Single Intron Research Articles

Short and narrow flag leaf1, a GATA zinc finger domain-containing protein, regulates flag leaf size in rice (Oryza sativa)

BackgroundThe flag leaf of rice (Oryza sativa L.) is an important determinant of plant type characteristics and grain yield. Identification of flag leaf mutants of rice is crucial to elucidate the molecular mechanism of flag-leaf development, and for exploitation of rice germplasm resources.ResultsIn this study, we describe a mutant designated short and narrow flag leaf 1 (snfl1). Histological analysis showed that the length of epidermal cells and number of longitudinal veins were decreased in the flag leaf of the snfl1 mutant. Map-based cloning indicated that a member of the GATA family of transcription factors is a candidate gene for SNFL1. A single-nucleotide transition at the last base in the single intron of snfl1 led to variation in alternative splicing and early termination of translation. Complemented transgenic plants harbouring the candidate SNFL1 gene rescued the snfl1 mutant. Analysis of RT-PCR and the SNFL1 promoter by means of a GUS fusion expression assay showed that abundance of SNFL1 transcripts was higher in the culm, leaf sheath, and root. Expression of the SNFL1-GFP fusion protein in rice protoplasts showed that SNFL1 was localized in nucleus.ConclusionsWe conclude that SNFL1 is an important regulator of leaf development, the identification of which might have important implications for future research on GATA transcription factors.

An intronic single nucleotide polymorphism in the MUTYH gene is associated with increased risk for HCV-induced hepatocellular carcinoma

Background & aimsThe role of base excision repair genes in human hepatocarcinogenesis has not yet been explored. Here, we investigated relationships between variants of these genes and the risk of developing hepatocellular carcinoma (HCC). MethodsNineteen tagging SNPs in base excision repair genes (including MUTYH, OGG1 and MTH1) were genotyped using iPLEX assays; one significant SNP was found and confirmed in Japanese patients with chronic hepatitis C (CHC) (n = 38 HCC and 55 controls). The effects of modifying the intronic variants were determined by luciferase assays. MUTYH-null mice were used to examine the involvement of oxidative stress and DNA repair enzymes in hepatocarcinogenesis. ResultsSignificant associations were found for a single intron SNP (rs3219487) in the MUTYH gene. The risk of developing HCC in patients with A/A or G/A genotypes was higher than in those with the G/G genotype (OR = 9.27, 95% CI = 2.39 −32.1, P = 0.0005). MUTYH mRNA levels in both peripheral mononuclear cells were significantly lower in G/A or A/A genotyped subjects (P = 0.0157 and 0.0108, respectively). We found that −2000 in the MUTYH promoter region is involved in enhanced expression of MUTYH by insertion of a major allele sequence of rs3219487. Liver tumors were observed in MUTYH-null mice after 12 months´ high iron diet, but no tumors developed when dietary anti-oxidant (N-Acetyl-L-cysteine) was also provided. ConclusionsCHC patients with the rs3219487 adenine allele had a significantly increased risk of developing HCC. MUTYH-null mice with iron-associated oxidative stress were susceptible to development of liver tumors unless prevented by dietary anti-oxidants.

P057 Long range next generation DNA sequencing of HLA from a registry population from the Netherlands

