Contiguous Sequence Research Articles

The transcriptome of Trichuris suis--first molecular insights into a parasite with curative properties for key immune diseases of humans.

BackgroundIatrogenic infection of humans with Trichuris suis (a parasitic nematode of swine) is being evaluated or promoted as a biological, curative treatment of immune diseases, such as inflammatory bowel disease (IBD) and ulcerative colitis, in humans. Although it is understood that short-term T. suis infection in people with such diseases usually induces a modified Th2-immune response, nothing is known about the molecules in the parasite that induce this response.Methodology/Principal FindingsAs a first step toward filling the gaps in our knowledge of the molecular biology of T. suis, we characterised the transcriptome of the adult stage of this nematode employing next-generation sequencing and bioinformatic techniques. A total of ∼65,000,000 reads were generated and assembled into ∼20,000 contiguous sequences ( = contigs); ∼17,000 peptides were predicted and classified based on homology searches, protein motifs and gene ontology and biological pathway mapping.ConclusionsThese analyses provided interesting insights into a number of molecular groups, particularly predicted excreted/secreted molecules (n = 1,288), likely to be involved in the parasite-host interactions, and also various molecules (n = 120) linked to chemokine, T-cell receptor and TGF-β signalling as well as leukocyte transendothelial migration and natural killer cell-mediated cytotoxicity, which are likely to be immuno-regulatory or -modulatory in the infected host. This information provides a conceptual framework within which to test the immunobiological basis for the curative effect of T. suis infection in humans against some immune diseases. Importantly, the T. suis transcriptome characterised herein provides a curated resource for detailed studies of the immuno-molecular biology of this parasite, and will underpin future genomic and proteomic explorations.

Characterization of genomic sequence showing strong association with polyembryony among diverse Citrus species and cultivars, and its synteny with Vitis and Populus

Polyembryony, in which multiple somatic nucellar cell-derived embryos develop in addition to the zygotic embryo in a seed, is common in the genus Citrus. Previous genetic studies indicated polyembryony is mainly determined by a single locus, but the underlying molecular mechanism is still unclear. As a step towards identification and characterization of the gene or genes responsible for nucellar embryogenesis in Citrus, haplotype-specific physical maps around the polyembryony locus were constructed. By sequencing three BAC clones aligned on the polyembryony haplotype, a single contiguous draft sequence consisting of 380 kb containing 70 predicted open reading frames (ORFs) was reconstructed. Single nucleotide polymorphism genotypes detected in the sequenced genomic region showed strong association with embryo type in Citrus, indicating a common polyembryony locus is shared among widely diverse Citrus cultivars and species. The arrangement of the predicted ORFs in the characterized genomic region showed high collinearity to the genomic sequence of chromosome 4 of Vitis vinifera and linkage group VI of Populus trichocarpa, suggesting that the syntenic relationship among these species is conserved even though V. vinifera and P. trichocarpa are non-apomictic species. This is the first study to characterize in detail the genomic structure of an apomixis locus determining adventitious embryony.

Floral heteromorphy in Primula vulgaris: progress towards isolation and characterization of the S locus

The common primrose, Primula vulgaris, along with many other species of the Primulaceae, exhibits floral heteromorphy in which different individuals develop one of two possible forms of flower, known as pin and thrum. Both flower types are hermaphrodite and exhibit reciprocal positions of male and female reproductive structures, which together with a sporophytic incompatibility system, prevent self-pollination and promote out-crossing. The development of the two different forms of flower is controlled by a co-adapted linkage group of genes known as the S locus. Here progress towards identification and characterization of these genes is described to provide a molecular genetic explanation of the different floral characteristics that define heterostyly in Primula as observed and described by Charles Darwin. Previous work to identify and characterize developmental mutations linked to the P. vulgaris S locus, together with the isolation of S locus-linked genes and polymorphic DNA sequences markers, is summarized. The development of tools are described which will facilitate isolation and characterization of the S locus and its environs, including the creation of two expressed sequence tag libraries from pin and thrum flowers, as well as the construction and screening of two bacterial artificial chromosome (BAC) libraries containing thrum genomic DNA. Screening of these libraries with four S locus-linked sequences has enabled us to assemble four BAC contigs representing over 40 individual overlapping BAC clones which represent over 2·2 Mb of S locus-linked genomic sequence. PCR-based approaches for identification of the allelic origin of these BACs are described as well as identification of an additional 14 S locus-linked genes within BAC-end sequences. On-going work to assemble the four S locus-linked contigs into one contiguous sequence spanning the S locus is outlined in preparation for sequence analysis and characterization of the genes located within this region.

