How plants make light work of growth

Entry to medical schools; Harry Grenville

Nuffleld Advanced Biology

Environmental politics and policy

Grants for school nature areas

Biology in TVEI/CPVE courses - new handbook

New AV catalogue     

How student teachers use scientific conceptions to discuss a complex environmental issue

This paper focuses upon the extent to which student teachers develop conceptual understanding about key scientific principles through their training, and the extent to which they can deploy this knowledge in discussions of complex environmental issues.

All students involved in the teacher-training programme answered a questionnaire — before and after their first term of the programme — in which their conceptions of respiration and photosynthesis were tested. 15 students were also interviewed about a newspaper article that discusses the ethicality of using surplus heat from a crematorium in the far heating system. They were asked to comment on the article, to pose questions about the issue and explicitly asked what happens to the bodies if either combusted or buried. The first results show that some students, though not the majority, develop their ability to answer conceptual questions about scientific content as a result of their first science course. However, even among these students, the task of deploying this conceptual understanding in discussions of complex, socially relevant questions proved very difficult. Most students expressed personal opinions without using scientific arguments. It may be that the students have not developed the ability to recognise and distinguish different contexts or that the learning situation has not been challenging enough.     

Neutral adaptation of the genetic code to double-strand coding

Summary We lay new foundations to the hypothesis that the genetic code is adapted to evolutionary retention of information in the antisense strands of natural DNA/RNA sequences. In particular, we show that the genetic code exhibits, beyond the neutral replacement patterns of amino acid substitutions, optimal properties by favoring simultaneous evolution of proteins encoded in DNA/RNA sense-antisense strands. This is borne out in the sense-antisense transformations of the codons of every amino acid which target amino acids physicochemically similar to each other. Moreover, silent mutations in the sense strand generate conservative ones in its antisense counterpart and vice versa. Coevolution of proteins coded by complementary strands is shown to be a definite possibility, a result which does not depend on any physical interaction between the coevolving proteins. Likewise, the degree to which the present genetic code is dedicated to evolutionary sense-antisense tolerance is demonstrated by comparison with many randomized codes. Double-strand coding is quantified from an information-theoretical point of view.     

Dual functions of codons in the genetic code

The discovery of the genetic code provided one of the basic foundations of modern molecular biology. Most organisms use the same genetic language, but there are also well-documented variations representing codon reassignments within specific groups of organisms (such as ciliates and yeast) or organelles (such as plastids and mitochondria). In addition, duality in codon function is known in the use of AUG in translation initiation and methionine insertion into internal protein positions as well as in the case of selenocysteine and pyrrolysine insertion (encoded by UGA and UAG, respectively) in competition with translation termination. Ambiguous meaning of CUG in coding for serine and leucine is also known. However, a recent study revealed that codons in any position within the open reading frame can serve a dual function and that a change in codon meaning can be achieved by availability of a specific type of RNA stem-loop structure in the 3′-untranslated region. Thus, duality of codon function is a more widely used feature of the genetic code than previously known, and this observation raises the possibility that additional recoding events and additional novel features have evolved in the genetic code.     

Different types of secondary information in the genetic code

Whole-genome and functional analyses suggest a wealth of secondary or auxiliary genetic information (AGI) within the redundancy component of the genetic code. Although there are multiple aspects of biased codon use, we focus on two types of auxiliary information: codon-specific translational pauses that can be used by particular proteins toward their unique folding and biased codon patterns shared by groups of functionally related mRNAs with coordinate regulation. AGI is important to genetics in general and to human disease; here, we consider influences of its three major components, biased codon use itself, variations in the tRNAome, and anticodon modifications that distinguish synonymous decoding. AGI is plastic and can be used by different species to different extents, with tissue-specificity and in stress responses. Because AGI is species-specific, it is important to consider codon-sensitive experiments when using heterologous systems; for this we focus on the tRNA anticodon loop modification enzyme, CDKAL1, and its link to type 2 diabetes. Newly uncovered tRNAome variability among humans suggests roles in penetrance and as a genetic modifier and disease modifier. Development of experimental and bioinformatics methods are needed to uncover additional means of auxiliary genetic information.     

Pattern and chance in the use of the genetic code

Summary The use of triplet code words inE. coli,X174, MS2, and rabbit globin was examined. A significant deficiency of purines in the third position of fourfold degenerate codons was noted, although its significance is not understood. There has been no consistent selection against uracil in pyrimidine restricted codons. For many amino acids the choice between code words appears random, while for arginine, isoleucine, and probably glycine, distinct biases exist which can be explained in terms of tRNA availability.     

