Multiple levels of meaning in DNA sequences, and one more

If we define a genetic code as a widespread DNA sequence pattern that carries a message with an impact on biology, then there are multiple genetic codes. Sequences involved in these codes overlap and, thus, both interact with and constrain each other, such as for the triplet code, the intron-splicing code, the code for amphipathic alpha helices, and the chromatin code. Nucleosomes preferentially are located at the ends of exons, thus protecting splice junctions, with the N9 positions of guanines of the GT and AG junctions oriented toward the histones. Analysis of protein-coding sequences reveals numerous traces of tandem repeats, apparently formed by triplet expansion, which in effect is a genome inflation ``code'. Our data are consistent with the hypothesis that expansion of simple tandem repetition of certain aggressive triplets has been a characteristic of life from its emergence. Such expanding triplets appear to be the major factor underlying observed codon usage biases.     

Code domains in tandem repetitive DNA sequence structures

Traditionally, many people doing research in molecular biology attribute coding properties to a given DNA sequence if this sequence contains an open reading frame for translation into a sequence of amino acids. This protein coding capability of DNA was detected about 30 years ago. The underlying genetic code is highly conserved and present in every biological species studied so far. Today, it is obvious that DNA has a much larger coding potential for other important tasks. Apart from coding for specific RNA molecules such as rRNA, snRNA and tRNA molecules, specific structural and sequence patterns of the DNA chain itself express distinct codes for the regulation and expression of its genetic activity. A chromatin code has been defined for phasing of the histone-octamer protein complex in the nucleosome. A translation frame code has been shown to exist that determines correct triplet counting at the ribosome during protein synthesis. A loop code seems to organize the single stranded interaction of the nascent RNA chain with proteins during the splicing process, and a splicing code phases successive 5' and 3' splicing sites. Most of these DNA codes are not exclusively based on the primary DNA sequence itself, but also seem to include specific features of the corresponding higher order structures. Based on the view that these various DNA codes are genetically instructive for specific molecular interactions or processes, important in the nucleus during interphase and during cell division, the coding capability of tandem repetitive DNA sequences has recently been reconsidered.     

Genome-wide comparative analysis of simple sequence coding repeats among 25 insect species

We present a detailed genome-scale comparative analysis of simple sequence repeats within protein coding regions among 25 insect genomes. The repetitive sequences in the coding regions primarily represented single codon repeats and codon pair repeats. The CAG triplet is highly repetitive in the coding regions of insect genomes. It is frequently paired with the synonymous codon CAA to code for polyglutamine repeats. The codon pairs that are least repetitive code for polyalanine repeats. The frequency of hexanucleotide and dinucleotide motifs of codon pair repeats is significantly (p<0.001) different in the Drosophila species compared to the non-Drosophila species. However, the frequency of synonymous and non-synonymous codon pair repeats varies in a correlated manner (r(2)=0.79) among all the species. Results further show that perfect and imperfect repeats have significant association with the trinucleotide and hexanucleotide coding repeats in most of these insects. However, only select species show significant association between the numbers of perfect/imperfect hexamers and repeat coding for single amino acid/amino acid pair runs. Our data further suggests that genes containing simple sequence coding repeats may be under negative selection as they tend to be poorly conserved across species. The sequences of coding repeats of orthologous genes vary according to the known phylogeny among the species. In conclusion, the study shows that simple sequence coding repeats are important features of genome diversity among insects.     

On the Evolution of the Standard Genetic Code: Vestiges of Critical Scale Invariance from the RNA World in Current Prokaryote Genomes

Herein two genetic codes from which the primeval RNA code could have originated the standard genetic code (SGC) are derived. One of them, called extended RNA code type I, consists of all codons of the type RNY (purine-any base-pyrimidine) plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. In order to test if putative nucleotide sequences in the RNA World and in both extended RNA codes, share the same scaling and statistical properties to those encountered in current prokaryotes, we used the genomes of four Eubacteria and three Archaeas. For each prokaryote, we obtained their respective genomes obeying the RNA code or the extended RNA codes types I and II. In each case, we estimated the scaling properties of triplet sequences via a renormalization group approach, and we calculated the frequency distributions of distances for each codon. Remarkably, the scaling properties of the distance series of some codons from the RNA code and most codons from both extended RNA codes turned out to be identical or very close to the scaling properties of codons of the SGC. To test for the robustness of these results, we show, via computer simulation experiments, that random mutations of current genomes, at the rates of 10⁻¹⁰ per site per year during three billions of years, were not enough for destroying the observed patterns. Therefore, we conclude that most current prokaryotes may still contain relics of the primeval RNA World and that both extended RNA codes may well represent two plausible evolutionary paths between the RNA code and the current SGC.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属(Eucalyptus)的10 000条EST序列进行分析, 在其中的1 499条序列上共发现1 775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外, 还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关; 微卫星的丰度与重复单元长度也呈负相关(三碱基重复微卫星除外)。在桉树EST序列中, 重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面, 桉树EST序列与杨树(Populus trichocarpa)基因组注释的转录序列随重复单元长度的变化呈现出相同的规律, 但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低, 推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计, 并对设计的引物进行PCR检测, 结果表明所设计的引物具有极高的扩增成功率。     