Synganglion transcriptome and developmental global gene expression in adult females of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

454 Pyrosequencing was used to characterize the expressed genes from the synganglion and associated neurosecretory organs of unfed and partially fed virgin and mated replete females of the American dog tick, Dermacentor variabilis. A total of 14,881 contiguous sequences (contigs) was assembled, with an average size of 229 bp. Gene ontology terms for Level 2 biological processes were assigned to 4366 contigs. Seven acetylcholinesterases, a muscarinic acetylcholine (ACh) receptor, two nicotinic ACh receptor β-subunits, two ACh unc-18 regulators, two dopamine receptors, two gamma aminobutyric acid (GABA) receptors, two GABA transporters, two norepinephrine transporters and an octopamine receptor are described. Microarrays were conducted to examine global gene expression and quantitative real-time polymerase chain reaction was used to verify expression of selected neuropeptides. Hierarchical clustering of all differentially expressed transcripts grouped part-fed and replete ticks as being more similar in terms of differentially expressed genes with unfed ticks as the outgroup. Nine putative neuropeptides (allatostatin, bursicon-β, preprocorazonin, glycoprotein hormone α, insulin-like peptide, three orcokinins, preprosulphakinin) and a gonadotropin releasing hormone receptor were differentially expressed, and their developmental expression and role in reproduction was investigated. The presence of eclosion hormone, corazonin and bursicon in the synganglion, which in insects regulate behaviour and cuticle development associated with moulting, suggest that this system may be used in ticks to regulate blood feeding, cuticle expansion and development related to female reproduction; adult ticks do not moult.

Transcriptome sequencing of an ecologically important graminivorous sawfly: a resource for marker development

Sawfly larvae (Hymenoptera: Symphyta) are an important, highly nutritious, invertebrate food source for farmland birds. Reduced numbers of farmland invertebrates are a possible factor contributing to the observed declines in many farmland bird populations associated with post-1950s intensification of agriculture. To date, studies on sawfly populations have been census-based and, therefore, the genetic factors underlying their declines are unknown. We have produced the first genetic resource for any sawfly species in the form of a de novo transcriptome assembly comprising 18,539 contiguous sequences (contigs) and 260 singletons. The assembly was sequenced using 454 pyrosequencing technology and produced 1,284 microsatellite markers for the common farmland sawfly Dolerus aeneus, which may also be useful in other closely-related species. These markers will facilitate monitoring of changes in genetic diversity and gene flow in sawfly populations therefore helping to predict extinction risk.

Analysis of BAC-end sequences in rainbow trout: Content characterization and assessment of synteny between trout and other fish genomes

