U6 Gene Research Articles

The miR-338-3p expression level in pemphigus diagnosis

BACKGROUND: Pemphigus is a group of potentially fatal chronic cutaneous diseases in which blisters appear on the skin and mucous membranes as a result of IgG autoantibodies binding to desmosomes in the epidermis, leading to keratinocytes acantholysis. Currently, methods to monitor disease activity and therapy efficiency using various biomarkers are being investigated. MicroRNA expression, in particular miR-338-3p, has been one of these biomarkers, as changes in miR-338-3p expression may trigger the Th1/Th2 cell imbalance and possibly be involved in the pathogenesis of the disease. AIM: This study aimed to design a protocol to evaluate the level of miR-338-3p expression in peripheral blood mononuclear cells and verify the diagnostic value of miR-338-3p expression in pemphigus. MATERIALS AND METHODS: Experimental prospective comparative study was conducted from February 2023 to February 2024 at the Dermatology Department of Sechenov University. The study included 10 patients with pemphigus in the active stage of the disease, 3 patients in remission, and 9 participants of the control group. The expression of miRNA-338-3p was analyzed by real-time polymerase chain reaction, cDNA was obtained using StemLoop method. The evaluation of miRNA-338-3p expression level was based on its comparison with the expression of U6 gene using 2-ΔΔСt method. RESULTS: The expression level of miR-338-3p was analyzed in 10 patients in the active stage of the disease (5 men, 50%; 5 women, 50%; mean age 46±10.7 years), 3 patients in remission (2 women, 66.7%; 1 man, 33.3%; mean age 57±8 years), 9 control group (8 women, 88.9%; 1 man, 11.1%; mean age 36±16.8 years). The mean expression level of miR-338-3p was 8.64 (SD±5.72) in patients with active disease, 3.38 (SD±1.44) in patients in remission, and 1.48 (SD±1.12) in controls. A statistically significant increase in the expression level of miR-338-3p was found in patients in the active disease stage compared to the control group (p=0.002). A statistically significant correlation was found between the level of miR-338-3p expression and the PDAI index score (p 0.001). CONCLUSION: Based on the data obtained in this study, it can be assumed that microRNAs are important in pemphigus, and miR-338-3p expression in particular may serve as a key element in pemphigus pathogenesis. More detailed study of microRNAs and analysis of expression variability according to clinical data may provide the basis for developing new diagnostic methods and severity scoring, allowing more accurate and less invasive diagnostic methods, as well as monitoring and predicting disease progression.

Avian leukosis virus usurps the cellular SERBP1 protein to enhance its transcription and promote productive infections in avian cells

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which cluster separately from the well-characterized subgroups A, B, C, D, E, and J in sequence analysis, and exhibits a specific host range and a unique pattern of superinfection interference. Avian leukosis virus subgroup K replicate more slowly in avian cells than other ALV strains, leading to escaped detection during ALV eradication, but the underlying mechanism are largely unknown. In our previous study, we have reported that JS11C1 and most of other suspected ALV-K strains possessed unique mutations in the U3 region. Here, we selected 5 mutations in some important transcriptional regulation elements to explore the possible factor contributing for the lower activity of LTR, including CA-TG mutation in the CAAT box, 21 nt deletion in the CAAT box, A-G and A-T mutations in the CArG boxes, 11 nt insertion in the PRE boxes, and C-T mutation in the TATA box. On the basis of infectious clone of JS11C1, we demonstrated that the 11 nt fragment in the PRE boxes was associated with the transcription activity of LTR, the enhancer ability of U3, and the replication capacity of the virus. Notably, we determined the differential U3-protein interaction profile of ALVs and found that the 11 nt fragment specifically binds to cellular SERPINE1 mRNA binding protein 1 (SERBP1) to increase the LTR activity and enhance virus replication. Collectively, these findings reveal that a 11 nt fragment in the U3 gene contributed to its binding ability to the cellular SERBP1 to enhance its transcription and the infectious virus productions in avian cells. This study highlighted the vital role of host factor in retrovirus replication and thus provides a new perspective to elucidate the interaction between retrovirus and its host and a molecular basis to develop efficient strategies against retroviruses.