Amino Acid Repeats Cause Extraordinary Coding Sequence Variation in the Social Amoeba Dictyostelium discoideum

Protein sequences are normally the most conserved elements of genomes owing to purifying selection to maintain their functions. We document an extraordinary amount of within-species protein sequence variation in the model eukaryote Dictyostelium discoideum stemming from triplet DNA repeats coding for long strings of single amino acids. D. discoideum has a very large number of such strings, many of which are polyglutamine repeats, the same sequence that causes various human neurological disorders in humans, like Huntington’s disease. We show here that D. discoideum coding repeat loci are highly variable among individuals, making D. discoideum a candidate for the most variable proteome. The coding repeat loci are not significantly less variable than similar non-coding triplet repeats. This pattern is consistent with these amino-acid repeats being largely non-functional sequences evolving primarily by mutation and drift.     

Codon Size Reduction as the Origin of the Triplet Genetic Code

The genetic code appears to be optimized in its robustness to missense errors and frameshift errors. In addition, the genetic code is near-optimal in terms of its ability to carry information in addition to the sequences of encoded proteins. As evolution has no foresight, optimality of the modern genetic code suggests that it evolved from less optimal code variants. The length of codons in the genetic code is also optimal, as three is the minimal nucleotide combination that can encode the twenty standard amino acids. The apparent impossibility of transitions between codon sizes in a discontinuous manner during evolution has resulted in an unbending view that the genetic code was always triplet. Yet, recent experimental evidence on quadruplet decoding, as well as the discovery of organisms with ambiguous and dual decoding, suggest that the possibility of the evolution of triplet decoding from living systems with non-triplet decoding merits reconsideration and further exploration. To explore this possibility we designed a mathematical model of the evolution of primitive digital coding systems which can decode nucleotide sequences into protein sequences. These coding systems can evolve their nucleotide sequences via genetic events of Darwinian evolution, such as point-mutations. The replication rates of such coding systems depend on the accuracy of the generated protein sequences. Computer simulations based on our model show that decoding systems with codons of length greater than three spontaneously evolve into predominantly triplet decoding systems. Our findings suggest a plausible scenario for the evolution of the triplet genetic code in a continuous manner. This scenario suggests an explanation of how protein synthesis could be accomplished by means of long RNA-RNA interactions prior to the emergence of the complex decoding machinery, such as the ribosome, that is required for stabilization and discrimination of otherwise weak triplet codon-anticodon interactions.     

Role of Everlasting Triplet Expansions in Protein Evolution

Analysis of occurrence of simple amino acid repeats in large ensemble of prokaryotic and eukaryotic sequences reveals that nearly all amino acids found in the repeats belong to those which have in their codon repertoires aggressively expanding triplets, all of three known pathologically expanding classes GCU (GCU, CUG, UGC, AGC, GCA, CAG), GCC (GCC, CCG, CGC, GGC, GCG, CGG), and AAG (AAG, AGA, GAA, CTT, TTC, TCT). This is observed especially clear in the first exons of proteins of higher eukaryotes. The data are interpreted as manifestation of everlasting triplet expansions, which, presumably, started from the very origin of the triplet code. The spontaneous expansions continued to occur all the way during evolution, leaving their footprints in the protein-coding sequences as still visible simple amino acid repeats, as preferred triplets encoding the repeats, and as preferred codons in the codon usage tables.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属（Eucalyptus）的10000条EST序列进行分析,在其中的1499条序列上共发现1775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外,还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关;微卫星的丰度与重复单元长度也呈负相关（三碱基重复微卫星除外）。在桉树EST序列中,重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面,桉树EST序列与杨树（Populus trichocarpa）基因组注释的转录序列随重复单元长度的变化呈现出相同的规律,但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低,推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计,并对设计的引物进行PCR检测,结果表明所设计的引物具有极高的扩增成功率。     

Trinucleotide repeats associated with human disease.