BackgroundRainbow trout (Oncorhynchus mykiss) are cultivated worldwide for aquaculture production and are widely used as a model species to gain knowledge of many aspects of fish biology. The common ancestor of the salmonids experienced a whole genome duplication event, making extant salmonids such as the rainbow trout an excellent model for studying the evolution of tetraploidization and re-diploidization in vertebrates. However, the lack of a reference genome sequence hampers research progress for both academic and applied purposes. In order to enrich the genomic tools already available in this species and provide further insight on the complexity of its genome, we sequenced a large number of rainbow trout BAC-end sequences (BES) and characterized their contents.ResultsA total of 176,485 high quality BES, were generated, representing approximately 4% of the trout genome. BES analyses identified 6,848 simple sequence repeats (SSRs), of which 3,854 had high quality flanking sequences for PCR primers design. The first rainbow trout repeat elements database (INRA RT rep1.0) containing 735 putative repeat elements was developed, and identified almost 59.5% of the BES database in base-pairs as repetitive sequence. Approximately 55% of the BES reads (97,846) had more than 100 base pairs of contiguous non-repetitive sequences. The fractions of the 97,846 non-repetitive trout BES reads that had significant BLASTN hits against the zebrafish, medaka and stickleback genome databases were 15%, 16.2% and 17.9%, respectively, while the fractions of the non-repetitive BES reads that had significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 10.7%, 9.5% and 9.5%, respectively. Comparative genomics using paired BAC-ends revealed several regions of conserved synteny across all the fish species analyzed in this study.ConclusionsThe characterization of BES provided insights on the rainbow trout genome. The discovery of specific repeat elements will facilitate analyses of sequence content (e.g. for SNPs discovery and for transcriptome characterization) and future genome sequence assemblies. The numerous microsatellites will facilitate integration of the linkage and physical maps and serve as valuable resource for fine mapping QTL and positional cloning of genes affecting aquaculture production traits. Furthermore, comparative genomics through BES can be used for identifying positional candidate genes from QTL mapping studies, aid in future assembly of a reference genome sequence and elucidating sequence content and complexity in the rainbow trout genome.

Transcriptional Activators of Human Genes with Programmable DNA-Specificity

TAL (transcription activator-like) effectors are translocated by Xanthomonas bacteria into plant cells where they activate transcription of target genes. DNA target sequence recognition occurs in a unique mode involving a central domain of tandem repeats. Each repeat recognizes a single base pair in a contiguous DNA sequence and a pair of adjacent hypervariable amino acid residues per repeat specifies which base is bound. Rearranging the repeats allows the design of novel TAL proteins with predictable DNA-recognition specificities. TAL protein-based transcriptional activation in plant cells is mediated by a C-terminal activation domain (AD). Here, we created synthetic TAL proteins with designed repeat compositions using a novel modular cloning strategy termed “Golden TAL Technology”. Newly programmed TAL proteins were not only functional in plant cells, but also in human cells and activated targeted expression of exogenous as well as endogenous genes. Transcriptional activation in different human cell lines was markedly improved by replacing the TAL-AD with the VP16-AD of herpes simplex virus. The creation of TAL proteins with potentially any desired DNA-recognition specificity allows their versatile use in biotechnology.

Exploiting Nucleotide Composition to Engineer Promoters

The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a “proof-of-concept” for custom-designing promoters that are suitable for biotechnological and medical applications.

Sequencing of high G+C microbial genomes using the ultrafast pyrosequencing technology

Next generation pyrosequencing of high G+C content genomes still poses problems to automated sequencing and assembly processes which necessitates cost and time intensive manual work in order to finish such genomes completely. The sequencing of the high G+C actinomycete Actinoplanes sp. SE50/110 was performed with standard pyrosequencing technology (454 Life Sciences) and revealed a high number of gaps. The reasons for the introduction of gaps were analyzed on a previously known 41kb long DNA reference sequence from Actinoplanes sp. SE50/110, hosting the acarbose biosynthesis gene cluster. Mapping of the sequencing results on the reference gene cluster sequence revealed a fragmentation into 30 contiguous sequences of different lengths. The gaps between these sequences were characterized by extremely low read coverage which strongly correlated with the G+C content in the gap regions in a negative manner. Furthermore, the gap-sequences contained strong stem-loop structures which hindered the amplification of these sequences during the emulsion PCR. Being significantly underrepresented or absent in the subsequent sequencing process, these sequences lead to weakly or uncovered genomic regions which forces the assembly algorithm to output multiple contiguous sequences instead of one finished genome. However, by applying a different pyrosequencing protocol, it was possible to sequence the complete acarbose biosynthesis gene cluster. The changes to the protocol include longer read length and addition of chemicals to the amplification chemistry, which reduces the self-annealing of DNA fragments during the amplification process and enables the complete reconstruction of high G+C content genomes without manual intervention.