Aim Long range next generation sequencing was used to characterize the HLA class I (HLA-A,B,C) and class II (DRB1, DRB3/4/5, DQB1) alleles of a hematopoietic stem cell registry population (N = 643) from The Netherlands (Matchis). Methods The in-house typing protocol included several new strategies to reduce errors, add automation, and balance the yield of all loci when 96 samples are sequenced in one 600 cycle paired-end Illumina MiSeq sequence run. Results Of the 22 new class I alleles, three contain a nonsynonymous substitution in an exon (1, 4 or 7). The remainder are single nucleotide intron variants. Thirty-two class I alleles are incomplete in the IMGT/HLA database. The majority of the alleles in the population are common; the three most common alleles at each locus are listed in the table. Alleles not previously listed as common or well-documented include mainly 4th field variations; exceptions included single copies of B*51:20, C*07:100, and DRB1*04:92. Alleles encoding nonsynonymous substitutions compared to the primary allele in the G group include, as examples, A*23:17 (1 vs. 20 A*23:01:01:01), B*44:27:01 (1 vs 83 B*44:02:01:01), C*07:06 (3 vs 190 C*07:01:01:01), DRB1*14:54:01 (44 vs 3 DRB1*14:01:01), DQB1*02:02:01 (84 vs 175 DQB1*02:01:01). Non-expressed alleles were not observed. DRB3/4/5 associations were identified. Conclusions Long-range sequencing of HLA alleles from world-wide populations will continue to build our understanding of HLA diversity and its impact on the immune response. Download : Download high-res image (231KB) Download : Download full-size image C.K. Hurley: 7. Other (Identify); Company/Organization; Intellectual property sequencing Thermo Fisher.

Numerous recursive sites contribute to accuracy of splicing in long introns in flies.

Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates that are inherently unstable, making it difficult to study. Here we developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. A subset of recursive sites were validated by RT-PCR and sequencing. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, we observe that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.

Three new pentatricopeptide repeat proteins facilitate the splicing of mitochondrial transcripts and complex I biogenesis in Arabidopsis.

Group II introns are common features of most angiosperm mitochondrial genomes. Intron splicing is thus essential for the expression of mitochondrial genes and is facilitated by numerous nuclear-encoded proteins. However, the molecular mechanism and the protein cofactors involved in this complex process have not been fully elucidated. In this study, we characterized three new pentatricopeptide repeat (PPR) genes, called MISF26, MISF68, and MISF74, of Arabidopsis and showed they all function in group II intron splicing and plant development. The three PPR genes encode P-type PPR proteins that localize in the mitochondrion. Transcript analysis revealed that the splicing of a single intron is altered in misf26 mutants, while several mitochondrial intron splicing defects were detected in misf68 and misf74 mutants. To our knowledge, MISF68 and MISF74 are the first two PPR proteins implicated in the splicing of more than one intron in plant mitochondria, suggesting that they may facilitate splicing differently from other previously identified PPR splicing factors. The splicing defects in the misf mutants induce a significant decrease in complex I assembly and activity, and an overexpression of mRNAs of the alternative respiratory pathway. These results therefore reveal that nuclear encoded proteins MISF26, MISF68, and MISF74 are involved in splicing of a cohort of mitochondrial group II introns and thereby required for complex I biogenesis.

The complete chloroplast genome and phylogeny of Artemisia selengensis in Dongting Lake

Artemisia selengensis Turcz (Louhao in Chinese) is a widely used health food and a well-known traditional Chinese medicine. However, only a small part of the chloroplast genome data of Artemisia has been reported and there was no report for A. selengensis. In this study, we presented the complete chloroplast genome of A. selengensis and analysed its phylogenetic relationship with other 28 related species belonging to the Asteraceae family. The result showed that the whole genome was 151 215 bp in length with a typical conserved quadripartite structure. The total GC content of whole genome, LSC, SSC, and IRa/IRb regions was 37.46, 35.55, 30.81, and 43.09%, respectively. A total of 133 genes were identified, including 88 protein-coding, 37 transfer RNA, and eight ribosome RNA genes. Among these genes, nineteen genes contained a single intron and two genes contained two introns. The phylogenetic relationship showed that A. selengensis was similar to A. gmelinii. The complete chloroplast genome presented here will enrich the genetic resources of medicinal plant and promote our understanding of the phylogeny of Artemisia within the Asteraceae family.

