Estrogen receptor (esr) mediates the effects of estrogen on the expression of related genes, thereby regulating the growth and reproduction of mammals. To investigate the effect of retrotransposon insertion polymorphism (RIP) of the porcine esr gene on porcine growth performance, retrotransposon insertion polymorphism of the esr gene were predicted by comparative genomics and bioinformatics, and PCR was used to verify the insertion polymorphisms in different porcine breeds. Finally, the correlation analysis between the genotypes and performance of Large White pigs was conducted. The results showed that four retrotransposon polymorphic sites were identified in the esr1 and esr2 genes, which are esr1-SINE- RIP1 located in intron 2 of the esr1 gene, esr1-LINE-RIP2 and RIP3-esr1- SINE located in intron 5 of the gene, and esr2-LINE-RIP located in intron 1 of the esr2 gene, respectively. Among them, insertion of a 287 bp of SINE into intron 2 of the esr1 gene significantly affected (P<0.05) the live back fat thickness and 100 kg body weight back fat thickness of Large White pigs. Moreover, the live back fat thickness and back fat thickness at 100 kg body weight of homozygous with insertion (SINE+/+) was significantly greater than that of heterozygous with insertion (SINE+/-) and homozygous without insertion (SINE-/-). Therefore, esr1-SINE-RIP1 could be used as a molecular marker to assist the selection of deposition traits in Large White pigs.
Animals ; Genotype ; Introns/genetics* ; Phenotype ; Polymorphism, Genetic/genetics* ; Retroelements/genetics* ; Swine/genetics*

The dynamic activity of transposable elements (TEs) contributes to the vast diversity of genome size and architecture among plants. Here, we examined the genomic distribution and transposition activity of long terminal repeat retrotransposons (LTR-RTs) in Arabidopsis thaliana (Ath) and three of its relatives, Arabidopsis lyrata (Aly), Eutrema salsugineum (Esa), and Schrenkiella parvula (Spa), in Brassicaceae. Our analyses revealed the distinct evolutionary dynamics of Gypsyretrotransposons, which reflects the different patterns of genome size changes of the four species over the past million years. The rate of Gypsy transposition in Aly is approximately five times more rapid than that of Ath and Esa, suggesting an expanding Aly genome. Gypsy insertions in Esa are strictly confined to pericentromeric heterochromatin and associated with dramatic centromere expansion. In contrast, Gypsy insertions in Spa have been largely suppressed over the last million years, likely as a result of a combination of an inherent molecular mechanism of preferential DNA removal and purifying selection at Gypsy elements. Additionally, species-specific clades of Gypsy elements shaped the distinct genome architectures of Aly and Esa.
Brassicaceae/genetics* ; Evolution, Molecular ; Genome Size ; Genome, Plant ; Genomics ; Phylogeny ; Retroelements ; Species Specificity

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.
Coculture Techniques ; DNA ; Endogenous Retroviruses ; Gene Expression ; Genome ; HeLa Cells ; Humans ; Kidney ; MicroRNAs ; Parents ; Proviruses ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; RNA-Directed DNA Polymerase ; Selective Breeding ; Terminal Repeat Sequences ; Transfection

