Creating new genes by plasmid recombination in Escherichia coli and Bacillus subtilis

Gene shuffling is a way of creating proteins with interesting new characteristics, starting from diverged sequences. We tested an alternative to gene shuffling based on plasmid recombination and found that Bacillus subtilis efficiently recombines sequences with 4% divergence, and Escherichia coli mutS is more appropriate for sequences with 22% divergence.     

Correcting errors in synthetic DNA through consensus shuffling

Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ~60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ~1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.     

Evolutionary history of exon shuffling

Exon shuffling has been characterized as one of the major evolutionary forces shaping both the genome and the proteome of eukaryotes. This mechanism was particularly important in the creation of multidomain proteins during animal evolution, bringing a number of functional genetic novelties. Here, genome information from a variety of eukaryotic species was used to address several issues related to the evolutionary history of exon shuffling. By comparing all protein sequences within each species, we were able to characterize exon shuffling signatures throughout metazoans. Intron phase (the position of the intron regarding the codon) and exon symmetry (the pattern of flanking introns for a given exon or block of adjacent exons) were features used to evaluate exon shuffling. We confirmed previous observations that exon shuffling mediated by phase 1 introns (1-1 exon shuffling) is the predominant kind in multicellular animals. Evidence is provided that such pattern was achieved since the early steps of animal evolution, supported by a detectable presence of 1-1 shuffling units in Trichoplax adhaerens and a considerable prevalence of them in Nematostella vectensis. In contrast, Monosiga brevicollis, one of the closest relatives of metazoans, and Arabidopsis thaliana, showed no evidence of 1-1 exon or domain shuffling above what it would be expected by chance. Instead, exon shuffling events are less abundant and predominantly mediated by phase 0 introns (0-0 exon shuffling) in those non-metazoan species. Moreover, an intermediate pattern of 1-1 and 0-0 exon shuffling was observed for the placozoan T. adhaerens, a primitive animal. Finally, characterization of flanking intron phases around domain borders allowed us to identify a common set of symmetric 1-1 domains that have been shuffled throughout the metazoan lineage.     

Barcoding forest insect pests in South Korea: Constructing a basic endemic species dataset

A total of 103 barcode (mitochondrial COI) sequences were newly provided for 77 forest insect pests from 66 genera belonging to Coleoptera, Hemiptera, and Lepidoptera. All 77 species had distinct COI sequences, revealing low intraspecific genetic divergence (< 1.20%) and high interspecific genetic divergence (> 7.30%). Among the 66 genera, 32 COI sequences of 25 species belonging to 16 genera were compared with 280 COI sequences of 117 species belonging to the same 16 genera archived in GenBank, showing that most species were clearly distinguished by barcode sequences. Based on these results, we conclude that a DNA barcode is effective for identifying forest insect pest species.     

In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling

Fungal immunomodulatory proteins (FIPs) found in a wide variety of mushrooms hold significant therapeutic potential. Despite much research, the structural determinants for their immunomodulatory functions remain unknown. In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between FIP-glu (FIP gene isolated from Ganoderma lucidum) and FIP-gsi (FIP gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between FIP-glu, FIP-fve (FIP gene isolated from Flammulina velutipes), and FIP-vvo (FIP gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and testing of their protein products revealed that they maintained functional abilities to agglutinate blood cells and induce cytokine production by splenocytes from Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by using DNA shuffling, a powerful tool for the generation of novel therapeutic proteins and for their structural and functional studies.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.     

Relationships of osmerid fishes (Osmeridae) of Russia: divergence of nucleotide sequences of mitochondrial and nuclear genes

Relationships of seven species of smelts (family Osmeridae) inhabiting Russian waters were analyzed on the basis of nucleotide sequence divergence of cytb, COI and intron 1 of rpS7 genes. Nuclear sequence divergence between the species within of the genera Osmerus, Mallotus and Hypomesus was 1.6–2.6, 2.9 and 6.6–11.8 %, respectively, and mitochondrial sequence divergence was 8.9–9.7, 3.9 and 15.1–18.3 %, respectively. The mtDNA divergence was 20.4 % between the genera Hypomesus and Mallotus, 18.47 % between Hypomesus and Osmerus, and 17.62 % between Mallotus and Osmerus. Nuclear DNA divergence was 11.58, 11.19, and 11.37 %, respectively. MP, ML and Bayesian analyses coupled with inclusion in analyses of GenBank-derived sequences of osmerid species that do not inhabit this region suggest that the lineage of the genus Mallotus was the first to separate from the common hypothetic ancestor of the smelts and is sister to the Osmerus–Hypomesus clade. The position of Hypomesus olidus is not resolved and points to the need for further research.     

