Characterization of the complete chloroplast genome of Polygonatum franchetii Hua, a Chinese endemic medicinal species, and phylogenetic relationships of Polygonatum / 药学学报

italic>Polygonatum franchetii Hua is a medicinal plant endemic to China from Polygonatum Mill. The chloroplast genomes of two P. franchetii individuals sampled from two different habitats were sequenced by using the DNBSEQ-T7 high-throughput sequencing platform. After assembly and annotation, the two complete chloroplast genomes were characterized, and then comparative and phylogenetic analyses were performed with other published chloroplast genome sequences from Polygonatum. The whole chloroplast genomes of the two P. franchetii individuals were 155 942 and 155 962 bp in length, with a large single copy region (LSC, 84 670 and 84 722 bp), a small single copy region (SSC, 18 564 and 18 566 bp) and a pair of reverse repeats (IRa/IRb, 26 354 and 26 337 bp), respectively. Both of them contained 113 genes, including 79 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Comparative analyses showed that the genome length, the guanine and cytosine (GC) content, genes content and order were highly conserved between the two P. franchetii individuals and among different Polygonatum species. The detected repeat sequences, including dispersed repeats, tandem repeats and simple sequence repeats (SSRs), were also relatively similar in types and positions, though showing a slightly difference in number. No significant expansion or contraction of the inverted repeat regions was found. Sequences variation between the two P. franchetii individuals was lower than that among different Polygonatum species. Besides, coding sequences (CDS) showed less divergence than noncoding sequences, and sequence divergence of IRs regions was lower than that of the LSC and SSC regions, both intraspecifically and interspecifically. Eight sequences with high nucleotide diversity among different species were screened, all of which were found located in the LSC and SSC regions. Phylogenetic inference showed that all Polygonatum species clustered into a monophyletic clade with a 100% bootstrap value, within which, species in section Verticillata formed a distinct group, section Sibirica and section Polygonatum were sister groups. The two P. franchetii individuals grouped together and showed the closest phylogenetic affinity to P. stenophyllum Maxim., belonging to the section Verticillata. The chloroplast genome of P. franchetii and its phylogenetic position in Polygonatum were comprehensively investigated and clearly elucidated in this study, the results may lay a foundation for the resource development and utilization of P. franchetii, as well as further molecular identification and phylogenetic studies of medicinal Polygonatum species.

Genomic variations of SARS-CoV-2 and molecular diagnosis / 西安交通大学学报(医学版)

Rapid and accurate detection of contagium virus is a key tool for controlling the outbreak. As the number of patients infected with 2019 novel coronavirus (SARS-CoV-2) pneumonia is increasing and the epidemic is spreading, many hospitals, laboratories and pharmaceutical companies have developed diagnostic reagents that can detect SARS-CoV-2. Currently, genome sequencing or real-time PCR is a method for detecting SARS-CoV-2. However, there have been reported cases of negative results by nucleic acid detection but confirmed on the radiological CT scan. How to improve the diagnostic efficiency and the sensitivity of molecular detection remains to be solved. To analyze the variations in viral genomic sequence may be helpful in guiding the prevention and treatment of diseases infected by SARS-CoV-2. At present, controversy still exists over the source of SARS-CoV-2 even though the probable origin is bat in nature. This article summarizes the recent findings of SARS-CoV-2 from genetic, virological and evolutionary biological perspectives. By comparing the genomic variations among SARS-CoV-2 infected patients, we hope the findings can be used in the viral detection and potential antiviral therapy. Also, it will be great if we can trace the spread of the virus from one person to another, which will be an much effective way to predict and control the spread from the virus-carrier without symptoms at the incubation period to the others.

Sequence Variation in Superoxide Dismutase Gene of Toxoplasma gondii among Various Isolates from Different Hosts and Geographical Regions

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.

Genetic Diversity of Toxoplasma gondii Strains from Different Hosts and Geographical Regions by Sequence Analysis of GRA20 Gene

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.

Sequence Generation and Genotyping of 15 Autosomal STR Markers Using Next Generation Sequencing / 대한법의학회지

Recently, next generation sequencing (NGS) has received attention as the ultimate genotyping method to overcome the limitations of capillary electrophoresis (CE)-based short tandem repeat (STR) analysis, such as the limited number of STR loci that can be measured simultaneously using fluorescent-labeled primers and the maximum size of STR amplicons. In this study, we analyzed 15 autosomal STR markers via the NGS method and evaluated their effectiveness in STR analysis. Using male and female standard DNA as single-sources and their 1:1 mixture, we sequentially generated sample amplicons by the multiplex polymerase chain reaction (PCR) method, constructed DNA libraries by ligation of adapters with a multiplex identifier (MID), and sequenced DNA using the Roche GS Junior Platform. Sequencing data for each sample were analyzed via alignment with pre-built reference sequences. Most STR alleles could be determined by applying a coverage threshold of 20% for the two single-sources and 10% for the 1:1 mixture. The structure of the STR in each allele was accurately determined by examining the sequences of the target STR region. The mixture ratio of the mixed sample was estimated by analyzing the coverage ratios between assigned alleles at each locus and the reference/variant ratios from the observed sequence variations. In conclusion, the experimental method used in this study allowed the successful generation of NGS data. In addition, the NGS data analysis protocol enables accurate STR allele call and repeat structure determination at each locus. Therefore, this approach using the NGS system will be helpful to interpret and analysis the STR profiles from singe-source and even mixed samples in forensic investigation.