G691S/S904S polymorphism in the RET protooncogene of a 25-year-old medical student with bilateral pheochromocytoma

The case of a 25-year-old medical student with bilateral pheochromocytoma is described. Following diagnostic testing, tumors were surgically removed. Genetic analysis revealed that the patient is a heterozygote with the following mutations on opposite homologs: G691S (exon 11) and S904S (TCC-TCG, exon 15), suggesting the diagnosis of multiple endocrine neoplasia 2A (MEN2A). A diagnosis of MEN2 would be an indication of thyroidectomy in this patient. Although this mutation is described in the literature, it has no known connection to pheochromocytomas. Therefore, it is unknown whether there is a causal connection between the G691S genotype and the pheochromocytomas in this patient. If so, G691S is to be added to the list of genotypes causing MEN2A. Here, the procedure of sequencing the RET protooncogene is described and a possible association between the G691S genotype and MEN2A is discussed.     

Doublet frequencies in the DNA of genetic code limit organisms

Summary Unrelated organisms with DNA of extreme G + C content (25% or 70%) are found to share very specific patterns of nearest neighbour base doublet frequency in their DNAs. This is shown to be a result of restrictions on the extremity of amino acid composition in their proteins, combined with a maximisation of the use of one type of base pair in redundant codon positions. Inferences are made about the universal nature of the genetic code and the proportion of DNA used for specifying protein in different species. The composition of coding DNA strands in these organisms is also discussed.     

Evolution of the genetic code: partial optimization of a random code for robustness to translation error in a rugged fitness landscape

Background  

The standard genetic code table has a distinctly non-random structure, with similar amino acids often encoded by codons series that differ by a single nucleotide substitution, typically, in the third or the first position of the codon. It has been repeatedly argued that this structure of the code results from selective optimization for robustness to translation errors such that translational misreading has the minimal adverse effect. Indeed, it has been shown in several studies that the standard code is more robust than a substantial majority of random codes. However, it remains unclear how much evolution the standard code underwent, what is the level of optimization, and what is the likely starting point.     

Environmental PCR survey to determine the distribution of a non-canonical genetic code in uncultivable oxymonads

The universal genetic code is conserved throughout most living systems, but a non-canonical code where TAA and TAG encode glutamine has evolved in several eukaryotes, including oxymonad protists. Most oxymonads are uncultivable, so environmental RT-PCR and PCR was used to examine the distribution of this rare character. A total of 253 unique isolates of four protein-coding genes were sampled from the hindgut community of the cockroach, Cryptocercus punctulatus , an environment rich in diversity from two of the five subgroups of oxymonad, saccinobaculids and polymastigids. Four α-tubulins were found with non-canonical glutamine codons. Environmental RACE confirmed that these and related genes used only TGA as stop codons, as expected for the non-canonical code, whereas other genes used TAA or TAG as stop codons, as expected for the universal code. We characterized α-tubulin from manually isolated Saccinobaculus ambloaxostylus , confirming it uses the universal code and suggesting, by elimination, that the non-canonical code is used by a polymastigid. HSP90 and EF-1α phylogenies also showed environmental sequences falling into two distinct groups, and are generally consistent with previous hypotheses that polymastigids and Streblomastix are closely related. Overall, we propose that the non-canonical genetic code arose once in a common ancestor of Streblomastix and a subgroup of polymastigids.     

Characterisation of a non-canonical genetic code in the oxymonad Streblomastix strix

The genetic code is one of the most highly conserved characters in living organisms. Only a small number of genomes have evolved slight variations on the code, and these non-canonical codes are instrumental in understanding the selective pressures maintaining the code. Here, we describe a new case of a non-canonical genetic code from the oxymonad flagellate Streblomastix strix. We have sequenced four protein-coding genes from S.strix and found that the canonical stop codons TAA and TAG encode the amino acid glutamine. These codons are retained in S.strix mRNAs, and the legitimate termination codons of all genes examined were found to be TGA, supporting the prediction that this should be the only true stop codon in this genome. Only four other lineages of eukaryotes are known to have evolved non-canonical nuclear genetic codes, and our phylogenetic analyses of alpha-tubulin, beta-tubulin, elongation factor-1 alpha (EF-1 alpha), heat-shock protein 90 (HSP90), and small subunit rRNA all confirm that the variant code in S.strix evolved independently of any other known variant. The independent origin of each of these codes is particularly interesting because the code found in S.strix, where TAA and TAG encode glutamine, has evolved in three of the four other nuclear lineages with variant codes, but this code has never evolved in a prokaryote or a prokaryote-derived organelle. The distribution of non-canonical codes is probably the result of a combination of differences in translation termination, tRNAs, and tRNA synthetases, such that the eukaryotic machinery preferentially allows changes involving TAA and TAG.     

Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNA^Pyl pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNA^Pyl in Chinese hamster ovary cells. When tRNA^Pyl was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA^Pyl pair facilitated the most efficient incorporation of a pyrrolysine analog, N^ε-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNA^Pyl system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to N^ε-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N^ε-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.     

