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.

RPS4Y gene family evolution in primates

Background: The RPS4 gene codifies for ribosomal protein S4, a very well-conserved protein present in all kingdoms. In primates, RPS4 is codified by two functional genes located on both sex chromosomes: the RPS4X and RPS4Y genes. In humans, RPS4Y is duplicated and the Y chromosome therefore carries a third functional paralog: RPS4Y2, which presents a testis-specific expression pattern. Results: DNA sequence analysis of the intronic and cDNA regions of RPS4Y genes from species covering the entire primate phylogeny showed that the duplication event leading to the second Y-linked copy occurred after the divergence of New World monkeys, about 35 million years ago. Maximum likelihood analyses of the synonymous and non-synonymous substitutions revealed that positive selection was acting on RPS4Y2 gene in the human lineage, which represents the first evidence of positive selection on a ribosomal protein gene. Putative positive amino acid replacements affected the three domains of the protein: one of these changes is located in the KOW protein domain and affects the unique invariable position of this motif, and might thus have a dramatic effect on the protein function.Conclusion: Here, we shed new light on the evolutionary history of RPS4Y gene family, especially on that of RPS4Y2. The results point that the RPS4Y1 gene might be maintained to compensate gene dosage between sexes, while RPS4Y2 might have acquired a new function, at least in the lineage leading to humans.

Genome sequence of the pea aphid Acyrthosiphon pisum

Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems.

Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision.

The elucidation of mechanisms underlying telencephalic neural development has been limited by the lack of knowledge regarding the molecular and cellular aspects of the ganglionic eminence (GE), an embryonic structure that supplies the brain with diverse sets of GABAergic neurons. Here, we report a comprehensive transcriptomic analysis of this structure including its medial (MGE), lateral (LGE) and caudal (CGE) subdivisions and its temporal dynamics in 12.5 to 16 day-old rat embryos. Surprisingly, comparison across subdivisions showed that CGE gene expression was the most unique providing unbiased genetic evidence for its differentiation from MGE and LGE. The molecular signature of the CGE comprised a large set of genes, including Rwdd3, Cyp26b1, Nr2f2, Egr3, Cpta1, Slit3, and Hod, of which several encode cell signaling and migration molecules such as WNT5A, DOCK9, VSNL1 and PRG1. Temporal analysis of the MGE revealed differential expression of unique sets of cell specification and migration genes, with early expression of Hes1, Lhx2, Ctgf and Mdk, and late enrichment of Olfm3, SerpinE2 and Wdr44. These GE profiles reveal new candidate regulators of spatiotemporally governed GABAergic neuronogenesis.

A proteomics approach to decipher the molecular nature of planarian stem cells.

Background In recent years, planaria have emerged as an important model system for research into stem cells and regeneration. Attention is focused on their unique stem cells, the neoblasts, which can differentiate into any cell type present in the adult organism. Sequencing of the Schmidtea mediterranea genome and some expressed sequence tag projects have generated extensive data on the genetic profile of these cells. However, little information is available on their protein dynamics. Results We developed a proteomic strategy to identify neoblast-specific proteins. Here we describe the method and discuss the results in comparison to the genomic high-throughput analyses carried out in planaria and to proteomic studies using other stem cell systems. We also show functional data for some of the candidate genes selected in our proteomic approach. Conclusions We have developed an accurate and reliable mass-spectra-based proteomics approach to complement previous genomic studies and to further achieve a more accurate understanding and description of the molecular and cellular processes related to the neoblasts.

SA2RAGE: a new sequence for fast B1+ -mapping.

At high magnetic field strengths (≥ 3T), the radiofrequency wavelength used in MRI is of the same order of magnitude of (or smaller than) the typical sample size, making transmit magnetic field (B1+) inhomogeneities more prominent. Methods such as radiofrequency-shimming and transmit SENSE have been proposed to mitigate these undesirable effects. A prerequisite for such approaches is an accurate and rapid characterization of the B1+ field in the organ of interest. In this work, a new phase-sensitive three-dimensional B1+-mapping technique is introduced that allows the acquisition of a 64 × 64 × 8 B1+-map in ≈ 20 s, yielding an accurate mapping of the relative B1+ with a 10-fold dynamic range (0.2-2 times the nominal B1+). Moreover, the predominant use of low flip angle excitations in the presented sequence minimizes specific absorption rate, which is an important asset for in vivo B1+-shimming procedures at high magnetic fields. The proposed methodology was validated in phantom experiments and demonstrated good results in phantom and human B1+-shimming using an 8-channel transmit-receive array.

PPAR alpha structure-function relationships derived from species-specific differences in responsiveness to hypolipidemic agents.

The nuclear receptor PPAR alpha is a key regulatory transcription factor in lipid homeostasis, some liver detoxification processes and the control of inflammation. Recent findings suggest that many hypolipidemic drugs and anti-inflammatory agents can potentially act by binding to PPAR alpha and inducing its activity. Here, we identify some structure-function relationships in PPAR alpha, by using the species-specific responsiveness to the two hypolipidemic agents, Wy 14,643 and 5,8,11,14-eicosatetraynoic acid (ETYA). We first show that the species-specific differences are mediated primarily via the ligand binding domain of the receptor and that these two drugs are indeed ligands of PPAR alpha. By mutagenesis analyses we identify amino acid residues in the ligand binding domains of Xenopus, mouse and human PPAR alpha, that confer preferential responsiveness to ETYA and Wy 14,643. These findings will aid in the development of new synthetic PPAR alpha ligands as effective therapeutics for lipid-related diseases and inflammatory disorders.

Comprehensive molecular profiling of lung adenocarcinoma.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.

