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.

The polymorphic nature of HIV type 1 env V4 affects the patterns of potential N-glycosylation sites in proviral DNA at the intrahost level.

We have previously shown that env V4 from HIV-1 plasma RNA is highly heterogeneous within a single patient, due to indel-associated polymorphism. In this study, we have analyzed the variability of V4 in proviral DNA from unfractionated PBMC and sorted T and non-T cell populations within individual patients. Our data show that the degree of sequence variability and length polymorphism in V4 from HIV provirus is even higher than we previously reported in plasma. The data also show that the sequence of V4 depends largely on the experimental approach chosen. We could observe no clear trend for compartmentalization of V4 variants in specific cell types. Of interest is the fact that some variants that had been found to be predominant in plasma were not detected in any of the cell subsets analyzed. Consistently with our observations in plasma, V3 was found to be relatively conserved at both interpatient and intrapatient level. Our data show that V4 polymorphism involving insertions and deletions in addition to point mutations results in changes in the patterns of sequons in HIV-1 proviral DNA as well as in plasma RNA. These rearrangements may result in the coexistence, within the same individual, of a swarm of different V4 regions, each characterized by a different carbohydrate surface shield. Further studies are needed to investigate the mechanism responsible for the variability observed in V4 and its role in HIV pathogenesis.

Familial evaluation in catecholaminergic polymorphic ventricular tachycardia: disease penetrance and expression in cardiac ryanodine receptor mutation-carrying relatives.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with mutations in the cardiac ryanodine receptor gene (Ryr2) in the majority of patients. Previous studies of CPVT patients mainly involved probands, so current insight into disease penetrance, expression, genotype-phenotype correlations, and arrhythmic event rates in relatives carrying the Ryr2 mutation is limited. METHODS AND RESULTS: One-hundred sixteen relatives carrying the Ryr2 mutation from 15 families who were identified by cascade screening of the Ryr2 mutation causing CPVT in the proband were clinically characterized, including 61 relatives from 1 family. Fifty-four of 108 antiarrhythmic drug-free relatives (50%) had a CPVT phenotype at the first cardiological examination, including 27 (25%) with nonsustained ventricular tachycardia. Relatives carrying a Ryr2 mutation in the C-terminal channel-forming domain showed an increased odds of nonsustained ventricular tachycardia (odds ratio, 4.1; 95% CI, 1.5-11.5; P=0.007, compared with N-terminal domain) compared with N-terminal domain. Sinus bradycardia was observed in 19% of relatives, whereas other supraventricular dysrhythmias were present in 16%. Ninety-eight (most actively treated) relatives (84%) were followed up for a median of 4.7 years (range, 0.3-19.0 years). During follow-up, 2 asymptomatic relatives experienced exercise-induced syncope. One relative was not being treated, whereas the other was noncompliant. None of the 116 relatives died of CPVT during a 6.7-year follow-up (range, 1.4-20.9 years). CONCLUSIONS: Relatives carrying an Ryr2 mutation show a marked phenotypic diversity. The vast majority do not have signs of supraventricular disease manifestations. Mutation location may be associated with severity of the phenotype. The arrhythmic event rate during follow-up was low.

Colour-polymorphic snake species are older

Many characteristics, for example life-history traits, physiological tolerance to heat and cold, and energy requirements, contribute to a population's ability to persist in the face of climatic variation. Recent studies have suggested that the presence of intraspecific colour polymorphism could be another potential contributor to population resilience (e.g. to climate change) in ectothermic vertebrates such as reptiles. In the present study, we tested for a relationship between the presence of intraspecific colour polymorphism and the age of snake species. Using phylogenetic comparative methods, we demonstrate that the presence of intraspecific colour polymorphism is correlated with the age of a species, with polymorphic snake species being significantly older than monomorphic species. Understanding how species have dealt with past environmental modifications, such as climate change, can provide important insights into how they are likely to respond in the future to ongoing climate warming.

Sudden death: ethical and legal problems of post-mortem forensic genetic testing for hereditary cardiac diseases.

Hereditary non-structural diseases such as catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT, and the Brugada syndrome as well as structural disease such as hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) cause a significant percentage of sudden cardiac deaths in the young. In these cases, genetic testing can be useful and does not require proxy consent if it is carried out at the request of judicial authorities as part of a forensic death investigation. Mutations in several genes are implicated in arrhythmic syndromes, including SCN5A, KCNQ1, KCNH2, RyR2, and genes causing HCM. If the victim's test is positive, this information is important for relatives who might be themselves at risk of carrying the disease-causing mutation. There is no consensus about how professionals should proceed in this context. This article discusses the ethical and legal arguments in favour of and against three options: genetic testing of the deceased victim only; counselling of relatives before testing the victim; counselling restricted to relatives of victims who tested positive for mutations of serious and preventable diseases. Legal cases are mentioned that pertain to the duty of geneticists and other physicians to warn relatives. Although the claim for a legal duty is tenuous, recent publications and guidelines suggest that geneticists and others involved in the multidisciplinary approach of sudden death (SD) cases may, nevertheless, have an ethical duty to inform relatives of SD victims. Several practical problems remain pertaining to the costs of testing, the counselling and to the need to obtain permission of judicial authorities.

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.