POLR3A-mutated Wiedemann-Rautenstrauch fibroblasts display differential profile of intron retention and expression of TP53 isoforms

BACKGROUND: Wiedemann-Rautenstrauch Syndrome (WRS) is a neonatal progeroid syndrome for which biallelic pathogenic variants in RNA polymerase III subunit A (POLR3A) have recently been described. POLR3 is a 17 subunits protein complex responsible for the transcription of short RNAs including all the transfer RNAs (tRNAs), the 5 S subunit of ribosomal RNA, the short nuclear RNA U6, among other regulatory RNAs. OBJECTIVE: We aim to evaluate the impact of POLR3A pathogenic variants on the relative expression of the short nuclear RNA U6 and on the differential profile of intron retention RNA U6, p53 isoforms and in fibroblasts derived from patients with WRS and control fibroblasts. METHODS: RNA was extracted by the TRIzol method; intron retention analysis was performed by using IRFinder from an mRNA sequencing (RNA-Seq) platform; P53 isoforms, short nuclear RNA U6 and additional genes related to cell senescence were measured by RT-PCR. RESULTS: No significant differences were found in the percentage of intron retention (control: 7.8%, WRS1 : 6.3%and WRS2 : 8.14%). Genes showing higher intron retention profile in both groups were mainly related to RNA binding pathways, cell cycle regulation, positive regulation of transcription, positive regulation of inflammatory pathways, negative regulation of apoptosis, RNA transcription, mitochondria, and regulation of translation initiation. However, in WRS fibroblasts the genes with more intron retention were those related to the immune response and mitochondrial function; while in control those related to the response to oxidative stress had the most introns retained. WRS1 showed higher expression of short nuclear RNA U6 compared to control and WRS2; while both WRS cells showed higher expression of p53β and lower percentage of Δ133p63α, consistent with a higher expression of the cellular senescence markers p16 and p21. CONCLUSIONS: These results demonstrated the important role of POLR3A in the maintenance of cellular homeostasis and highlight its potential role in cell senescence in WRS.

Assessing miR-146a-5P with IL-6 and TLR-4 serum levels in diabetic mellitus patients with or without urinary tract infection

Introduction and Aim: Diabetes is a chronic metabolic disease that affects glucose levels. This condition increases blood sugar and weakens immunity, damaging neutrophil function and urine antibacterial activity. IL-6 and TLR-4 are involved in inflammation and immune system control, while miR-146-5p may be a biomarker for diabetes. This study examines IL-6, TLR-4, and miR-146-5p in diabetics with and without UT infection. Materials and Methods: In this study, blood samples were collected from 100 diabetic mellitus patients suffering from UTI in addition to 100 individuals apparently healthy as control. The serum levels of IL-6 and TLR-4 were detected using commercial BTlab brand ELISA kits. The plasma RNA expression level of the miR-146a-5p was determined by real time-PCR using a specific primer for miR-146a-5p with the U6 gene utilized as a reference gene. Results: The results showed that diabetic patient’s group had a significantly higher response of IL-6 than controls (75.6 vs. 27.6). TLR-4 levels showed high levels in diabetic patients’ group (1.3) in comparison with the control group (0.441). Both IL-6 and TLR-4 levels showed a significant difference between the diabetic patients with UTI compared to control. The results revealed no-significant differences in the levels among both diabetic patients with UTI and patients without UTI. TLR-4 and IL-6 showed a significant inverse relationship while the relation between TLR-4 level and miR-146a-5p expression level showed a negative relationship. Conclusion: The levels of IL-6, TLR-4, and miR-146-5p expression were found to be considerably greater in individuals with diabetes having UTIs compared to patients with diabetes alone. An inverse relationship existed between IL-6 and TLR-4 levels as well as between TLR-4 and miR-146a-5p. The expression levels of IL-6 and miR-146-5p were nearly equal.

