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Possible steps to the emergence of life: the [GADV]-protein world hypothesis.

Based on the fact that RNA has not only a genetic function but also a catalytic function, the RNA world theory on the origin of life was first proposed about 20 years ago. The theory assumes that RNA was amplified by self-replication to increase RNA diversity on the primitive earth. Since then, the theory has been widely accepted as the most likely explanation for the emergence of life. In contrast, we reached another hypothesis, the [GADV]-protein world hypothesis, which is based on pseudo-replication of [GADV]-proteins. We reached this hypothesis during studies on the origins of genes and the genetic code, where [G], [A], [D], and [V] refer to Gly, Ala, Asp, and Val, respectively. In this review, possible steps to the emergence of life are discussed from the standpoint of the [GADV]-protein world hypothesis, comparing it in parallel with the RNA world theory. It is also shown that [GADV]-peptides, which were produced by repeated dry-heating cycles and by solid phase peptide synthesis, have catalytic activities, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. These experimental results support the [GADV]-protein world hypothesis for the origin of life.

Alanine↗

A dopamine transporter gene functional variant associated with cocaine abuse in a Brazilian sample.

The dopamine (DA) transporter DAT1 is a major target bound by cocaine in brain. We examined the influence of functional genetic variants in DAT1 on cocaine addiction. Repeat polymorphisms, including a 30-bp variable-number tandem repeat (VNTR) in intron 8 (Int8 VNTR) with two common alleles, were genotyped in cocaine-dependent abusers (n = 699) and in controls with no past history of drug abuse (n = 866) from São Paulo, Brazil. Positive association was observed with allele 3 of the Int8 VNTR and cocaine abuse (allele odds ratio = 1.2, 95% confidence interval = 1.01-1.37, P = 0.036; 3/3 homozygote odds ratio = 1.45, 95% confidence interval = 1.18-1.78, P = 0.0008). Population stratification was assessed and did not affect the results. Haplotypic analyses using additional polymorphisms indicated that the Int8 VNTR is responsible for the observed association. Functional analyses in reporter-gene constructs, demonstrated that allele 3 mediates significant (P < 0.05) but small reduced expression compared with the "protective" allele 2. This difference increased when 1 and 10 muM cocaine was added to the cell culture ( approximately 40% reduction of the 3 allele expression versus the 2 allele). The 3 allele also demonstrated approximately 3-fold-increased expression over the 2 allele in response to KCl plus forskolin challenge. We demonstrate a robust association between cocaine dependence and a VNTR allele in SLC6A3, conferring a small but detectable effect, and we show that this VNTR may be functional. This study suggests that DAT1 gene variation may play a role in cocaine dependence etiology.

Adolescent↗

Molecular genetics of long QT syndrome from genes to patients.

Recently, there has been intense excitement in the field of cardiac arrhythmias. Molecular genetic studies have led to significant progress in characterizing molecular mechanisms underlying long QT syndrome, an inherited cardiac disorder that causes syncope, seizures, and sudden death from ventricular arrhythmias. Three long QT syndrome genes have been identified: SCN5A on 3p21-24, HERG on 7q35-36, and KVLQT1 on 11p15.5; all encode cardiac mycote ion channels. Molecular and electrophysiological characterization of these three long QT syndrome genes has led to identification of three critical electrical currents in the human heart (INa, IKr, IKa) and provides insight into our fundamental understanding of cardiac function. Genetic testing and gene-specific therapies are now available for some families with long QT syndrome.

Chromosome Mapping↗

In vivo genome-wide CRISPR screens identify FOXR1 as a suppressor of CD8+ T cell antitumor immunity.

T cell dysfunction critically limits the efficacy of T cell-based immunotherapies in solid tumors, yet the intrinsic regulators of T cell dysfunction remain incompletely understood. Through an in vivo genome-wide CRISPR screen in tumor-infiltrating CD8+ T cells, we identified Forkhead Box R1 (FOXR1) as a potent transcriptional suppressor of CD8+ T cell effector functions. Genetic ablation of FOXR1 significantly enhanced cytokine production and cytotoxic capacity in both murine and human CD8+ T cells, whereas its overexpression impaired T cell activation and effector molecule expression. Mechanistically, multiomics integration of RNA-seq, CUT&Tag-seq, and ATAC-seq revealed that FOXR1 binds directly to promoter regions of key effector genes, including IL2, GZMB, and PRF1, and represses their expression. Importantly, FOXR1 deletion in human anti-CD19 CAR T cells improved their efficacy against solid tumors, demonstrating that FOXR1 is a checkpoint of T cell effector function and targeting FOXR1 is a promising strategy to enhance CAR T cell efficacy against solid tumors.