Evolutionary Drivers of Diversification and Distribution of a Southern Temperate Stream Fish Assemblage: Testing the Role of Historical Isolation and Spatial Range Expansion

This study used phylogenetic analyses of mitochondrial cytochrome b sequences to investigate genetic diversity within three broadly co-distributed freshwater fish genera (Galaxias, Pseudobarbus and Sandelia) to shed some light on the processes that promoted lineage diversification and shaped geographical distribution patterns. A total of 205 sequences of Galaxias, 177 sequences of Pseudobarbus and 98 sequences of Sandelia from 146 localities across nine river systems in the south-western Cape Floristic Region (South Africa) were used. The data were analysed using phylogenetic and haplotype network methods and divergence times for the clades retrieved were estimated using *BEAST. Nine extremely divergent (3.5–25.3%) lineages were found within Galaxias. Similarly, deep phylogeographic divergence was evident within Pseudobarbus, with four markedly distinct (3.8–10.0%) phylogroups identified. Sandelia had two deeply divergent (5.5–5.9%) lineages, but seven minor lineages with strong geographical congruence were also identified. The Miocene-Pliocene major sea-level transgression and the resultant isolation of populations in upland refugia appear to have driven widespread allopatric divergence within the three genera. Subsequent coalescence of rivers during the Pleistocene major sea-level regression as well as intermittent drainage connections during wet periods are proposed to have facilitated range expansion of lineages that currently occur across isolated river systems. The high degree of genetic differentiation recovered from the present and previous studies suggest that freshwater fish diversity within the south-western CFR may be vastly underestimated, and taxonomic revisions are required.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons

The level of sequence heterogeneity among rrn operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related rrn operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of rrn copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but ~40% of the organisms analyzed have either one or two operons. Among the Archaea, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For Archaea, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single rrn operon may lower this correction factor to ~2.5. Divergence among operons appears to be small overall for both Bacteria and Archaea, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and Thermoanaerobacter tengcongensis exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.     

HIV-1 Clade B pol Evolution following Primary Infection

Objective

Characterize intra-individual HIV-1 subtype B pol evolution in antiretroviral naive individuals.

Design

Longitudinal cohort study of individuals enrolled during primary infection.

Methods

Eligible individuals were antiretroviral naïve participants enrolled in the cohort from December 1997-December 2005 and having at least two blood samples available with the first one collected within a year of their estimated date of infection. Population-based pol sequences were generated from collected blood samples and analyzed for genetic divergence over time in respect to dual infection status, HLA, CD4 count and viral load.

Results

93 participants were observed for a median of 1.8 years (Mean = 2.2 years, SD = 1.9 years). All participants classified as mono-infected had less than 0.7% divergence between any two of their pol sequences using the Tamura-Nei model (TN93), while individuals with dual infection had up to 7.0% divergence. The global substitution rates (substitutions/nucleotide/year) for mono and dually infected individuals were significantly different (p<0.001); however, substitution rates were not associated with HLA haplotype, CD4 or viral load.

Conclusions

Even after a maximum of almost 9 years of follow-up, all mono-infected participants had less than 1% divergence between baseline and longitudinal sequences, while participants with dual infection had 10 times greater divergence. These data support the use of HIV-1 pol sequence data to evaluate transmission events, networks and HIV-1 dual infection.     

High Level of Interspecific Divergence in the <Emphasis Type="Italic">Salamandrella</Emphasis> Genus Based on Variability of the <Emphasis Type="Italic">RAG2</Emphasis> Gene

Previously, a very low level of divergence between the species of the genus Salamandrella—S. keyserlingii and S. schrenckii—was detected on the basis of variability of the nucleotide sequences of three genes of the nuclear genome (BDNF, POMC, and RAG1). Fixed interspecific differences were detected only in one nucleotide position of the RAG1 gene, and the level of interspecific divergence for this gene was only 0.07%. In this paper, we present the results of a study of the variability of the ENC1, MGAT4C, and RAG2 nuclear genes. The level of interspecific divergence for the MGAT4C gene was 0.14%, and for the RAG2 gene, it was 0.8%. The results of a phylogenetic analysis of the nucleotide sequences of the RAG2 gene in representatives of the family Hynobiidae indicate that the separation of the Salamandrella branch, which is basal for the genera Batrachuperus, Liua, Hynobius, and Pseudohynobius, occurred approximately 55 million years ago. The time of divergence between species of the Salamandrella genus was approximately 21 million years ago.     