PURPOSE: Helicobacter pylori infection induces phenotype-stabilizing methylation and promotes gastric mucosal atrophy that can inhibit CpG-island methylation. Relationship between the progression of gastric mucosal atrophy and the initiation of CpG-island methylation was analyzed to delineate epigenetic period for neoplastic transformation. MATERIALS AND METHODS: Normal-appearing gastric mucosa was biopsied from 110 H. pylori–positive controls, 95 H. pylori–negative controls, 99 gastric cancer patients, and 118 gastric dysplasia patients. Gastric atrophy was assessed using endoscopic-atrophic-border score. Methylation-variable sites of eight CpG-island genes adjacent to Alu (CDH1, ARRDC4, PPARG, and TRAPPC2L) or LTR (MMP2, CDKN2A, RUNX2, and RUNX3) retroelements and stomach-specific TFF3 gene were analyzed using radioisotope-labeled methylation-specific polymerase chain reaction. RESULTS: Mean ages of H. pylori–positive controls with mild, moderate, and severe atrophy were 51, 54, and 65 years and those of H. pylori–associated TFF3 overmethylation at the three atrophic levels (51, 58, and 63 years) tended to be periodic. Alu-adjacent overmethylation (50 years) was earlier than TFF3 overmethylation (58 years) in H. pylori–positive controls with moderate atrophy. Cancer patients with moderate atrophy showed late Alu-adjacent (58 years) overmethylation and frequent LTR-adjacent overmethylation. LTR-adjacent overmethylation was frequent in cancer (66 years) and dysplasia (68 years) patients with severe atrophy. CONCLUSION: Atrophic progression is associated with gastric cancer at moderate level by impeding the initiation of Alu-adjacent methylation. LTR-adjacent methylation is increased in cancer patients and subsequently in dysplasia patients.
Atrophy* ; DNA Methylation ; Epigenomics ; Gastric Mucosa ; Gastritis, Atrophic ; Genes, Essential* ; Helicobacter pylori ; Housekeeping* ; Humans ; Methylation* ; Polymerase Chain Reaction ; Retroelements ; Stomach Neoplasms*

To develop more active LTR retrotransposons in Phyllostachys edulis, a Ph. edulis LTR retrotransposon (Ph-LTR2) was identified, and the expression pattern of the transposon under stress was systematically analyzed. Ph-LTR2 transposon is 6 030 bp in length and belongs to the Reina subfamily in the Ty3-Gypsy family. With the similarity of 96.41% of both LTR sequences, the Ph-LTR2 transposon inserted the moso bamboo genome about 61.92 thousand years ago. There are 5 copies identified in the genome. The Ph-LTR2 transposon domain includes GAG (gag protein) protein domain, PR (Proteases) protein domain, RT (Reverse transcriptase) protein domain, RH (Ribonuclease H) protein domain, INT (Integrase) protein domain and CHR (Chromatin organization modifier) protein domain. The expression patterns of INT, RT and RH were detected by real-time quantitative PCR. The three domains were found to have specific expression patterns at different tissues of the bamboo. Under the conditions of low/high temperature, methylation inhibitors treatments, irradiation and high salt stress, transcription levels of the three domains of the Ph-LTR2 transposon increased with different degrees. Specifically, after treatment with low/high temperature and methylation inhibitors, the transcription level was up-regulated; after low dose radiation treatment and low concentration of salt solution treatment, the transcription level was also increased, but the expression level decreased with increasing dose of radiation and concentration of salt solution. These results indicate that the expression pattern of the Ph-LTR2 transposon responds to the changes of the external environment, but the exact mechanism is not yet known. The results of this study laid a certain theoretical foundation for the development of the genetic tool based on Ph-LTR2 transposons.
Genome ; Phylogeny ; Poaceae ; Retroelements

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.
Alleles ; Animals ; Endogenous Retroviruses ; Genes, MHC Class I ; Genes, MHC Class II ; Genome ; Heterografts ; Leukocytes ; Mass Screening ; Recombination, Genetic ; Retroviridae ; RNA ; Swine ; Transgenes ; Transplantation, Heterologous ; Viremia

