Gene expression defines natural changes in mammalian lifespan.

Mammals differ more than 100-fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney, and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life-history traits, and identified the processes and pathways involved. These findings provide direct insights into how nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages.

Genome sequencing reveals insights into physiology and longevity of the naked mole rat.

The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the 'queen', who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat's exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.

Association between common variation in genes encoding sweet taste signaling components and human sucrose perception.

Variation in taste perception of different chemical substances is a well-known phenomenon in both humans and animals. Recent advances in the understanding of sweet taste signaling have identified a number of proteins involved in this signal transduction. We evaluated the hypothesis that sequence variations occurring in genes encoding taste signaling molecules can influence sweet taste perception in humans. Our population consisted of unrelated individuals (n = 160) of Caucasian, African-American, and Asian descent. Threshold and suprathreshold sensitivities of participants for sucrose were estimated using a sorting test and signal detection analysis that produced cumulative R-index area under the curve (AUC) scores. Genetic association analysis revealed significant correlation of sucrose AUC scores with genetic variation occurring in the GNAT3 gene (single point P = 10(-3) to 10(-4)), which encodes the taste-specific G(alpha) protein subunit gustducin. Subsequent sequencing identified additional GNAT3 variations having significant association with sucrose AUC scores. Collectively, GNAT3 polymorphisms explain 13% of the variation in sucrose perception. Our findings underscore the importance of common genetic variants influencing human taste perception.

MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR.

BACKGROUND: Multiple approaches for the site-directed mutagenesis (SDM) have been developed. However, only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. In addition, many of the existing multiple SDM methods have technical limitations associated with type and number of mutations that can be introduced, or are technically demanding and require special chemical reagents. RESULTS: In this study we developed a quick and efficient strategy for introduction of multiple complex mutations in a target DNA without intermediate subcloning by using a combination of connecting SDM and suppression PCR. The procedure consists of sequential rounds, with each individual round including PCR amplification of target DNA with two non-overlapping pairs of oligonucleotides. The desired mutation is incorporated at the 5' end of one or both internal oligonucleotides. DNA fragments obtained during amplification are mixed and ligated. The resulting DNA mixture is amplified with external oligonucleotides that act as suppression adapters. Suppression PCR limits amplification to DNA molecules representing full length target DNA, while amplification of other types of molecules formed during ligation is suppressed. To create additional mutations, an aliquot of the ligation mixture is then used directly for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA. CONCLUSION: A wide variety of complex multiple mutations can be generated in a short period of time. The procedure is rapid, highly efficient and does not require special chemical reagents. Thus, MALS represents a powerful alternative to the existing methods for multiple SDM.

Allelic polymorphism within the TAS1R3 promoter is associated with human taste sensitivity to sucrose.

Human sweet taste perception is mediated by the heterodimeric G protein-coupled receptor encoded by the TAS1R2 and TAS1R3 genes. Variation in these genes has been characterized, but the functional consequences of such variation for sweet perception are unknown. We found that two C/T single-nucleotide polymorphisms (SNPs) located at positions -1572 (rs307355) and -1266 (rs35744813) upstream of the TAS1R3 coding sequence strongly correlate with human taste sensitivity to sucrose and explain 16% of population variability in perception. By using a luciferase reporter assay, we demonstrated that the T allele of each SNP results in reduced promoter activity in comparison to the C alleles, consistent with the phenotype observed in humans carrying T alleles. We also found that the distal region of the TAS1R3 promoter harbors a composite cis-acting element that has a strong silencing effect on promoter activity. We conclude that the rs307355 and rs35744813 SNPs affect gene transcription by altering the function of this regulatory element. A worldwide population survey reveals that the T alleles of rs307355 and rs35744813 occur at lowest frequencies in European populations. We propose that inherited differences in TAS1R3 transcription account for a substantial fraction of worldwide differences in human sweet taste perception.

Genomic fingerprinting of Burkholderia pseudomallei and B. mallei pathogens with DNA array based on interspecies sequence differences obtained by subtractive hybridization.

The ability to rapidly and efficiently identify causative agents of dangerous human and animal diseases is a prerequisite to diagnosis, prophylaxis and therapy. Such identification systems can be developed based on DNA markers enabling differentiation between various bacterial strains. One source of these markers is genetic polymorphism. An efficient method for detecting the most stable polymorphisms without knowledge of genomic sequences is subtractive hybridization. In this work we report an approach to typing of Burkholderia pseudomallei and B. mallei that cause melioidosis and glanders, respectively. Typing is based on hybridization of bacterial genomes with a DNA array of genomic markers obtained using subtractive hybridization. The array comprised 55 DNA fragments which distinguished the genomes of B. pseudomallei C-141 and B. mallei C-5 strains, and it was used to test 28 radioactively labeled B. pseudomallei strains and 8 B. mallei strains. Each strain was characterized by a specific hybridization pattern, and the results were analyzed using cluster analysis. 18 patterns specific to B. pseudomallei and 6 patterns specific to B. mallei were found to be unique. The data allowed us to differentiate most studied B. pseudomallei variants from one another and from B. mallei strains. It was concluded that DNA markers obtained by subtractive hybridization can be potentially useful for molecular typing of B. pseudomallei and B. mallei strains, as well as for their molecular diagnosis. The method reported can be easily adapted for use both with DNA arrays and DNA microarrays with fluorescent probes.

A genome-wide sequence-independent comparative analysis of insertion-deletion polymorphisms in multiple Mycobacterium tuberculosis strains.