Genome shuffling amplifies the carbon source spectrum and improves arachidonic acid production in Diasporangium sp.

The aim of this study was to develop a new fungal strain that simultaneously amplifies the carbon source spectrum and increases arachidonic acid (AA) productivity using genome shuffling between Diasporangium sp. and inactive Aspergillus niger. Through three rounds of genome shuffling, one of the stable daughter strains (F₁) acquired the ability to produce arachidonic acid and utilize various carbon sources. Compared to the parental Diasporangium sp., which could only use four out of eight carbon sources tested, F₁ could utilize all eight carbon sources. During fermentation with CMC-Na as the carbon source, F₁ was able to obtain 30.16% of lipid effectively whereas the parental Diasporangium sp. was not able to grow at all. When glucose was used as the carbon source, the CMCase activity of F₁ was 879.36 U, 298.23% higher than that of the parental Diasporangium sp. Under optimized fermentation conditions in a 5-L fermentation container, the AA yield of F₁ reached 0.81 g/l, 94.78% higher than that of the parental generation. These results indicate that inter-kingdom genome shuffling can be used successfully in eukaryotic microorganisms and that it can effectively improve the production of desired metabolites within a short period of time. The findings of this study may be useful for extending the application of genome shuffling in eukaryotic microbial breeding.     

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 ± 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure–function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.     

Genetic divergence analysis of the Common Barn Owl Tyto alba (Scopoli, 1769) and the Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile using COI sequence

In this paper new mitochondrial COI sequences of Common Barn Owl Tyto alba (Scopoli, 1769) and Short-eared Owl Asio flammeus (Pontoppidan, 1763) from southern Chile are reported and compared with sequences from other parts of the World. The intraspecific genetic divergence (mean p-distance) was 4.6 to 5.5% for the Common Barn Owl in comparison with specimens from northern Europe and Australasia and 3.1% for the Short-eared Owl with respect to samples from north America, northern Europe and northern Asia. Phylogenetic analyses revealed three distinctive groups for the Common Barn Owl: (i) South America (Chile and Argentina) plus Central and North America, (ii) northern Europe and (iii) Australasia, and two distinctive groups for the Short-eared Owl: (i) South America (Chile and Argentina) and (ii) north America plus northern Europe and northern Asia. The level of genetic divergence observed in both species exceeds the upper limit of intraspecific comparisons reported previously for Strigiformes. Therefore, this suggests that further research is needed to assess the taxonomic status, particularly for the Chilean populations that, to date, have been identified as belonging to these species through traditional taxonomy.     

Identifying and Quantifying Orphan Protein Sequences in Fungi

For large regions of many proteins, and even entire proteins, no homology to known domains or proteins can be detected. These sequences are often referred to as orphans. Surprisingly, it has been reported that the large number of orphans is sustained in spite of a rapid increase of available genomic sequences. However, it is believed that de novo creation of coding sequences is rare in comparison to mechanisms such as domain shuffling and gene duplication; hence, most sequences should have homologs in other genomes.To investigate this, the sequences of 19 complete fungi genomes were compared. By using the phylogenetic relationship between these genomes, we could identify potentially de novo created orphans in Saccharomyces cerevisiae. We found that only a small fraction, < 2%, of the S. cerevisiae proteome is orphan, which confirms that de novo creation of coding sequences is indeed rare. Furthermore, we found it necessary to compare the most closely related species to distinguish between de novo created sequences and rapidly evolving sequences where homologs are present but cannot be detected.Next, the orphan proteins (OPs) and orphan domains (ODs) were characterized. First, it was observed that both OPs and ODs are short. In addition, at least some of the OPs have been shown to be functional in experimental assays, showing that they are not pseudogenes. Furthermore, in contrast to what has been reported before and what is seen for older orphans, S. cerevisiae specific ODs and proteins are not more disordered than other proteins. This might indicate that many of the older, and earlier classified, orphans indeed are fast-evolving sequences. Finally, > 90% of the detected ODs are located at the protein termini, which suggests that these orphans could have been created by mutations that have affected the start or stop codons.     

Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k_cat/K_m ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.