Divergence of 3' ends as a driver of short interspersed nuclear element (SINE) evolution in the Salicaceae.

Short interspersed nuclear elements (SINEs) are small, non-autonomous and heterogeneous retrotransposons that are widespread in plants. To explore the amplification dynamics and evolutionary history of SINE populations in representative deciduous tree species, we analyzed the genomes of the six following Salicaceae species: Populus deltoides, Populus euphratica, Populus tremula, Populus tremuloides, Populus trichocarpa, and Salix purpurea. We identified 11 Salicaceae SINE families (SaliS-I to SaliS-XI), comprising 27 077 full-length copies. Most of these families harbor segmental similarities, providing evidence for SINE emergence by reshuffling or heterodimerization. We observed two SINE groups, differing in phylogenetic distribution pattern, similarity and 3' end structure. These groups probably emerged during the 'salicoid duplication' (~65 million years ago) in the Salix-Populus progenitor and during the separation of the genus Salix (45-65 million years ago), respectively. In contrast to conserved 5' start motifs across species and SINE families, the 3' ends are highly variable in sequence and length. This extraordinary 3'-end variability results from mutations in the poly(A) tail, which were fixed by subsequent amplificational bursts. We show that the dissemination of newly evolved 3' ends is accomplished by a displacement of older motifs, leading to various 3'-end subpopulations within the SaliS families.

Human leukocyte antigen-G 3' untranslated region polymorphism +3142G/C (rs1063320) and haplotypes are associated with manifestations of the American Tegumentary Leishmaniasis in a Northeastern Brazilian population.

While the role of cytokine genes has been well documented in the context of Leishmania (Viannia) braziliensis infection, no studies have addressed the influence of human leukocyte antigen-G (HLA-G) in susceptibility/resistance to American Tegumentary Leishmaniasis (ATL). Here, we evaluated the influences of HLA-G, IL-10, TNF-A and IFN-G in the susceptibility and clinical manifestations of ATL. DNA of 114 ATL patients and 346 healthy individuals were sequenced for well-documented polymorphisms in HLA-G 3' untranslated region (UTR), in IL-10 and TNF-A promoters and in IFN-G intron 1. Soluble HLA-G (sHLA-G) and cytokine levels were evaluated by ELISA and flow cytometry, respectively. Analyses were performed using GraphPad and R-package software. Individuals bearing HLA-G +3142G/G showed an association with increased risk for ATL, whereas those carrying the HLA-G +3142C/G and one copy of UTR6 haplotype, showed an association with decreased risk for ATL. sHLA-G was overexpressed in "susceptible" patients compared to the "resistant'' one, and also in patients bearing +3142G/G genotype. From these results, HLA-G +3142G/G may be considered as genotype of susceptibility and UTR6 as marker of protection to ATL. Our findings showed a participation of HLA-G in the pathogenesis of the ATL.

Distinct Clinical Courses of Epithelial Ovarian Cancer with Mutations in BRCA1 5' and 3' Exons.

BACKGROUND/AIM: This study aimed to determine the effect of different BRCA1 exonal mutations on the clinical course of epithelial ovarian cancer (EOC). PATIENTS AND METHODS: Clinicopathological variables and survival outcomes were compared among 53 primary EOC patients with pathogenic BRCA1 mutations in exons 1-11 (5' mutations) and in exons 12-24 (3' mutations). RESULTS: BRCA1 5' exonal mutations were found in 35 (66.0%) patients. The median follow-up period was 40 months. Clinicopathological variables remained unchanged between the two groups. Patients with 5' mutations had a significantly longer progression-free survival than those with C-terminal mutations (p=0.034), better predicting progression-free survival [2.923 (1.402-6.093), p=0.004], but not overall survival in cases of multiple relapses (p=0.497). CONCLUSION: N-terminal BRCA1 mutations in EOC patients are associated with favourable primary progression-free survival, a trend observed only in primary progression-free survival, not in overall survival.

Short fish-origin DNA elements served as flanking sequences in a knockdown cloning vector enabling the generation of a functional siRNA molecule in mammalian cells and fish embryos.

