遗传密码扩充技术

遗传密码的扩充打破了天然生命体基因只能编码天然氨基酸的限制.遗传密码扩充技术使人们可以将具有特殊性质的非天然氨基酸通过基因表达到生物活体的蛋白质中.该文对遗传密码扩充的原理、方法及意义进行简述.     

Coding Rules for Amino Acids in the Genetic Code: The Genetic Code is a Minimal Code of Mutational Deterioration

Coding rules for amino acids in the genetic code are discussed from the point that the genetic code is a minimal code ofmutational deterioration. The global mutational deterioration(GMD) function is defined through several parameters describingsingle base mutations and amino acid distances. The problem ofsearching for the global minimum of the GMD function is discussedin some detail. From GMD minimization under initial constraintswe have succeeded in deducing the standard genetic code.     

Regional Context in the Alignment of Biological Sequence Pairs

Sequence divergence derives from either point substitution or indel (insertion or deletion) processes. We investigated the rates of these two processes both in protein and non-protein coding DNA. We aligned sequence pairs using two pair-hidden Markov models (PHMMs) conjoined by one silent state. The two PHMMs had their own set of parameters to model rates in their respective regions. The aim was to test the hypothesis that the indel mutation rate mimics the point mutation rate. That is, indels are found less often in conserved regions (slow point substitution rate) and more often in non-conserved regions (fast point substitution rate). Both polypeptides and rRNA molecules in our data exhibited a clear distinction between slow and fast rates of the two processes. These two rates served as surrogates to conserved and non-conserved secondary structure components, respectively. With polypeptides we found both the fast indel rate and the fast replacement rate were co-located with hydrophilic residues. We also found that the average concordance, of our alignments with corresponding curated alignments, improves markedly when the model allows either of the two fast rates to colocate with hydrophilic residues. With rRNA molecules, our model did not detect colocation between the fast indel rate and the fast substitution rate. Nevertheless, coupling the indel rates with the point substitution rates across the two regions markedly increased model fit. This result suggests that rRNA pairwise alignments should be modeled after allowing for the two processes to vary simultaneously and independently in the two regions.     

Genetic Code Variations in Mitochondria: tRNA as a Major Determinant of Genetic Code Plasticity

Characteristic features of tRNA such as the anticodon sequence and modified nucleotides in the anticodon loop are thought to be crucial effectors for promoting or restricting codon reassignment. Our recent findings on basepairing rules between anticodon and codon in various metazoan mitochondria suggest that the complete loss of a codon is not necessarily essential for codon reassignment to take place. We postulate that a possible competition between two tRNAs with cognate anticodon sequences towards the relevant codon to be varied has a potential role in codon reassignment. Our proposition can be viewed as an expanded version of the codon capture theory proposed by Osawa and Jukes (J Mol Evol 28: 271–278, 1989). Received: 28 December 2000 / Accepted: 12 March 2001     

New Unnatural L-Nucleoside Enantiomers:From Their Stereospecific Synthesis to Their Biological Activities

Abstract

Several purine and pyrimidine β-L-dideoxynucleosides were stereospecifically synthesized and their antiviral properties examined. Two of them, namely β-L-2′,3′-dideoxyadenosine (β-L-ddA) and its 2′,3′-didehydro derivative (β-L-d4A) were found to have significant anti-human immunodeficiency virus (HIV) and anti-hepatitis B virus (HBV) activities in cell culture.     

Error-reducing Structure of the Genetic Code Indicates Code Origin in Non-thermophile Organisms

During the RNA World, organisms experienced high rates of genetic errors, which implies that there was strong evolutionary pressure to reduce the errors’ phenotypical impact by suitably structuring the still-evolving genetic code. Therefore, the relative rates of the various types of genetic errors should have left characteristic imprints in the structure of the genetic code. Here, we show that, therefore, it is possible to some extent to reconstruct those error rates, as well as the nucleotide frequencies, for the time when the code was fixed. We find evidence indicating that the frequencies of G and C in the genome were not elevated. Since, for thermodynamic reasons, RNA in thermophiles tends to possess elevated G+C content, this result indicates that the fixation of the genetic code occurred in organisms which were either not thermophiles or that the code’s fixation occurred after the rise of DNA. Supplementary Materials Original data and programs are available at the author’s web site: .     

