Biomedical subjects

Anders Fuglsang

Publications and source records attributed to Anders Fuglsang.

At least 19 recordsLinked to original sources

PubMed2004

Compositional nonrandomness upstream of start codons in archaebacteria.

Since the regions directing transcriptional and translational initiation in archaebacteria are poorly characterized, the purpose of this study was to characterize them using measurements of nonrandomness upstream of start codons on eight fully sequenced archaebacterial genomes. Two distinctly different regions with conservation were identified. The location of the first corresponded well to the classical Shine-Dalgarno region (phi peak), and the other was located approximately 20-35 nucleotides upstream of start codons (alpha region), but both regions are not present in all strains. The composition of the region around the phi peak showed an overrepresentation of guanine, whereas composition in the alpha region had an overrepresentation of adenine and thymine. It is furthermore shown that the alpha region surprisingly is associated with start codon usage and other characteristics of the genes. The alpha region is likely to correspond to TATA-boxes and thereby indicates use of leaderless (or short-leadered) transcripts.

5' Flanking Region↗

PubMed2004

The 'effective number of codons' revisited.

Frank Wright [Gene 87 (1990) 23] derived a formula for calculation of a quantity termed the 'effective number of codons' (Nc) based on codon homozygosities. This quantity is a number between 20 and 61 and tells to what degree the codon usage in a gene is biased, i.e., it approaches 20 codons for the extremely biased genes, and approaches 61 for the genes where all possible codons are used with no preference. Among the different measures of codon bias Nc is considered the most useful and has found widespread use in papers dealing with codon usage phenomena. In this paper, the mathematical behaviours of codon homozygosities and Nc are evaluated, using Escherichia coli as the model organism. The results indicate that the classical formula for calculation of Nc could appropriately be substituted under circumstances, where there is bias discrepancy, i.e., when one amino acid (or more) within a degeneracy group is associated with strong codon bias while at the same time others in the same degeneracy group have little bias. An alternative estimator, termed Nc, is proposed and tested against Nc, and performs better when there is such bias discrepancy.

Codon↗

PubMed2004

The relationship between palindrome avoidance and intragenic codon usage variations: a Monte Carlo study.

Several studies have shown that codon usage within genes varies, as it seems dependent on both codon context and codon position within the gene. Given that palindromes in addition often are avoided in genomes, this study aimed at finding out if intragenic variations in codon usage may be a way to control the amount and location of palindromes. A Monte Carlo algorithm was written which resampled the codons in genes while keeping the amino acid sequence of the translation product constant. On the resampled sequences, palindromes were counted and their intragenic positions mapped. Escherichia coli K12 uses type II restriction-modification systems and displays pronounced codon usage phenomena. Using this as a reference organism it was clearly shown that the number of palindromes in genes is generally lower than the amount of palindromes in resampled genes; thus, the succession of codons seems to be a way to decrease the number of palindromes. The intragenic position of palindromes in resampled sequences, however, was largely equal to the position in the native genes, so codon usage phenomena are unlikely to be a way to control the intragenic position of palindromes. The analysis was repeated on two bacteriophages and gave similar same results, even though the virus genomes are much smaller. Studies on the endosymbionts Buchnera sp. APS and Wigglesworthia sp., which seemingly have no type II restriction-modification systems, showed that in these species there is only weak evidence for codon usage acting to control the number of palindromes.

Algorithms↗

PubMed2004

Evolution of prokaryotic DNA: intragenic and extragenic divergences observed with orthologs from three related species.

This study compared orthologous gene pairs from Escherichia coli K12, E. coli O157:H7 EDL933, Salmonella typhimurium LT2, and Yersinia pestis CO92 using only homologs of equal length, and differing nucleotides were counted and mapped. The data showed very clearly how the rates of divergence change with intragenic and extragenic position. The rate of synonymous mutation is lowest near start codons and near stop codons, and, a little surprisingly, the opposite seemed to be true for nonsynonymous substitutions. Analysis outside genes reveals that nucleotide divergences occur less frequently upstream of start codons than downstream of stop codons, and a remarkable drop in divergences is seen for two of the data sets around N = 9 nucleotides upstream of start codons; that is, the Shine-Dalgarno region changes at a lower rate. The explanation is likely to be the link between expressivity and sequence complementarity to the 3' end of 16S ribosomal rRNA. The latter is highly conserved across many bacterial and archaebacterial species.