Animals↗

Organization of phenylalanine ammonia lyase (PAL), acidic PR-5 and osmotin-like (OSM) defence-response gene families in the potato genome.

Defence-response (DR) genes are candidates for the genetic functions underlying quantitative resistance to plant pathogens. The organization of three DR gene families encoding phenylalanine ammonia lyase (PAL), acidic PR-(pathogenesis-related) protein 5, and basic PR-5, or osmotin-like (OSM), proteins was studied in the potato genome. A bacterial artificial chromosome (BAC) library containing approximately 50,000 clones was constructed from high-molecular weight genomic DNA of the diploid potato clone PD59, a hybrid between Solanum tuberosum and S. phureja. BAC clones carrying one or more copies of the DR genes were identified and characterized by Southern hybridization, sequence analysis and genetic mapping. PAL, acidic PR-5 and OSM (basic PR-5) genes were all organized into gene families of varying complexity. The PAL gene family consisted of at least 16 members, several of which were physically linked. Four acidic PR-5 homologous were localized to a 45-kb segment on potato chromosome XII. One of these, PR-5/319, codes for the acidic thaumatin-like protein C found in intercellular fluids of potato. Nine OSM genes were organized at two loci: eight form a 90-kb cluster on chromosome VIII, and a single gene was found on chromosome XI. The topology of a phylogenetic tree based on PR-5 and OSM protein sequences from Solanaceae suggests a mode of evolution for these gene families. The results will form the basis for further studies on the potential role of these defence-related loci in quantitative resistance to pathogens.

Amino Acid Sequence↗

Eating disorders, serotonin transporter polymorphisms and potential treatment response.

Anorexia nervosa, bulimia nervosa, and binge eating disorder are eating disorders with common clinical and psychological features, potentially shared mechanisms, significant morbidity and, at least for anorexia nervosa, a high mortality rate. Among the numerous risk factors involved, the importance of a genetic vulnerability has been demonstrated, and the heritability, in the broad sense, has being estimated to be between 50 and 70%. Studies have thus focused on different candidate genes. Serotonin transmission and regulation has been extensively studied with regard to its role in core mechanisms such as feeding and fasting, but also in different clinical characteristics of eating disorders. The serotonin transporter (5-HTT), encoded by the SLC6A4 gene, may also have an important role in eating disorders, as its availability is decreased in patients with bulimia nervosa and binge eating disorder. The promoter region contains a functional insertion/deletion polymorphism with two common alleles that have been designated the short (*S) and long (*L) alleles. The frequency of the SLC6A4*S allele has been assessed in four independent samples of patients with anorexia nervosa, but gave discrepant results. A meta-analysis was performed, which showed that the *S allele could represent a moderate but significant risk factor that increases the risk of anorexia nervosa (odds ratio [OR] = 1.38, 95% confidence interval [CI] 1.16-1.72). Eating disorders are treated using different types of psychotherapy and pharmacotherapy with antidepressants; serotonin reuptake inhibitors being the most frequently prescribed. High doses of selective serotonin reuptake inhibitors (SSRIs) are usually prescribed in eating disorders. The prevalence of non-responders (roughly one out of two), and the presence of a functional genetic polymorphism in the promotor region of SLC6A4, emphasizes the potential utility of psychopharmacogenetics in prescribing SSRIs in the treatment of patients with weight-restored anorexia nervosa. Information about genetic variations of cytochrome P450 could also facilitate pharmacotherapy by preventing the administration of high doses in poor metabolizers and identify rapid metabolizes who may require higher doses for efficacy. SLC6A4 genotyping would allow physicians to individualize selective serotonin reuptake therapy for their patients.

Carrier Proteins↗

Characteristics of the nopaline catabolic plasmid in Agrobacterium strains K84 and K1026 used for biological control of crown gall disease.

Wild-type Agrobacterium radiobacter strain 84 and its Tra- derivative K1026, used for biological control of crown gall disease, each contain the plasmid pAtK84b. It confers incompatibility to tumor-inducing (Ti) plasmids of pathogenic A. tumefaciens, thus preventing transfer of Ti plasmids into K84 and K1026, and the consequent development of pathogens resistant to the specific antibiotic, agrocin 84 produced by K84 and K1026. pAtK84b also resembles one group of Ti plasmids in its capacity for directing nopaline catabolism. A study of the DNA homology among pAtK84b, pTiC58, and pTiAch5 was carried out. pAtK84b was transferred by conjugation to a plasmidless recipient and, after isolation, was hybridized with Ti plasmid DNA. Areas of DNA homology were located on published maps of pTiC58 and pTiAch5, a restriction enzyme map of pAtK84b was constructed, and areas of homology with DNA of known genetic function were located on the map. Strong and extensive (over 50%) homology was found between pAtK84b and pTiC58 (nopaline catabolic, Noc), but much less between pAtK84b and pTiAch5 (octopine catabolic). There was no detectable homology between pAtK84b and the oncogenic T-DNA and virulence (Vir) regions of either Ti plasmid. The size of pAtK84b was 173 kb and the orientation of regions of identified gene function (Noc, incompatability/origin of replication, and conjugal transfer) on pTiC58 was matched by the locations of homologous areas on pAtK84b. It is concluded that pAtK84b may be a deletion product of a pTiC58-type plasmid which has been disarmed in the oncogenic T-DNA and Vir regions.

Arginine↗

AppA, a redox regulator of photosystem formation in Rhodobacter sphaeroides 2.4.1, is a flavoprotein. Identification of a novel fad binding domain.

The AppA protein is required for increased photosystem gene expression upon transition of the facultatively photoheterotrophic bacterium Rhodobacter sphaeroides 2.4.1 from aerobic to anaerobic photosynthetic conditions. AppA shows no obvious similarity to proteins with established function. Genetic evidence suggests that its effect is exerted through modulation of the activity of the repressor PpsR, which controls expression of multiple photosystem genes. To gain insight into the nature of AppA involvement in redox-dependent photosystem gene expression, the appA gene was overexpressed in Escherichia coli. AppA was produced as insoluble inclusion bodies. The purified inclusion bodies were found to contain FAD. By overexpressing various deletion derivatives, we were able to localize the region of AppA sufficient for FAD binding to approximately 120 amino-terminal residues. To assess the role of FAD binding in AppA function, we constructed an AppA derivative lacking the entire FAD binding domain. Surprisingly, this derivative complemented the AppA null mutant undergoing transition from aerobic to anaerobic photosynthetic growth conditions almost to the same extent as the full-length AppA protein. When the sequence of the amino-terminal portion of AppA was examined, it was shown not to contain any known flavin binding motifs. However, two open reading frames of unknown function, showing significant similarity to the amino terminus of AppA, were identified, i.e. Synechocystis sp. Srl1694 and E. coli F403. The latter gene was amplified and overexpressed in E. coli, and the partially purified F403 protein was found to contain FAD as a cofactor. We have therefore concluded that the amino terminus of AppA represents a novel FAD binding domain present in a small group of bacterial proteins. The binding of FAD by AppA may be the first clue as to how this regulatory protein is involved in redox-regulated reactions.

Amino Acid Sequence↗

Genetic characterization of Mu-like bacteriophage D108.

Infection of Escherichia coli by bacteriophage D108 was shown to result in the generation of apparently random chromosomal mutations. Approximately 1% of the cells lysogenized by D108, as with Mu, acquired new auxotrophic mutations. D108-induced mutations were nonreverting and were most probably the result of insertion of the D108 genome into regions of genetic function. D108 and Mu shared many similar properties but were heteroimmune and had different host ranges. Lytic infections of Mu lysogens with D108 and D108 lysogens with Mu resulted in 100-fold increases in release of phage with prophage markers over those due to spontaneous induction. Phenotypic mixing was common, with most phage carrying the prophage immunity being packaged in particles with the host range of the superinfecting phage. A fraction of the superinfecting phage genomes were, however, packaged in particles with the prophage-specified host range. Although 10% of the prophage progeny were D108-Mu genetic hybrids, superinfecting phage-induced release of the prophage with reciprocal phenotypic mixing occurred in recA hosts, in which the frequency of D108-Mu genetic hybrids was reduced 100-fold.

Coliphages↗

Seeking completeness in bacterial mutant hunts.

The identification of most or all of the genetic functions that are required for a particular biological process could be achieved through phenotypic studies of high genome-coverage mutant collections. Technologies for creating such collections, in the form of mixed populations or individually arrayed sequence-defined mutants, are now available for numerous bacterial species. The analysis of mixed mutant collections using microarray-based detection procedures appears to be particularly effective in identifying functions required for complex processes such as virulence. The phenotypic analysis of sequence-defined mutant libraries provides a virtually complete identification of nonessential genes required for processes for which suitable screens can be devised. Such libraries also serve as a source of individual mutants for examining the biological relevance of gene associations revealed by transcriptional profiling or homology.

Bacteria↗

Prediction and measurement of an autoregulatory genetic module.