Triplet repeat expansion diseases (TREDs) are characterized by the coincidence of disease manifestation with amplification of d(CAG. CTG), d(CGG.CCG) or d(GAA.TTC) repeats contained within specific genes. Amplification of triplet repeats continues in offspring of affected individuals, which generally results in progressive severity of the disease and/or an earlier age of onset, phenomena clinically referred to as 'anticipation'. Recent biophysical and biochemical studies reveal that five of the six [d(CGG)n, d(CCG)n, (CAG)n, d(CTG)n and d(GAA)n] complementary sequences that are associated with human disease form stable hairpin structures. Although the triplet repeat sequences d(GAC)n and d(GTC)n also form hairpins, repeats of the double-stranded forms of these sequences are conspicuously absent from DNA sequence databases and are not anticipated to be associated with human disease. With the exception of d(GAG)n and d(GTG)n, the remaining triplet repeat sequences are unlikely to form hairpin structures at physiological salt and temperature. The details of hairpin structures containing trinucleotide repeats are summarized and discussed with respect to potential mechanisms of triplet repeat expansion and d(CGG.CCG) n methylation/demethylation.     

Deletion errors generated during replication of CAG repeats.

Triplet repeat sequence instability is associated with hereditary neurological diseases and with certain types of cancer. Here we study one form of this instability, deletion of triplet repeats during replication of template (CAG)(n)sequences by DNA polymerases. To monitor loss of triplet codons, we inserted (CAG)(9)and (CAG)(17)repeats into the lacZ sequence in M13mp2 and changed one repeat to a TAG codon to yield DNA substrates with colorless plaque phenotypes. Templates containing these inserts within gaps were copied and errors were scored as blue plaque Lac revertants whose DNA was sequenced to determine if loss of the TAG codon resulted from substitutions or deletions. DNA synthesis by either DNA polymerase beta or exonuclease-deficient T7 DNA polymerase produced deletions involving loss of from 1 to 8 of 9 or 15 of 17 repeats. Thus, these polymerases utilize misaligned template-primers containing from 3 to 45 extra template strand nucleotides. Deletion frequencies were much higher than substitution frequencies at the TAG codon in certain repeats, indicating that triplet repeats are at high risk for mutation in the absence of error correction. Proofreading-proficient T7 DNA polymerase generated deletions at 2- to 10-fold lower frequencies than did its exonuclease-deficient derivative. This suggests that misaligned triplet repeat sequences are subject to proofreading, but at reduced efficiency compared to editing of single-base mismatches.     

Large cryptic internal sequence repeats in protein structures from <Emphasis Type="Italic">Homo sapiens</Emphasis>

Amino acid sequences are known to constantly mutate and diverge unless there is a limiting condition that makes such a change deleterious. However, closer examination of the sequence and structure reveals that a few large, cryptic repeats are nevertheless sequentially conserved. This leads to the question of why only certain repeats are conserved at the sequence level. It would be interesting to find out if these sequences maintain their conservation at the three-dimensional structure level. They can play an active role in protein and nucleotide stability, thus not only ensuring proper functioning but also potentiating malfunction and disease. Therefore, insights into any aspect of the repeats — be it structure, function or evolution — would prove to be of some importance. This study aims to address the relationship between protein sequence and its three-dimensional structure, by examining if large cryptic sequence repeats have the same structure.     

Genetic deletions between directly repeated sequences in bacteriophage T7

Summary DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10⁻¹⁰, while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10⁻⁶. The deletion events were independent of a hostrecA mutation.     

Simplification of the genetic code: restricted diversity of genetically encoded amino acids

At earlier stages in the evolution of the universal genetic code, fewer than 20 amino acids were considered to be used. Although this notion is supported by a wide range of data, the actual existence and function of the genetic codes with a limited set of canonical amino acids have not been addressed experimentally, in contrast to the successful development of the expanded codes. Here, we constructed artificial genetic codes involving a reduced alphabet. In one of the codes, a tRNA^Ala variant with the Trp anticodon reassigns alanine to an unassigned UGG codon in the Escherichia coli S30 cell-free translation system lacking tryptophan. We confirmed that the efficiency and accuracy of protein synthesis by this Trp-lacking code were comparable to those by the universal genetic code, by an amino acid composition analysis, green fluorescent protein fluorescence measurements and the crystal structure determination. We also showed that another code, in which UGU/UGC codons are assigned to Ser, synthesizes an active enzyme. This method will provide not only new insights into primordial genetic codes, but also an essential protein engineering tool for the assessment of the early stages of protein evolution and for the improvement of pharmaceuticals.