Chromosomal organization of multigene families and meiotic analysis in species of Loricariidae (Siluriformes) from Brazilian Amazon, with description of a new cytotype for genus Spatuloricaria.

In Amazon, some species of Loricariidae are at risk of extinction due to habitat loss and overexploitation by the ornamental fish market. Cytogenetic data related to the karyotype and meiotic cycle can contribute to understanding the reproductive biology and help management and conservation programs of these fish. Additionally, chromosomal mapping of repetitive DNA in Loricariidae may aid comparative genomic studies in this family. However, cytogenetics analysis is limited in Amazonian locariids. In this study, chromosomal mapping of multigenic families was performed in Scobinancistrus aureatus, Scobinancistrus pariolispos and Spatuloricaria sp. Meiotic analyzes were performed in Hypancistrus zebra and Hypancistrus sp."pão". Results showed new karyotype for Spatuloricaria sp. (2n=66, NF=82, 50m-10sm-6m). Distinct patterns of chromosomal organization of histone H1, histone H3 and snDNA U2 genes were registered in the karyotypes of the studied species, proving to be an excellent cytotaxonomic tool. Hypotheses to explain the evolutionary dynamics of these sequences in studied Loricariidae were proposed. Regarding H. zebra and H. sp. "pão", we describe the events related to synapse and transcriptional activity during the meiotic cycle, which in both species showed 26 fully synapsed bivalents, with high gene expression only during zygotene and pachytene. Both Hypancistrus species could be used may be models for evaluating changes in spermatogenesis of Loricariidae.

Identification and functional analysis of Cochliomyia hominivorax U6 gene promoters.

The New World screwworm, Cochliomyia hominivorax, is an obligate parasite, which is a major pest of livestock. While the sterile insect technique was used very successfully to eradicate C. hominivorax from North and Central America, more cost-effective genetic methods will likely be needed in South America. The recent development of CRISPR/Cas9-based genetic approaches, such as homing gene drive, could provide a very efficient means for the suppression of C. hominivorax populations. One component of a drive system is the guide RNA(s) driven by a U6 gene promoter. Here, we have developed an in vivo assay to evaluate the activity of the promoters from seven C. hominivorax U6 genes. Embryos from the related blowfly Lucilia cuprina were injected with plasmid DNA containing a U6-promoter-guide RNA construct and a source of Cas9, either protein or plasmid DNA. Activity was assessed by the number of site-specific mutations in the targeted gene in hatched larvae. One promoter, Chom U6_b, showed the highest activity. These U6 gene promoters could be used to build CRISPR/Cas9-based genetic systems for the control of C. hominivorax.

Expression of miR-let7b and miR-19b in progressive familial intrahepatic cholestasis (PFIC) children

BackgroundMicroRNAs (miRNAs) are a group of small non-coding RNAs that bind to the target mRNA and regulate gene expression. Recently circulating microRNAs were investigated as markers of diseases and therapeutic targets. Although various studies analyze the miRNA expression in liver disease, these studies on PFIC are few. Progressive familial intrahepatic cholestasis (PFIC) is a rare liver disease with autosomal recessive inheritance. Most children with PFIC progress to cirrhosis and liver failure and consequently need to have a liver transplant. The aim of this study is the investigation of the miR-19b and miR-let7b expression levels in Iranian PFIC children. Methods25 PFIC patients, 25 healthy children and 25 Biliary Atresia patients were considered as case and two control groups respectively. Blood samples were obtained and Liver function tests (LFTs) were measured. After RNA extraction and cDNA synthesis, quantitative PCR was performed using specific primers for miR-19b and miR-let7b. The U6 gene is used as an internal control. ResultsqPCR on PFIC patients’ samples demonstrated that the miR-19b and the miR-let7b expression were significantly decreased in patients compared to the control groups, with a p-value<0.0001 and p-value=0.0006 receptively. ConclusionIn conclusion, circulating micro-RNA like miR-19b and miR-let7b have a potential opportunity to be a non-invasive diagnostic marker or therapeutic target for PFIC in the future.

Functional verification of U6 promoters from Agaricus bisporus and the feasibility of target gene silencing by shRNA

CRISPR/Cas9 system has been proven to be an efficient tool to generate gene mutation. The expression of gRNA guiding Cas9 nuclease to target gene of interest was commonly driven by RNA polymerase III promoter in several organisms, most of which generally took advantage of U6 promoter. For identification of U6 promoter which could guide the expression of gRNA in Agaricus bisporus, we aligned U6 genes retrieved from NCBI against A. bisporus genome. A total of two U6 genes with the typical eukaryotic U6 functional sites and high sequence conservation were obtained. And then shRNA-mediated adenylate cyclase (AC) gene silencing cassette with ∼500 bp upstream of U6 genes was transformed into A. bisporus to explore the function of U6 promoters. Changes in expression of target gene AC and phenotype demonstrated that the two U6 promoters could effectively drive the expression of shRNA to silence the target gene implying that U6 promoters might be capable of driving the expression of gRNA of CRISPR/Cas9 system in A. bisporus. The identification of U6 genes and functional evaluation of U6 promoters would facilitate the application of shRNA and CRISPR/Cas9 system for investigation of function of target gene in A. bisporus in which targeted gene knockouts were likely to be difficult to be implemented because of multinucleate and heterokaryon.

MicroRNA signature of leukocytes in the context of chronic systemic inflammation in vascular dementia

Chronic low-level inflammation during the aging process is a key risk factor for the activation of resident cells of the brain innate immune system of the (microglia and astrocytes). Such activation leads to the development of neuroinflammation and cognitive impairment which are typical to neurodegenerative diseases such as Alzheimers disease, vascular dementia, Parkinson disease etc. Currently, there is a lack of minimally invasive, affordable methods for diagnosing age-related neurodegenerative diseases and drugs that could slow down or prevent their progression. Hence, a search for new peripheral biomarkers is required, both for diagnostics and monitoring the efficiency of drug therapy. The option of using microRNAs as such biomarkers is under discussion. Our goal was to identify a leukocyte microRNA signature in vascular dementia as compared with healthy aging and reproductive age, in view of inflammation and cognitive deficits. We have examined 54 persons from young to senile age who were classified into the following groups: Vascular dementia, Healthy aging and Reproductive age. Expression of miRNAs known as regulators of communications between the immune and nervous systems (let-7d, let-7g, miR-21, miR-124, miR-146a, miR-155, miR-342-3p) was measured in peripheral blood leukocytes. The decision to study leukocytes was made, since these blood cells are responsible for immune functions, and, especially, cytokine production during aging. Total RNA was isolated by phenol-chloroform technique. The microRNA expression was determined by quantitative polymerase chain reaction with SYBRGreen. The U6 gene of small nuclear DNA was used as a reference housekeeping gene. The differences between groups were determined using the KruskalWallis test with post hoc pairwise comparisons according to ConoverInman. As a result of the study, it was found that the expression of microRNA-21 and microRNA-342 in leukocytes of elderly/senile people, both in healthy aging and in vascular dementia, was increased when compared to the persons in their reproductive age. In the persons with vascular dementia, the expression level of miRNA-124 and miRNA-342 in peripheral blood leukocytes was higher than in healthy aging group. Hence,, microRNA-124 and microRNA-342 may be informative biomarkers for the diagnostics of vascular dementia. However, large-scale studies of their biomarker potential are warranted.

Activation of the SigE-SigB signaling pathway by inhibition of the respiratory electron transport chain and its effect on rifampicin resistance in Mycobacterium smegmatis.

Using a mutant of Mycobacterium smegmatis lacking the major aa3 cytochrome c oxidase of the electron transport chain (Δaa3), we demonstrated that inhibition of the respiratory electron transport chain led to an increase in antibiotic resistance of M. smegmatis to isoniazid, rifampicin, ethambutol, and tetracycline. The alternative sigma factors SigB and SigE were shown to be involved in an increase in rifampicin resistance of M. smegmatis induced under respiration-inhibitory conditions. As in Mycobacterium tuberculosis, SigE and SigB form a hierarchical regulatory pathway in M. smegmatis through SigE-dependent transcription of sigB. Expression of sigB and sigE was demonstrated to increase in the Δaa3 mutant, leading to upregulation of the SigB-dependent genes in the mutant. The pho U2 (MSMEG_1605) gene implicated in a phosphate-signaling pathway and the MSMEG_1097 gene encoding a putative glycosyltransferase were identified to be involved in the SigB-dependent enhancement of rifampicin resistance observed for the Δaa3 mutant of M. smegmatis. The significance of this study is that the direct link between the functionality of the respiratory electron transport chain and antibiotic resistance in mycobacteria was demonstrated for the first time using an electron transport chain mutant rather than inhibitors of electron transport chain.

CAPTURE of the Human U2 snRNA Genes Expands the Repertoire of Associated Factors.

In order to identify factors involved in transcription of human snRNA genes and 3′ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with these human snRNA genes including RNA polymerase II (pol II), Cyclin-Dependent Kinase 7 (CDK7), Negative Elongation Factor (NELF), Suppressor of Ty 5 (SPT5), Mediator 23 (MED23) and several subunits of the Integrator Complex. Suppressor of Ty 6 (SPT6); Cyclin K, the partner of Cyclin-Dependent Kinase 12 (CDK12) and Cyclin-Dependent Kinase 13 (CDK13); and SWI/SNF chromatin remodelling complex-associated SWI/SNF-related, Matrix-associated, Regulator of Chromatin (SMRC) factors were also enriched. Several polyadenylation factors, including Cleavage and Polyadenylation Specificity Factor 1 (CPSF1), Cleavage Stimulation Factors 1 and 2 (CSTF1,and CSTF2) were enriched by U2 gene CAPTURE. We have already shown by chromatin immunoprecipitation (ChIP) that CSTF2—and Pcf11 and Ssu72, which are also polyadenylation factors—are associated with the human U1 and U2 genes. ChIP-seq and ChIP-qPCR confirm the association of SPT6, Cyclin K, and CDK12 with the U2 genes. In addition, knockdown of SPT6 causes loss of subunit 3 of the Integrator Complex (INTS3) from the U2 genes, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes and helped to identify a functional role for SPT6.

Diversity and Evolution of DNA Transposons Targeting Multicopy Small RNA Genes from Actinopterygian Fish.

Simple SummaryDNA transposons are parasitic DNA segments that can move or duplicate themselves from one site to another in the genome. Dada is a unique group of DNA transposons, which specifically insert themselves into multicopy RNA genes such as transfer RNA (tRNA) genes or small nuclear RNA (snRNA) genes to avoid the disruption of single-copy functional genes. However, only a few Dada families have been characterized along with their target sequences. Here, vertebrate genomes were surveyed to characterize new Dada transposons, and over 120 Dada families were characterized from diverse fishes. They were classified into 12 groups with confirmed target specificities. Various tRNA genes, as well as 5S ribosomal RNA (rRNA) genes were inserted by Dada transposons. Phylogenetic analysis revealed that Dada transposons inserted in the same RNA genes are closely related. Phylogenetically related Dada transposons inserted in different RNA genes show the sequence similarity around their insertion sites, indicating Dada proteins recognize DNA nucleotide sequences to find their targets. Understanding how Dada discovers the targets would help develop target-specific insertions of foreign DNA segments.Dada is a unique superfamily of DNA transposons, inserted specifically in multicopy RNA genes. The zebrafish genome harbors five families of Dada transposons, whose targets are U6 and U1 snRNA genes, and tRNA-Ala and tRNA-Leu genes. Dada-U6, which is inserted specifically in U6 snRNA genes, is found in four animal phyla, but other target-specific lineages have been reported only from one or two species. Here, vertebrate genomes and transcriptomes were surveyed to characterize Dada families with new target specificities, and over 120 Dada families were characterized from the genomes of actinopterygian fish. They were classified into 12 groups with confirmed target specificities. Newly characterized Dada families target tRNA genes for Asp, Asn, Arg, Gly, Lys, Ser, Tyr, and Val, and 5S rRNA genes. Targeted positions inside of tRNA genes are concentrated in two regions: around the anticodon and the A box of RNA polymerase III promoter. Phylogenetic analysis revealed the relationships among actinopterygian Dada families, and one domestication event in the common ancestor of carps and minnows belonging to Cyprinoidei, Cypriniformes. Sequences targeted by phylogenetically related Dada families show sequence similarities, indicating that the target specificity of Dada is accomplished through the recognition of primary nucleotide sequences.

Human spliceosomal snRNA sequence variants generate variant spliceosomes.

Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4, and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than 1000-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1% to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing noncanonical snRNAs may contribute to different tissue- and cell-type–specific alternative splicing patterns.

Large-scale comparative analysis of cytogenetic markers across Lepidoptera

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

Cytogenetic markers using single-sequence probes reveal chromosomal locations of tandemly repetitive genes in scleractinian coral Acropora pruinosa

The short and similar sized chromosomes of Acropora pose a challenge for karyotyping. Conventional methods, such as staining of heterochromatic regions, provide unclear banding patterns that hamper identification of such chromosomes. In this study, we used short single-sequence probes from tandemly repetitive 5S ribosomal RNA (rRNA) and core histone coding sequences to identify specific chromosomes of Acropora pruinosa. Both the probes produced intense signals in fluorescence in situ hybridization, which distinguished chromosome pairs. The locus of the 5S rDNA probe was on chromosome 5, whereas that of core histone probe was on chromosome 8. The sequence of the 5S rDNA probe was composed largely of U1 and U2 spliceosomal small nuclear RNA (snRNA) genes and their interspacers, flanked by short sequences of the 5S rDNA. This is the first report of a tandemly repetitive linkage of snRNA and 5S rDNA sequences in Cnidaria. Based on the constructed tentative karyogram and whole genome hybridization, the longest chromosome pair (chromosome 1) was heteromorphic. The probes also hybridized effectively with chromosomes of other Acropora species and population, revealing an additional core histone gene locus. We demonstrated the applicability of short-sequence probes as chromosomal markers with potential for use across populations and species of Acropora.

Chromosome Mapping of U2 snDNA in Species of Leptodactylus (Anura, Leptodactylidae)

Small nuclear RNA (snRNA) is a class of molecules involved in the processing of pre-mRNA and in regulatory cell processes. snRNAs are always associated with a set of specific proteins. The complexes are referred to as small nuclear ribonucleoproteins, and spliceosome U RNAs are their most common snRNA components. The repetitive sequences of U snDNAs have been cytogenetically mapped in several species of Arthropoda, fishes, and mammals; however, their distribution remains unknown in amphibians. Here, we show results of FISH mapping of U2 snDNA repetitive sequences in species of the amphibian genus Leptodactylus to reveal the distribution patterns of this sequence in their karyotypes. The probe hybridized in the metacentric chromosome pair 6 in Leptodactylus fuscus, L. gracilis, L. latrans, L. chaquensis, L. petersii, L. podicipinus, and L. brevipes. A different pattern was observed in L. labyrinthicus with hybridization signals in 4 chromosome pairs. The same localization of U2 gene sequences in most of the species analyzed suggests a relatively conserved pattern and a similarity of the chromosome 6 among these species of Leptodactylus.

SEQUENCING OF THE U6 PROMOTERS IN CASTOR BEANS AND VECTOR CONSTRUCTION FOR CRISPR/Cas9 GENOMIC EDITING ON THEIR BASIS

Castor bean is an important crop in many countries. It is mainly used to obtain the castor oil, which is widely applied in various industries. The rich protein cakes and meals are remains after the oil is pressed. They are promising for use as protein additives in the fodder production. However, it is limited due to the presence of toxins. These are ricin protein and ricinine alkaloid. One of such methods can be genomic editing using the CRISPR/Cas9 system. The technology consists in cutting the target region of the intact DNA by the Cas9 enzyme with the assistance of a short guide RNA fragment. The delivery of the Cas9 and guide RNA genes into the cell of the edited plant is often carried out by plasmid vectors. For efficient synthesis of the guide RNA in such vectors, the promoters of small nuclear RNA genes are usually used. In dicotyledonous plants editing, the promoter of the Arabidopsis U6 gene is most often used. In this work, the amplification, sequencing and analysis of the castor bean U6 promoters were carried out at the first time. The obtained sequences were analyzed and used in the construction of CRISPR/Cas9 vectors for the ricin gene editing. Our aim was to study castor bean U6 promoters with help by bioinformatics and molecular genetic approaches. Zanzibar Green and Gibzonskaya varieties of castor bean plants were used in this work. DNA was isolated from young leaves. The preparation of sequences, alignments and homology level calculation were carried out by GenDoc program (http://www.nrbsc.org/gfx/genedoc/index.html). Primers for amplification of castor bean U6 promoters were designed by Primer3 program (http://bioinfo.ut.ee/primer3-0.4.0/). PCR was performed using a C-100 PCR machine (Bio-Rad Laboratories, Inc., USA). PCR products were separated in 1.5 % agarose gel at 6 B/cm in the Sub-Cell GT electrophoresis camera (Bio-Rad Laboratories, Inc., USA). The amplicons were purified with the GeneJET PCR Purification Kit (Thermo Fisher Scientific, Inc., USA). The purified amplicons were cloned into the pAL2-T vector (Evrogen, Russia) according to protocol of manufacturer. In the CRISPR/Cas9 vector construction, the pRGE31, HindIII and SbfI restriction enzymes (Sibenzyme, Russia) and nucleotides were used. Purification of the digested products was carried out with the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Inc., USA). Bioinformatic search were conducted in the GenBank base, and 12 scaffolds with the castor bean U6 gene were found. Six promoters with intact USE and TATA-box elements of regulation were used in the primer design for amplification with cv. Zanzibar Green and cv. Gibzonskaya DNA matrices. One fragment PCR products were cloned and sequenced. The analysis of the obtained amplicon sequences reveled that promoter regions of two studied varieties were similar. The level of promoter identity from different amplicons ranged within 51-77 %. In comparison of these promoters with ones from other plants, the level of homology was 42-64 %. The promoter sequences with intact USE and TATA-box motifs were used in construction of the CRISPR/Cas9 vectors which can be used for efficient editing of the castor bean genes involved in the ricin and ricinine synthesis.

Impact of miR-155, miR-145 and miR-328 on pigmentary Process in Iraqi Patients with vitiligo