Characterization of a gene cluster and its putative promoter region for violacein biosynthesis in Pseudoalteromonas sp. 520P1

Violacein, a purple pigment produced by some Gram-negative bacteria, has various physiological properties, such as antitrypanosomal and antitumoral activities. A gene cluster that encodes five enzymes, VioA-VioE, is responsible for synthesizing violacein. The expression of these enzymes is known to be regulated by a quorum sensing mechanism in Chromobacterium violaceum and Pseudoalteromonas sp. 520P1. To clarify the molecular mechanism of regulation of violacein synthesis, we cloned and characterized the gene cluster from Pseudoalteromonas sp. 520P1. A fosmid library of strain 520P1 was constructed and clones containing the gene cluster were isolated. The gene cluster was 7383bp in length and encoded five enzyme genes, vioA-vioE. A putative promoter sequence was predicted in the upstream region of the cluster. In the promoter region, two contiguous palindromic sequences, a possible quorum sensing regulatory site, were found. However, the isolated Escherichia coli clones harboring the gene cluster and its upstream region were unable to produce violacein probably due to the lack of quorum sensing machinery for expression. To further examine the ability of vioA-vioE genes to synthesize violacein in vivo, the upstream promoter region was removed from the cluster and heterologous expression of the treated cluster was performed in E. coli using a recombinant pET vector with T7 promoter. Purple pigment was expressed, and the pigment was identified to be violacein using ultraviolet and visible light and HPLC analysis. These results will contribute to further studies regarding violacein biosynthesis and its mass production.

Transcriptome Analysis of Female and Male Xiphophorus maculatus Jp 163 A

Background Xiphophorus models are important for melanoma, sex determination and differentiation, ovoviviparity and evolution. To gain a global view of the molecular mechanism(s) whereby gene expression may influence sexual dimorphism in Xiphophorus and to develop a database for future studies, we performed a large-scale transcriptome study.Methodology/Principal FindingsThe 454-FLX massively parallel DNA sequencing platform was employed to obtain 742,771 and 721,543 reads from 2 normalized cDNA libraries generated from whole adult female and male X. maculatus Jp 163 A, respectively. The reads assembled into 45,538 contigs (here, a "contig" is a set of contiguous sequences), of which, 11,918 shared homology to existing protein sequences. These numbers estimate that the contigs may cover 53% of the total number of Xiphophorus transcriptome. Putative translations were obtained for 11,918 cDNA contigs, of which, 3,049 amino acid sequences contain Pfam domains and 11,064 contigs encode secretory proteins. A total of 3,898 contigs were associated with 2,781 InterPro (IPR) entries and 5,411 contigs with 132 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. There were 10,446 contigs annotated with 69,778 gene ontology (GO) terms and the three corresponding organizing principles. Fifty-four potential sex differentially expressed genes have been identified from these contigs. Eight and nine of these contigs were confirmed by real-time PCR as female and male predominantly expressed genes respectively. Based on annotation results, 34 contigs were predicted to be differentially expressed in male and female and 17 of them were also confirmed by real-time PCR.Conclusions/SignificanceThis is the first report of an annotated overview of the transcriptome of X. maculatus and identification of sex differentially expressed genes. These data will be of interest to researchers using the Xiphophorus model. This work also provides an archive for future studies in molecular mechanisms of sexual dimorphism and evolution, and can be used in comparative studies of other fish.

