Retrotransposon insertion polymorphism of the porcine esr gene and its association with production performances of Large White pigs / 生物工程学报

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.

Genome Size Evolution Mediated by Gypsy Retrotransposons in Brassicaceae / 基因组蛋白质组与生物信息学报·英文版

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.

Analysis on biological functions of restriction factor SAMHD1 of primates / 吉林大学学报(医学版)

Objective: To discuss the antiviral effect, the inhibitory effect on LINE-1 retrotransposon activity and the redection of interferon production signal pathway of restriction factor SAMHD1 of the primates, and to provide the basis for further study of the SAMHD1 of the primates. Methods: The U937 cells stably expressing the SAMHD1 of primates were established; the U937-control cells established with pLVX -puro were used as negative control group, and the U937-SAMHD1 cells stably expressing the SAMHD1 protein of the different primates were used as experimental groups; the cells were treated with PMA to induce cell differentiation. The virus infection rates of HIV-1 in the cells in various groups were determined by flow cytometry. The HEK293T cells transfected with the expression plasmid of SAMDH1 were used as control group, and the cells co-transfected with the SAMHD1 and HIV-2/SIV Vpx expression plasmids were used as experimental groups. The cells were obtained 48 h after transfection, and the expression levels of SAMHD1 protein were determined by Western blotting method. The intracellular location of SAMHD1 protein was determined by immunofluorescence. The HEK293T cells transfected with LINE-1-GFP report plasmid were used as control group, and the cells co-transfected with LINE-1-GFP and SAMHD1 expression plasmids were used as experimental groups. The rates of GFP positive cells (activity of SAMHD1 to LINE-1 transposon) were determined by flow cytometry. The HEK293T cells transfected with IFN- Luc report plasmid were used as control group, and the cells co-transfected with IFN-Luc and pSAMHDl expression plasmids were used as experimental groups. The expression levels of luciferase in HEK293T cells were determined by chemiluminescence instrument. Results: Compared with negative control group, the virus infection rates of HIV-1 in experimental groups with stable expression of SAMHD1 in the primates were significantly decreased (P<0. 01). Compared with control group, the expression levels of SAMHD1 protein of the primates in experimental groups were decreased (P<0. 05 or P<0. 01). The immunofluorescence results showed that the SAMHD1 protein of the primates was localized in the nucleus. Compared with control group, the rates of GFP positive cells (activity of SAMHD1 to LINE-1 transposon) in experimental groups were significantly decreased (P< 0.05 or P<0.01). Compared with control group, the expression levels of luciferase in the HEK293T cells in experimental groups were significantly decreased (P<0. 05). Conclusion: The SAMHD1 protein of the different primates can resist the HIV-1 infection, inhibit the LINE-1 retrotransposon and antagonize the IFN production by natural immune system.

Identification and expression pattern analysis of a Moso Bamboo LTR retrotransposon / 生物工程学报

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.

Molecular diversity analysis of Tetradium ruticarpum (WuZhuYu) in China based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers / 中国天然药物

"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum.

Molecular diversity analysis of Tetradium ruticarpum (WuZhuYu) in China based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers / 中国天然药物

"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum.

Application of new markers in genetic analysis for hair stem without follicles / 复旦学报（医学版）

Objective To solve the problems of low success rate in detecting complete DNA typing of biological samples,such as naturally fallen hair,severely degraded bones and teeth which is difficult in forensic identification.Methods We applyed a specific retrotransposon (RE) DNA sequences in identification,with DNA fragment length of 60-125 bp.A RE combined with 21 loci were used in individual discrimination to compute the likelihood (LR) values and combined paternity index (CPI)values in phylogenetic identification.Results This research obtained high success rate in detection of hair stem without follicles and severely degraded biological samples.We found that most of the LR values were 108-1012,which met the requirement of identity establishing.Through the CPI values,it was difficult to support the assumptions,with most of the CPI values less than 10 000.Conclusions The RE with 21 loci can be used for individual discrimination,but more loci are need for applying in phylogenetic identification.

Validation of a PCR-based test for the genetic diagnosis of Filipino patients with X-linked dystonia parkinsonism (Xdp)

BACKGROUND AND OBJECTIVE: X-linked dystonia parkinsonism (XDP, DYT3, MIM #314250) is a neurodegenerative movement disorder found endemically in the Philippines. An SVA retrotransposon insertion mutation has been described in patients with XDP, which requires Southern analysis for detection. However, this method is costly and time-consuming. Hence we developed a PCR-based method and validated it among our local population. METHODS AND RESULTS: A total of 58 samples from 58 patients with a clinical diagnosis of XDP were collected. Other samples were from an obligate female carrier, two unaffected male relatives, and two patients with typical Parkinson’s disease. Primers designed to amplify the SVA retrotransposon found in the DYT3-TAF1 gene (NCBI Accession Number AB191243) were used. All patients were positive for the expected 3229-bp product after PCR amplification. The normal control showed a 599-bp product, while the female carrier showed both the 3229 and 599-bp product. Subsequent RFLP analysis using BamHI verified the presence of the SVA retrotransposon insertion mutation. CONCLUSION: Our results show that large-scale PCR-based testing to screen for genetic diseases with a relatively high prevalence such as XDP is possible in our setting. When followed by RFLP analysis, this can provide genetic confirmation of the diagnosis of XDP and facilitate proper genetic counselling and therapy.