Bacteria↗

PubMed2004

Using spectrophotometry to determine in vitro turnover rates of peptides in plasma.

A new method for determination of first-order elimination constants for dipeptides is presented. The peptides are hydrolysed by plasma enzymes into amino acids, and ortho-phthaldialdehyde (OPA) is used to react with free primary amino groups. The concentration of free amino groups can, thus, be followed using simple spectrophotometry. A mathematical model for the concentration of free primary amino groups with time is presented through which the elimination constant, and thus the half life, can be determined by curve fitting. The method is applied to inhibitors of angiotensin-converting enzyme derived from the primary structure of milk proteins. The results show that these dipeptides have in vitro half lives ranging from 4.3-64 min, when incubated with 50% rat plasma. This explains why these casokinins in vivo only cause a very moderate and short-lasting inhibition. The model for calculation of elimination constant is limited to dipeptides that do not contain a C-terminal proline. The derivatization method can be applied to longer peptides as a crude indicator of peptide hydrolysis, but does not allow calculation of their elimination constants per se.

Animals↗

PubMed2004

Bioinformatic analysis of the link between gene composition and expressivity in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The compositional non-randomness was studied in genes of Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both species, codon usage is well correlated with expressivity (measured as the codon adaptation index). Both species generally display higher nucleotide non-randomness in the group of highly expressed genes than in the lowly expressed genes. The highly expressed genes in both species are furthermore characterized by marked peaks in non-randomness at N=3 upstream of start codons, N=2 downstream of start codons and at N=1 and N=7 downstream of stop codons, indicating that these nucleotides may be key elements in translational regulation. Intragenic variation in codon usage was also observed to be linked to expressivity. It is suggested that the firm link between expressivity and codon usage calls for codon optimization. Based on bioinformatic calculations, examples of proteins are given for which codon optimizations might be relevant.

Base Sequence↗

PubMed2004

Nucleotides downstream of start codons show marked non-randomness in Escherichia coli but not in Bacillus subtilis.

This study aimed at measuring the nucleotide non-randomness in the region downstream of start codons in bacterial genes and to see if the non-randomness differs between biased and unbiased genes, in terms of the effective number of codons (Nc) and the codon adaptation index (CAI). In Escherichia coli, there was a marked elevation in nucleotide conservation for the genes having low Nc-values compared to the genes having high Nc-values, i.e the more biased genes showed a higher level of non-randomness. Likewise, the genes displaying high CAI-values showed stronger nucleotide conservation than the genes of low CAI-values. This elevated conservation is visible up to approximately 15-17 nucleotides downstream of the start codon, after which there is little difference. This indicates that there may be distinct selectional mechanisms acting upon the first 5-6 codons within genes in E. coli. In B. subtilis, these effects are less pronounced, if present at all. Furthermore, analyses of codons used in this region were not in support of the hypothesis that the elevation in nucleotide non-randomness is a question of selection for certain optimal codons.

Bacillus subtilis↗

PubMed2003

Lactic acid bacteria as prime candidates for codon optimization.

In species having a strong correlation of expressivity and codon bias it has been shown that heterologous expression can be optimized by changing codons of the introduced gene towards the set of codons that the host organism naturally uses in its highly expressed genes. Even though two lactic acid bacteria are fully sequenced, there are no reports on attempts of codon optimization in the literature. In this report it is demonstrated that codons used in highly expressed genes tend to differ from the codons in lowly expressed genes, and that there is a strong correlation of codon bias and empirical expressivity (codon adaptation index) in Lactococcus lactis and Lactobacillus plantarum. This strongly suggests that codon optimization strategies could be applied to expression systems with lactic acid bacteria as producer strains. A good example of a candidate for codon optimization is the mouse interleukin-2 gene, which in its natural form has an extremely low codon adaptation index for expression in Lc. lactis.

Algorithms↗

PubMed2003

The effective number of codons for individual amino acids: some codons are more optimal than others.

The aim of this study was to evaluate the codon bias using the effective number of codons for individual amino acids (N(c)(AA)) and to assess the codon bias in relation to the definition of optimal codons, using Escherichia coli as a model organism. We show that a general correlation exists between the effective number of codons (Ncirc(c)) or codon adaptation index (CAI) and N(c)(AA), but that this correlation is not equally strong for all amino acids within a degeneracy group. For example, leucine codons contribute more to Ncirc(c) and the codon adaptation index than serine codons. A possible explanation is that some optimal codons are more optimal than others, in terms of the selectional advantage they offer. This hypothesis is confirmed by further analysis on the correlations that exist between values for relative synonymous codon usage (RSCU), N(c)(AA), and the codon adaptation index.