Improving the quality of a siRNA-knockdown cloning vector requires simpler, shorter, and more effective flanking sequences. In this study, we designed such flanking sequences based on those found in zebrafish pre-miR3906, namely, internal element (IE) 1 and IE2. We engineered a vegf-shRNA fragment flanked by an 80-bp IE1/IE2 and then inserted into the 3' UTR of GFP reporter cDNA driven by a cytomegalovirus promoter to obtain a plasmid containing gfp-IE-vegf-shRNA-polA. Upon microinjection of this plasmid into zebrafish embryos, we found that IE flanking sequences could effectively induce the production of vegf-shRNA fragment, which was then processed into a functional siRNA to silence the target vegf121 gene. Northern blot showed that the vegf-shRNA fragment was cleaved from gfp-IE-vegf-shRNA-polA, resulting in the loss of polyA tails, subsequently degrading the remaining RNA-containing GFP. Moreover, Western blot revealed that addition of IE-based vegf-shRNA fragment could markedly decrease the expression of VEGF. Finally, to facilitate a more versatile application of the IE-based knockdown vector, we generated an inducible expression vector in which IE-vegf-shRNA was constructed downstream in a Tet-on system to generate a Tet-on-IE-vegf-shRNA construct. After doxycycline induction, the protein level of VEGF in SW620 cells harboring the Tet-on-IE-vegf-shRNA construct was decreased 77%. Interestingly, when SW620 cells harboring Tet-on-IE-vegf-shRNA cells were induced and transplanted into zebrafish embryos, we found that abnormal branch of the sub-intestinal vessels was reduced in the recipient embryos, suggesting that vegf-shRNA cleaved from Tet-on-IE-vegf-shRNA-polA was processed into a functional vegf-siRNA in embryos suppressing endogenous VEGF and reducing tumor angiogenesis. Therefore, we conclude that fish-origin IEs are flanking sequences with short, simple, and effective DNA elements. This IE-based knockdown cloning vector provides a new alternative material to facilitate the generation of functional siRNA with which to perform loss-of-function experiments, both in vitro (mammalian cells) and in vivo (zebrafish embryos).

Cotranscriptional 3'-End Processing of T7 RNA Polymerase Transcripts by a Smaller HDV Ribozyme.

In vitro run-off transcription by T7 RNA polymerase generates heterogeneous 3'-ends because the enzyme tends to add untemplated adenylates. To generate homogeneous 3'-termini, HDV ribozymes have been used widely. Their sequences are added to the 3'-terminus such that co-transcriptional self-cleavage generates homogeneous 3'-ends. A shorter HDV sequence that cleaves itself efficiently would be advantageous. Here we show that a recently discovered, small HDV ribozyme is a good alternative to the previously used HDV ribozyme. The new HDV ribozyme is more efficient in some sequence contexts, and less efficient in other sequence contexts than the previously used HDV ribozyme. The smaller size makes the new HDV ribozyme a good alternative for transcript 3'-end processing.

Animal Hen1 2'-O-methyltransferases as tools for 3'-terminal functionalization and labelling of single-stranded RNAs.

S-adenosyl-L-methionine-dependent 2'-O-methylati-on of the 3'-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22-80 nt) of single-stranded RNAs permitting their 3'-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3'-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.

Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars.

KEY MESSAGE: Data obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads. The reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25 S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S 5 , S 6a , S 6b , S 8 , S 11 , S 23 , S 39 , S 46 , S 50 and S 58 ). Moreover, sequence divergence clearly suggests that alleles S 6a and S 6b , proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.

Chimeric 3' flanking regions strongly enhance gene expression in plants.

Plants represent a promising platform for the highly scalable production of recombinant proteins. Previously, we identified the tobacco extensin terminator lacking its intron as an element that reduced transcript read-through and improved recombinant protein production in a plant-based system. In this study, we systematically compared nonreplicating plant expression vectors containing over 20 commonly used or newly identified terminators from diverse sources. We found that eight gene terminators enhance reporter gene expression significantly more than the commonly used 35S and NOS terminators. The intronless extensin terminator provided a 13.6-fold increase compared with the NOS terminator. Combining terminators in tandem produced large synergistic effects, with many combinations providing a >25-fold increase in expression. Addition of the tobacco Rb7 or TM6 matrix attachment region (MAR) strongly enhanced protein production when added to most terminators, with the Rb7 MAR providing the greatest enhancement. Using deletion analysis, the full activity of the 1193 bp Rb7 MAR was found to require only a 463-bp region at its 3' end. Combined terminators and MAR together provided a >60-fold increase compared with the NOS terminator alone. These combinations were then placed in a replicating geminiviral vector, providing a total of >150-fold enhancement over the original NOS vector, corresponding to an estimated yield of 3-5 g recombinant protein per kg leaf fresh weight or around 50% of the leaf total soluble protein. These results demonstrate the importance of 3' flanking regions in optimizing gene expression and show great potential for 3' flanking regions to improve DNA-based recombinant protein production systems.

Characterizing Helicobacter pylori cagA in Myanmar.

BACKGROUND/AIMS: Differences in the Helicobacter pylori infection rate are not sufficient to clarify the dissimilarity of gastric cancer incidence between Myanmar and its neighboring countries. To better understand this trend, the H. pylori virulence gene cagA was characterized in Myanmar. METHODS: Glutamate-proline-isoleucine-tyrosine-alanine (EPIYA) patterns and CagA multimerization (CM) motifs of cagA genotypes were examined by performing polymerase chain reactions and DNA sequencing. RESULTS: Of 69 tested H. pylori strains, cagA-positive patients had significantly more severe histological scores in their antrum than cagA-negative patients. Sequence analysis revealed that 94.1% of strains had Western-type cagA containing an EPIYA motif (92.6%) or EPIYT motif (6.4%). The intestinal metaplasia scores in the antral of patients infected with the ABC and ABCC types of cagA were significantly higher than those of patients with AB-type cagA. Interestingly, in patients infected with H. pylori, 46.3% of strains with three EPIYA motifs contained two identical Western-typical CM motifs, and these patients showed significantly higher antrum inflammation scores than patients infected with two identical nontypical-CM motif strains (p=0.02). CONCLUSIONS: In Myanmarese strains, Western-type cagA was predominant. The presence of CM motifs and the proportion of multiple EPIYA-C segments might partially explain the intermediate gastric cancer risk found in Myanmar.

Elucidating the 16S rRNA 3' boundaries and defining optimal SD/aSD pairing in Escherichia coli and Bacillus subtilis using RNA-Seq data.

Bacterial translation initiation is influenced by base pairing between the Shine-Dalgarno (SD) sequence in the 5' UTR of mRNA and the anti-SD (aSD) sequence at the free 3' end of the 16S rRNA (3' TAIL) due to: 1) the SD/aSD sequence binding location and 2) SD/aSD binding affinity. In order to understand what makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3' TAIL and 2) extent of the core aSD sequence within the 3' TAIL. Our approach to characterize these components in Escherichia coli and Bacillus subtilis involves 1) mapping the 3' boundary of the mature 16S rRNA using high-throughput RNA sequencing (RNA-Seq), and 2) identifying the segment within the 3' TAIL that is strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous discrepancies in the reported 3' TAIL in B. subtilis and recovered the established 3' TAIL in E. coli. Furthermore, we extend previous studies to suggest that both highly and lowly expressed genes favor SD sequences with intermediate binding affinity, but this trend is exclusive to SD sequences that complement the core aSD sequences defined herein.

Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression.

Chlamydomonas reinhardtii is a unicellular green alga that has attracted interest due to its potential biotechnological applications, and as a model for algal biofuel and energy metabolism. Despite all the advantages that this unicellular alga offers, poor and inconsistent expression of nuclear transgenes remains an obstacle for basic and applied research. We used a data-mining strategy to identify highly expressed genes in Chlamydomonas whose flanking sequences were tested for the ability to drive heterologous nuclear transgene expression. Candidates identified in this search included two ribosomal protein genes, RPL35a and RPL23, and ferredoxin, FDX1, whose flanking regions including promoters, terminators and untranslated sequences could drive stable luciferase transgene expression to significantly higher levels than the commonly used Hsp70A-RBCS2 (AR) hybrid promoter/terminator sequences. The RPL23 flanking sequences were further tested using the zeocin resistance gene sh-ble as a reporter in monocistronic and dicistronic constructs, and consistently yielded higher numbers of zeocin-resistant transformants and higher levels of resistance than AR- or PSAD-based vectors. Chlamydomonas RPL23 sequences also enabled transgene expression in Volvox carteri. Our study provides an additional benchmark for strong constitutive expression of transgenes in Chlamydomonas, and develops a general approach for identifying flanking sequences that can be used to drive transgene expression for any organism where transcriptome data are available.

Effect of the fourth nucleotide at the 3' end of neuraminidase and matrix viral genomic RNA on the pathogenicity of influenza virus A/PR/8/34.

Twelve nucleotides located at the 3' end of viral genomic RNA (vRNA) are conserved among influenza A viruses (IAV) and have a promoter function. Hoffmann's 8-plasmid reverse genetics vector system introduced mutations at position 4, C nucleotide (C4) to U nucleotide (U4), of the 3' ends of neuraminidase (NA) and matrix (M) vRNAs of wild-type A/PR/8/34 (PR8). This resulted in a constellation of C4 and U4 vRNAs coding for low (polymerases) and relatively high (all others) copy number proteins, respectively. U4 has been reported to increase promoter activity in comparison to C4, but the constellation effect on the replication efficiency and pathogenicity of reverse genetics PR8 (rgPR8) has not been fully elucidated. In the present study, we generated 3 recombinant viruses with C4 in the NA and/or M vRNAs and rgPR8 by using reverse genetics and compared their pathobiological traits. The mutant viruses showed lower replication efficiency than rgPR8 due to the low transcription levels of NA and/or M genes. Furthermore, C4 in the NA and/or M vRNAs induced lower PR8 virus pathogenicity in BALB/c mice. The results suggest that the constellation of C4 and U4 among vRNAs may be one of the multigenic determinants of IAV pathogenicity.

The IgH locus 3' cis-regulatory super-enhancer co-opts AID for allelic transvection.

Immunoglobulin heavy chain (IgH) alleles have ambivalent relationships: they feature both allelic exclusion, ensuring monoallelic expression of a single immunoglobulin (Ig) allele, and frequent inter-allelic class-switch recombination (CSR) reassembling genes from both alleles. The IgH locus 3' regulatory region (3'RR) includes several transcriptional cis-enhancers promoting activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM) and CSR, and altogether behaves as a strong super-enhancer. It can also promote deregulated expression of translocated oncogenes during lymphomagenesis. Besides these rare, illegitimate and pathogenic interactions, we now show that under physiological conditions, the 3'RR super-enhancer supports not only legitimate cis- , but also trans-recruitment of AID, contributing to IgH inter-allelic proximity and enabling the super-enhancer on one allele to stimulate biallelic SHM and CSR. Such inter-allelic activating interactions define transvection, a phenomenon well-known in drosophila but rarely observed in mammalian cells, now appearing as a unique feature of the IgH 3'RR super-enhancer.

Rare pseudoautosomal copy-number variations involving SHOX and/or its flanking regions in individuals with and without short stature.

Pseudoautosomal region 1 (PAR1) contains SHOX, in addition to seven highly conserved non-coding DNA elements (CNEs) with cis-regulatory activity. Microdeletions involving SHOX exons 1-6a and/or the CNEs result in idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). Here, we report six rare copy-number variations (CNVs) in PAR1 identified through copy-number analyzes of 245 ISS/LWD patients and 15 unaffected individuals. The six CNVs consisted of three microduplications encompassing SHOX and some of the CNEs, two microduplications in the SHOX 3'-region affecting one or four of the downstream CNEs, and a microdeletion involving SHOX exon 6b and its neighboring CNE. The amplified DNA fragments of two SHOX-containing duplications were detected at chromosomal regions adjacent to the original positions. The breakpoints of a SHOX-containing duplication resided within Alu repeats. A microduplication encompassing four downstream CNEs was identified in an unaffected father-daughter pair, whereas the other five CNVs were detected in ISS patients. These results suggest that microduplications involving SHOX cause ISS by disrupting the cis-regulatory machinery of this gene and that at least some of microduplications in PAR1 arise from Alu-mediated non-allelic homologous recombination. The pathogenicity of other rare PAR1-linked CNVs, such as CNE-containing microduplications and exon 6b-flanking microdeletions, merits further investigation.

Rhoptry-associated protein (rap-1) genes in the sheep pathogen Babesia sp. Xinjiang: Multiple transcribed copies differing by 3' end repeated sequences.

Sheep babesiosis occurs mainly in tropical and subtropical areas. The sheep parasite Babesia sp. Xinjiang is widespread in China, and our goal is to characterize rap-1 (rhoptry-associated protein 1) gene diversity and expression as a first step of a long term goal aiming at developing a recombinant subunit vaccine. Seven different rap-1a genes were amplified in Babesia sp. Xinjiang, using degenerate primers designed from conserved motifs. Rap-1b and rap-1c gene types could not be identified. In all seven rap-1a genes, the 5' regions exhibited identical sequences over 936 nt, and the 3' regions differed at 28 positions over 147 nt, defining two types of genes designated α and ß. The remaining 3' part varied from 72 to 360 nt in length, depending on the gene. This region consists of a succession of two to ten 36 nt repeats, which explains the size differences. Even if the nucleotide sequences varied, 6 repeats encoded the same stretch of amino acids. Transcription of at least four α and two ß genes was demonstrated by standard RT-PCR.

Characterization of leptin receptor gene in Bubalus bubalis and association analysis with body measurement traits.

Leptin has a pleiotropic effect on regulating appetite, energy metabolism, growth, reproduction, body composition and immunity. This property supports leptin and its receptor as candidate genes for evaluating genetic polymorphisms to associate with growth, milk yield and other economic traits. The aim of this study is to characterize the leptin receptor gene in Bubalus bubalis, to identify single-nucleotide polymorphism (SNP) sites in different coding and non-coding regions and to analyse potential associations between SNPs identified and the body measurements traits of growing buffalo heifers. A group of 64 animals were genotyped by direct sequencing and twenty-eight SNPs were detected. A sequence analysis revealed the presence of nine interesting SNPs in gene sequence. The association analysis of polymorphisms with the body measurements traits of growing buffalo heifers shows significant statistical effects on chest depth and sacrum height. Therefore according to the results obtained from this study, the leptin receptor gene appears to have potential effects on the body measurement traits of Bubalus bubalis.

Association of the 3' region of the neuregulin 1 gene with bipolar I disorder in the Chinese Han population.

BACKGROUND: Based on the function of neuregulin 1 (NRG1) in neurodevelopment, susceptibility to bipolar disorder presumably involves this gene. The 3' region of NRG1 contains the majority of the coding exons, and transcripts from this region encode 8 of the 9 known NRG1 isoforms; therefore, this region is likely to be predominant versus the 5' region in terms of their relative contributions to NRG1 function. We investigated the association between the 3' region of the NRG1 gene and bipolar I disorder (BPI) in the Chinese Han population and performed further analyses depending on the presence or absence of psychotic features. METHODS: A total of 385 BPI patients and 475 healthy controls were recruited for this study. Thirty tag single nucleotide polymorphisms (SNPs) across the 3' region of the NRG1 gene were genotyped for allelic and haplotypic associations with BPI and subgroups with psychotic features (BPI-P) or without psychotic features (BPI-NP). RESULTS: Individual marker analysis showed that 2 SNPs (rs12547858 and rs6468121) in this region were significantly associated with BPI. Moreover, subgroup analyses showed significant but marginal associations of rs6468121 with BPI-P and rs3757933 with BPI-NP. Haplotype analyses showed that 6 haplotypes were associated with BPI only. LIMITATIONS: The sample size was relatively small. The investigated tag SNPs only represented 83% of the information on the targeted region. There might be a retrospective bias in the subgroup analyses. CONCLUSION: The results suggest that the 3' region of the NRG1 gene plays a role in BPI susceptibility in the Chinese Han population. In addition, the preliminary results show that BPI with psychotic features and BPI without psychotic features may constitute different sub-phenotypes; however, this finding should be confirmed in a larger population sample.