Structural characteristic and phylogenetic analysis of the complete chloroplast genome of Dianthus Caryophyllus

Chloroplast genomes are widely used in genetic engineering, molecular marker development, and phylogeny. In order to analyze the complete chloroplast genome of Dianthus caryophyllus, the complete chloroplast genome of D. caryophyllus was sequenced and annotated. On the other hand, phylogenetic analysis of the chloroplast genome of D. caryophyllus was carried out. The results showed that the whole length of the chloroplast genome of D. caryophyllus was 147,604 bp, and had a typical conserved quadripartite structure. The G and C basic content of D. caryophyllus chloroplast was 36.3%. The genome contained 83 protein-coding genes, 34 tRNA genes, and 6 rRNA genes. Among the protein-coding genes, 10 genes contain a single intron and 2 genes contain two introns. The phylogeny of D. caryophyllus indicated that the closest phylogenetic relationship was D. longicalyxanus. This study provides materials for the molecular study of D. caryophyllus may improve the carnation industry.

Characterization of the complete chloroplast genome of Lycium barbarum (Solanales: Solanaceae), a unique economic plant to China

Lycium barbarum (Solanaceae) is a unique economic plant to China. The complete chloroplast (cp)genome was sequenced and assembled by using Illumina paired-end reads data. The circular cp genome is 155,656 bp in size, including a pair of inverted repeat (IR) regions of 25,451 bp, a large single copy (LSC) region of 86,554 bp and a small single copy (SSC) region of 18,200 bp. Besides, 15 genes possess a single intron, while another three genes (clpP, rps12 and ycf3) have a couple of introns. The GC content of entire L. barbarum cp genome, LSC, SSC and IR regions are 37.8%, 35.9%, 32.3%, and 43.2%, respectively. Phylogenetic analysis based on the concatenated coding sequences of cp PCGs showed that L. barbarum and Atropa belladonna are closely related with each other within the family Solanaceae.

Molecular characterization of β-endoglucanase from antagonistic Trichoderma saturnisporum isolate GITX-Panog (C) induced under mycoparasitic conditions

The endoglucanase belonging to glycoside hydrolase family 61 are little studied. In present study, a β-endoglucanase of ~37 kDa induced on autoclaved mycelium of Fusarium oxysporum was cloned and characterized. The molecular characterization of β-endoglucanase encoding gene revealed presence of a single intron and an open reading frame of 1044-bp which encoded a protein of 347 amino acid residues. The phylogenetic analysis of Eglu revealed its similarity to endo-β-glucanases of other Trichoderma spp. The catalytic site of β-endoglucanase contained Asp, Asn, His and Tyr residues. The cDNA encoding β-glucanase was cloned into E. coli and Pichia pastoris using pQUA-30 and pPIC9K vector system, respectively. The comparison of structure revealed that most similar structure to Eglu is Hypocrea jecorina template 5o2w.1.A of glycoside hydrolase family 61.The biochemical characterization of β-endoglucanase purified from T. saturnisporum isolate and the recombinant protein expressed in E. coli and P. pastoris was active under acidic conditions with a pH optima of 5 and temperature optima of 60 °C. The purified and expressed enzyme preparation was able to inhibit growth of F.oxysporum at 1 × 105 spores/mL which clearly revealed its significance in plant pathogen suppression.

The glyceraldehyde-3-phosphate dehydrogenase of the shrimp Litopenaeus vannamei: Molecular cloning, characterization and expression during hypoxia

Some marine crustaceans like the white shrimp Litopenaeus vannamei are tolerant to environmental hypoxia. Under oxygen deprivation, shrimp tissues obtain energy by enhancing anaerobic glycolysis. In mammals, hypoxia increases the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown a “moonlighting” role in cells. However, the effect of hypoxia on the GAPDH expression has not been studied in crustaceans. In the present work, we obtained a 2744 bp gene sequence with a 999 bp ORF split by a single intron. The deduced protein is 332 amino acids and corresponds to the L. vannamei GAPDH (LvGAPDH), which is highly similar in sequence and structure to other animal GAPDHs. During hypoxia, LvGAPDH expression is significantly induced in gills but not in hepatopancreas, suggesting that it may play a role in the molecular and cellular response of shrimp to hypoxia.

