Evolutionary and Comparative Genomics to Drive Rational Drug Design, with Particular Focus on Neuropeptide Seven-Transmembrane Receptors

Seven transmembrane receptors (7TMRs), also known as G protein-coupled receptors, are popular targets of drug development, particularly 7TMR systems that are activated by peptide ligands. Although many pharmaceutical drugs have been discovered via conventional bulk analysis techniques the increasing availability of structural and evolutionary data are facilitating change to rational, targeted drug design. This article discusses the appeal of neuropeptide-7TMR systems as drug targets and provides an overview of concepts in the evolution of vertebrate genomes and gene families. Subsequently, methods that use evolutionary concepts and comparative analysis techniques to aid in gene discovery, gene function identification, and novel drug design are provided along with case study examples.

Characterization of the acetohydroxyacid synthase multigene family in the tetraploide plant Chenopodium quinoa

Background Currently, the technology called Clearfield® is used in the development of crops resistant to herbicides that inhibit the enzyme acetohydroxy acid synthase (AHAS, EC 2.2.1.6). AHAS is the first enzyme of the biosynthetic pathway that produces the branched-chain of the essential amino acids valine, leucine, and isoleucine. Therefore, multiple copies of the AHAS gene might be of interest for breeding programs targeting herbicide resistance. In this work, the characterization of the AHAS gene was accomplished for the Chenopodium quinoa Regalona-Baer cultivar. Cloning, sequencing, and Southern blotting were conducted to determine the number of gene copies. Results The presence of multiple copies of the AHAS gene as has been shown previously in several other species is described. Six copies of the AHAS gene were confirmed with Southern blot analyses. CqHAS1 and CqAHAS2 variants showed the highest homology with AHAS mRNA sequences found in the NR Database. A third copy, CqAHAS3, shared similar fragments with both CqAHAS1 and CqAHAS2, suggesting duplication through homeologous chromosomes pairing. Conclusions The presence of multiple copies of the gene AHAS shows that gene duplication is a common feature in polyploid species during evolution. In addition, to our knowledge, this is the first report of the interaction of sub-genomes in quinoa.

Evolution of Rodent Pheromones: A Review of the ABPs with Comparison to the ESPs and the MUPs.

Three proteinaceous pheromone families, the androgen-binding proteins (ABPs), the exocrine-gland secreting peptides (ESPs) and the major urinary proteins (MUPs) are encoded by large gene families in the Mus musculus and Rattus norvegicus genomes. The purpose of this article is to review what is known about the evolutionary histories of the the Abp gene family expansions in rodents and, where appropriate, to compare them to what is known of the expansions of the Mup and Esp gene families. The issues important to these histories are the extent of the gene family expansions, the timing of their expansions and the roles played by selection, gene conversion and non-allelic homologous recombination (NAHR). I also compare and contrast the evolutionary histories of all three mouse gene families in light of the proposed functions of their pheromones in mouse communication.

Clinical Phenotype of Peripheral Myelin Protein 22 Gene Duplication Abnormality in Charcot-Marie-Tooth Disease / 中国康复理论与实践

@#: Objective To analyse the characteristics of symptoms, signs and electrophysiology in Charcot-Marie-Tooth disease (CMT) with peripheral myelin protein 22 (PMP22) gene duplication abnormality.Methods 61 patients with CMT, 14 patients with family history and 47 sporadic patients were included. PMP22 gene duplication fragment was detected with PCR-double enzyme cutting assay. Medical history, signs were collected. Some of them received lumbar puncture and sural nerve pathological examination. Results The main clinical manifestation of the patients with PMP22 gene duplication abnormlity were asthenia of both lower extremities, especially dorsiflexion of foot, accompanied with distal atrophy (especially bilateral legs), some with upper extremity distal atrophy, ankle hyporeflexia or vanished and sensory disturbance. Protein in cerebrospinal fluid may increase, giant potential and conduction velocity of sensory and motor nerve decreased. Sural nerve biopsies revealed demyelination accompanied with axonal degeneration.Conclusion The main clinical manifestation of patients with PMP22 gene duplication abnormlity is charactered as the distal atrophy and asthenia of lower limbs, accompanied with sensory abnormlity. Myelin sheath and axonal alteration were found in electromyogram and peripheral nerve pathology.

Detection of hydin Gene Duplication in Personal Genome Sequence Data

Human personal genome sequencing can be done with high efficiency by aligning a huge number of short reads derived from various next generation sequencing (NGS) technologies to the reference genome sequence. One of the major obstacles is the incompleteness of human reference genome. We tried to analyze the effect of hidden gene duplication on the NGS data using the known example of hydin gene. Hydin2 , a duplicated copy of hydin on chromosome 16q22, has been recently found to be localized to chromosome 1q21, and is not included in the current version of standard human genome reference. We found that all of eight personal genome data published so far do not contain hydin2, and there is large number of nsSNPs in hydin. The heterozygosity of those nsSNPs was significantly higher than expected. The sequence coverage depth in hydin gene was about two fold of average depth. We believe that these unique finding of hydin can be used as useful indicators to discover new hidden multiplication in human genome.

Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes): isolated isoforms and kinetics properties

Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37) and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

Rapid sequence divergence rates in the 5 prime regulatory regions of young Drosophila melanogaster duplicate gene pairs

While it remains a matter of some debate, rapid sequence evolution of the coding sequences of duplicate genes is characteristic for early phases past duplication, but long established duplicates generally evolve under constraint, much like the rest of the coding genome. As for coding sequences, it may be possible to infer evolutionary rate, selection, and constraint via contrasts between duplicate gene divergence in the 5 prime regions and in the corresponding synonymous site divergence in the coding regions. Finding elevated rates for the 5 prime regions of duplicated genes, in addition to the coding regions, would enable statements regarding the early processes of duplicate gene evolution. Here, 1 kb of each of the 5 prime regulatory regions of Drosophila melanogaster duplicate gene pairs were mapped onto one another to isolate shared sequence blocks. Genetic distances within shared sequence blocks (d5’) were found to increase as a function of synonymous (dS), and to a lesser extend, amino-acid (dA) site divergence between duplicates. The rate d5’/dS was found to rapidly decay from values > 1 in young duplicate pairs (dS < 0.3) to 0.28 or less in older duplicates (dS > 0.8). Such rapid rates of 5 prime evolution exceeding 1 (~neutral) predominantly were found to occur in duplicate pairs with low amino-acid site divergence and that tended to be co-regulated when assayed on microarrays. Conceivably, functional redundancy and relaxation of selective constraint facilitates subsequent positive selection on the 5 prime regions of young duplicate genes. This might promote the evolution of new functions (neofunctionalization) or division of labor among duplicate genes (subfunctionalization). In contrast, similar to the vast portion of the non-coding genome, the 5 prime regions of long-established gene duplicates appear to evolve under selective constraint, indicating that these long-established gene duplicates have assumed critical functions.

Management of Upper Moiety in Complete Duplex System / 대한비뇨기과학회지

PURPOSE: Our experience of upper moiety in the complete duplex system was retrospectively analyzed to determine its optimum management. MATERIALS AND METHODS: Between 1988 and 2003, 27 patients were treated with the complete duplex system. Fifteen patients had ureterocele (9 intravesical, 6 ectopic) and the other 12 had an ectopic ureter. In all cases, excretory urography, ultrasonography, voiding cysto-urethrogram (VCUG) and dimercaptosuccinic acid (DMSA) renal scan were performed. The initial treatment was performed using salvage (transurethral incision or ureteropyelostomy) or non-salvage procedures (upper pole nephrectomy or nephrectomy). The median follow-up was 30 (13-48) months. RESULTS: The 27 patients were divided into three groups based on the function of the upper moiety from the DMSA renal scan - or=15%; 11 patients (group C). In group A, upper pole nephrectomy was performed in 9 patients, a total reconstruction in 3 and a nephrectomy in 1. In group B, an upper pole nephrectomy was initially performed, with an ureteropyelostomy and transurethral incision (TUI). In group C, all patients received a transurethral incision as the initial treatment. The ipsilateral renal function was well conserved in the cases of upper pole nephrectomy, with no complications. Patients initially receiving salvage procedures showed a significant improvement and conservation of the ipsilateral renal function, but 4 patients required additional operative management due to moderate to severe vesicoureteral reflux (VUR), recurrent urinary tract infection and decreased renal function of the upper moiety. CONCLUSIONS: Salvage procedures are a preferable adequate therapeutic modality for the complete duplex system with a well conserved renal function.

Cloning of Squalene Synthase Gene from Saccharomyces cerevisiae and Construction of Yeast Expression Vector / 广州中医药大学学报

Objective To lay the foundation for studying the synthesis of artemisinin in microorganism,squalene synthase(SS) gene,a key enzyme gene from Saccharomyces cerevisiae,was cloned and a yeast expression vector was constructed.Methods After amplification of SS gene by polymerase chain reaction(PCR),ligation to T-vector and analysis of the cloned sequence,enzyme digestion and reconfirmation of the target gene,the antisense yeast expression vector was constructed by inverted insertion of the target gene into a yeast expression vector,pGAPZ?A,and digested with two restriction enzymes for vertifying the recombinant.Results The length of SS gene was 1335bp.The preliminary sequence data indicated that SS gene obtained from the experiment had a high sequence homology with that from GenBank,except for a few base pairs.The antisense yeast expression vector has been constructed and vertified by digesting with two enzymes.Conclusion SS gene from Saccharomyces cerevisiae has been successfully cloned and sequenced.An antisense yeast expression vector has been also constructed.

Cloning of Human Ro60 cDNA and Construction of Baculovirus Expression Vector / 中华皮肤科杂志

Objective To construct a baculovirus expression vector for expression of human Ro60 cDNA. Methods Ro60 cDNA was cloned from Hela cell RNA with RT-PCR and was inserted into plasmid pFastBac HTc. Plasmid pFastBac HTc-Ro60 was then transformed into E. coli DH10Bac competent cells for transposition. The constructed recombinant was determined by both blue-white colony screening and PCR analysis. Results A 1.56kb Ro60 cDNA was obtained from Hela cell RNA and was successfully inserted to plasmid pFastBac HTc. DNA sequencing and restriction enzyme analysis revealed that both open reading frame and sequence of the cDNA were identical to Ro60 sequence previously published. Specific transposition and virus recombination were obtained in the vector Bacmid-Ro60. Conclusion Ro60 cDNA has been successfully cloned and constructed to baculovirus expression vector, which is useful for protein expression and further study of this autoantigen in the pathogenesis of lupus erythematosus.