Non-segmental vitiligo is a developed depigmentation causing acquired, depigmented, and progressive lesions of the areas in the skin. The present study was designed to shed light on genetic and hormonal factors that may play a critical role in the pigmentary process of the non-segmented vitiligo (NSV) patients. In the first part, the levels of melanocytes stimulating hormone were measured by ELISA technique, results revealed that the serum level of Alpha-Melanocytes Stimulating hormone was dramatically increased in NSV patients compared to the controls (p < 0.05). The second part focused on determining the roles of miR-155, miR145, and miR-328 that associated with vitiligo by measuring the gene expression of related microRNAs in peripheral blood samples taken from NSV patients and healthy subjects. The total RNA was extracted and converted to cDNA using the Stem-Loop technique, by real-time PCR using SYBR Green and folding analysis. The gene expression was standardized to the level of a housekeeping gene (U6 gene) and quantified by the ∆Ct value and folding (2-∆∆Ct) method. Analysis of the associated miRNAs exposed a higher expression of mir-155 and miR-145 in NSV (P = 0.0001). While mir-328 was significantly down-regulated in vitiligo compared to the controls (P = 0.0001). Based on these results, it was concluded that an elevated level of α-MSH could protect melanocytes by inhibiting the pro-inflammatory activity and oxidative stress. Overexpression miR-155, miR-145, and down-regulation of miR-328 in peripheral blood of vitiligo patients suggest their importance as targeting indicators for vitiligo.

Evaluation of miR-21 Expression Level in Helicobacter pylori-Infected Gastric Mucosa

Background: Gastric cancer is one of the main causes of death worldwide. In this regard, Helicobacter pylori infection is considered as the main risk factor for gastric cancer. MicroRNA (mirNA) can interface with mRNA molecules as well as blocking their translation into proteins or inducing degradation. Objectives: The aim of this study was to compare the expression of mir-21 in biopsy samples of gastritis and healthy adjacent tissues. Methods: Between Feb-Dec 2017, 70 patients with dyspeptic symptoms from Taleghani Hospital were enrolled in this study. Accordingly, the expression level of mir-21 was evaluated using semi-quantitative RT-PCR in mucosal biopsy samples from those well-characterized patients. Moreover, the U6 gene was used as an internal control. Results: Our data indicated that mir-21 expression was significantly up-regulated in the infected samples with H. pylori compared to healthy samples. Conclusions: Our results confirm that H. pylori infection can alter the expression of mir-21 in gastric epithelial cells and gastric mucosal tissues. However, the exact role of the miRNA changes in H. pylori infection will require further experiments.

Molecular characterization of avian leukosis virus subgroup J in Chinese local chickens between 2013 and 2018

Avian leukosis virus subgroup J (ALV-J) was first isolated from broiler chickens in China in 1999; subsequently, it was rapidly introduced into layer chickens and Chinese local chickens. Recently, the incidence of ALV-J in broiler and layer chickens has significantly decreased. However, it has caused substantial damage to Chinese local chickens, resulting in immense challenges to their production performance and breeding safety. To systematically analyze the molecular characteristics and the epidemic trend of ALV-J in Chinese local chickens, 260 clinical samples were collected for the period of 2013–2018; 18 ALV-J local chicken isolates were identified by antigen-capture enzyme-linked immunosorbent assay and subgroup A-, B-, and J-specific multiplex PCR. The whole genomic sequences of 18 isolates were amplified with PCR and submitted to GenBank. Approximately, 55.5% (10/18) of the 18 isolates demonstrated a relatively high homology (92.3–95.4%) with 20 ALV-J early-isolated local strains (genome sequences obtained from GenBank) in gp85 genes clustering in a separated branch. The 3ʹ untranslated region (3ʹ UTR) of the 18 isolates showed a 195–210 and 16–28 base pair deletion in the redundant transmembrane region and in direct repeat 1, respectively; 55.5% (10/18) of the 18 isolates retained the 147 residue E element. The U3 gene of 61.1% (11/18) of the 18 isolates shared high identity (94.6–97.3%) with ALV-J early-isolated local strains. These results implied that the gp85 and U3 of ALV-J local chicken isolates have rapidly evolved and formed a unique local chicken branch. In addition, it was determined that the gene deletion in the 3′UTR region currently serves as a unique molecular characteristic of ALV-J in China. Hence, the obtained results built on the existing ALV-J molecular epidemiological data and further elucidated the genetic evolution trend of ALV-J in Chinese local chickens.