The deduction of phenotypic cellular responses from the structure and behavior of complex gene regulatory networks is one of the defining challenges of systems biology. This goal will require a quantitative understanding of the modular components that constitute such networks. We pursued an integrated approach, combining theory and experiment, to analyze and describe the dynamics of an isolated genetic module, an in vivo autoregulatory gene network. As predicted by the model, temperature-induced protein destabilization led to the existence of two expression states, thus elucidating the trademark bistability of the positive feedback-network architecture. After sweeping the temperature, observed population distributions and coefficients of variation were in quantitative agreement with those predicted by a stochastic version of the model. Because model fluctuations originated from small molecule-number effects, the experimental validation underscores the importance of internal noise in gene expression. This work demonstrates that isolated gene networks, coupled with proper quantitative descriptions, can elucidate key properties of functional genetic modules. Such an approach could lead to the modular dissection of naturally occurring gene regulatory networks, the deduction of cellular processes such as differentiation, and the development of engineered cellular control.

Cell Differentiation↗

Voltage-gated calcium channels as targets for the treatment of chronic pain.

This review focuses on the importance of voltage-gated calcium channels in modulating and controlling the function of peripheral and central neurons involved in nociceptive processing. We describe the different families of voltage-gated calcium channels that are expressed in pain pathway neurons, how the expression levels of calcium channel currents change in chronic pain conditions, and the validation of N-type, T-type, and P-type calcium channels as targets for the treatment of pain. The molecular mechanism of action is reviewed for the most prominent calcium channel-targeted drugs including gabapentin and ziconotide as well as antiepileptics administered off-label for the treatment of pain. We discuss how the major genetic, functional, and pharmacological differences between subtypes of neuronal calcium channels can be leveraged to identify new molecular targets and to discover and develop new therapeutic agents for the treatment of chronic pain syndromes.

Afferent Pathways↗

Genetic analysis of the proximal region of chromosome 2 of Drosophila melanogaster. I. Detachment products of compound autosomes.

To examine the genetic composition of proximal heterochromatin in chromosome 2, the detachment of compound second autosomes, for generating proximal deficiencies, appeared a promising method. Compound seconds were detached by gamma radiation. A fraction of the detachment products were recessive lethals owing to proximal deficiencies. Analysis by inter se complementation, pseudo-dominance tests with proximal mutations and allelism tests with known deficiencies provided evidence for at least two loci between the centromere and the light locus in 2L and one locus in 2R between the rolled locus and the centromere. The data further demonstrate that rolled, and probably light, are located within the proximal heterochromatin. Thus, functional genetic loci are found in heterochromatin, albeit at low density.

Alleles↗

Genetic synthetic lethality screen at the single gene level in cultured human cells.

Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.

Flow Cytometry↗

Potent metalloporphyrin peroxynitrite decomposition catalyst protects against the development of doxorubicin-induced cardiac dysfunction.

BACKGROUND: Increased oxidative stress and dysregulation of nitric oxide have been implicated in the cardiotoxicity of doxorubicin (DOX), a commonly used antitumor agent. Peroxynitrite is a reactive oxidant produced from nitric oxide and superoxide in various forms of cardiac injury. Using a novel metalloporphyrinic peroxynitrite decomposition catalyst, FP15, and nitric oxide synthase inhibitors or knockout mice, we now delineate the pathogenetic role of peroxynitrite in rodent models of DOX-induced cardiac dysfunction. METHODS AND RESULTS: Mice received a single injection of DOX (25 mg/kg IP). Five days after DOX administration, left ventricular performance was significantly depressed, and high mortality was noted. Treatment with FP15 and an inducible nitric oxide synthase inhibitor, aminoguanidine, reduced DOX-induced mortality and improved cardiac function. Genetic deletion of the inducible nitric oxide synthase gene was also accompanied by better preservation of cardiac performance. In contrast, inhibition of the endothelial isoform of nitric oxide synthase with N-nitro-L-arginine methyl ester increased DOX-induced mortality. FP15 reduced the DOX-induced increase in serum LDH and creatine kinase activities. Furthermore, FP15 prevented the DOX-induced increase in lipid peroxidation, nitrotyrosine formation, and metalloproteinase activation in the heart but not NAD(P)H-driven superoxide generation. Peroxynitrite neutralization did not interfere with the antitumor effect of DOX. FP15 also decreased ischemic injury in rats and improved cardiac function and survival of mice in a chronic model of DOX-induced cardiotoxicity. CONCLUSIONS: Thus, peroxynitrite plays a key role in the pathogenesis of DOX-induced cardiac failure. Targeting peroxynitrite formation may represent a new cardioprotective strategy after DOX exposure or in other conditions associated with peroxynitrite formation, including myocardial ischemia/reperfusion injury.