A genome-to-genome analysis of associations between human genetic variation, HIV-1 sequence diversity, and viral control.

HIV-1 sequence diversity is affected by selection pressures arising from host genomic factors. Using paired human and viral data from 1071 individuals, we ran >3000 genome-wide scans, testing for associations between host DNA polymorphisms, HIV-1 sequence variation and plasma viral load (VL), while considering human and viral population structure. We observed significant human SNP associations to a total of 48 HIV-1 amino acid variants (p<2.4 × 10(-12)). All associated SNPs mapped to the HLA class I region. Clinical relevance of host and pathogen variation was assessed using VL results. We identified two critical advantages to the use of viral variation for identifying host factors: (1) association signals are much stronger for HIV-1 sequence variants than VL, reflecting the 'intermediate phenotype' nature of viral variation; (2) association testing can be run without any clinical data. The proposed genome-to-genome approach highlights sites of genomic conflict and is a strategy generally applicable to studies of host-pathogen interaction. DOI:http://dx.doi.org/10.7554/eLife.01123.001.

Mapping the transcription start points of the Staphylococcus aureus eap, emp, and vwb promoters reveals a conserved octanucleotide sequence that is essential for expression of these genes.

Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp.

Molecular evidence of Pleistocene bidirectional faunal exchange between Europe and the Near East: the case of the bicoloured shrew (Crocidura leucodon, Soricidae).

We sequenced 1077 bp of the mitochondrial cytochrome b gene and 511 bp of the nuclear Apolipoprotein B gene in bicoloured shrew (Crocidura leucodon, Soricidae) populations ranging from France to Georgia. The aims of the study were to identify the main genetic clades within this species and the influence of Pleistocene climatic variations on the respective clades. The mitochondrial analyses revealed a European clade distributed from France eastwards to north-western Turkey and a Near East clade distributed from Georgia to Romania; the two clades separated during the Middle Pleistocene. We clearly identified a population expansion after a bottleneck for the European clade based on mitochondrial and nuclear sequencing data; this expansion was not observed for the eastern clade. We hypothesize that the western population was confined to a small Italo-Balkanic refugium, whereas the eastern population subsisted in several refugia along the southern coast of the Black Sea.

The expansion of amino-acid repeats is not associated to adaptive evolution in mammalian genes.

BACKGROUND: The expansion of amino acid repeats is determined by a high mutation rate and can be increased or limited by selection. It has been suggested that recent expansions could be associated with the potential of adaptation to new environments. In this work, we quantify the strength of this association, as well as the contribution of potential confounding factors. RESULTS: Mammalian positively selected genes have accumulated more recent amino acid repeats than other mammalian genes. However, we found little support for an accelerated evolutionary rate as the main driver for the expansion of amino acid repeats. The most significant predictors of amino acid repeats are gene function and GC content. There is no correlation with expression level. CONCLUSIONS: Our analyses show that amino acid repeat expansions are causally independent from protein adaptive evolution in mammalian genomes. Relaxed purifying selection or positive selection do not associate with more or more recent amino acid repeats. Their occurrence is slightly favoured by the sequence context but mainly determined by the molecular function of the gene.

Changes of the mucosal N3 and N6 fatty acid status occur early in the colorectal adenoma-carcinoma sequence

Despite data favouring a role of dietary fat in colonic carcinogenesis, no study has focused on tissue n3 and n6 fatty acid (FA) status in human colon adenoma-carcinoma sequence. Thus, FA profile was measured in plasma phospholipids of patients with colorectal cancer (n = 22), sporadic adenoma (n = 27), and normal colon (n = 12) (control group). Additionally, mucosal FAs were assessed in both diseased and normal mucosa of cancer (n = 15) and adenoma (n = 21) patients, and from normal mucosa of controls (n = 8). There were no differences in FA profile of both plasma phospholipids and normal mucosa, between adenoma and control patients. There were considerable differences, however, in FAs between diseased and paired normal mucosa of adenoma patients, with increases of linoleic (p = 0.02), dihomogammalinolenic (p = 0.014), and eicosapentaenoic (p = 0.012) acids, and decreases of alpha linolenic (p = 0.001) and arachidonic (p = 0.02) acids in diseased mucosa. A stepwise reduction of eicosapentaenoic acid concentrations in diseased mucosa from benign adenoma to the most advanced colon cancer was seen (p = 0.009). Cancer patients showed lower alpha linolenate (p = 0.002) and higher dihomogammalinolenate (p = 0.003) in diseased than in paired normal mucosa. In conclusion changes in tissue n3 and n6 FA status might participate in the early phases of the human colorectal carcinogenesis.

Sequence homologies within the 5' end region of the estrogen-controlled vitellogenin gene in Xenopus and chicken.

In oviparous vertebrates vitellogenin, the precursor of the major yolk proteins, is synthesized in the liver of mature females under the control of estrogen. We have established the organization and primary structure of the 5' end region of the Xenopus laevis vitellogenin A2 gene and of the major chicken vitellogenin gene. The first three homologous exons have exactly the same length in both species, namely 53, 21 and 152 nucleotides, and present an overall sequence homology of 60%. In both species, the 5'-non-coding region of the vitellogenin mRNA measures only 13 nucleotides, nine of which are conserved. In contrast, the corresponding introns of the Xenopus and the chicken vitellogenin gene show no significant sequence homology. Within the 500 nucleotides preceding the 5' end of the genes, at least six blocks with sequence homologies of greater than 70% were detected. It remains to be demonstrated which of these conserved sequences, if any, are involved in the hormone-regulated expression of the vitellogenin genes.