Comparative analysis of Gp120 sequences of enterotropic and neurotropic SIV strains / 中国比较医学杂志

Objective To study the mutations of Env sequence of SIVmac239 after infection of Chinese rhesus monkeys, and compare the differences and characteristics of Gp120 sequences of enterotropic and neurotropic SIV strains. Methods Six strains of simian immunodeficiency virus were analyzed in this study: four separated from peripheral blood mononuclear cells of SIVmac239-infected monkeys and two neurotropic SIVmac251 strains.Isolated and cultured monoclonal virus was obtained by limiting dilution assay.Gp120 sequences were amplified after the RNA extraction and phylogenetic analysis was processed thereafter.So did the Gp120 amino acid sequence and N-glycosylation sites analysis of the enterotropic and neurotropic strains.Results SIVmac239 had different mutations in four rhesus monkeys.The diversity in amino acid sequences of the enterotropic and neurotropic strains concentrated in the V1 and V4 regions of Gp120.The enterotropic strains had an addition of glycosylation site in V4 but the glycosylation site changes of neurotropic strains were located in the conservative regions of C1, C2 and C3.Conclusions The addition of one glycosylation site in V4 region of GP120 and loss of one glycosylation site in C1 region are associated with enhanced enterotropism and neurotropism.The differences between the enterotropic and neurotropic strains are not dipicted in Gp120 V3 region which is closely related with the tropism of strains.

Sequence variation data of F8 and F9 genes in functionally validated control individuals: implications on the molecular diagnosis of hemophilia

BACKGROUND: The F8 and F9 genes encode for coagulation factor VIII (FVIII) and FIX, respectively, and mutations in these genes are the genetic basis of hemophilia A/B. To determine whether a sequence variation in F8/F9 is a disease-causing mutation, frequency data from a control population is needed. This study aimed to obtain data on sequence variation in F8/F9 in a set of functionally validated control chromosomes of Korean descent. METHODS: We re-sequenced F8 and F9 from DNA samples of 100 Korean male control individuals with normal PT, aPTT, and FVIII activity. PCR and direct sequencing analyses were performed using primer pairs to cover all coding regions and the flanking intronic sequences. RESULTS: Thirteen individuals (13%) were hemizygous for sequence variations in the coding region of F8. Six (6%) had c.3780C>G (p.Asp1260Glu), five (5%) had c.3864A>C (p.Ser1288=). One each individual (1%) had c.4794G>T (p.Glu1598Asp) and c.5069 A>G (p.Glu1690Gly). Asp1260Glu and Ser1288= were known SNPs (rs1800291 and rs1800292, respectively). Glu1598Asp was assigned as a missense mutation in public databases (HGMD and HAMSTeRS), and Glu1690Gly was a novel variation. Based on the normal FVIII activities in control individuals carrying these variations (109% and 148%, respectively), they were considered to be rare SNPs. No variation was observed in F9 of control individuals. CONCLUSION: A significant proportion of control individuals carried sequence variations in F8, but not in F9. These results can be used as a reference dataset for molecular diagnosis of hemophilia A and B, particularly in Korea.

Identification of a shared F8 mutation in the Korean patients with acquired hemophilia A

Although uncommon, acquired hemophilia A (HA) is associated with a high rate of mortality due to severe bleeding. In spite of many hypotheses regarding the cause of acquired HA, there is as yet no established theory. In this study, we investigated the possibility that mutation(s) in the F8 gene may be correlated with the development of inhibitory autoantibodies. Direct sequencing analysis was performed on all 26 exons of the F8 gene of 2 patients exhibiting acquired HA. Both patients were found to share a common point mutation (c.8899G>A) in the 3'-untranslated region (3'-UTR) of exon 26. This is the first report on the genotyping of F8 in the context of acquired HA.

Detection of miRNA gene sequence variations in multiple myeloma and its significance / 白血病·淋巴瘤

Objective To investigate sequence variations of 12 miRNA genes in multiple myeloma(MM) in order to find whether sequence variations in miRNA genes are associated with tumorigenesis and discuss the clinical significance of MM associated with miRNA genes mutations. Methods The miRNA gene mutations in 20 cases of MM, 4 MM-derived cell lines and 20 controls were detected by the methods of polymerase chain reaction single stranded conformation polymorphism (PCR-SSCP) and silver staining technique. Both clinical features and laboratory results were analyzed simultaneously. Results The electrophoretic patterns showed a total of three variations in miR-19a, miR-19b and miRNA-335,which were observed in 3 MM cells (15 %, 3/20). We also found variations of miRNA-335 in MM-derived cell lines KM-3and RPMI8226. However, no sequence alteration in the miRNA genes was observed in our set of controls. One of the three MM patients died, and two of them were detected mutations at the terminal stage of the disease.Conclusion A relative high frequency of miRNA gene mutation was found in MM and MM derived cell lines, which suggests possibility of a main mechanism underlying tumorigenesis. And, detecting miRNA gene mutations in MM might be benefit to evaluate the progression and prognosis of disease.

In Silico Functional Assessment of Sequence Variations: Predicting Phenotypic Functions of Novel Variations

A multitude of protein-coding sequence variations (CVs) in the human genome have been revealed as a result of major initiatives, including the Human Variome Project, the 1000 Genomes Project, and the International Cancer Genome Consortium. This naturally has led to debate over how to accurately assess the functional consequences of CVs, because predicting the functional effects of CVs and their relevance to disease phenotypes is becoming increasingly important. This article surveys and compares variation databases and in silico prediction programs that assess the effects of CVs on protein function. We also introduce a combinatorial approach that uses machine learning algorithms to improve prediction performance.

cDNA-AFLP Reveals Variation of Sequence Region trnF-ndhJ in Cytoplasmic Male Sterile Chinese Cabbage-pak-choi / 生物化学与生物物理进展

By means of cDNA amplified fragment length polymorphism(cDNA-AFLP) technique, a fragment P1708 was amplified from Polima cytoplasmic male sterile Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinenesis Makino, syn, B. rapa L. ssp. chinenesis) 'Bpol97-05A'. RT-PCR showed that this fragment was specifically expressed in male sterile material. Sequencing and BLAST search in GenBank database indicated that P1708 had 100% homolog with chloroplast ndhJ-trnF gene region except a 54 bp insertion. Gene specific primer pairs were synthesized according to ndhJ-trnF gene region and two fragments about 1 900 bp were amplified respectively using genomic DNA templates of Polima cabbage and male fertile oilseed rape. The sequencing results showed that the gene region ndhJ-trnF of Polima cabbage contained two 54 bp repeats and some variation sites. The repeat part shared the same sequence as trnF gene except three bases at 5′ ends. For the insertion of 108 bp sequence, a new open reading frame was created.

Analysis of human papillomavirus type 16 E6, E7 sequence variation in primary cervical cancer from Korean women and its relationship to the expression of immunomodulatory gene / 대한산부인과학회잡지

OBJECTIVES: As one of the possible mechanisms of viral evasion in the HPV-infected cervical cancer cells, the role of amino acid sequence change in epitope region has not been reported yet. In this study, sequence variations of HPV 16 E6, E7 gene, especially focused on epitope region, were analysed, the status of immunomodulatory factors were documented, and finally the possible correlation between the sequence variations and the loss of HLA class I expression was examined. METHODS: The entire ORF(open reading frame)s of HPV 16 E6, E7 were sequenced by the fluorescent dideoxy termination method. In addition proteins and transcripts of HLA-ABC, beta2-microglobulin(beta2-m), TAP (transporter associated with antigen processing), and LMP(large multi-functional proteasome) were evaluated by immunohistochemistry and RT-PCR, respectively in 40 clinical specimens of primary cervical cancer and 6 cervical cancer cell-lines. Medical records including pathologic reports were reviewed. RESULTS: Among the 27 cases confirmed as harboring HPV 16 DNA, only one(3.7%) found as a prototype. Among 11 kind of variants identified in total, 4 variants(5 nucleotide sites) which were never reported before has been found, registered firstly to GenBank. The most frequently found one(16 cases, 59.3%) contains D25E, N29S in E6, E7 region, respectively and the most common variation in E6, E7 ORFs found concurrently(p<0.05). Down-regulation of HLA-ABC and beta2-m was identified in 32(86.5%) and 35 cases(89.7%), respectively and transcripts of TAP, LMP were identified in over 85% of cases. However, there was no significant difference in HPV 16 infection, D25E in E6 and so on between HLA-ABC, beta2-m positive and negative groups. The well-known clinicopathologic parameters did not correlate with sequence variations and immunomodulatory factors. Five sequence variations in HPV 16 E6, E7 ORFs that were not previously reported worldwide were found, registered firstly to GenBank. CONCLUSION: It seems that multiple mechanisms are operated in down-regulation of HLA class I molecules and the phenotypic profile of immunomodulatory factors seems to be unrelated in vivo to the naturally occurring HPV 16 E6, E7 variations in epitope region.