SNP-based susceptibility-resistance association and mRNA expression regulation analyses of tlr7 to grass carp Ctenopharyngodon idella reovirus.

Eleven single nucleotide polymorphisms (SNP) in Ctenopharyngodon idella toll-like receptor 7 (citlr7) gene, containing two in the 5'-flanking region, three within the single intron and six distributed in the coding sequence (CDS), were identified. A case-control study of 73 susceptible individuals and 67 resistant individuals was conducted to test the SNPs-based susceptibility-resistance association and mRNA expression of citlr7 to grass carp reovirus (GCRV), showing that both 820 A/G and 1726 A/G were significantly correlative sites in genotype (P < 0·05). Multifactor dimensionality reduction (MDR) analysis suggested the exertion of antiviral effects of 820 A/G might rely on SNPs interactions of citlr7 and C. idella toll-like receptor 8 (citlr8). Combining the mortality rate and citlr7 mRNA expression, it was suggested that 1726 GG-genotyped individuals might be more resistant than 1726 A/G genotyped individuals, indicating the selection on synonymous mutations in 1726 A/G might be susceptibility-resistance-type specific. In addition, haplotype analysis uncovered no significantly correlative haplotypes in citlr7. These findings may provide an in-depth insight for the further functional research of citlr7. The potential genetic markers identified may contribute to the molecular and transgenic breeding of C. idella.

Characterization of the complete chloroplast genome of Cephalotaxus sinensis (Cupressales: Taxaceae), an endangered Tertiary relict tree endemic to China

Cephalotaxus sinensis is an endangered Tertiary relict tree endemic to China with high medicinal values. To contribute to its conservation genetics studies, its complete chloroplast (cp) genome was assembled using Illumina high-throughput sequencing technology in this study. The cp genome is 135,646 bp long with an asymmetric base composition (32.5% A, 17.6% C, 17.5% G and 32.4% T). It encodes a set of 113 unique genes (incl. 82 protein coding, 27 tRNA and 4 rRNA genes) with one of them (trnQ-UUG) being duplicated. Besides, 14 genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps12, rps16, trnA-UGC, trnG-UCC, trnI-GAU and trnK-UUU) possess a single intron, and another gene (ycf3) has a couple of introns. Phylogenetic analysis generally supported the current family-level taxonomy of the order Cupressales, and suggested that C. sinensis is closely related to the congeneric C. oliveri.

The complete chloroplast genome of Taiwania cryptomerioides (Cupressales: Cupressaceae), an endangered relict conifer species endemic to East Asia

Taiwania cryptomerioides is an endangered relict conifer species native to East Asia with a variety of practical applications. To contribute to its conservation and sustainable utilization, its complete chloroplast genome was assembled from Illumina sequencing reads. The circular genome is 131,427 bp in size with a biased nucleotide composition (33.5% A, 17.5% C, 17.2% G & 31.8% T). It encodes a panel of 114 gene species, including 83 protein-coding genes (PCGs) and 31 tRNAs. Gene duplication was detected for three tRNA gene species (trnI-CAU, trnQ-UUG & trnV-GAC). Besides, 15 gene species (atpF, ndhA, ndhB, petB, petD, rpl16, rpl2, rpoC1, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA & trnV-UAC) possess a single intron, and another one (ycf3) has a couple of introns. Phylogenetic analysis suggests that T. cryptomerioides and T. flousiana are closely related to each other within the family Cupressaceae.