Transcriptome analysis and SNP/SSR marker information of red pepper variety YCM334 and Taean

Transcriptome analysis is becoming a powerful tool for the identification and characterization of molecular markers, splicing-specific mRNA mutants, and differentially expressed genes. In this study, we present the first comprehensive characterization of the transcriptomes of the mature fruits of two red pepper ( Capsicum annuum) accessions, YCM334 and Taean, using the 454 Genome Sequencer (GS)-FLX Titanium System. For YCM334, sequencing runs yielded 121,152 reads (average length = 409 nucleotides), which were assembled into 11,770 contiguous sequences (contigs) and 13,827 singletons. Sequencing runs for Taean yielded 154,088 reads (average length = 395 nucleotides) assembled into 14,200 contigs and 15,135 singletons. Using a BLAST search against the NCBI non-redundant (NR) and UniProt databases with an arbitrary expectation value of E-5, over 10,000 unigenes were functionally annotated and classified using the FunCat functional annotation scheme, and were found to distribute in 18 FunCat categories for YCM334 and 17 for Taean. Expression pattern analysis identified 540 Tentative Consensus (TC) sequences that were specifically detected in YCM334. Most of these differential TCs assigned with specific annotations covered 16 FunCat categories, of which 4 were annotated with resistance terms. Polymorphic marker discovery revealed a total of 1265 and 1436 simple sequence repeat (SSR) motifs for YCM334 and Taean, respectively, and a total of 3218 single nucleotide polymorphisms (SNPs) with high confidence. The information generated in this study provides valuable resources for characterization of some important agronomic traits and identification of molecular mechanism such as phytopathogen resistance.

Asymptotic power of tests of normality under local alternatives

Seven tests of univariate normality are studied in view of their asymptotic power under local alternatives. The procedures under consideration are either based on the empirical skewness and/or kurtosis, including the popular Jarque–Bera statistic, as well as Cramér–von Mises, Anderson–Darling and Kolmogorov–Smirnov functionals of an empirical process with estimated parameters. The large-sample behavior of these test statistics under contiguous sequences is obtained; this allows for the computation of their associated local power curves and of their asymptotic relative efficiency in the light of a measure proposed by Berg and Quessy (2009). Comparisons are made under four classes of local alternatives, including those used by Thadewald and Büning (2007) in a recent Monte-Carlo power study. These theoretical results are related to empirical ones and many recommendations are formulated.

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens.

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea.A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ∼20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs.Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue.This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.

Highly Parallel Gene-To-BAC Addressing Using Microarrays

Second-generation sequencing now provides the potential for low-cost generation of whole-genome sequences. However, for large-genome organisms with high repetitive DNA content, genome-wide short read sequence assembly is currently impossible, with accurate ordering and localization of genes still relying heavily on integration with physical and genetic maps. To facilitate this process, we have used Agilent microarrays to simultaneously address thousands of gene sequences to individual BAC clones and contiguous sequences that form part of an emerging physical map of the large and currently unsequenced 5.3-Gb barley genome. The approach represents a cost-effective, highly parallel alternative to traditional addressing methods. By coupling the gene-to-BAC address data with gene-based molecular markers, thousands of BACs can be anchored directly to the genetic map, thereby generating a framework for orientating and ordering genes, and providing direct links to phenotypic traits.

Integration of linkage maps for the Amphidiploid Brassica napus and comparative mapping with Arabidopsis and Brassica rapa

BackgroundThe large number of genetic linkage maps representing Brassica chromosomes constitute a potential platform for studying crop traits and genome evolution within Brassicaceae. However, the alignment of existing maps remains a major challenge. The integration of these genetic maps will enhance genetic resolution, and provide a means to navigate between sequence-tagged loci, and with contiguous genome sequences as these become available.ResultsWe report the first genome-wide integration of Brassica maps based on an automated pipeline which involved collation of genome-wide genotype data for sequence-tagged markers scored on three extensively used amphidiploid Brassica napus (2n = 38) populations. Representative markers were selected from consolidated maps for each population, and skeleton bin maps were generated. The skeleton maps for the three populations were then combined to generate an integrated map for each LG, comparing two different approaches, one encapsulated in JoinMap and the other in MergeMap. The BnaWAIT_01_2010a integrated genetic map was generated using JoinMap, and includes 5,162 genetic markers mapped onto 2,196 loci, with a total genetic length of 1,792 cM. The map density of one locus every 0.82 cM, corresponding to 515 Kbp, increases by at least three-fold the locus and marker density within the original maps. Within the B. napus integrated map we identified 103 conserved collinearity blocks relative to Arabidopsis, including five previously unreported blocks. The BnaWAIT_01_2010a map was used to investigate the integrity and conservation of order proposed for genome sequence scaffolds generated from the constituent A genome of Brassica rapa.ConclusionsOur results provide a comprehensive genetic integration of the B. napus genome from a range of sources, which we anticipate will provide valuable information for rapeseed and Canola research.