We applied an enhanced version of subtractive hybridization for comparative analyses of indel differences between genomes of several Mycobacterium tuberculosis strains widespread in Russian regions, and the H37Rv reference strain. A number of differences were detected and partially analyzed, thus demonstrating the practicality of the approach. A majority of the insertions found were shared by all Russian strains, except for strain 1540 that revealed the highest virulence in animal tests. This strain possesses a number of genes absent from other clinical strains. Two of the differential genes were found to encode putative membrane proteins and are presumed to affect mycobacterial interaction with the host cell, thus enhancing virulent properties of the isolate. The method used is of general application, and enables the elaboration of a catalogue of indel polymorphic genomic differences between closely related strains.

Genome-wide identification and mapping of variable sequences in the genomes of Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei and Burkholderia pseudomallei, closely related Gram-negative bacteria, are the causative agents of such serious infectious diseases of humans and animals as glanders and melioidosis, respectively. Despite numerous studies of these pathogens, the detailed mechanisms of their pathogenesis is still poorly understood. One of the serious obstacles to revealing factors responsible for pathogenicity lies in the considerable natural variability of B. pseudomallei and B. mallei, which is also a challenge to development of rapid and efficient diagnostic tools facilitating unambiguous identification of the infectious agents. To gain a deeper insight into B. mallei and B. pseudomallei interspecies divergence and intraspecies polymorphism, we compared the genomes of B. mallei C-5 and B. pseudomallei C-141 strains using a subtractive hybridization technique. A library of DNA fragments specific for B. mallei C-5 and absent from B. pseudomallei C-141 was obtained and analyzed. Some of the differential sequences detected were also not found in the recently sequenced genome of B. pseudomallei K96243. However, a multitude of B. mallei C-5 sequences absent from the B. pseudomallei C-141 genome were detected in the genome of B. pseudomallei K96243. On the other hand, some sequences identified as constituents of the B. mallei C-5 genome were not found in the genome of B. mallei ATCC 23344. Some of the differential DNA fragments displayed similarity to different mobile elements that have not yet been described for B. mallei, whereas the others matched fragments of various prophages, or, when translated into protein sequences, components of active transport systems and different enzymes. A substantial proportion of the differential clones had no database matches either at the nucleotide or amino acid sequence level. The results suggest great genome-wide intra- and interspecies variability of B. mallei and B. pseudomallei. The differences identified may be useful as molecular signatures for identification of B. mallei strains.

Genome-wide comparison reveals great inter- and intraspecies variability in B. pseudomallei and B. mallei pathogens.

Burkholderia mallei and B. pseudomallei, closely related Gram-negative bacteria, are causative agents of serious infectious diseases of humans and animals: glanders and melioidosis, respectively. Despite numerous studies of these pathogens, the detailed mechanism of their pathogenesis is still unknown. The problem is even more complicated due to natural variability of B. pseudomallei and B. mallei strains, the understanding of which is a prerequisite for rational design of tools for diagnostics, prophylaxis and therapy of the diseases. Using a subtractive hybridization technique, we compared the genomes of B. pseudomallei C-141 and B. mallei C-5 strains. A subtracted library of DNA fragments specific for B. pseudomallei C-141 and absent from B. mallei C-5 was obtained and analyzed. A variety of differences have been detected and mapped on the recently sequenced genome of B. pseudomallei K96243. A comparative sequence analysis also revealed considerable genomic differences between B. pseudomallei C-141 and B. mallei ATCC 23344 strains sequenced at The Institute for Genomic Research (TIGR). We also observed significant genomic differences between B. pseudomallei C-141 and B. pseudomallei K96243. Some of the differential DNA fragments displayed similarity to different mobile elements which have not yet been described for B. pseudomallei, whereas the others matched various prophage components, components of active transport systems, different enzymes and transcription regulators. A substantial proportion of the differential clones had no database matches either at the nucleotide or protein level. The results provide evidence for great genome-wide variability of B. pseudomallei, further confirmed by Southern blot analysis of various B. pseudomallei strains. The data obtained can be useful for future development of efficient diagnostic tools allowing rapid identification of species, strains and isolates of B. mallei and B. pseudomallei.

The human genome contains many types of chimeric retrogenes generated through in vivo RNA recombination.

L1 retrotransposons play an important role in mammalian genome shaping. In particular, they can transduce their 3'-flanking regions to new genomic loci or produce pseudogenes or retrotranscripts through reverse transcription of different kinds of cellular RNAs. Recently, we found in the human genome an unusual family of chimeric retrotranscripts composed of full-sized copies of U6 small nuclear RNAs fused at their 3' termini with 5'-truncated, 3'-poly(A)-tailed L1s. The chimeras were flanked by 11-21 bp long direct repeats, and contained near their 5' ends T2A4 hexanucleotide motifs, preferably recognized by L1 nicking endonuclease. These features suggest that the chimeras were formed using the L1 integration machinery. Here we report the identification of 81 chimeras consisting of fused DNA copies of different RNAs, including mRNAs of known human genes. Based on their structural features, the chimeras were subdivided into nine distinct families. 5' Parts of the chimeras usually originated from different nuclear RNAs, whereas their 3' parts represented cytoplasmic RNAs: mRNAs, including L1 mRNA and Alu RNA. Some of these chimeric retrotranscripts are expressed in a variety of human tissues. These findings suggest that RNA-RNA recombination during L1 reverse transcription followed by the integration of the recombinants into the host genome is a general event in genome evolution.