Natural human tRNAAla anticodon variants mistranslate the genetic code.

Characterization of Mutant MUTYH Proteins Associated With Familial Colorectal Cancer

RNA Determinants for Translational Editing: Mischarging a Minihelix Substrate by a tRNA Synthetase (Nordin, B. E., and Schimmel, P. (1999) <i>J. Biol. Chem.</i> 274, 6835–6838) Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid (Beebe, K., Merriman, E., and Schimmel, P. (2003) <i>J. Biol. Chem.</i> 278, 45056–45061)

I believe that the genetic code was the greatest discovery of the 20th century (Fig. 1). The code was worked out during the period that I was a student: a medical student at Tufts University (I dropped out after a year), a graduate student at MIT and Cornell (with Gordon Hammes), and a postdoctoral student at Stanford University (with Paul Flory). My research as a student was centered on theoretical and experimental physical chemistry, as applied to biological systems. With Hammes, I had done a spectroscopic and thermodynamic analysis of ribonuclease and its interaction with one of its ligands. I also had done both experimental and theoretical analyses of kinetic relaxation spectroscopy, inspired in part by the pioneering work of Eigen, who himself had been a mentor of Hammes. (Eigen gave lectures at Cornell that I attended, and we spent time in conversation as well. In more recent years, we have seen each other on a regular basis and continued our conversations.) With Flory at Stanford, I worked on statistical mechanics of polypeptides. All of that graduate and postdoctoral work was exciting, particularly the mathematical side that I especially loved because of its elegance, rigor, and abstraction. However, while this sort of mental activity was going on, I kept thinking about the newly discovered genetic code and aminoacyl-tRNA synthetases.

Non-synonymous cSNPs that occurred in the protein-coding region could potentially alter the amino acid composition and thus the functionality of gene product. Protein variants generated by these non-synonymous cSNPs are thought to be responsible for the diversity of human population and the cause of human diseases associated with inheritance. It is essential to identify these non-synonymous cSNPs/functional variants for functional genomic studies. In addition to direct genome sequencing methods, dbEST has been used for SNP discovery due to its rich information of millions ESTs. Previous SNP discovery methods from dbEST needed to include many ESTs per cluster, which greatly reduce the effectiveness of dbEST. Such approaches would be useful in identifying SNPs for polymorphic markers. We would like to identify these sequence variations as either disease association mutations or functional variants. Therefore, it is necessary to use alternative bioinformatic tools to extract these low frequency functional variants. We have used our CGI bioinformatic tools to data-mining functional variants from ESTs, especially low frequency non-synonymous cSNPs. We have established the CGI tools for comparative genomic studies in identifying novel human genes. With 9848 human reference protein as starting alignment scaffolds and 2.2 million human ESTs, we have now identified more than 50,000 potential functional variants. In addition, our approach can be used to validate and correct amino acid sequences of human reference proteins.

Proteomic genotyping for individual human identification: Inferring SNPs in the absence of DNA evidence.

It has been known for decades that codon usage contributes to translation efficiency and hence to protein production levels. However, its role in protein synthesis is still only partly understood. This lack of understanding hampers the design of synthetic genes for efficient protein production. In this study, we generated a synonymous codon-randomized library of the complete coding sequence of red fluorescent protein. Protein production levels and the full coding sequences were determined for 1459 gene variants in Escherichia coli. Using different machine learning approaches, these data were used to reveal correlations between codon usage and protein production. Interestingly, protein production levels can be relatively accurately predicted (Pearson correlation of 0.762) by a Random Forest model that only relies on the sequence information of the first eight codons. In this region, close to the translation initiation site, mRNA secondary structure rather than Codon Adaptation Index (CAI) is the key determinant of protein production. This study clearly demonstrates the key role of codons at the start of the coding sequence. Furthermore, these results imply that commonly used CAI-based codon optimization of the full coding sequence is not a very effective strategy. One should rather focus on optimizing protein production via reducing mRNA secondary structure formation with the first few codons.

A panel of 89 local commercial cultivars of bread wheat was tested in field trials in the dry conditions of Northern Kazakhstan. Two distinct groups of cultivars (six cultivars in each group), which had the highest and the lowest grain yield under drought were selected for further experiments. A dehydration test conducted on detached leaves indicated a strong association between rates of water loss in plants from the first group with highest grain yield production in the dry environment relative to the second group. Modern high-throughput Amplifluor Single Nucleotide Polymorphism (SNP) technology was applied to study allelic variations in a series of drought-responsive genes using 19 SNP markers. Genotyping of an SNP in the TaDREB5 (DREB2-type) gene using the Amplifluor SNP marker KATU48 revealed clear allele distribution across the entire panel of wheat accessions, and distinguished between the two groups of cultivars with high and low yield under drought. Significant differences in expression levels of TaDREB5 were revealed by qRT-PCR. Most wheat plants from the first group of cultivars with high grain yield showed slight up-regulation in the TaDREB5 transcript in dehydrated leaves. In contrast, expression of TaDREB5 in plants from the second group of cultivars with low grain yield was significantly down-regulated. It was found that SNPs did not alter the amino acid sequence of TaDREB5 protein. Thus, a possible explanation is that alternative splicing and up-stream regulation of TaDREB5 may be affected by SNP, but these hypotheses require additional analysis (and will be the focus of future studies).

