Intron Sequences Research Articles

Distinguishing Genetic Drift from Selection in Papillomavirus Evolution.

Pervasive purifying selection on non-synonymous substitutions is a hallmark of papillomavirus genome history, but the role of selection on and the drift of non-coding DNA motifs on HPV diversification is poorly understood. In this study, more than a thousand complete genomes representing Alphapapillomavirus types, lineages, and SNP variants were examined phylogenetically and interrogated for the number and position of non-coding DNA sequence motifs using Principal Components Analyses, Ancestral State Reconstructions, and Phylogenetic Independent Contrasts. For anciently diverged Alphapapillomavirus types, composition of the four nucleotides (A, C, G, T), codon usage, trimer usage, and 13 established non-coding DNA sequence motifs revealed phylogenetic clusters consistent with genetic drift. Ancestral state reconstruction and Phylogenetic Independent Contrasts revealed ancient genome alterations, particularly for the CpG and APOBEC3 motifs. Each evolutionary analytical method we performed supports the unanticipated conclusion that genetic drift and different evolutionary drivers have structured Alphapapillomavirus genomes in distinct ways during successive epochs, even extending to differences in more recently formed variant lineages.

PCSK9 Involvement in Autism Etiology: Sequence Variations, Protein Concentration, and Promoter Methylation

Besides its main role in the control of blood cholesterol, PCSK9 has a role in the regulation of neuronal development and apoptosis. We suggest, for the first time, the possible involvement of PCSK9 in autism. In this case-control study, Sanger sequencing was used to analyze sequence variations in the PCSK9 gene exons and their flanking intronic sequences. ELISA assay was used to determine the plasma concentration of PCSK9. The methylation percentage of the PCSK9 gene promoter was assessed by methylation-specific PCR (MSP). Forty-three variants were found; out of them, seven showed differential frequency between patients and controls. rs.45448095, rs.45613943, rs.630431, rs.529500286, and rs.45439391 are risk factors for autism, while rs.11800231 and rs.483462 are protective variants. The concentration of plasma PCSK9 protein was significantly elevated and the methylation percentage of PCSK9 gene promoter was significantly lower in cases than in controls (P <0.001 and = 0.002, respectively). ROC curve analysis identified an area under the curve (AUC) of 0.915 for plasma protein concentration and 0.693 for percent gene promoter methylation. In addition, two new variants were identified (g.23809C>T in intron 11 and g.24071T>G in 3' UTR). This is the first study to investigate the correlation between PCSK9 protein and autism and suggests the potential involvement of PCSK9 as one of the susceptibility genes for autism. Further studies with a larger number of subjects are recommended.

Epigenetic and Metabolic Regulation of Macrophages during Gout

The analysis of metabolite mediators has allowed a broader understanding of disease mechanisms. Experimental evidence indicates that metabolic rewiring is a key feature of inflammatory cells to restore tissue homeostasis upon damage. Over the last two decades, next-generation sequencing techniques have offered the possibility of looking at the genome-wide effect of the exposure of inflammatory cells to external stimuli. During gout flares, monosodium urate crystals activate a distinct metabolic profile and inflammatory transcriptional program in inflammatory cells. The extracellular signals are transduced through distinct signalling pathways, which are regulated by non-coding RNA and DNA sequences, and modification of histones. During response to inflammatory stimuli, changes in the abundance of metabolic mediators can regulate the activation of histones and of chromatin remodellers. The interplay between metabolic changes by MSUc, the regulation of epigenetic changes and the activation of transcription factor networks in inflammatory cells remains unknown. A better understanding of the interplay between metabolites and how it alters inflammatory response may provide novel insights into disease mechanisms during gout. In this review, we aim to provide a deeper understanding of the current view of how metabolic deregulation could alter the epigenetic landscape of inflammatory cells during gout.

