies that enable the rigorous assessment of epigenetic changes and their phenotypic consequences may provide the basis for explaining the missing heritability of arterial hypertension and offer new possibilities for treatment and/or prevention.

Introgressive hybridization of threatened European tree frogs (Hyla arborea) by introduced H. intermedia in Western Switzerland

Hybridization by introduced taxa is a major threat to native species. Characterizing human introductions is thus one of the missions of conservation geneticists. Here we survey a declining population of the regionally endangered European tree frog (Hyla arborea) in the Grangettes natural reserve (Rhone valley, Western Switzerland), where previous evidence indicated human introduction of the Italian taxon H. intermedia. We combined fast-evolving mitochondrial and nuclear markers and an extended sampling to conduct population genetic analyses of the Grangettes and putative source areas. We show that the Grangettes population is a hybrid swarm, with all individuals featuring recent nuclear admixture and mitochondrial DNA of introduced H. intermedia, most likely of proximate south Alpine origin. In contrast, H. arborea and H. intermedia hardly introgress in their natural parapatric ranges, consistent with an advanced reproductive isolation. Thus, potential hybrid incompatibilities may account for the strong decline of this population, despite important conservation efforts. Although their hybrid nature makes them a priori unworthy of any protection, we propose specific measures to recover local H. arborea gene pool and preserve tree frogs in the Grangettes, the last population remaining from this heavily impacted part of the Alps.

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml- 1). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

Distribution and population genetic variation of cryptic species of the Alpine mayfly Baetis alpinus (Ephemeroptera: Baetidae) in the Central Alps.

BACKGROUND: Many species contain evolutionarily distinct groups that are genetically highly differentiated but morphologically difficult to distinguish (i.e., cryptic species). The presence of cryptic species poses significant challenges for the accurate assessment of biodiversity and, if unrecognized, may lead to erroneous inferences in many fields of biological research and conservation. RESULTS: We tested for cryptic genetic variation within the broadly distributed alpine mayfly Baetis alpinus across several major European drainages in the central Alps. Bayesian clustering and multivariate analyses of nuclear microsatellite loci, combined with phylogenetic analyses of mitochondrial DNA, were used to assess population genetic structure and diversity. We identified two genetically highly differentiated lineages (A and B) that had no obvious differences in regional distribution patterns, and occurred in local sympatry. Furthermore, the two lineages differed in relative abundance, overall levels of genetic diversity as well as patterns of population structure: lineage A was abundant, widely distributed and had a higher level of genetic variation, whereas lineage B was less abundant, more prevalent in spring-fed tributaries than glacier-fed streams and restricted to high elevations. Subsequent morphological analyses revealed that traits previously acknowledged as intraspecific variation of B. alpinus in fact segregated these two lineages. CONCLUSIONS: Taken together, our findings indicate that even common and apparently ecologically well-studied species may consist of reproductively isolated units, with distinct evolutionary histories and likely different ecology and evolutionary potential. These findings emphasize the need to investigate hidden diversity even in well-known species to allow for appropriate assessment of biological diversity and conservation measures.