Adaptive Evolution Hotspots at the GC-Extremes of the Human Genome: Evidence for Two Functionally Distinct Pathways of Positive Selection

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

A molecular systematic survey of cultured microbial associates of deep-water marine invertebrates

BackgroundAn industrial approach to protein production demands maximization of cloned gene expression, balanced with the recombinant host’s viability. Expression of toxic genes from thermophiles poses particular difficulties due to high GC content, mRNA secondary structures, rare codon usage and impairing the host’s coding plasmid replication.TaqII belongs to a family of bifunctional enzymes, which are a fusion of the restriction endonuclease (REase) and methyltransferase (MTase) activities in a single polypeptide. The family contains thermostable REases with distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI and a few enzymes found in mesophiles. While not being isoschizomers, the enzymes exhibit amino acid (aa) sequence homologies, having molecular sizes of ~120 kDa share common modular architecture, resemble Type-I enzymes, cleave DNA 11/9 nt from the recognition sites, their activity is affected by S-adenosylmethionine (SAM).ResultsWe describe the taqIIRM gene design, cloning and expression of the prototype TaqII. The enzyme amount in natural hosts is extremely low. To improve expression of the taqIIRM gene in Escherichia coli (E. coli), we designed and cloned a fully synthetic, low GC content, low mRNA secondary structure taqIIRM, codon-optimized gene under a bacteriophage lambda (λ) PR promoter. Codon usage based on a modified ‘one amino acid–one codon’ strategy, weighted towards low GC content codons, resulted in approximately 10-fold higher expression of the synthetic gene. 718 codons of total 1105 were changed, comprising 65% of the taqIIRM gene. The reason for we choose a less effective strategy rather than a resulting in high expression yields ‘codon randomization’ strategy, was intentional, sub-optimal TaqII in vivo production, in order to decrease the high ‘toxicity’ of the REase-MTase protein.ConclusionsRecombinant wt and synthetic taqIIRM gene were cloned and expressed in E. coli. The modified ‘one amino acid–one codon’ method tuned for thermophile-coded genes was applied to obtain overexpression of the ‘toxic’ taqIIRM gene. The method appears suited for industrial production of thermostable ‘toxic’ enzymes in E. coli. This novel variant of the method biased toward increasing a gene’s AT content may provide economic benefits for industrial applications.

Much effort and interest have focused on assessing the importance of naturalselection, particularly positive natural selection, in shaping the human genome.Although scans for positive selection have identified candidate loci that may beassociated with positive selection in humans, such scans do not indicate whetheradaptation is frequent in general in humans. Studies based on the reasoning ofthe MacDonald–Kreitman test, which, in principle, can be used toevaluate the extent of positive selection, suggested that adaptation isdetectable in the human genome but that it is less common than in Drosophila orEscherichia coli. Both positive and purifying naturalselection at functional sites should affect levels and patterns of polymorphismat linked nonfunctional sites. Here, we search for these effects by analyzingpatterns of neutral polymorphism in humans in relation to the rates ofrecombination, functional density, and functional divergence with chimpanzees.We find that the levels of neutral polymorphism are lower in the regions oflower recombination and in the regions of higher functional density ordivergence. These correlations persist after controlling for the variation in GCcontent, density of simple repeats, selective constraint, mutation rate, anddepth of sequencing coverage. We argue that these results are most plausiblyexplained by the effects of natural selection at functionalsites—either recurrent selective sweeps or backgroundselection—on the levels of linked neutral polymorphism. Naturalselection at both coding and regulatory sites appears to affect linked neutralpolymorphism, reducing neutral polymorphism by 6% genome-wide and by11% in the gene-rich half of the human genome. These findings suggestthat the effects of natural selection at linked sites cannot be ignored in thestudy of neutral human polymorphism.

