Genetic mappings in artificial genomes

This paper concerns processing of genomes of artificial (computer-simulated) organisms. Of special interest is the process of translation of genotypes into phenotypes, and utilizing the mapping information obtained during such translation. If there exists more than one genetic encoding in a single artificial life model, then the translation may also occur between different encodings. The obtained mapping information allows to present genes-phenes relationships visually and interactively to a person, in order to increase understanding of the genotype-tophenotype translation process and genetic encoding properties. As the mapping associates parts of the source sequence with the translated destination, it may be also used to trace genes, phenes, and their relationships during simulated evolution. A mappings composition procedure is formally described, and a simple method of visual mapping presentation is established. Finally, advanced visualizations of gene-phene relationships are demonstrated as practical examples of introduced techniques. These visualizations concern genotypes expressed in various encodings, including an encoding which exhibits polygenic and pleiotropic properties.     

Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset

A method of supervised classification using two available structure templates was applied to investigate the possible heterogeneity existing in a large cryo-EM dataset of an Escherichia coli 70S ribosome-EF-G complex. Two subpopulations showing the ribosome in distinct conformational states, related by a ratchet-like rotation of the 30S subunit with respect to the 50S subunit, were extracted from the original dataset. The possible presence of additional intermediate states is discussed.     

Codon Usage Decreases the Error Minimization Within the Genetic Code

The genetic code is not random but instead is organized in such a way that single nucleotide substitutions are more likely to result in changes between similar amino acids. This fidelity, or error minimization, has been proposed to be an adaptation within the genetic code. Many models have been proposed to measure this adaptation within the genetic code. However, we find that none of these consider codon usage differences between species. Furthermore, use of different indices of amino acid physicochemical characteristics leads to different estimations of this adaptation within the code. In this study, we try to establish a more accurate model to address this problem. In our model, a weighting scheme is established for mistranslation biases of the three different codon positions, transition/transversion biases, and codon usage. Different indices of amino acids physicochemical characteristics are also considered. In contrast to pervious work, our results show that the natural genetic code is not fully optimized for error minimization. The genetic code, therefore, is not the most optimized one for error minimization, but one that balances between flexibility and fidelity for different species.     

Purification and characterization of yeast mitochondrial initiation factor 2

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.     

Comprehensive sequence analysis of translation termination sites in various eukaryotes

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.     

Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin

In contrast to canonical mRNAs, translation of leaderless mRNA has been previously reported to continue in the presence of the antibiotic kasugamycin. Here, we have studied the effect of the antibiotic on determinants known to affect translation of leadered and leaderless mRNAs. Kasugamycin did not affect the Shine-Dalgarno (SD)-anti-SD (aSD) interaction or the function of translation initiation factor 3 (IF3). Thus, the preferential translation of leaderless mRNA in the presence of kasugamycin can neither be attributed to an expanding pool of 30S subunits with a "blocked" aSD nor to a lack of action of IF3, which has been shown to discriminate against translation initiation at 5'-terminal start codons. Using toeprinting, we observed that on leaderless mRNA 70S in contrast to 30S translation initiation complexes are comparatively resistant to the antibiotic. These results taken together with the known preference of 70S ribosomes for 5'-terminal AUGs lend support to the hypothesis that translation of leaderless mRNAs may as well proceed via an alternative initiation pathway accomplished by intact 70S ribosomes.     

Limitations on the recombinant plasmid selection by Lac(+)/Lac(-) colony phenotype detection

Lac(+)/Lac(-) selection of recombinant plasmids based on the insertional inactivation of LacZalpha gene cannot differentiate recombinant clones in some cases. Several fragments of exon 11 of human brca1 gene were cloned in LacZalpha-containing plasmids so that frameshift appeared at the 5(')-end of the fragments tested but these fragments were in frame with the part of LacZalpha situated downstream of the polylinker. All plasmids except one caused blue colonies formation after being transformed in Escherichia coli LacZDeltaM15 cells in spite of the frameshift. The fact may be explained by reinitiation of translation within the mRNA transcribed from the inserted DNA fragments at in-frame AUG, GUG, and UUG. The data demonstrated limitations on the Lac(+)/Lac(-) selection of LacZalpha-based recombinant plasmids.     

Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1 (EF-1), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1. An intron is located in the 5 untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1tef-c (97%) than to the P. purpurea EF-1tef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.