Amino Acids↗

PubMed2003

Distribution of potential type II restriction sites (palindromes) in prokaryotes.

Restriction-modification systems are used as a defensive mechanism against inappropriate invasion of foreign DNA. The recognition sequences for the common type II restriction enzymes and their corresponding methylases are usually palindromes. In this study, we identified the most over- and underrepresented words in DNA of four bacteria: Escherichia coli, Bacillus subtilis, Clostridium perfringens, and Pseudomonas aeruginosa. Using maximum order Markov chain analysis, we found that palindromic words were most often more underrepresented than their non-palindromic counterparts. No strict rule for the intragenic palindrome content could be derived, but for three of the bacteria there was a weak correlation between codon usage bias and palindrome content. A clear drop in palindrome counts was observed in the Shine-Dalgarno region for B. subtilis and C. perfringens, but not in E. coli or P. aeruginosa. It was also shown that palindromes in eubacteria and archaebacteria seem to occur slightly more infrequently than expected on the basis of the genomic GC-content, but some exceptions to this principle exist.

Bacteria↗

PubMed2003

Patterns of context-dependent codon biases.

The association of codon context and codon usage was studied in seven bacteria as well as Schizosaccharomyces pombe and Encephalitozoon cuniculi. The association is strongest in magnitude closest to the codons of interest but there is apparently no rule about which of the two contexts is generally strongest associated to codon usage. In all bacterial species and in the intron-rich Sch. pombe it was furthermore observed from plots of chi2 versus N that the wobble positions of codons in the proximity cause regular peaks both upstream and downstream. This observation is discussed in relation to a possible effect of mutational pressure on the association of codon usage and codon context. Absence of peaks corresponding to the wobble positions in the intron-poor En. cuniculi, and presence in Sch. pombe, may indicate that the role of introns in the context-dependent codon bias is negligible.

Animals↗

PubMed2003

Non-randomness in Shine-Dalgarno regions: links to gene characteristics.

A probabilistic approach to the study of the Shine-Dalgarno region was used to identify the most non-random positions based on parsing of genomes in four species: Escherichia coli, Bacillus subtilis, the AT-rich Clostridium perfringens, and the GC-rich Streptomyces coelicolor. The compositional non-randomness shows a clear peak centered around 9-11 nucleotides upstream of the start codon. This peak was in all species associated with guanine as the most abundant nucleotide, flanked by guanine in the closest proximity and adenines farther away (cytosine in case of S. coelicolor). Using contingency tables, the nucleotides in the Shine-Dalgarno region were shown to have a strong association to the choice of start codons. We also show that gene characteristics such as length, aromaticity, and lipophilicity are related to the nucleotide at this peak position upstream of the start codon.

Conserved Sequence↗

PubMed2003

Codon optimizer: a freeware tool for codon optimization.

Selection plays a major role in the determination of codon usage in all organisms studied so far. In highly expressed genes, a narrow set of codons is used and these codons correspond to the more abundant tRNA species. This minimizes the risk of tRNA depletion during translation. In fact, the codons in a gene may be true bottlenecks, especially in cases where foreign genes are expressed in a host in which the usage of codons in highly expressed genes does not resemble the usage of codons in the species from which the foreign gene originates. In such cases, it has been shown that substitution of rare codons in the introduced gene may increase the yield dramatically. In addition, replacement of rare codons might decrease the chance of misincorporation and protect the protein from premature turnover. Here, a piece of software is announced that calculates a codon-optimized sequence of any gene based on knowledge of highly expressed genes of a host. In addition, it calculates the codon adaptation index of the gene and identifies internal type II restriction sites of the optimized sequence. The program runs under Windows and is available as freeware for use in academia.

Animals↗

PubMed2003

Lactic acid bacteria: inhibition of angiotensin converting enzyme in vitro and in vivo.