Differentiation block in acute myeloid leukemia regulated by intronic sequences of FTO

Iroquois transcription factor gene IRX3 is highly expressed in 20-30% of acute myeloid leukemia (AML) and contributes to the pathognomonic differentiation block. Intron 8 FTO sequences ∼220kB downstream of IRX3 exhibit histone acetylation, DNA methylation, and contacts with the IRX3 promoter, which correlate with IRX3 expression. Deletion of these intronic elements confirms a role in positively regulating IRX3. RNAseq revealed long non-coding (lnc) transcripts arising from this locus. FTO-lncAML knockdown (KD) induced differentiation of AML cells, loss of clonogenic activity, and reduced FTO intron 8:IRX3 promoter contacts.While both FTO-lncAML KD and IRX3 KD induced differentiation, FTO-lncAML but not IRX3 KD led to HOXA downregulation suggesting transcript activity in trans. FTO-lncAMLhigh AML samples expressed higher levels of HOXA and lower levels of differentiation genes. Thus, a regulatory module in FTO intron 8 consisting of clustered enhancer elements and a long non-coding RNA is active in human AML, impeding myeloid differentiation.

Intronic small nucleolar RNAs regulate host gene splicing through base pairing with their adjacent intronic sequences

BackgroundSmall nucleolar RNAs (snoRNAs) are abundant noncoding RNAs best known for their involvement in ribosomal RNA maturation. In mammals, most expressed snoRNAs are embedded in introns of longer genes and produced through transcription and splicing of their host. Intronic snoRNAs were long viewed as inert passengers with little effect on host expression. However, a recent study reported a snoRNA influencing the splicing and ultimate output of its host gene. Overall, the general contribution of intronic snoRNAs to host expression remains unclear.ResultsComputational analysis of large-scale human RNA-RNA interaction datasets indicates that 30% of detected snoRNAs interact with their host transcripts. Many snoRNA-host duplexes are located near alternatively spliced exons and display high sequence conservation suggesting a possible role in splicing regulation. The study of the model SNORD2-EIF4A2 duplex indicates that the snoRNA interaction with the host intronic sequence conceals the branch point leading to decreased inclusion of the adjacent alternative exon. Extended SNORD2 sequence containing the interacting intronic region accumulates in sequencing datasets in a cell-type-specific manner. Antisense oligonucleotides and mutations that disrupt the formation of the snoRNA-intron structure promote the splicing of the alternative exon, shifting the EIF4A2 transcript ratio away from nonsense-mediated decay.ConclusionsMany snoRNAs form RNA duplexes near alternative exons of their host transcripts, placing them in optimal positions to control host output as shown for the SNORD2-EIF4A2 model system. Overall, our study supports a more widespread role for intronic snoRNAs in the regulation of their host transcript maturation.

RNA Transcript Diversity in Neuromuscular Research.

Three decades since the Human Genome Project began, scientists have now identified more then 25,000 protein coding genes in the human genome. The vast majority of the protein coding genes (> 90%) are multi-exonic, with the coding DNA being interrupted by intronic sequences, which are removed from the pre-mRNA transcripts before being translated into proteins, a process called splicing maturation. Variations in this process, i.e. by exon skipping, intron retention, alternative 5' splice site (5'ss), 3' splice site (3'ss), or polyadenylation usage, lead to remarkable transcriptome and proteome diversity in human tissues. Given its critical biological importance, alternative splicing is tightly regulated in a tissue- and developmental stage-specific manner. The central nervous system and skeletal muscle are amongst the tissues with the highest number of differentially expressed alternative exons, revealing a remarkable degree of transcriptome complexity. It is therefore not surprising that splicing mis-regulation is causally associated with a myriad of neuromuscular diseases, including but not limited to amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), and myotonic dystrophy type 1 and 2 (DM1, DM2). A gene's transcript diversity has since become an integral and an important consideration for drug design, development and therapy. In this review, we will discuss transcript diversity in the context of neuromuscular diseases and current approaches to address splicing mis-regulation.