Variable Temperature Infrared Spectroscopy of Cytosine-Guanine Base Pairs: Tautomerism Versus Polarization

Abstract

This report describes an infrared (IR) spectroscopic study of a model cytosine - guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O⁶-methylguanosine (O⁶MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35, 724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw, J. Am. Chem. Soc. 112, 829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C=O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O⁶-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.     

Out of the Randomness: Correlating Noise in Biological Systems

The study of the dynamics of biological systems requires one to follow relaxation processes in time with micron-size spatial resolution. This need has led to the development of different fluorescence correlation techniques with high spatial resolution and a tremendous (from nanoseconds to seconds) temporal dynamic range. Spatiotemporal information can be obtained even on complex dynamic processes whose time evolution is not forecast by simple Brownian diffusion. Our discussion of the most recent applications of image correlation spectroscopy to the study of anomalous sub- or superdiffusion suggests that this field still requires the development of multidimensional image analyses based on analytical models or numerical simulations. We focus in particular on the framework of spatiotemporal image correlation spectroscopy and examine the critical steps in getting information on anomalous diffusive processes from the correlation maps. We point out how a dual space-time correlative analysis, in both the direct and the Fourier space, can provide quantitative information on superdiffusional processes when these are analyzed through an empirical model based on intermittent active dynamics. We believe that this dual space-time analysis, potentially amenable to mathematical treatment and to the exact fit of experimental data, could be extended to include the rich phenomenology of subdiffusive processes, thereby quantifying relevant parameters for the various motivating biological problems of interest.     

The Evolution of the Genetic Code Revisited

The evolution of the genetic code in terms of the adoption of new codons has previously been related to the relative thermostability of codon–anticodon interactions such that the most stable interactions have been hypothesised to represent the most ancient coding capacity. This derivation is critically dependent on the accuracy of the experimentally determined stability parameters. A new set of parameters recently determined for B-DNA reveals that the codon–anticodon pairs for the codes in non-plant mitochondria on the one hand and prokaryotic and eukaryotic organisms on the other can be unequivocally divided into two classes – the most stable base steps define a common code specified by the first two bases in a codon while the less stable base steps correlate with divergent usage and the adoption of a 3-letter code. This pattern suggests that the fixation of codons for A, G, P, V, S, T, D/E, R may have preceded the divergence of the non-plant mitochondrial line from other organisms. Other variations in the code correlate with the least stable codon–anticodon pairs. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Metabolic Basis for the Self-Referential Genetic Code

An investigation of the biosynthesis pathways producing glycine and serine was necessary to clarify an apparent inconsistency between the self-referential model (SRM) for the formation of the genetic code and the model of coevolution of encodings and of amino acid biosynthesis routes. According to the SRM proposal, glycine was the first amino acid encoded, followed by serine. The coevolution model does not state precisely which the first encodings were, only presenting a list of about ten early assignments including the derivation of glycine from serine—this being derived from the glycolysis intermediate glycerate, which reverses the order proposed by the self-referential model. Our search identified the glycine-serine pathway of syntheses based on one-carbon sources, involving activities of the glycine decarboxylase complex and its associated serine hydroxymethyltransferase, which is consistent with the order proposed by the self-referential model and supports its rationale for the origin of the genetic code: protein synthesis was developed inside an early metabolic system, serving the function of a sink of amino acids; the first peptides were glycine-rich and fit for the function of building the early ribonucleoproteins; glycine consumption in proteins drove the fixation of the glycine-serine pathway.     

Genetic Incorporation of Unnatural Amino Acids into Proteins in Mycobacterium tuberculosis

New tools are needed to study the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), to facilitate new drug discovery and vaccine development. We have developed methodology to genetically incorporate unnatural amino acids into proteins in Mycobacterium smegmatis, BCG and Mtb, grown both extracellularly in culture and inside host cells. Orthogonal mutant tRNA^Tyr/tyrosyl-tRNA synthetase pairs derived from Methanococcus jannaschii and evolved in Escherichia coli incorporate a variety of unnatural amino acids (including photocrosslinking, chemically reactive, heavy atom containing, and immunogenic amino acids) into proteins in response to the amber nonsense codon. By taking advantage of the fidelity and suppression efficiency of the MjtRNA/pIpaRS pair in mycobacteria, we are also able to use p-iodophenylalanine to induce the expression of proteins in mycobacteria both extracellularly in culture and inside of mammalian host cells. This provides a new approach to regulate the expression of reporter genes or mycobacteria endogenous genes of interest. The establishment of the unnatural amino acid expression system in Mtb, an intracellular pathogen, should facilitate studies of TB biology and vaccine development.