Mutations in retrotransposon AtCOPIA4 compromises resistance to Hyaloperonospora parasitica in Arabidopsis thaliana

Retrotransposons (RTEs) are a principal component of most eukaryotic genomes, representing 50 percent-80 percent of some grass genomes. RTE sequences have been shown to be preferentially present in disease resistance gene clusters in plants. Arabidopsis thaliana has over 1,600 annotated RTE sequences and 56 of these appear to be expressed because of the exact expressed sequence tag (EST) matches and the presence of intact open reading frames. Of the 22 represented in the Affymetrix ATH1 array, AtCOPIA4 was found to be expressed at a higher level than all other RTEs across different developmental stages. Since AtCOPIA4 is located in the RPP5 gene cluster and is adjacent to RPP4 which confers resistance to the downy mildew oomycete Hyaloperonospora parasitica isolate EMWA1, we evaluated AtCOPIA4 mutants for resistance to this pathogen. T-DNA insertional and antisense knockout of AtCOPIA4 was found to reduce the resistance of wild type plants by 2-4 folds. Our results suggest that retrotransposon can be exapted to participate in plant defense response.

Retrotransposon Microsatellite Amplified Polymorphism Strain Fingerprinting Markers Applicable to Various Mushroom Species

The retrotransposon marY1 is a gypsy family retroelement, which is detected ubiquitously within the fungal taxonomic groups in which mushrooms are included. To utilize marY1 as a molecular marker for the DNA fingerprinting of mushrooms, oligonucleotides marY1-LTR-L and marY1-LTR-R were designed on the basis of highly conserved regions from the multiple sequence alignment of 30 marY1 sequences retrieved from a nucleotide sequence database. In accordance with Retrotransposon Microsatellite Amplified Polymorphism (REMAP) fingerprinting methodology, the two oligonucleotides were utilized together with the short sequence repeat primers UBC807 and UBC818 for polymerase chain reaction using templates from different mushroom genomic DNAs. Among the tested oligonucleotides, the marY1-LTR-L and UBC807 primer set yielded the greatest amount of abundance and variation in terms of DNA band numbers and patterns. This method was successfully applied to 10 mushroom species, and the primer set successfully discriminated between different commercial mushroom cultivars of the same strains of 14 Pleurotus ostreatus and 16 P. eryngii. REMAP reproducibility was superior to other popular DNA fingerprinting methodologies including the random amplified polymorphic DNA method.

Quantitative PCR analysis for methylation level of genome: clinical implications in cancer.

Background: Cancer cells are frequently characterized by hypomethylation of the genome including repetitive sequences. This epigenetic process is believed to be associated with several biological causes and consequences in cancer. Therefore, LINE-1 repetitive sequences demethylation in cancer should result in different clinical outcomes. Objective: Recently, we have developed an improved quantitative combined bisulfite restriction analysis PCR protocol that efficiently evaluates the methylation status of LINE-1s; the method is referred to as PCR “COBRALINE-1”. This article reviewed what have been learned by applying this technique to study methylation level of repetitive sequences from several sources of genomic DNA. Results: We have found that LINE-1 methylation patterns among normal tissues are distinct. Therefore, this epigenetic event may be continuously altered in adult tissues by the process of cellular differentiation. Moreover, we confirmed that global hypomethylation is an ongoing process that develops during tumor progression, in addition to previous evidence of genomic and LINE-1 hypomethylation occurring as an early event in carcinogenesis. COBRALINE-1 is a highly effective technique for evaluating the genome-wide level of methylation, in particular from tissue samples with minute amounts of low quality DNA. The technique has been applied to study samples from micro-dissected archived paraffin-embedded tissues and sera of several types of cancer. Conclusion: The COBRALINE-1 technique demonstrated its potential to be a tumor marker and a great tool to explore the biology of global hypomethylation.

Evolutionary course of CsRn1 long-terminal-repeat retrotransposon and its heterogeneous integrations into the genome of the liver fluke, Clonorchis sinensis

The evolutionary course of the CsRn1 long-terminal-repeat (LTR) retrotransposon was predicted by conducting a phylogenetic analysis with its paralog LTR sequences. Based on the clustering patterns in the phylogenetic tree, multiple CsRn1 copies could be grouped into four subsets, which were shown to have different integration times. Their differential sequence divergences and heterogeneous integration patterns strongly suggested that these subsets appeared sequentially in the genome of C. sinensis. Members of recently expanding subset showed the lowest level of divergence in their LTR and reverse transcriptase gene sequences. They were also shown to be highly polymorphic among individual genomes of the trematode. The CsRn1 element exhibited a preference for repetitive, agenic chromosomal regions in terms of selecting integration targets. Our results suggested that CsRn1 might induce a considerable degree of intergenomic variation and, thereby, have influenced the evolution of the C. sinensis genome.

Divergent long-terminal-repeat retrotransposon families in the genome of Paragonimus westermani

To gain information on retrotransposons in the genome of Paragonimus westermani, PCR was carried out with degenerate primers, specific to protease and reverse transcriptase (rt) genes of long-terminal-repeat (LTR) retrotransposons. The PCR products were cloned and sequenced, after which 12 different retrotransposon-related sequences were isolated from the trematode genome. These showed various degrees of identity to the polyprotein of divergent retrotransposon families. A phylogenetic analysis demonstrated that these sequences could be classified into three different families of LTR retrotransposons, namely, Xena, Bel, and Gypsy families. Of these, two mRNA transcripts were detected by reverse transcriptase-PCR, showing that these two elements preserved their mobile activities. The genomic distributions of these two sequences were found to be highly repetitive. These results suggest that there are diverse retrotransposons including the ancient Xena family in the genome of P. westermani, which may have been involved in the evolution of the host genome.