Understanding WT1 Alterations and Expression Profiles in Hematological Malignancies

WT1 is a true chameleon, both acting as an oncogene and tumor suppressor. As its exact role in leukemogenesis is still ambiguous, research with model systems representing natural conditions surrounding the genetic alterations in WT1 is necessary. In a cohort of 59 leukemia/lymphoma cell lines, we showed aberrant expression for WT1 mRNA, which does not always translate into protein levels. We also analyzed the expression pattern of the four major WT1 protein isoforms in the cell lines and primary AML blasts with/without WT1 mutations and demonstrated that the presence of mutations does not influence these patterns. By introduction of key intronic and exonic sequences of WT1 into a lentiviral expression vector, we developed a unique tool that can stably overexpress the four WT1 isoforms at their naturally occurring tissue-dependent ratio. To develop better cellular model systems for WT1, we sequenced large parts of its gene locus and also other important myeloid risk factor genes and revealed previously unknown alterations. Functionally, inhibition of the nonsense-mediated mRNA decay machinery revealed that under natural conditions, the mutated WT1 alleles go through a robust degradation. These results offer new insights and model systems regarding the characteristics of WT1 in leukemia and lymphoma.

STUDY OF NON-CODING AREAS OF THE DNA GENOME OF DIFFERENT ANIMAL SPECIES

STUDY OF NON-CODING AREAS OF THE DNA GENOME OF DIFFERENT ANIMAL SPECIES

Cancer-Related Mutations Alter RNA-Driven Functional Cross-Talk Underlying Premature-Messenger RNA Recognition by Splicing Factor SF3b.

The pillar of faithful premature-messenger (pre-mRNA) splicing is the precise recognition of key intronic sequences by specific splicing factors. The heptameric splicing factor 3b (SF3b) recognizes the branch point sequence (BPS), a key part of the 3' splice site. SF3b contains SF3B1, a protein holding recurrent cancer-associated mutations. Among these, K700E, the most-frequent SF3B1 mutation, triggers aberrant splicing, being primarily implicated in hematologic malignancies. Yet, K700E and the BPS recognition site are 60 Å apart, suggesting the existence of an allosteric cross-talk between the two distal spots. Here, we couple molecular dynamics simulations and dynamical network theory analysis to unlock the molecular terms underpinning the impact of SF3b splicing factor mutations on pre-mRNA selection. We establish that by weakening and remodeling interactions of pre-mRNA with SF3b, K700E scrambles RNA-mediated allosteric cross-talk between the BPS and the mutation site. We propose that the altered allostery contributes to cancer-associated missplicing by mutated SF3B1. This finding broadens our comprehension of the elaborate mechanisms underlying pre-mRNA metabolism in eukaryotes.

Disruption of regulatory domains and novel transcripts as disease-causing mechanisms.

Deletions, duplications, insertions, inversions, and translocations, collectively called structural variations (SVs), affect more base pairs of the genome than any other sequence variant. The recent technological advancements in genome sequencing have enabled the discovery of tens of thousands of SVs per human genome. These SVs primarily affect non-coding DNA sequences, but the difficulties in interpreting their impact limit our understanding of human disease etiology. The functional annotation of non-coding DNA sequences and methodologies to characterize their three-dimensional (3D) organization in the nucleus have greatly expanded our understanding of the basic mechanisms underlying gene regulation, thereby improving the interpretation of SVs for their pathogenic impact. Here, we discuss the various mechanisms by which SVs can result in altered gene regulation and how these mechanisms can result in rare genetic disorders. Beyond changing gene expression, SVs can produce novel gene-intergenic fusion transcripts at the SV breakpoints.

A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila.

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.

Globally reduced N6-methyladenosine (m6A) in C9ORF72-ALS/FTD dysregulates RNA metabolism and contributes to neurodegeneration.

Repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we show that N6-methyladenosine (m6A), the most prevalent internal mRNA modification, is downregulated in C9ORF72-ALS/FTD patient-derived induced pluripotent stem cell (iPSC)-differentiated neurons and postmortem brain tissues. The global m6A hypomethylation leads to transcriptome-wide mRNA stabilization and upregulated gene expression, particularly for genes involved in synaptic activity and neuronal function. Moreover, the m6A modification in the C9ORF72 intron sequence upstream of the expanded repeats enhances RNA decay via the nuclear reader YTHDC1, and the antisense RNA repeats can also be regulated through m6A modification. The m6A reduction increases the accumulation of repeat RNAs and the encoded poly-dipeptides, contributing to disease pathogenesis. We further demonstrate that, by elevating m6A methylation, we could significantly reduce repeat RNA levels from both strands and the derived poly-dipeptides, rescue global mRNA homeostasis and improve survival of C9ORF72-ALS/FTD patient iPSC-derived neurons.

Limb girdle muscular dystrophy 23 caused by compound heterozygous mutations of LAMA2 gene.

Mutations of LAMA2 gene are associated with congenital muscular dystrophy (CMD). The LAMA2-related CMD mainly consists of two diseases, merosin deficient congenital muscular dystrophies type 1A (MDC1A) and limb girdle muscular dystrophy 23 (LGMD23). LGMD23 is characterized by slowly progressive proximal muscle weakness, which primarily affects the lower limbs and results in gait difficulties. Additional clinical features include increased serum creatine kinase, abnormal electromyography with or without white matter abnormalities on brain imaging. Clinical data were collected from a Chinese Han family. Whole-exome sequencing, Sanger sequencing, RT-PCR and TA clone sequencing were performed on the family members. Compound heterozygous mutations of LAMA2: c.1693C > T (p. Q565*) (maternally inherited) and c.9212-6T > G (paternally inherited) were identified and confirmed in the proband. The mutation c.1693C > T (p. Q565*) was classified as pathogenic according to American College of Medical Genetics and Genomics (ACMG) guidelines. By performing RT-PCR and TA clone sequencing, an insertion of 40-bp intronic sequence (intron 64) was found in the transcripts of the proband and her father, which resulted in a frameshift and premature truncation codon of the LAMA2. In particular, the variant truncated the LamG domain of the LAMA2. Therefore, the c.9212-6T>G was classified as likely pathogenic according to American College of Medical Genetics and Genomics (ACMG) guidelines. Our findings described two novel mutations in a girl with LGMDR23, which contributes to the genetic counseling of the family and expands the clinical and molecular spectrums of the rare disease.

Circular stable intronic RNAs possess distinct biological features and are deregulated in bladder cancer.

Until recently, intronic lariats were regarded as short-lasting splicing byproducts with no apparent function; however, increasing evidence of stable derivatives suggests regulatory roles. Yet little is known about their characteristics, functions, distribution, and expression in healthy and tumor tissue. Here, we profiled and characterized circular stable intronic sequence RNAs (sisRNAs) using total RNA-Seq data from bladder cancer (BC; n= 457, UROMOL cohort), healthy tissue (n= 46), and fractionated cell lines (n = 5). We found that the recently-discovered full-length intronic circles and the stable lariats formed distinct subclasses, with a surprisingly high intronic circle fraction in BC (∼45%) compared to healthy tissues (0-20%). The stable lariats and their host introns were characterized by small transcript sizes, highly conserved BP regions, enriched BP motifs, and localization in multiple cell fractions. Additionally, circular sisRNAs showed tissue-specific expression patterns. We found nine circular sisRNAs as differentially expressed across early-stage BC patients with different prognoses, and sisHNRNPK expression correlated with progression-free survival. In conclusion, we identify distinguishing biological features of circular sisRNAs and point to specific candidates (incl. sisHNRNPK, sisWDR13and sisMBNL1) that were highly expressed, had evolutionary conserved sequences, or had clinical correlations, which may facilitate future studies and further insights into their functional roles.

Novel RNA epigenetics-driven technologies for rapid prediction of cancer drug resistance.

e19001 Background: The human genome comprises ~99% non-coding DNA sequences (ncDNAs), most of which are actively transcribed into various forms of non-coding RNAs (ncRNAs) that undergo extensive chemical modifications to regulate various functions of the subcellular organelles in human cells ( Statello, et al. Nat Rev Mol Cell Biol 2020). However, the clinical potential of RNA modifications and RNA modifying proteins, i.e., RNA epigenetics, is largely unexplored. In a recent study ( Cheng et al. Nat Commun 2018), we first demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, directly interact with elongating RNA polymerase II (eRNAPII) and the bromodomain-containing protein 4 (BRD4) to form distinct active chromatin structures (ACS) that regulate drug resistance in leukemia cells. This proof-of-concept study enabled us to develop the first-generation of RNA epigenetics-driven drug-predictive system. The primary goal of this study is to validate and optimize this novel drug-predictive system with clinical myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methods: We developed new technologies, including 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). Immunohistochemistry (IHC), western blot (WB), co-immunoprecipitation (co-IP), and dot blot were also used in this study. Results: Our data demonstrated significantly increased expression of the NSUN1, NSUN2 and other key components of the drug-resistant ACS in MDS/AML, which is correlated with disease progression and drug resistance in MDS and AML. We have developed novel technologies, i.e., EC- and PL-RCA-coupled CM and FCM (EC-/PL-RCA/CM &amp; FCM) to visualize and quantify RCMTs-mediated ACS and nascent RNA synthesis in MDS/AML. We discovered nascent RNA synthesis as a highly sensitive and super-fast drug-predictive marker that is independent of cell death and apoptosis. Our data demonstrated that PL-RCA/FCM and EC-/CM have a very high sensitivity to detect the NSUN1/2-mediated ACS in leukemia cell lines and clinical MDS/AML specimens. Our preliminary data demonstrated that increased levels of NSUN1/2-mediated ACS detected by PL-RCA/FCM in pre-treatment MDS/AML can rapidly predict the responses to venetoclax and/or azacitidine. We are actively pursuing RNA-seq targeting NSUN1/2-associated nascent RNAs and ACS, which provides a novel strategy to dissect the functional genomics and RNA epigenetics underlying cancer drug resistance. Conclusions: We have developed a novel RCMTs/NSUN1/2-targeting system for rapid prediction and in-depth analysis of drug resistance in MDS/AML. A large sized clinical study is needed to further validate its usefulness in clinical settings.

Inhibition of microRNA-33b in humanized mice ameliorates nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma in their advanced stages; however, there are currently no approved therapies. Here, we show that microRNA (miR)-33b in hepatocytes is critical for the development of NASH. miR-33b is located in the intron of sterol regulatory element-binding transcription factor 1 and is abundantly expressed in humans, but absent in rodents. miR-33b knock-in (KI) mice, which have a miR-33b sequence in the same intron of sterol regulatory element-binding transcription factor 1 as humans and express miR-33b similar to humans, exhibit NASH under high-fat diet feeding. This condition is ameliorated by hepatocyte-specific miR-33b deficiency but unaffected by macrophage-specific miR-33b deficiency. Anti-miR-33b oligonucleotide improves the phenotype of NASH in miR-33b KI mice fed a Gubra Amylin NASH diet, which induces miR-33b and worsens NASH more than a high-fat diet. Anti-miR-33b treatment reduces hepatic free cholesterol and triglyceride accumulation through up-regulation of the lipid metabolism-related target genes. Furthermore, it decreases the expression of fibrosis marker genes in cultured hepatic stellate cells. Thus, inhibition of miR-33b using nucleic acid medicine is a promising treatment for NASH.

Functional mapping of microRNA promoters with dCas9 fused to transcriptional regulators.

MicroRNAs are small non-coding RNAs that control gene expression during development, physiology, and disease. Transcription is a key factor in microRNA abundance and tissue-specific expression. Many databases predict the location of microRNA transcription start sites and promoters. However, these candidate regions require functional validation. Here, dCas9 fused to transcriptional activators or repressors - CRISPR activation (CRISPRa) and inhibition (CRISPRi)- were targeted to the candidate promoters of two intronic microRNAs, mmu-miR-335 and hsa-miR-3662, including the promoters of their respective host genes Mest and HBS1L. We report that in mouse embryonic stem cells and brain organoids, miR-335 was downregulated upon CRISPRi of its host gene Mest. Reciprocally, CRISPRa of Mest promoter upregulated miR-335. By contrast, CRISPRa of the predicted miR-335-specific promoter (located in an intron of Mest) did not affect miR-335 levels. Thus, the expression of miR-335 only depends on the promoter activity of its host gene Mest. By contrast, miR-3662 was CRISPR activatable both by the promoter of its host gene HBS1L and an intronic sequence in HEK-293T cells. Thus, CRISPRa and CRISPRi are powerful tools to evaluate the relevance of endogenous regulatory sequences involved in microRNA transcription in defined cell types.

Single-nucleus RNA sequencing of human pancreatic islets identifies novel gene sets and distinguishes β-cell subpopulations with dynamic transcriptome profiles

BackgroundSingle-cell RNA sequencing (scRNA-seq) provides valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo. On the other hand, single-nucleus RNA sequencing (snRNA-seq) has compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and affords enhanced information from intronic sequences that can be leveraged to identify pre-mRNA transcripts.MethodsWe obtained nuclear preparations from fresh human islet cells and generated snRNA-seq datasets. We compared these datasets to scRNA-seq output obtained from human islet cells from the same donor. We employed snRNA-seq to obtain the transcriptomic profile of human islets engrafted in immunodeficient mice. In both analyses, we included the intronic reads in the snRNA-seq data with the GRCh38-2020-A library.ResultsFirst, snRNA-seq analysis shows that the top four differentially and selectively expressed genes in human islet endocrine cells in vitro and in vivo are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT (β-cells); PTPRT, FAP, PDK4, LOXL4 (α-cells); LRFN5, ADARB2, ERBB4, KCNT2 (δ-cells); and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 (γ-cells). Second, by integrating information from scRNA-seq and snRNA-seq of human islet cells, we distinguish three β-cell sub-clusters: an INS pre-mRNA cluster (β3), an intermediate INS mRNA cluster (β2), and an INS mRNA-rich cluster (β1). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA-rich cluster (β1) becomes the predominant sub-cluster in vivo.ConclusionsIn summary, snRNA-seq and pre-mRNA analysis of human islet cells can accurately identify human islet cell populations, subpopulations, and their dynamic transcriptome profile in vivo.

Genetic Variations of Fourth Exon Region of Prolactin Gene and Its Associations with Growth Traits in European Sea Bass Dicentrarchus labrax (Moronidae)

European sea bass Dicentrarchus labrax (Moronidae), which has a high commercial value, is mainly produced in the Mediterranean region. Prolactin (PRL) is a member of the helix bundle protein family that is secreted in the anterior pituitary gland and has a significant role in osmoregulation and growth in teleost fish. In this study, we investigated the genetic variation of the partial sequence of 3rd, 4th introns, and 4th exon regions of the PRL gene in Dicentrarchus labrax and its association with growth traits that were reared in cage conditions. Two SNPs (g.484 A C, g.529 T C) and one 15 bp indel (insertion/deletion) have been found in the 3rd intron region of the PRL gene of Dicentrarchus labrax. A significant association has been identified between the total body weight and the genotypes for PRL g.529 T C locus (р 0.05). The heterozygote genotype of the PRL g.529 T C locus has a significantly higher total body weight than the other genotypes in the population (p 0.05). Association analysis indicated that this SNP in the PRL gene is correlated with growth traits and could be used for genomic selection programs in Dicentrarchus labrax breeding.

Influence of the 135 bp Intron on Stilbene Synthase VaSTS11 Transgene Expression in Cell Cultures of Grapevine and Different Plant Generations of Arabidopsis thaliana

Modern plant biotechnology often faces the problem of obtaining a stable and powerful vector for gene overexpression. It is known that introns carry different regulatory elements whose effects on transgene expression have been poorly studied. To study the effect of an intron on transgene expression, the stilbene synthase 11 (VaSTS11) gene of grapevine Vitis amurensis Rupr. was selected and overexpressed in grapevine callus cell cultures and several plant generations of Arabidopsis thaliana as two forms, intronless VaSTS11c and intron-containing VaSTS11d. The STS genes play an important role in the biosynthesis of stilbenes, valuable plant secondary metabolites. VaSTS11d contained two exons and one intron, while VaSTS11c contained only two exons, which corresponded to the mature transcript. It has been shown that the intron-containing VaSTS11d was better expressed in several generations of transgenic A. thaliana than VaSTS11c and also exhibited a lower level of cytosine methylation. As a result, the content of stilbenes in the VaSTS11d-transgenic plants was much higher than in the VaSTS11c-transgenic plants. Similarly, the best efficiency in increasing the content of stilbenes was also observed in grapevine cell cultures overexpressing the intron-containing VaSTS11d transcript. Thus, the results indicate that an intron sequence with regulatory elements can have a strong positive effect on both transgene expression level and its biological functions in plants and plant cell cultures.