Characterization of the complete chloroplast genome of Juniperus microsperma (Cupressaceae), a rare endemic from the Qinghai-Tibet Plateau

The complete chloroplast genome of Juniperus microsperma, a rare endemic conifer from the Qinghai-Tibet Plateau whose current status satisfies the IUCN Red List criteria of endangered species, was determined in this study. The genome size is 127,409 bp in length. The genome contained 119 genes, including 82 protein-coding genes, four ribosomal RNA genes and 33 transfer RNA genes. The most of genes occur as a single copy, while two genes occur in double copies (trnL-CAU and trnQ-UUG). In these genes, eight genes (atpF, ndhA, ndhB, rpoC1, petD, petB, rpl16 and rpl2) have a single intron, while two genes (rps12 and ycf3) harbor two introns. There are no inverted repeat sequences in this genome. The GC content of this cp genome is 35.0%. Phylogenomic analysis revealed a close relationship among J. microsperma, J. virginiana, J. scopulorum and J. bermudiana.

Self-incompatibility allele identification in Ukrainian sweet cherry (Prunus avium L.) cultivars

Aim. Ukrainian breeders have created a large number of sweet cherry cultivars, which still remain almost unexplored at the molecular level. The aim of our study was to identify the self-incompatibility alleles (S-alleles) in Ukrainian sweet cherry cultivars and landraces, and to elucidate, to which cross-incompatibility group the cultivars belong. Methods. The PCR was conducted using consensus primers to the first and second introns of S-RNAse gene and to the single intron of SFB gene. The electrophoretic analysis of the PCR products of the second intron of S-RNAse was carried out in agarose gel, whereas detection of fluorescently labeled DNA fragments of the first S-RNAse intron and the SFB intron was performed using a genetic analyzer. Results. The S-alleles of 25 Ukrainian sweet cherry cultivars and 10 landraces were identified. The S-alleles frequencies and affiliation of cultivars and landraces to the groups of cross-incompatibility were determined. The obtained data can be used in breeding programs and by planning of industrial plantings. Conclusions. In the study, 12 different S-alleles and 79 S-haplotypes were identified. The S1, S3, S4, S5, S6 and S9 alleles are the most widespread among Ukrainian sweet cherry cultivars and landraces. The high frequencies of S5 and especially of S9 alleles are characteristic for the Ukrainian cultivars and distinguish them from other European ones. For the Ukrainian sweet cherry cultivars, the XXXVII (S5S9) cross-incompatibility group appeared to be the most numerous.Keywords: Ukrainian sweet cherry cultivars, S-locus, Sgenotypes, self- and cross-incompatibility, Prunus avium.

Characterization of the complete plastid genome sequence of eleutherococcus brachypus (araliaceae), an endangered shrub in China

Eleutherococcus brachypus: (Harms) Nakai (Araliaceae) is an endangered shrub that it is endemic to western China. In this study, we characterized the complete nucleotide sequence of the chloroplast (cp) genome in E. brachypus. The total length of the cp genome was 156,981 bp, including a large single copy (LSC) region of 86,921 bp, a small single copy (SSC) region of 18,184 bp, and a pair of inverted repeats (IRs) comprising 25,938 bp. The cp genome of E. brachypus contained 114 genes, including 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. In total, 15 genes comprising tRNA-Lys (UUU), tRNA-Gly (UCC), tRNA-Leu (UAA), tRNA-Val (UAC), tRNA-Ile (GAU), tRNA-Ala (UGC), rps16, atpF, rpoC1, petB, petD, rps16, rpl2, ndhB, and ndhA contained a single intron, and three genes contained two introns, i.e., ycf3, clpP, and rps12. The GC contents of the whole cp genome, LSC region, SSC region, and IR region were 38.0, 36.2, 32.0, and 43.0% respectively, which are similar to those in other Araliaceae plants. Phylogenetic analysis suggested that E. brachypus has a close relationship with congeneric E. gracilistylus.

The complete chloroplast genome of Cupressus jiangeensis (cupressaceae), a critically endangered conifer species in China

Cupressus jiangeensis is a critically endangered conifer listed in the IUCN Red List of Threatened Species. Illumina pair-end reads of this species was used to character the whole chloroplast (cp) genome sequence. The circular genome with 128,286 bp in length contains two duplicated genes (trnL and trnQ) and 115 single copy genes, including 82 protein-coding genes, 31 tRNA and four ribosomal RNA genes. Eight genes (atpF, ndhA, ndhB, rpoC1, petD, petB, rpl16 and rpl2) have a single intron, while two genes (rps12 and ycf3) have two introns. There are no inverted repeat sequences in this genome. The GC content of this cp genome is 34.68%. Phylogenomic analysis strongly supported the close relationship of C. jiangeensis and C. chengiana.

Insertion of Multiple Artificial Introns of Universal Design into cDNA during Minigene Construction Assures Correct Transgene Splicing

The presence of introns is often required for efficient transgene expression. The use of full-length genes for transgenesis is associated with technical difficulties due to the large size of the genetic construct. To solve this problem, we recently suggested a universal design of small artificial introns that ensures efficient splicing. However, the insertion of more than one intron into cDNA might result in the aberrant splicing of the minigene with exon skipping. Here, we showed that the insertion of two artificial introns of universal design into cDNA resulted in a splicing pattern that corresponds to the excision of each intron with an exon between them remaining in the transcript. No transcript formation with exon skipping was detected. Therefore, the developed design of small artificial introns assures splicing solely between the donor and the acceptor splice sites of each single intron and results in the generation of a correct transcript from minigene pre-mRNA. These findings enable the construction of minigenes for transgenesis with more than one artificial intron, with no additional cis-elements required to prevent aberrant splicing.

The complete chloroplast genome sequences of Larix kaempferi and Larix olgensis var. koreana (Pinaceae)

The complete chloroplast (cp) genome sequence of Larix kaempferi and L. olgensis var. koreana were determined by Ion torrent Platform sequencing in this study. The L. kaempferi cp genome was 122,158bp consists of two inverted repeat (IR) regions of 436bp each, a large single-copy (LSC) region of 65,394bp, and a small single-copy (SSC) region of 55,892bp. The chloroplast genome sequence of L. olgensis var. koreana was 122,573bp in length, consisting of two IRs (436bp), one LSC (65,597bp), and one SSC (56,104bp), and is longer than that of L. kaempferi. The genome contained 110 genes, including 71 protein-coding genes, 35 tRNA genes, and 4 ribosomal RNA genes. The 13 genes contain introns, including 12 genes with a single intron each and ycf3 gene with two introns. And, the rps12 gene is a trans-spliced gene. The phylogenetic analysis revealed that all sampled species in Pinaceae formed a monophyletic clade with high bootstrap value. The genus Larix is closely related to Pseudotsuga.

Complete Chloroplast Genome Sequencing and Genetic Relationship Analysis of Capsicum chinense Jacq

Capsicum chinense is one of the five domesticated pepper species belonging to the Solanaceae family. Capsicum sp. have been used as model systems in comparative and evolutionary genomics because their superior availability of chloroplast genome in the solanaceae family. Similarly, molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. So far however, only partial taxonomic and phylogenetic analyses have been carried out for the genus. Thus, the complete chloroplast genome sequence of a cultivated pepper (C. chinense) has been reported here. The total length of the chloroplast genome is 156,936 bp, with 37.7% overall GC content. A pair of inverted repeats (IRs) of 25,847 bp was separated by a small single copy (SSC) region of 17,912 bp and a large single copy (LSC) region of 87,330 bp. The chloroplast genome harbors 113 known genes, including 79 protein-coding genes, four ribosomal RNA genes, and 30 transfer RNA (tRNA) genes. In all, 21 of these genes are duplicated in the inverted repeat regions, 15 genes and six tRNA genes contain a single intron, while two genes have two introns. Analysis revealed 117 simple sequence repeat (SSR) loci, which are mostly located in the intergenic regions. The complete chloroplast genome reported here enriches our knowledge of the genetic complement of C. chinense, and contributes to our understanding of the genetic relationships within the genus Capsicum.