纳豆激酶基因的优化设计、合成及其在烟草叶片中的瞬时表达

根据植物偏爱密码子优化设计、合成纳豆激酶基因sNK,利用重叠延伸PCR法在其中插入番茄果实特异性表达基因E8的第一内含子构成sNKi基因,通过农杆菌渗透法将这两种基因渗入到烟草NC89叶片中并实现瞬时表达。通过RT-qPCR法将两种基因在烟草叶片中转录水平的表达量进行比较,结果表明两种基因在烟草叶片中均表达,且sNKi基因的表达量显著高于sNK基因;通过纤维蛋白平板法在两种基因的瞬时表达样品中均能检测到纤溶酶活性,表明目的基因在烟草叶片中可正常翻译并表现出溶栓活性,且sNKi基因在翻译水平的表达量显著高于sNK基因。表明内含子对人工合成的纳豆激酶基因的瞬时表达具有显著的促进作用。     

Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.     

Looking for Organization Patterns of Highly Expressed Genes: Purine-Pyrimidine Composition of Precursor mRNAs

We analyzed precursor messenger RNAs (pre-mRNAs) of 12 eukaryotic species. In each species, three groups of highly expressed genes, ribosomal proteins, heat shock proteins, and amino-acyl tRNA synthetases, were compared with a control group (randomly selected genes). The purine-pyrimidine (R-Y) composition of pre-mRNAs of the three targeted gene groups proved to differ significantly from the control. The exons of the three groups tested have higher purine contents and R-tract abundance and lower abundance of Y-tracts compared to the control (R-tract—tract of sequential purines with R _n ≥ 5; Y-tract—tract of sequential pyrimidines with Y _n ≥ 5). In species widely employing “intron definition” in the splicing process, the Y content of introns of the three targeted groups appeared to be higher compared to the control group. Furthermore, in all examined species, the introns of the targeted genes have a lower abundance of R-tracts compared to the control. We hypothesized that the R-Y composition of the targeted gene groups contributes to high rate and efficiency of both splicing and translation, in addition to the mRNA coding role. This is presumably achieved by (1) reducing the possibility of the formation of secondary structures in the mRNA, (2) using the R-tracts and R-biased sequences as exonic splicing enhancers, (3) lowering the amount of targets for pyrimidine tract binding protein in the exons, and (4) reducing the amount of target sequences for binding of serine/arginine-rich (SR) proteins in the introns, thereby allowing SR proteins to bind to proper (exonic) targets. (Reviewing Editor: Dr. Axel Meyer)     

Prediction of protein coding regions by the 3-base periodicity analysis of a DNA sequence

With the exponential growth of genomic sequences, there is an increasing demand to accurately identify protein coding regions (exons) from genomic sequences. Despite many progresses being made in the identification of protein coding regions by computational methods during the last two decades, the performances and efficiencies of the prediction methods still need to be improved. In addition, it is indispensable to develop different prediction methods since combining different methods may greatly improve the prediction accuracy. A new method to predict protein coding regions is developed in this paper based on the fact that most of exon sequences have a 3-base periodicity, while intron sequences do not have this unique feature. The method computes the 3-base periodicity and the background noise of the stepwise DNA segments of the target DNA sequences using nucleotide distributions in the three codon positions of the DNA sequences. Exon and intron sequences can be identified from trends of the ratio of the 3-base periodicity to the background noise in the DNA sequences. Case studies on genes from different organisms show that this method is an effective approach for exon prediction.     

Internal genomic sequence of human CYP1A1 gene is involved in superinduction of dioxin-induced CYP1A1 transcription by cycloheximide

Guanylate cyclase-activating protein 2 (GCAP2) is expressed in vertebrate photoreceptors cells where it regulates the activity of membrane bound guanylate cyclases in a Ca(2+)-dependent manner. The essential trigger step involves a Ca(2+)-induced conformational change in GCAP2. We investigated these Ca(2+)-dependent changes by probing the cysteine accessibility in wild type and mutant GCAP2 forms with the thiol-modifying reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Cysteine residues in position 35 and 111 displayed a restricted accessibility in the presence of Ca(2+), whereas cysteine in position 131 reacted with DTNB in the presence and absence of Ca(2+). Our data indicate that the Ca(2+)-sensitivity of GCAP2 is significantly controlled by its third Ca(2+)-binding site, EF-hand 3.     

Indel evolution of mammalian introns and the utility of non-coding nuclear markers in eutherian phylogenetics

Nuclear DNA intron sequences are increasingly used to investigate evolutionary relationships among closely related organisms. The phylogenetic usefulness of intron sequences at higher taxonomic levels has, however, not been firmly established and very few studies have used these markers to address evolutionary questions above the family level. In addition, the mechanisms driving intron evolution are not well understood. We compared DNA sequence data derived from three presumably independently segregating introns (THY, PRKC I and MGF) across 158 mammalian species. All currently recognized extant eutherian mammalian orders were included with the exception of Cingulata, Dermoptera and Scandentia. The total aligned length of the data was 6366 base pairs (bp); after the exclusion of autapomorphic insertions, 1511 bp were analyzed. In many instances the Bayesian and parsimony analyses were complementary and gave significant posterior probability and bootstrap support (>80) for the monophyly of Afrotheria, Euarchontoglires, Laurasiatheria and Boreoeutheria. Apart from finding congruent support when using these methods, the intron data also provided several indels longer than 3 bp that support, among others, the monophyly of Afrotheria, Paenungulata, Ferae and Boreoeutheria. A quantitative analysis of insertions and deletions suggested that there was a 75% bias towards deletions. The average insertion size in the mammalian data set was 16.49 bp +/- 57.70 while the average deletion was much smaller (4.47 bp +/- 14.17). The tendency towards large insertions and small deletions is highlighted by the observation that out of a total of 17 indels larger than 100 bp, 15 were insertions. The majority of indels (>60% of all events) were 1 or 2 bp changes. Although the average overall indel substitution rate of 0.00559 per site is comparable to that previously reported for rodents and primates, individual analyses among different evolutionary lineages provide evidence for differences in the formation rate of indels among the different mammalian groups.     

Functional splice sites in a zebrafish LINE and their influence on zebrafish gene expression

Long interspersed elements (LINEs) are transposable elements that exist in many kinds of eukaryotic genomes, where they have a large effect on genome evolution. There are several thousands to hundreds of thousands of LINE copies in each eukaryotic genome. LINE elements are amplified by a mechanism called retrotransposition, in which a LINE-encoded protein reverse transcribes (copies) its own RNA. We previously isolated two retrotransposition-competent LINEs, ZfL2-1 and ZfL2-2, from zebrafish. Although it has generally been thought that LINEs do not have ‘introns’ (because the LINE RNA is used as the template during retrotransposition), we now show that these two LINEs contain multiple putative functional splice sites. We further show that at least one pair of these splice sites is actually functional in zebrafish cells. Moreover, some of these splice sites are coupled with the splicing signal of a host endogenous gene, thereby generating a new chimeric spliced mRNA variant for this gene. Our results suggest the possible role of these LINE splice sites in modulating retrotransposition and host gene expression.     

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.     

Population structure of mitochondrial genomes in Saccharomyces cerevisiae

Background

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.

Results

To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.

Conclusions

Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1664-4) contains supplementary material, which is available to authorized users.