During SARS-CoV-2 proliferation, the translation of viral RNAs is usually the rate-limiting step. Understanding the molecular details of this step is beneficial for uncovering the origin and evolution of SARS-CoV-2 and even for controlling the pandemic. To date, it is unclear how SARS-CoV-2 competes with host mRNAs for ribosome binding and efficient translation. We retrieved the coding sequences of all human genes and SARS-CoV-2 genes. We systematically profiled the GC content and folding energy of each CDS. Considering that some fixed or polymorphic mutations exist in SARS-CoV-2 and human genomes, all algorithms and analyses were applied to both pre-mutate and post-mutate versions. In SARS-CoV-2 but not human, the 5-prime end of CDS had lower GC content and less RNA structure than the 3-prime part, which was favorable for ribosome binding and efficient translation initiation. Globally, the fixed and polymorphic mutations in SARS-CoV-2 had created an even lower GC content at the 5-prime end of CDS. In contrast, no similar patterns were observed for the fixed and polymorphic mutations in human genome. Compared with human RNAs, the SARS-CoV-2 RNAs have less RNA structure in the 5-prime end and thus are more favorable of fast translation initiation. The fixed and polymorphic mutations in SARS-CoV-2 are further amplifying this advantage. This might serve as a strategy for SARS-CoV-2 to adapt to the human host.

Previously we described a PCR protocol for detecting the inversion in the factor VIII gene, which is a common cause of Hemophilia A. This PCR assay is challenging due to the size of the amplification (10-12 kb), the varying GC content (30%-80%) and the multiplex PCR products involved (four for carrier female). Efficient amplification of the four segments depends on three unusual modifications to standard long-distance PCR protocols: (i) very high concentrations of dimethyl sulfoxide, (ii) addition of deaza-dGTP and (iii) high concentration of Taq and Pwo DNA polymerases. One of the segments was amplified much more efficiently than the others under standard three-temperature cycling conditions (12 s at 94 degrees C, 30 s at 65 degrees C and 14 min at 68 degrees C). To facilitate the uniform amplification of the multiple regions, subcycling-PCR (S-PCR) was developed. In S-PCR, the combined annealing/elongation step is composed of subcycles of shuttling between a low and a high temperature, e.g., shuttling four times between 60 degrees and 65 degrees C. S-PCR produces consistent robust amplification of the various segments produced by wild-type, mutant and carrier individuals. S-PCR is a simple generalization of PCR, which generally may be advantageous in three contexts: (i) amplification of long segments in which the GC content varies within the segment, (ii) multiplex amplification of long segments and (iii) multiplex amplification of short segments in which the GC content varies among the segments.

The housekeeping (ubiquitously expressed) genes in the mammal genome were shown here to be on average slightly GC-richer than tissue-specific genes. Both housekeeping and tissue-specific genes occupy similar ranges of GC content, but the former tend to concentrate in the upper part of the range. In the human genome, tissue-specific genes show two maxima, GC-poor and GC-rich. The strictly tissue-specific human genes tend to concentrate in the GC-poor region; their distribution is left-skewed and thus reciprocal to the distribution of housekeeping genes. The intermediately tissue-specific genes show an intermediate GC content and the right-skewed distribution. Both in the human and mouse, genes specific for some tissues (e.g., parts of the central nervous system) have a higher average GC content than housekeeping genes. Since they are not transcribed in the germ line (in contrast to housekeeping genes), and therefore have a lower probability of inheritable gene conversion, this finding contradicts the biased gene conversion (BGC) explanation for elevated GC content in the heavy isochores of mammal genome. Genes specific for germ-line tissues (ovary, testes) show a low average GC content, which is also in contradiction to the BGC explanation. Both for the total data set and for the most part of tissues taken separately, a weak positive correlation was found between gene GC content and expression level. The fraction of ubiquitously expressed genes is nearly 1.5-fold higher in the mouse than in the human. This suggests that mouse tissues are comparatively less differentiated (on the molecular level), which can be related to a less pronounced isochoric structure of the mouse genome. In each separate tissue (in both species), tissue-specific genes do not form a clear-cut frequency peak (in contrast to housekeeping genes), but constitute a continuum with a gradually increasing degree of tissue-specificity, which probably reflects the path of cell differentiation and/or an independent use of the same protein in several unrelated tissues.

In the last two decades, our understanding of human gene regulation has improved tremendously. There are plentiful computational methods which focus on integrative data analysis of humans, and model organisms, like mouse and drosophila. However, these tools are not directly employable by researchers working on non-model organisms to answer fundamental biological, and evolutionary questions. We aimed to develop new tools, and adapt existing software for the analysis of transcriptomic and epigenomic data of one such non-model organism, Paramecium tetraurelia, an unicellular eukaryote. Paramecium contains two diploid (2n) germline micronuclei (MIC) and a polyploid (800n) somatic macronuclei (MAC). The transcriptomic and epigenomic regulatory landscape of the MAC genome, which has 80% protein-coding genes and short intergenic regions, is poorly understood. We developed a generic automated eukaryotic short interfering RNA (siRNA) analysis tool, called RAPID. Our tool captures diverse siRNA characteristics from small RNA sequencing data and provides easily navigable visualisations. We also introduced a normalisation technique to facilitate comparison of multiple siRNA-based gene knockdown studies. Further, we developed a pipeline to characterise novel genome-wide endogenous short interfering RNAs (endo-siRNAs). In contrary to many organisms, we found that the endo-siRNAs are not acting in cis, to silence their parent mRNA. We also predicted phasing of siRNAs, which are regulated by the RNA interference (RNAi) pathway. Further, using RAPID, we investigated the aberrations of endo-siRNAs, and their respective transcriptomic alterations caused by an RNAi pathway triggered by feeding small RNAs against a target gene. We find that the small RNA transcriptome is altered, even if a gene unrelated to RNAi pathway is targeted. This is important in the context of investigations of genetically modified organisms (GMOs). We suggest that future studies need to distinguish transcriptomic changes caused by RNAi inducing techniques and actual regulatory changes. Subsequently, we adapted existing epigenomics analysis tools to conduct the first comprehensive epigenomic characterisation of nucleosome positioning and histone modifications of the Paramecium MAC. We identified well positioned nucleosomes shifted downstream of the transcription start site. GC content seems to dictate, in cis, the positioning of nucleosomes, histone marks (H3K4me3, H3K9ac, and H3K27me3), and Pol II in the AT-rich Paramecium genome. We employed a chromatin state segmentation approach, on nucleosomes and histone marks, which revealed genes with active, repressive, and bivalent chromatin states. Further, we constructed a regulatory association network of all the aforementioned data, using the sparse partial correlation network technique. Our analysis revealed subsets of genes, whose expression is positively associated with H3K27me3, different to the otherwise reported negative association with gene expression in many other organisms. Further, we developed a Random Forests classifier to predict gene expression using genic (gene length, intron frequency, etc.) and epigenetic features. Our model has a test performance (PR-AUC) of 0.83. Upon evaluating different feature sets, we found that genic features are as predictive, of gene expression, as the epigenetic features. We used Shapley local feature explanation values, to suggest that high H3K4me3, high intron frequency, low gene length, high sRNA, and high GC content are the most important elements for determining gene expression status. In this thesis, we developed novel tools, and employed several bioinformatics and machine learning methods to characterise the regulatory landscape of the Paramecium’s (epi)genome.

Background:Leishmaniasis is a disease caused by the Leishmania sp. and can be classified into two major types: cutaneous and visceral leismaniasis. Visceral leishmaniasis is the deadlier type and is mediated by Leishmania donovani and involves the establishment of persistent infection and causes damage to the liver, spleen and bone marrow. With no vaccine yet available against leishmaniasis and the current therapeutic drugs of leishmaniasis being toxic and expensive; an alternative treatment is necessary.Objective:Surface glycocalyx protein gp63, plays a major role in the virulence and resulting pathogenicity associated with the disease. Henceforth, silencing the gp63 mRNA through the RNA interference system was the aim of this study.Methods:In this study two competent siRNAs and three miRNAs have been designed against gp63 for five different strains of L. donovani by using various computational methods. Target specific siRNAs were designed using siDirect 2.0 and to design possible miRNA, another tool named IDT (IntegratedDNA Technology). Screening for off-target similarity was done by BLAST and the GC contents and the secondary structures of the designed RNAs were determined. RNA-RNA interaction was calculated by RNAcofold and IntraRNA, followed by the determination of heat capacity and the concentration of duplex by DNAmelt web server.Results:The selected RNAi molecules; two siRNA and three miRNA had no off-target in human genome and the ones with lower GC content were selected for efficient RNAi function. The selected ones showed proper thermodynamic characteristics to suppress the expression of the pathogenic gene of gp63.

Decision letter: Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates

We recently classified 23 bacteria into two types based on their complete genomes; "S-type" as represented by Staphylococcus aureus and "E-type" as represented by Escherichia coli. Classification was characterized by concentrations of Arg, Ala or Lys in the amino acid composition calculated from the complete genome. Based on these previous classifications, not only prokaryotic but also eukaryotic genome structures were investigated by amino acid compositions and nucleotide contents. Organisms consisting of 112 bacteria, 15 archaea and 18 eukaryotes were classified into two major groups by cluster analysis using GC contents at the three codon positions calculated from complete genomes. The 145 organisms were classified into "AT-type" and "GC-type" represented by high A or T (low G or C) and high G or C (low A or T) contents, respectively, at every third codon position. Reciprocal changes between G or C and A or T contents at the third codon position occurred almost synchronously in every codon among the organisms. Correlations between amino acid concentrations (Ala, Ile and Lys) and the nucleotide contents at the codon position were obtained in both "AT-type" and "GC-type" organisms, but with different regression coefficients. In certain correlations of amino acid concentrations with GC contents, eukaryotes, archaea and bacteria showed different behaviors; thus these kingdoms evolved differently. All organisms are basically classifiable into two groups having characteristic codon patterns; organisms with low GC and high AT contents at the third codon position and their derivatives, and organisms with an inverse relationship.

Free energy of mRNA positively correlates with GC content in chloroplast transcriptomes of edible legumes

Free-living bacteria in nutrient limited environments often exhibit traits which may reduce the cost of reproduction, such as smaller genome size, low GC content and fewer sigma (σ) factor and 16S rRNA gene copies. Despite the potential utility of these traits to detect relationships between microbial communities and ecosystem-scale properties, few studies have assessed these traits on a community-scale. Here, we analysed these traits from publicly available metagenomes derived from marine, soil, host-associated and thermophilic communities. In marine and thermophilic communities, genome size and GC content declined in parallel, consistent with genomic streamlining, with GC content in thermophilic communities generally higher than in marine systems. In contrast, soil communities averaging smaller genomes featured higher GC content and were often from low-carbon environments, suggesting unique selection pressures in soil bacteria. The abundance of specific σ-factors varied with average genome size and ecosystem type. In oceans, abundance of fliA, a σ-factor controlling flagella biosynthesis, was positively correlated with community average genome size-reflecting known trade-offs between nutrient conservation and chemotaxis. In soils, a high abundance of the stress response σ-factor gene rpoS was associated with smaller average genome size and often located in harsh and/or carbon-limited environments-a result which tracks features observed in culture and indicates an increased capacity for stress response in nutrient-poor soils. This work shows how ecosystem-specific constraints are associated with trade-offs which are embedded in the genomic features of bacteria in microbial communities, and which can be detected at the community level, highlighting the importance of genomic features in microbial community analysis.

On the relationship between GC content and the number of predicted microRNA binding sites by MicroInspector

The transmission of genetic information relies on a coordinated network of cell cycle controls. Abnormalities in this network can result in genomic instability and lead to the transformation of normal cells into cancer cells. Chromosomal DNA replication is not only central to cellular division but also plays a crucial role in the maintenance of genomic integrity. DNA replication errors increase genetic instability, and may be a causative factor in diseases such as cancer and neuronal disorders. Replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict, often tissue-specific, temporal program. The genetic program that controls activation of replication origins in mammalian cells has still not been elucidated. There is evidence that specification of replication sites and timing of replication are dynamic processes that are regulated by tissue-specific and developmental cues and that are responsive to epigenetic modifications. Here, we focus on the spatiotemporal regulation of DNA replication in the human genome. There is growing evidence that chromosome band patterns and epigenetic transformation of chromatin influence the timing of replication. On the basis of this evidence, we propose that the chromatin regions showing switches in replication timing from early to late in S phase are correlated with chromosome band boundaries. These chromatin regions generally display transitions in GC contents and include more non-B-form DNA structures than other genomic regions. We also examine here the effect of changes in replication timing on genomic stability and the possible role of replication timing in the etiology of diseases such as cancer. Replication timing assays are one of many promising techniques under investigation that may in future allow much earlier cancer detection than is possible today.