Functional diversification of duplicate genes through subcellular adaptation of encoded proteins.

BACKGROUND: Gene duplication is the primary source of new genes with novel or altered functions. It is known that duplicates may obtain these new functional roles by evolving divergent expression patterns and/or protein functions after the duplication event. Here, using yeast (Saccharomyces cerevisiae) as a model organism, we investigate a previously little considered mode for the functional diversification of duplicate genes: subcellular adaptation of encoded proteins. RESULTS: We show that for 24-37% of duplicate gene pairs derived from the S. cerevisiae whole-genome duplication event, the two members of the pair encode proteins that localize to distinct subcellular compartments. The propensity of yeast duplicate genes to evolve new localization patterns depends to a large extent on the biological function of their progenitor genes. Proteins involved in processes with a wider subcellular distribution (for example, catabolism) frequently evolved new protein localization patterns after duplication, whereas duplicate proteins limited to a smaller number of organelles (for example, highly expressed biosynthesis/housekeeping proteins with a slow rate of evolution) rarely relocate within the cell. Paralogous proteins evolved divergent localization patterns by partitioning of ancestral localizations ('sublocalization'), but probably more frequently by relocalization to new compartments ('neolocalization'). We show that such subcellular reprogramming may occur through selectively driven substitutions in protein targeting sequences. Notably, our data also reveal that relocated proteins functionally adapted to their new subcellular environments and evolved new functional roles through changes of their physico-chemical properties, expression levels, and interaction partners. CONCLUSION: We conclude that protein subcellular adaptation represents a common mechanism for the functional diversification of duplicate genes.

Specific duplication and dorsoventrally asymmetric expression patterns of Cycloidea-like genes in zygomorphic species of Ranunculaceae.

Floral bilateral symmetry (zygomorphy) has evolved several times independently in angiosperms from radially symmetrical (actinomorphic) ancestral states. Homologs of the Antirrhinum majus Cycloidea gene (Cyc) have been shown to control floral symmetry in diverse groups in core eudicots. In the basal eudicot family Ranunculaceae, there is a single evolutionary transition from actinomorphy to zygomorphy in the stem lineage of the tribe Delphinieae. We characterized Cyc homologs in 18 genera of Ranunculaceae, including the four genera of Delphinieae, in a sampling that represents the floral morphological diversity of this tribe, and reconstructed the evolutionary history of this gene family in Ranunculaceae. Within each of the two RanaCyL (Ranunculaceae Cycloidea-like) lineages previously identified, an additional duplication possibly predating the emergence of the Delphinieae was found, resulting in up to four gene copies in zygomorphic species. Expression analyses indicate that the RanaCyL paralogs are expressed early in floral buds and that the duration of their expression varies between species and paralog class. At most one RanaCyL paralog was expressed during the late stages of floral development in the actinomorphic species studied whereas all paralogs from the zygomorphic species were expressed, composing a species-specific identity code for perianth organs. The contrasted asymmetric patterns of expression observed in the two zygomorphic species is discussed in relation to their distinct perianth architecture.

Molecular genetics of charcot-marie-tooth disease: from genes to genomes.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders of the peripheral nervous system, mainly characterized by distal muscle weakness and atrophy leading to motor handicap. With an estimated prevalence of 1 in 2,500, this condition is one of the most commonly inherited neurological disorders. Mutations in more than 30 genes affecting glial and/or neuronal functions have been associated with different forms of CMT leading to a substantial improvement in diagnostics of the disease and in the understanding of implicated pathophysiological mechanisms. However, recent data from systematic genetic screening performed in large cohorts of CMT patients indicated that molecular diagnosis could be established only in ∼50-70% of them, suggesting that additional genes are involved in this disease. In addition to providing an overview of genetic and functional data concerning various CMT forms, this review focuses on recent data generated through the use of highly parallel genetic technologies (SNP chips, sequence capture and next-generation DNA sequencing) in CMT families, and the current and future impact of these technologies on gene discovery and diagnostics of CMTs.

Organization of lin genes and IS6100 among different strains of hexachlorocyclohexane-degrading Sphingomonas paucimobilis: evidence for horizontal gene transfer.

The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp+, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.

The JNK binding domain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not the transcription of key proapoptotic or protective genes in insulin-secreting cell lines.

The stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) is a central signal for interleukin-1beta (IL-1beta)-induced apoptosis in insulin-producing beta-cells. The cell-permeable peptide inhibitor of JNK (JNKI1), that introduces the JNK binding domain (JBD) of the scaffold protein islet-brain 1 (IB1) inside cells, effectively prevents beta-cell death caused by this cytokine. To define the molecular targets of JNK involved in cytokine-induced beta-cell apoptosis we investigated whether JNKI1 or stable expression of JBD affected the expression of selected pro- and anti-apoptotic genes induced in rat (RIN-5AH-T2B) and mouse (betaTC3) insulinoma cells exposed to IL-1beta. Inhibition of JNK significantly reduced phosphorylation of the specific JNK substrate c-Jun (p<0.05), IL-1beta-induced apoptosis (p<0.001), and IL-1beta-mediated c-fos gene expression. However, neither JNKI1 nor JBD did influence IL-1beta-induced NO synthesis or iNOS expression or the transcription of the genes encoding mitochondrial manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase rho (GSTrho), heat shock protein (HSP) 70, IL-1beta-converting enzyme (ICE), caspase-3, apoptosis-inducing factor (AIF), Bcl-2 or Bcl-xL. We suggest that the anti-apoptotic effect of JNK inhibition by JBD is independent of the transcription of major pro- and anti-apoptotic genes, but may be exerted at the translational or posttranslational level.