A Portrait of the Transcriptome of the Neglected Trematode, Fasciola gigantica—Biological and Biotechnological Implications

Fasciola gigantica (Digenea) is an important foodborne trematode that causes liver fluke disease (fascioliasis) in mammals, including ungulates and humans, mainly in tropical climatic zones of the world. Despite its socioeconomic impact, almost nothing is known about the molecular biology of this parasite, its interplay with its hosts, and the pathogenesis of fascioliasis. Modern genomic technologies now provide unique opportunities to rapidly tackle these exciting areas. The present study reports the first transcriptome representing the adult stage of F. gigantica (of bovid origin), defined using a massively parallel sequencing-coupled bioinformatic approach. From >20 million raw sequence reads, >30,000 contiguous sequences were assembled, of which most were novel. Relative levels of transcription were determined for individual molecules, which were also characterized (at the inferred amino acid level) based on homology, gene ontology, and/or pathway mapping. Comparisons of the transcriptome of F. gigantica with those of other trematodes, including F. hepatica, revealed similarities in transcription for molecules inferred to have key roles in parasite-host interactions. Overall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of F. gigantica, as well as a basis for applied outcomes such as the development of novel methods of intervention against this neglected parasite.

On Coding Non-Contiguous Letter Combinations

Starting from the hypothesis that printed word identification initially involves the parallel mapping of visual features onto location-specific letter identities, we analyze the type of information that would be involved in optimally mapping this location-specific orthographic code onto a location-invariant lexical code. We assume that some intermediate level of coding exists between individual letters and whole words, and that this involves the representation of letter combinations. We then investigate the nature of this intermediate level of coding given the constraints of optimality. This intermediate level of coding is expected to compress data while retaining as much information as possible about word identity. Information conveyed by letters is a function of how much they constrain word identity and how visible they are. Optimization of this coding is a combination of minimizing resources (using the most compact representations) and maximizing information. We show that in a large proportion of cases, non-contiguous letter sequences contain more information than contiguous sequences, while at the same time requiring less precise coding. Moreover, we found that the best predictor of human performance in orthographic priming experiments was within-word ranking of conditional probabilities, rather than average conditional probabilities. We conclude that from an optimality perspective, readers learn to select certain contiguous and non-contiguous letter combinations as information that provides the best cue to word identity.

Origin of biological information: Inherent occurrence of intron-rich split genes, coding for complex extant proteins, within pre-biotic random genetic sequences

AbstractThe origin of biological information is an unexplained phenomenon. Prior research in resolving the origin of proteins, based on the assumption that the first genes were contiguous prokaryotic sequences has not succeeded. Rather, it has been established that contiguous protein-coding genes do not exist in practically any amount of random genetic sequences. We found that complex eukaryotic proteins could be inherently encoded in split genes that could exist by chance within mere micrograms to milligrams of random DNA. Using protein amino acid sequence variability, codon degeneracy, and stringent exon-length restriction, we demonstrate that split genes for proteins of extant eukaryotes occur extensively in random genetic sequences. The results provide evidence that an abundance of split genes encoding advanced proteins in a small amount of prebiotic genetic material could have ignited the evolution of the eukaryotic genome.

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast.