GC content dependency of open reading frame prediction via stop codon frequencies

A frequently used approach for detecting potential coding regions is to search for stop codons. In the standard genetic code 3 out of 64 trinucleotides are stop codons. Hence, in random or non-coding DNA one can expect every 21st trinucleotide to have the same sequence as a stop codon. In contrast, the open reading frames (ORFs) of most protein-coding genes are considerably longer. Thus, the stop codon frequency in coding sequences deviates from the background frequency of the corresponding trinucleotides. This has been utilized for gene prediction, in particular, in detecting protein-coding ORFs. Traditional methods based on stop codon frequency are based on the assumption that the GC content is about 50%. However, many genomes show significant deviations from that value. With the presented method we can describe the effects of GC content on the selection of appropriate length thresholds of potentially coding ORFs. Conversely, for a given length threshold, we can calculate the probability of observing it in a random sequence. Thus, we can derive the maximum GC content for which ORF length is practicable as a feature for gene prediction methods and the resulting false positive rates. A rough estimate for an upper limit is a GC content of 80%. This estimate can be made more precise by including further parameters and by taking into account start codons as well. We demonstrate the feasibility of this method by applying it to the genomes of the bacteria Rickettsia prowazekii, Escherichia coli and Caulobacter crescentus, exemplifying the effect of GC content variations according to our predictions. We have adapted the method for predicting coding ORFs by stop codon frequency to the case of GC contents different from 50%. Usually, several methods for gene finding need to be combined. Thus, our results concern a specific part within a package of methods. Interestingly, for genomes with low GC content such as that of R. prowazekii, the presented method provides remarkably good results even when applied alone.     

A frequency-controlled random mutagenesis method for GC-rich genes

A novel random mutagenesis strategy was developed by combining sodium bisulfite modification with polymerase chain reaction (PCR). This method introduced the predominant substitution of GC to AT, meaning that it was more suitable for mutagenesis of GC-rich genes and helped to decrease the GC content of target DNA. Mutation efficiency correlated with modification time and different mutation frequency could easily be obtained by controlling modification time. The results indicated that this method could yield a desired and adequate frequency of random mutation to the DNA of interest, especially GC-rich genes, and provided a powerful tool for directed molecular evolution.     

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.     

Genomic GC content prediction in prokaryotes from a sample of genes

GC level is a key feature in prokaryotic genomes. Widely employed in evolutionary studies, new insights appear however limited because of the relatively low number of characterized genomes. Since public databases mainly comprise several hundreds of prokaryotes with a low number of sequences per genome, a reliable prediction method based on available sequences may be useful for studies that need a trustworthy estimation of whole genomic GC. As the analysis of completely sequenced genomes shows a great variability in distributional shapes, it is of interest to compare different estimators. Our analysis shows that the mean of GC values of a random sample of genes is a reasonable estimator, based on simplicity of the calculation and overall performance. However, usually sequences come from a process that cannot be considered as random sampling. When we analyzed two introduced sources of bias (gene length and protein functional categories) we were able to detect an additional bias in the estimation for some cases, although the precision was not affected. We conclude that the mean genic GC level of a sample of 10 genes is a reliable estimator of genomic GC content, showing comparable accuracy with many widely employed experimental methods.     

The HLA-DQ beta gene upstream region contains an immunoglobulin-like octamer motif that binds cell-type specific nuclear factors

A method is described for scanning relatively large fragments of DNA for sequences which bind nuclear factors. This method was used to identify an octamer (ATTTGTAT) in the DQ beta gene upstream region which differs from the immunoglobulin gene octamer (ATTTGCAT) by only 1 bp. The DQ beta gene octamer binds two proteins, one (B2) appears to be B cell specific while the other (B1) is not. These factors are either similar or identical to factors which bind to the octamer motif in immunoglobulin genes. All other class II MHC genes for which sequence information is available contain an octamer motif in their upstream region. Thus, the possibility that these sequences regulate B cell specific expression of class II MHC genes requires careful evaluation.     

Experimental designs for reliable detection of linkage disequilibrium in unstructured random population association studies

A method is given for design of experiments to detect associations (linkage disequilibrium) in a random population between a marker and a quantitative trait locus (QTL), or gene, with a given strength of evidence, as defined by the Bayes factor. Using a version of the Bayes factor that can be linked to the value of an F-statistic with an existing deterministic power calculation makes it possible to rapidly evaluate a comprehensive range of scenarios, demonstrating the feasibility, or otherwise, of detecting genes of small effect. The Bayes factor is advocated for use in determining optimal strategies for selecting candidate genes for further testing or applications. The prospects for fine-scale mapping of QTL are reevaluated in this framework. We show that large sample sizes are needed to detect small-effect genes with a respectable-sized Bayes factor, and to have good power to detect a QTL allele at low frequency it is necessary to have a marker with similar allele frequency near the gene.     