A total of 26 strains of wild-type lactic acid bacteria, mainly belonging to Lactococcus lactis and Lactobacillus helveticus, were assayed in vitro for their ability to produce a milk fermentate with inhibitory activity towards angiotensin converting enzyme (ACE). It was clear that the test strains in this study, in general, produce inhibitory substances in varying amounts. Using a spectrophotometric assay based on amino group derivatization with ortho-phthaldialdehyde as a measure of relative peptide content, it was shown that there is a significant correlation between peptide formation and ACE inhibition, indicating that peptide measurement constitutes a convenient selection method. The effect of active fermentates on in vivo ACE activity was demonstrated in normotensive rats. The pressor effect of angiotensin I (0.3 microg/kg) upon intravenous injection was significantly lower when rats were pre-fed with milks fermented using two strains of Lactobacillus helveticus. An increased response to bradykinin (10 microg/kg, intravenously injected) was observed using one of these fermented milks. It is concluded that Lactobacillus helveticus produces substances which in vivo can give rise to an inhibition of ACE. The inhibition in vivo was low compared to what can be achieved with classical ACE inhibitors. The clinical relevance of this finding is discussed. This work is the first in which an effect of fermented milk on ACE in vivo has been demonstrated, measured as decreased ability to convert angiotensin I to angiotensin II.

Angiotensin I↗

PubMed2003

The genome of Campylobacter jejuni: codon and amino acid usage.

The genes from the genome of the AT-rich bacterium Campylobacter jejuni were analysed and characterised with respect to usage and amino acid usage. Codon usage is generally biased for all amino acids having synonymous codons, so that AT-rich synonyms are most frequently used. Markov chain analysis showed that codon bias and over- or underrepresentation of the corresponding tri-letter words are not related. Predicted secondary structure, lipophilicity, codon position within the gene, strand, and position on the (+)-strand were all shown to be determinants of codon usage, and these effects were in part directly explained by compositional phenomena. Codon context and the GC-content at the wobble position of the fourfold degenerate sites exert indirect effects on codon usage. The factors that affect codon usage seem to affect all amino acids, rather than selected amino acids. The usage of amino acids correlates well with the GC-content of genes, i.e. usage of amino acids encoded by GC-rich codons increases with GC-content and vice versa.

Amino Acids↗

PubMed2003

Strong associations between gene function and codon usage.

The association between codon usage and gene function was analyzed in the complete genomes of Eschericia coli, Bacillus subtilis, Lactococcus lactis and Campylobacter jejuni, using the functional annotation provided by NCBI. Two distinctly different ways of quantifying codon usage were used in the analysis. By using contingency tables it was found that for most amino acids a highly significant association with gene function exists for all species, indicating that codon usage at the level of individual amino acids is generally closely coordinated with gene function. By computing the effective number of codons in the annotated genes and comparing the median values in groups of different gene functions it was shown for all species that codon bias gene by gene also differs.

Amino Acids↗

PubMed2003

Association of the phi nucleotide with codon bias, amino acid usage and expressivity: differences between Bacillus subtilis and Escherichia coli.

By measuring the non-randomness in Shine-Dalgarno regions it was recently shown that the compositional non-randomness peaks approximately 10 nucleotides upstream of the start codons. This position, termed the phi position, was furthermore shown to be associated with certain characteristics of the gene/protein and start codon usage. This raises the question whether codon usage in general is associated with the phi position. In this study, the connection between the phi nucleotide and general codon usage, both gene-wide and at the level of individual amino acids, was studied in Eschericia coli and Bacillus subtilis. E. coli but not B. subtilis shows a strong general association between the phi position and codon usage bias. In both species, the genes with higher expressivity show stronger conservation in the Shine-Dalgarno region compared to the genes with lower expressivity.

Amino Acids↗

PubMed2003

Characterization of new milk-derived inhibitors of angiotensin converting enzyme in vitro and in vivo.

Inhibition of angiotensin converting enzyme (ACE) has been observed with a variety of different peptides, and peptide fragments with inhibitory capabilities have been identified within many different proteins, including milk proteins. The purpose of this study therefore was to identify new short peptides with inhibitory properties from the primary structure of milk proteins and to characterize them in vitro and in vivo, since no milk derived ACE inhibitors have previously been evaluated for their ability to inhibit ACE in vivo. In vitro, 8 of 9 dipeptides were found to be competitive inhibitors of ACE. The IC50 was significantly lower when an angiotensin I-like substrate was used, than when a bradykinin-like substrate was used. Using three different in vivo models for ACE inhibition, a very moderate effect was observed for three of the new peptides, but only for up to 6 or 12 minutes. Nothing was observed with two reference compounds that are reported to be hypotensive ACE-inhibitors derived from milk proteins. This raises the question whether the mechanism of hypotensive action is straightforward inhibition of ACE in vivo.

Angiotensin-Converting Enzyme Inhibitors↗