Canine transmissible venereal tumor (CTVT) is a tumor that commonly occurs in genital and extragenital sites of both genders. Long interspersed nuclear elements (LINE-1) retrotransposon has a pivotal role in allogenic transfection among uncontrolled dog populations. This study aimed to perform pathomorphological, immunohistochemical, and in situ polymerase chain reaction (PCR) evaluation of CTVT (n = 18) in transfected dogs during chemotherapy. Immunohistochemically, tumor phases were investigated by using specific markers (CD3, CD4, CD8, CD79, and transforming growth factor beta [TGF-β]), and investigated an amplified specific sequence of TVT LINE-1 retrotransposon by in situ PCR. Polyhedral-shaped neoplastic cells that had large, round, hypo/hyperchromatic nuclei and eosinophilic cytoplasm were detected. All marker results were positive, especially in the early weeks of recovery. CD4 and TGF-β markers were conspicuously positive at the initial stage. In situ PCR LINE-1 sequence was initially positive in only four cases. It is believed that the CD and TGF-β markers provide phase identification at tumor initiation and during chemotherapy. It is thought that presence of T and B lymphocytes, which have roles in cellular and humoral immunity, is needed so that regression of the tumor is possible.
Animals ; B-Lymphocytes ; Cytoplasm ; Dogs ; Drug Therapy ; Eosinophils ; Immunity, Humoral ; Immunohistochemistry ; Polymerase Chain Reaction ; Retroelements ; Transfection ; Transforming Growth Factor beta ; Venereal Tumors, Veterinary

OBJECTIVETo prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells.

METHODSA genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1p, and rabbits were immunized with LINE1-ORF1p to prepare polyclonal antibody. Indirect ELISA was used to evaluate antibody titer, and Western blot and immunohistochemistry were used to evaluate the specific ability of antibody to recognize LINE1-ORF1p. The eukaryotic expression vector pEGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and qRT-PCR were used to measure the protein and mRNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone.

RESULTSThe LINE1-ORF1p antibody prepared had a titer of >1:16 000 and could specifically recognize LINE1-ORF1p in cells and tumor tissue. WT_CLS1 cells transfected with pEGFP-N1-LINE1-ORF1 had significant increases in the mRNA and protein expression of LINE1-ORF1 and significantly enhanced cell proliferation ability and colony formation ability (P<0.05).

CONCLUSIONSLINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.

Animals ; Antibodies ; analysis ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; Deoxyribonuclease I ; analysis ; genetics ; metabolism ; Humans ; Long Interspersed Nucleotide Elements ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Transfection ; Wilms Tumor ; genetics ; metabolism ; physiopathology

OBJECTIVE: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. METHODS: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. RESULTS: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. CONCLUSION: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.
Cumulus Cells* ; Digestion ; DNA ; DNA Methylation ; Down-Regulation ; Epigenomics* ; Fertilization in Vitro ; Gene Expression ; Genome, Human ; Humans ; Infertility ; Long Interspersed Nucleotide Elements ; Methylation ; Oocyte Retrieval ; Oocytes* ; Phenotype ; Polycystic Ovary Syndrome* ; Polymerase Chain Reaction

Transposable elements are one of major sources to cause genomic instability through various mechanisms including de novo insertion, insertion-mediated genomic deletion, and recombination-associated genomic deletion. Among them is Alu element which is the most abundant element, composing ~10% of the human genome. The element emerged in the primate genome 65 million years ago and has since propagated successfully in the human and non-human primate genomes. Alu element is a non-autonomous retrotransposon and therefore retrotransposed using L1-enzyme machinery. The 'master gene' model has been generally accepted to explain Alu element amplification in primate genomes. According to the model, different subfamilies of Alu elements are created by mutations on the master gene and most Alu elements are amplified from the hyperactive master genes. Alu element is frequently involved in genomic rearrangements in the human genome due to its abundance and sequence identity between them. The genomic rearrangements caused by Alu elements could lead to genetic disorders such as hereditary disease, blood disorder, and neurological disorder. In fact, Alu elements are associated with approximately 0.1% of human genetic disorders. The first part of this review discusses mechanisms of Alu amplification and diversity among different Alu subfamilies. The second part discusses the particular role of Alu elements in generating genomic rearrangements as well as human genetic disorders.
Alu Elements* ; DNA Transposable Elements ; Genetic Diseases, Inborn ; Genome ; Genome, Human ; Genomic Instability ; Humans* ; Nervous System Diseases ; Primates ; Recombination, Genetic ; Retroelements