On replication of nucleic acids in relation to the evolution of the genetic code and of proteins

Some properties of transitions generated during an imprecise replication of nucleic acids are described here. A possible role of these transitions in the evolution of the genetic code with respect to the evolution of the tertiary structure of proteins is suggested. The data supporting this hypothesis are obtained from the analysis of certain regularities present in the genetic code and in the proportions of amino acid residues buried in the interior of globular proteins.     

The evolution of the genetic code took place in an anaerobic environment

We have compared orthologous proteins from an aerobic organism, Cytophaga hutchinsonii, and from an obligate anaerobe, Bacteroides thetaiotaomicron. This comparison allows us to define the oxyphobic ranks of amino acids, i.e. a scale of the relative sensitivity to oxygen of the amino acid residues. The oxyphobic index (OI), which can be simply obtained from the amino acids' oxyphobic ranks, can be associated to any protein and therefore to the genetic code, if the number of synonymous codons attributed to the amino acids in the code is assumed to be the frequency with which the amino acids appeared in ancestral proteins. Sampling of the OI variable from the proteins of obligate anaerobes and aerobes has established that the OI value of the genetic code is not significantly different from the mean OI value of anaerobe proteins, while it is different from that of aerobe proteins. This observation would seem to suggest that the terminal phases of the evolution of genetic code organization took place in an anaerobic environment. This result is discussed in the framework of hypotheses suggested to explain the timing of the evolutionary appearance of the aerobic metabolism.     

The rules of variation: Amino acid exchange according to the rotating circular genetic code

General guidelines for the molecular basis of functional variation are presented while focused on the rotating circular genetic code and allowable exchanges that make it resistant to genetic diseases under normal conditions. The rules of variation, bioinformatics aids for preventative medicine, are: (1) same position in the four quadrants for hydrophobic codons, (2) same or contiguous position in two quadrants for synonymous or related codons, and (3) same quadrant for equivalent codons. To preserve protein function, amino acid exchange according to the first rule takes into account the positional homology of essential hydrophobic amino acids with every codon with a central uracil in the four quadrants, the second rule includes codons for identical, acidic, or their amidic amino acids present in two quadrants, and the third rule, the smaller, aromatic, stop codons, and basic amino acids, each in proximity within a 90 degree angle. I also define codifying genes and palindromati, CTCGTGCCGAATTCGGCACGAG.     

Challenges of the genetic code for exploring sequence space in directed protein evolution

Directed protein evolution is the most versatile method for studying protein structure-function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2-7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard.     

Challenges of the genetic code for exploring sequence space in directed protein evolution

Directed protein evolution is the most versatile method for studying protein structure–function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2–7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard.     

Evolution of the mitochondrial genetic code II. Reassignment of codon AUA from isoleucine to methionine

Summary The reassignment of codon AUA from isoleucine to methionine during mitochondrial evolution may be explained by the codon reassignment (capture) hypothesis without assuming direct replacement of isoleucine by methionine in mitochondrial proteins. According to this hypothesis, codon AUA would have disappeared from the reading frames of messenger RNA. AUA codons would have mutated mainly to AUU isoleucine codons because of constraints resulting from elimination of tRNA Ile with anticodon *CAU (in which *C is lysidine). Later, tRNA Met (CAU) would have undergone structural changes enabling it to pair with both AUG and AUA. AUA codons, formed by mutations of other codons, including AUG, would have reappeared and would have been translated as methionine.     

A rationale for the symmetries by base substitutions of degeneracy in the genetic code

The first symmetry by base substitutions of degeneracy in the genetic code was described by Rumer (1966) and the other symmetries were identified later by Jestin (2006) and Jestin and Soulé (2007). Here, a rationale accounting for these symmetries is reported. The number of non-synonymous substitutions over the replicated coding sequence is written as a function of the substitution matrix, whose elements are the number of substitutions from any codon to any other codon. The p-adic distance used as a similarity measure and applied to this matrix is shown to be biologically relevant. The rationale indicates that symmetries by base substitutions of degeneracy in the genetic code are symmetries of the measures of the number of non-synonymous substitutions for sets of synonymous codons.     

Critical roles for a genetic code alteration in the evolution of the genus Candida

During the last 30 years, several alterations to the standard genetic code have been discovered in various bacterial and eukaryotic species. Sense and nonsense codons have been reassigned or reprogrammed to expand the genetic code to selenocysteine and pyrrolysine. These discoveries highlight unexpected flexibility in the genetic code, but do not elucidate how the organisms survived the proteome chaos generated by codon identity redefinition. In order to shed new light on this question, we have reconstructed a Candida genetic code alteration in Saccharomyces cerevisiae and used a combination of DNA microarrays, proteomics and genetics approaches to evaluate its impact on gene expression, adaptation and sexual reproduction. This genetic manipulation blocked mating, locked yeast in a diploid state, remodelled gene expression and created stress cross-protection that generated adaptive advantages under environmental challenging conditions. This study highlights unanticipated roles for codon identity redefinition during the evolution of the genus Candida, and strongly suggests that genetic code alterations create genetic barriers that speed up speciation.