Simultaneous co-expression of memory- and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation

Purpose/Objective: Phenotypic and functional T cell properties are usually analyzed at the level of defined cell populations. However, large differences between individual T cells may have important functional consequences. To answer this issue, we performed highly sensitive single-cell gene expression profiling, which allows the direct ex vivo characterization of individual virus- and tumor-specific T cells from healthy donors and melanoma patients. Materials and methods: HLA-A*0201-positive patients with stage III/ IV metastatic melanoma were included in a phase I clinical trial (LUD- 00-018). Patients received monthly low-dose of the Melan-AMART- 1 26_35 unmodified natural (EAAGIGILTV) or the analog A27L (ELAGIGILTV) peptides, mixed CPG and IFA. Individual effector memory CD28+ (EM28+) and EM28- tetramer-specific CD8pos T cells were sorted by flow cytometer. Following direct cell lysis and reverse transcription, the resulting cDNA was precipitated and globally amplified. Semi-quantitative PCR was used for gene expression and TCR BV repertoire analyses. Results: We have previously shown that vaccination with the natural Melan-A peptide induced T cells with superior effector functions as compared to the analog peptide optimized for enhanced HLA binding. Here we found that natural peptide vaccination induced EM28+ T cells with frequent co-expression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3 and CCR5) and effector-related genes (IFNG, KLRD1, PRF1 and GZMB), comparable to protective EBV- and CMV-specific T cells. In contrast, memory/homing- and effectorassociated genes were less frequently co-expressed after vaccination with the analog peptide. Conclusions: These findings reveal a previously unknown level of gene expression diversity among vaccine- and virus-specific T cells with the simultaneous co-expression of multiple memory/homing- and effector- related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor- and virus-specific T cells.

Circadian clock genes and sleep homeostasis.

Circadian and sleep-homeostatic processes both contribute to sleep timing and sleep structure. Elimination of circadian rhythms through lesions of the suprachiasmatic nuclei (SCN), the master circadian pacemaker, leads to fragmentation of wakefulness and sleep but does not eliminate the homeostatic response to sleep loss as indexed by the increase in EEG delta power. In humans, EEG delta power declines during sleep episodes nearly independently of circadian phase. Such observations have contributed to the prevailing notion that circadian and homeostatic processes are separate but recent data imply that this segregation may not extend to the molecular level. Here we summarize the criteria and evidence for a role for clock genes in sleep homeostasis. Studies in mice with targeted disruption for core circadian clock genes have revealed alterations in circadian rhythmicity as well as changes in sleep duration, sleep structure and EEG delta power. Clock-gene expression in brain areas outside the SCN, in particular the cerebral cortex, depends to a large extent on prior sleep-wake history. Evidence for effects of clock genes on sleep homeostasis has also been obtained in Drosophila and humans, pointing to a phylogenetically preserved pathway. These findings suggest that, while within the SCN clock genes are utilized to set internal time-of-day, in the forebrain the same feedback circuitry may be utilized to track time spent awake and asleep. The mechanisms by which clock-gene expression is coupled to the sleep-wake distribution could be through cellular energy charge whereby clock genes act as energy sensors. The data underscore the interrelationships between energy metabolism, circadian rhythmicity, and sleep regulation.

Genetics for clinicians: from candidate genes to whole genome scans (technological advances).

Human genetics has progressed at an unprecedented pace during the past 10 years. DNA microarrays currently allow screening of the entire human genome with high level of coverage and we are now entering the era of high-throughput sequencing. These remarkable technical advances are influencing the way medical research is conducted and have boosted our understanding of the structure of the human genome as well as of disease biology. In this context, it is crucial for clinicians to understand the main concepts and limitations of modern genetics. This review will describe key concepts in genetics, including the different types of genetic markers in the human genome, review current methods to detect DNA variation, describe major online public databases in genetics, explain key concepts in statistical genetics and finally present commonly used study designs in clinical and epidemiological research. This review will therefore concentrate on human genetic variation analysis.

Genometric analyses of the organization of circular chromosomes: a universal pressure determines the direction of ribosomal RNA genes transcription relative to chromosome replication.