Transfer messenger RNA (tmRNA) directs the modification of proteins of which the biosynthesis has stalled or has been interrupted. Here, we report that aminoglycosides can interfere with this quality control system in bacteria, termed trans-translation. Neomycin B is the strongest inhibitor of tmRNA aminoacylation with alanine (K(i) value of approximately 35 micro m), an essential step during trans-translation. The binding sites of neomycin B do not overlap with the identity determinants for alanylation, but the aminoglycoside perturbs the conformation of the acceptor stem that contains the aminoacylation signals. Aminoglycosides reduce the conformational freedom of the transfer RNA-like domain of tmRNA. Additional contacts between aminoglycosides and tmRNA are within the tag reading frame, probably also disturbing reprogramming of the stalled ribosomes prior protein tagging. Aminoglycosides impair tmRNA aminoacylation in the presence of all of the transfer RNAs from Escherichia coli, small protein B, and elongation factor Tu, but when both proteins are present, the inhibition constant is 1 order of magnitude higher. SmpB and elongation factor Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation.

The Genetic Basis for Periodic Fever

Single nucleotide polymorphisms (SNPs) are loci on the genome where different alleles are observed in the population. It has been observed that there might be some patterns or context dependences in the sequence segments adjacent to SNPs sites. Discovering such dependences is very important for understanding possible origins of SNPs in evolution. We collected 519,767 bi-allelic SNPs of human in gene regions from HGBASE and separated them in 6 groups according to the types of alleles at the SNP loci. Bayesian network structure learning technique is applied to discovery of possible dependences in sequence segments around these sites as well as in reference sequences collected as comparison. Noticeable probabilistic correlations among some loci were detected in all the 6 SNP groups and nothing significant was found in the reference sequences. The dependence relations found with different SNP groups are different. These putative context dependences around SNP sites provide important hints for further analyzing SNP-related sequences patterns. The work also illustrates the powerfulness of the Bayesian network method as a tool for biological sequence analysis.

The Yeast Connection to Friedreich Ataxia

Sequence variation in human genes is largely confined to single-nucleotide polymorphisms (SNPs) and is valuable in tests of association with common diseases and pharmacogenetic traits. We performed a systematic and comprehensive survey of molecular variation to assess the nature, pattern and frequency of SNPs in 75 candidate human genes for blood-pressure homeostasis and hypertension. We assayed 28 Mb (190 kb in 148 alleles) of genomic sequence, comprising the 5' and 3' untranslated regions (UTRs), introns and coding sequence of these genes, for sequence differences in individuals of African and Northern European descent using high-density variant detection arrays (VDAs). We identified 874 candidate human SNPs, of which 22% were confirmed by DNA sequencing to reveal a discordancy rate of 21% for VDA detection. The SNPs detected have an average minor allele frequency of 11%, and 387 are within the coding sequence (cSNPs). Of all cSNPs, 54% lead to a predicted change in the protein sequence, implying a high level of human protein diversity. These protein-altering SNPs are 38% of the total number of such SNPs expected, are more likely to be population-specific and are rarer in the human population, directly demonstrating the effects of natural selection on human genes. Overall, the degree of nucleotide polymorphism across these human genes, and orthologous great ape sequences, is highly variable and is correlated with the effects of functional conservation on gene sequences.

Transfer RNA (tRNA)variants that alter the genetic code increaseprotein diversity and have many applications in synthetic biology.Since the tRNA variants can cause a loss of proteostasis, regulatingtheir expression is necessary to achieve high levels of novel protein.Mechanisms to positively regulate transcription with exogenous activatorproteins like those often used to regulate RNA polymerase II (RNAPII)-transcribed genes are not applicable to tRNAs as their expressionby RNA polymerase III requires elements internal to the tRNA. Here,we show that tRNA expression is repressed by overlapping transcriptionfrom an adjacent RNAP II promoter. Regulating the expression of theRNAP II promoter allows inverse regulation of the tRNA. Placing eitherGal4- or TetR–VP16-activated promoters downstream of a mistranslatingtRNASer variant that misincorporates serine at prolinecodons in Saccharomyces cerevisiae allowsmistranslation at a level not otherwise possible because of the toxicityof the unregulated tRNA. Using this inducible tRNA system, we explorethe proteotoxic effects of mistranslation on yeast cells. High levelsof mistranslation cause cells to arrest in the G1 phase. These cellsare impermeable to propidium iodide, yet growth is not restored uponrepressing tRNA expression. High levels of mistranslation increasecell size and alter cell morphology. This regulatable tRNA expressionsystem can be applied to study how native tRNAs and tRNA variantsaffect the proteome and other biological processes. Variations ofthis inducible tRNA system should be applicable to other eukaryoticcell types.

The stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.

Gibberellin signaling pathway genes encoding the DELLA protein and gibberellin receptor GID1 were sequenced in several varieties (Alpha, Valdai, Orlovskaya 9, Pracha) and one line of rye (EM-1) using next generation methods. The revealed multiple alleles of these genes differ mainly in single-nucleotide polymorphisms and less frequently in insertions and deletions. Most of the detected mutations turned out to be synonymous or located in the noncoding regions of the genes. Changes in the amino acid sequences of proteins associated with other mutations are probably functionally neutral. Mutations similar to wheat reduced-height gibberellin-insensitive alleles were not detected.