The inhibition of complement-dependent hemolysis of liposomes containing cerebroside sulfate

Cerebroside sulfate (CGS) was found to be capable of inhibiting complement-dependent hemolysis. The activity dependence of CGS-containing liposomes on their composition was studied. Mixtures of CGS with phosphatidylethanolamine, phosphatidylserine, sphingomyelin from cattle brain, cerebroside from cattle spinal cord (CG), and egg yolk phosphatidylcholine (ePC) were investigated. In the case of binary CGS/ePC mixtures, the antihemolytic activity varied nonlinearly with an increase in the mass part of CGS: it sharply increased with an increase in the CGS part from 0.3 to 0.5 and decreased by 20-30% of the maximum value with an increase in the CGS part from 0.9 to 1. On the basis of these experiments, the optimum distance between the charged groups of CGS was estimated to be 0.92-1.6 nm. In the ternary compositions of 4:3:3 CGS/ePC/polar lipid, only CG increased the activity of liposomes as compared to that of liposomes from the 4:6 CGS/ePC. The preliminary incubation of CGS-containing liposomes with complement decreased hemolysis more effectively than incubation with other components of the hemolytic system. This suggests that the interaction of CGS-containing liposomes with the complement proteins is responsible for their antihemolytic activity.     

Limitations of the poly(glutamic acid) reconstitution method in the reassembly of mono- and dinucleosomes

Reconstitution of mononucleosomes and dinucleosomes at physiological ionic strength by means of poly(glutamic acid) is not efficient at physiological histone octamer:DNA ratios, unlike that with the salt dialysis method. The shorter the DNA is, the less transfer of octamers from poly(glutamic acid) to DNA occurs. By increasing the octamer:DNA ratio it is possible to involve all the DNA in the assembly, but for DNA longer than core particle length, nucleoprotein particles containing extra histones are concomitantly generated. Except for core particle and chromatosome lengths of DNA reassembled at 0.6:1 or 1:1 octamer:DNA ratio (and thus with low yield), reconstituted nucleoprotein particles proved to be different from native nucleosomes by their insolubility upon isolation. In the aggregates, DNA ends seemed to be sufficiently loose to allow exonuclease III digestion up to a certain limit. This resulted in patterns that for some cloned DNA fragments could give the impression, without knowledge of the above, of resulting from a unique octamer position. In view of the small range of length of DNA and the low yield of faithful reconstitution, the assembly method using poly(glutamic acid) is only of limited use in mono- or dinucleosome reconstitution experiments, at least in our hands.     

Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori

We developed a microarray hybridization-based method, 'comparative genome sequencing' (CGS), to find mutations in bacterial genomes and used it to study metronidazole resistance in H. pylori. CGS identified mutations in several genes, most likely affecting metronidazole activation, and produced no false positives in analysis of three megabases. We conclude that CGS identifies mutations in bacterial genomes efficiently, should enrich understanding of systems biology and genome evolution, and help track pathogens during outbreaks.     

The inhibition of complement-dependent hemolysis by liposomes containing cerebroside sulfate

Cerebroside sulfate (CGS) was found to be capable of inhibiting complement-dependent hemolysis. The activity dependence of CGS-containing liposomes on their composition was studied. Mixtures of CGS with phosphatidylethanolamine, phosphatidylserine, sphingomyelin from cattle brain, cerebroside from cattle spinal cord (CG), and egg yolk phosphatidylcholine (ePC) were investigated. In the case of binary CGS/ePC mixtures, the antihemolytic activity varied nonlinearly with an increase in the mass part of CGS: it sharply increased with an increase in the CGS part from 0.3 to 0.5 and decreased by 20–30% of the maximum value with an increase in the CGS part from 0.9 to 1. On the basis of these experiments, the optimum distance between the charged groups of CGS was estimated to be 0.92–1.6 nm. In the ternary compositions of 4:3:3 CGS/ePC/polar lipid, only CG increased the activity of liposomes as compared to that of liposomes from the 4:6 CGS/ePC. The preliminary incubation of CGS-containing liposomes with complement decreased hemolysis more effectively than incubation with other components of the hemolytic system. This suggests that the interaction of CGS-containing liposomes with the complement proteins is responsible for their antihemolytic activity.