Selective pressures related to gene function and chromosomal architecture are acting on genome sequences and can be revealed, for instance, by appropriate genometric methods. Cumulative nucleotide skew analyses, i.e., GC, TA, and ORF orientation skews, predict the location of the origin of DNA replication for 88 out of 100 completely sequenced bacterial chromosomes. These methods appear fully reliable for proteobacteria, Gram-positives, and spirochetes as well as for euryarchaeotes. Based on this genome architecture information, coorientation analyses reveal that in prokaryotes, ribosomal RNA (rRNA) genes encoding the small and large ribosomal subunits are all transcribed in the same direction as DNA replication; that is, they are located along the leading strand. This result offers a simple and reliable method for circumscribing the region containing the origin of the DNA replication and reveals a strong selective pressure acting on the orientation of rRNA genes similar to the weaker one acting on the orientation of ORFs. Rate of coorientation of transfer RNA (tRNA) genes with DNA replication appears to be taxon-specific. Analyzing nucleotide biases such as GC and TA skews of genes and plotting one against the other reveals a taxonomic clusterization of species. All ribosomal RNA genes are enriched in Gs and depleted in Cs, the only so far known exception being the rRNA genes of deuterostomian mitochondria. However, this exception can be explained by the fact that in the chromosome of the human mitochondrion, the model of the deuterostomian organelle genome, DNA replication, and rRNA transcription proceed in opposite directions. A general rule is deduced from prokaryotic and mitochondrial genomes: ribosomal RNA genes that are transcribed in the same direction as the DNA replication are enriched in Gs, and those transcribed in the opposite direction are depleted in Gs.

Repression of flagellar genes in exponential phase by CsgD and CpxR, two crucial modulators of Escherichia coli biofilm formation.

Escherichia coli adapts its lifestyle to the variations of environmental growth conditions, swapping between swimming motility or biofilm formation. The stationary-phase sigma factor RpoS is an important regulator of this switch, since it stimulates adhesion and represses flagellar biosynthesis. By measuring the dynamics of gene expression, we show that RpoS inhibits the transcription of the flagellar sigma factor, FliA, in exponential growth phase. RpoS also partially controls the expression of CsgD and CpxR, two transcription factors important for bacterial adhesion. We demonstrate that these two regulators repress the transcription of fliA, flgM, and tar and that this regulation is dependent on the growth medium. CsgD binds to the flgM and fliA promoters around their -10 promoter element, strongly suggesting direct repression. We show that CsgD and CpxR also affect the expression of other known modulators of cell motility. We propose an updated structure of the regulatory network controlling the choice between adhesion and motility.

The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane.

Land plants need precise thermosensors to timely establish molecular defenses in anticipation of upcoming noxious heat waves. The plasma membrane-embedded cyclic nucleotide-gated Ca(2+) channels (CNGCs) can translate mild variations of membrane fluidity into an effective heat shock response, leading to the accumulation of heat shock proteins (HSP) that prevent heat damages in labile proteins and membranes. Here, we deleted by targeted mutagenesis the CNGCd gene in two Physcomitrella patens transgenic moss lines containing either the heat-inducible HSP-GUS reporter cassette or the constitutive UBI-Aequorin cassette. The stable CNGCd knockout mutation caused a hyper-thermosensitive moss phenotype, in which the heat-induced entry of apoplastic Ca(2+) and the cytosolic accumulation of GUS were triggered at lower temperatures than in wild type. The combined effects of an artificial membrane fluidizer and elevated temperatures suggested that the gene products of CNGCd and CNGCb are paralogous subunits of Ca(2+)channels acting as a sensitive proteolipid thermocouple. Depending on the rate of temperature increase, the duration and intensity of the heat priming preconditions, terrestrial plants may thus acquire an array of HSP-based thermotolerance mechanisms against upcoming, otherwise lethal, extreme heat waves.

Network-guided analysis of genes with altered somatic copy number and gene expression reveals pathways commonly perturbed in metastatic melanoma.

Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression ('SCNA-genes') in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.

Early maternal investment in mice: no evidence for compatible-genes sexual selection despite hybrid vigor.

Confronting a recently mated female with a strange male can induce a pregnancy block ('Bruce effect'). The physiology of this effect is well studied, but its functional significance is still not fully understood. The 'anticipated infanticide hypothesis' suggests that the pregnancy block serves to avoid the cost of embryogenesis and giving birth to offspring that are likely to be killed by a new territory holder. Some 'compatible-genes sexual selection hypotheses' suggest that the likelihood of a pregnancy block is also dependent on the female's perception of the stud's and the stimulus male's genetic quality. We used two inbred strains of mice (C57BL/6 and BALB/c) to test all possible combinations of female strain, stud strain, and stimulus strain under experimental conditions (N(total) = 241 mated females). As predicted from previous studies, we found increased rates of pregnancy blocks if stud and stimulus strains differed, and we found evidence for hybrid vigour in offspring of between-strain mating. Despite the observed heterosis, pregnancies of within-strain matings were not more likely to be blocked than pregnancies of between-strain matings. A power analysis revealed that if we missed an existing effect (type-II error), the effect must be very small. If a female gave birth, the number and weight of newborns were not significantly influenced by the stimulus males. In conclusion, we found no support for the 'compatible-genes sexual selection hypotheses'.

Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.