Acute Disease↗

Histone H4 and the maintenance of genome integrity.

The normal progression of Saccharomyces cerevisiae through nuclear division requires the function of the amino-terminal domain of histone H4. Mutations that delete the domain, or alter 4 conserved lysine residues within the domain, cause a marked delay during the G2+M phases of the cell cycle. Site-directed mutagenesis of single and multiple lysine residues failed to map this phenotype to any particular site; the defect was only observed when all four lysines were mutated. Starting with a quadruple lysine-to-glutamine substitution allele, the insertion of a tripeptide containing a single extra lysine residue suppressed the G2+M cell cycle defect. Thus, the amino-terminal domain of histone H4 has novel genetic functions that depend on the presence of lysine per se, and not a specific primary peptide sequence. To determine the nature of this function, we examined H4 mutants that were also defective for G2/M checkpoint pathways. Disruption of the mitotic spindle checkpoint pathway had no effect on the phenotype of the histone amino-terminal domain mutant. However, disruption of RAD9, which is part of the pathway that monitors DNA integrity, caused precocious progression of the H4 mutant through nuclear division and increased cell death. These results indicate that the lysine-dependent function of histone H4 is required for the maintenance of genome integrity, and that DNA damage resulting from the loss of this function activates the RAD9-dependent G2/M checkpoint pathway.

Amino Acid Sequence↗

Variant alleles of TGFB1 and TGFBR2 are associated with a decreased risk of gastric cancer in a Chinese population.

Growing evidence suggests that the transforming growth factor beta (TGF-beta) signaling pathway occupies a central position in the signaling networks that control cell growth and differentiation. TGF-beta1 and its receptor TGF-betaRII have been correlated with the development of certain forms of cancer, including gastric cancer. We hypothesized that functional genetic variants in TGFB1 and TGFBR2 are associated with gastric cancer risk. To test this hypothesis, we genotyped C-509T and Leu10Pro polymorphisms in TGFB1 and G-875A variant in TGFBR2, using the primer-introduced restriction analysis (PIRA)-PCR assay, in a case-control study of 675 gastric cancer cases and 704 healthy controls in a Chinese population. We found that the variant alleles of the promoter polymorphisms, TGFB1 C-509T and TGFBR2 G-875A, were associated with a significantly decreased risk of gastriccancer [adjusted odds ratio (OR) = 0.65, 95% confidence interval (CI) = 0.52-0.82 for -509CT/TT and adjusted OR = 0.67, 95% CI = 0.53-0.85 for -875GA/AA]. Furthermore, subjects with both variant genotypes of the TGFB1 C-509T and TGFBR2 G-875A were associated with a significantly (56%) decreased risk of gastric cancer (adjusted OR = 0.44, 95% CI = 0.32-0.62). These findings indicate, for the first-time, that the functional variants in the promoter of TGFB1 and TGFBR2 might contribute to gastric cancer susceptibility.

Alleles↗

Phylogeny of related functions: the case of polyamine biosynthetic enzymes.

Genome annotation requires explicit identification of gene function. This task frequently uses protein sequence alignments with examples having a known function. Genetic drift, co-evolution of subunits in protein complexes and a variety of other constraints interfere with the relevance of alignments. Using a specific class of proteins, it is shown that a simple data analysis approach can help solve some of the problems posed. The origin of ureohydrolases has been explored by comparing sequence similarity trees, maximizing amino acid alignment conservation. The trees separate agmatinases from arginases but suggest the presence of unknown biases responsible for unexpected positions of some enzymes. Using factorial correspondence analysis, a distance tree between sequences was established, comparing regions with gaps in the alignments. The gap tree gives a consistent picture of functional kinship, perhaps reflecting some aspects of phylogeny, with a clear domain of enzymes encoding two types of ureohydrolases (agmatinases and arginases) and activities related to, but different from ureohydrolases. Several annotated genes appeared to correspond to a wrong assignment if the trees were significant. They were cloned and their products expressed and identified biochemically. This substantiated the validity of the gap tree. Its organization suggests a very ancient origin of ureohydrolases. Some enzymes of eukaryotic origin are spread throughout the arginase part of the trees: they might have been derived from the genes found in the early symbiotic bacteria that became the organelles. They were transferred to the nucleus when symbiotic genes had to escape Muller's ratchet. This work also shows that arginases and agmatinases share the same two manganese-ion-binding sites and exhibit only subtle differences that can be accounted for knowing the three-dimensional structure of arginases. In the absence of explicit biochemical data, extreme caution is needed when annotating genes having similarities to ureohydrolases.

Amino Acid Sequence↗