ITS1 SEQUENCES OF NUCLEAR RIBOSOMAL DNA IN WILD RICES AND CULTIVATED RICES OF CHINA AND THEIR PHYLOGENETIC IMPLICATIONS

The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3% to 72.7%. In pairwise comparisons among the five taxa, sequence site divergence ranged from 1.5% to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA(mtDNA) and nuclear DNA (nDNA) of the genus Oryza .

p21^(WAF1/CIP1) Gene DNA Sequence Change and Their Relationship with the Phenotype of Human Osteosarcoma

Objective: To investigate the p21WAF1 /CIP1gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. Methods: p21WAF1 /CIP1gene DNA of 36 osteosarcoma spec- imens was examined by using polymerase chain reaction-single strand conformation polymorphism (PCR- SSCP) method. The PCR products were sequenced directly. Results: In p21WAF1 /CIP1 gene exon3 of 36 cases of human osteosarcoma, the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 17 cases with the incidence being 44.4%. In 10 normal blood samples, DNA sequence analysis showed the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 8 cases with the incidence being 80%. Conclusion: The novel location of p21WAF1 /CIP1gene polymorphism of osteosarcoma, but not mutation was de?ned, and this location might provide the meaningful reference for the further research of p21WAF1/CIP1 gene.p2lWAF1/CIP1基因DNA序列分析及其与骨肉瘤表型的关系

Cloning and Sequence Analysis on IGF-1 Gene of Hubei White Swine

[Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 （IGF-1） gene from liver of Hubei white swine. [Method] The total RNA was extracted by using Trizol from the liver of Hubei white swine and used as template to amplify IGF-1 gene cDNA by RT-PCR. The cDNA product was cloned into pCRII vector, screened with blue-white colonies, digested with double enzymes and sequenced. [Result] The sequencing result indicated that the IGF-1 gene consisted of 607 nucleotides, containing 5＇-untranslated region at nucleotides 1-145, a complete ORF at nucleotides 146-538 encoding 130 amino acids, and 3＇-untranslated region at nucleotides 539-607. It shared 100% homology with the porcine IGF-1 gene reported by Muller et al. [Conclusion] The successful cloning and sequencing of the Hubei white swine IGF-1 gene confirmed that IGF-I gene was highly conserved, which provided technical basis for the use of transgenic technology for breeding of Hubei white swine.

Detection of Novel BEST1 Variations in Autosomal Recessive Bestrophinopathy Using Third-generation Sequencing

Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on electrooculogram.The potential pathogenic mechanism involves mutations in the BEST1 gene,which encodes Ca2+-activated Cl−channels in the retinal pigment epithelium(RPE),resulting in degeneration of RPE and photoreceptor.In this study,the complete clinical characteristics of two Chinese ARB families were summarized.Methods:Pacific Biosciences(PacBio)single-molecule real-time(SMRT)sequencing was performed on the probands to screen for disease-causing gene mutations,and Sanger sequencing was applied to validate variants in the patients and their family members.Results:Two novel mutations,c.202T>C(chr11:61722628,p.Y68H)and c.867+97G>A,in the BEST1 gene were identified in the two Chinese ARB families.The novel missense mutation BEST1 c.202T>C(p.Y68H)resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1.Another novel variant,BEST1 c.867+97G>A(chr11:61725867),located in intron 7,might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators.Conclusion:Our findings represent the first use of third-generation sequencing(TGS)to identify novel BEST1 mutations in patients with ARB,indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes.The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population.

Alterative Expression and Sequence of Human Elongation Factor-1δ during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.

Accurate Diagnosis of SARS-CoV-2 JN.1 by Sanger Sequencing of Receptor-Binding Domain Is Needed for Clinical Evaluation of Its Immune Evasion

Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used SGTF of TaqPath COVID-19 Combo Kit RT-qPCR as proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥ 6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing instead of proxy indicator to diagnose JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The nested PCR amplicons were sequenced. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed R203K, G204R and Q229K, the 3 mutations associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 of the 26 known amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: Sanger sequencing of a 445-base segment of the SARS-CoV-2 RBD is useful for accurate determination of emerging variants. The CDC may consider using Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.

Special AT-rich sequence-binding protein 1 promotes cell growth and metastasis in colorectal cancer

AIM: To evaluate the expression of special AT-rich sequence-binding protein 1 (SATB1 ) gene in colorectal cancer and its role in colorectal cancer cell proliferation and invasion.METHODS: Immunohistochemistry was used to detect the protein expression of SATB1 in 30 colorectal cancer (CRC) tissue samples and pair-matched adjacent nontumor samples. Cell growth was investigated after enhancing expression of SATB1. Wound-healing assay and Transwell assay were used to investigate the impact of SATB1 on migratory and invasive abilities of SW480 cells in vitro . Nude mice that received subcutaneous implantation or lateral tail vein were used to study the effects of SATB1 on tumor growth or metastasis in vivo . RESULTS: SATB1 was over-expressed in CRC tissues and CRC cell lines. SATB1 promotes cell proliferation and cell cycle progression in CRC SW480 cells. SATB1 over-expression could promote cell growth in vivo . In addition, SATB1 could significantly raise the ability of cell migration and invasion in vitro and promote the ability of tumor metastasis in vivo . SATB1 could up-regulate matrix metalloproteases 2, 9, cyclin D1 and vimentin, meanwhile SATB1 could down-regulate E-cadherin in CRC. CONCLUSION: SATB1 acts as a potential growth and metastasis promoter in CRC. SATB1 may be useful as a therapeutic target for CRC.

Inference of Global HIV-1 Sequence Patterns and Preliminary FeatureAnalysis

The epidemiology of HIV-1 varies in different areas of the world, and it is possible that this complexity may leave unique footprints in the viral genome. Thus, we attempted to find significant patterns in global HIV-1 genome sequences. By applying the rule inference algorithm RIPPER (Repeated Incremental Pruning to Produce Error Reduction) to multiple sequence alignments of Env sequences from four classes of compiled datasets, we generated four sets of signature patterns. We found that these patterns were able to distinguish southeastern Asian from non- southeastern Asian sequences with 97.5% accuracy, Chinese from non-Chinese sequences with 98.3% accuracy, African from non-African sequences with 88.4% accuracy, and southern African from non-southern African sequences with 91.2% accuracy. These patterns showed different associations with subtypes and with amino acid positions. In addition, some signature patterns were characteristic of the geographic area from which the sample was taken. Amino acid features corresponding to the phylogenetic clustering of HIV-1 sequences were consistent with some of the deduced patterns. Using a combination of patterns inferred from subtypes B, C, and all subtypes chimeric with CRF01_AE worldwide, we found that signature patterns of subtype C were extremely common in some sampled countries (for example, Zambia in southern Africa), which may hint at the origin of this HIV-1 subtype and the need to pay special attention to this area of Africa. Signature patterns of subtype B sequences were associated with different countries. Even more, there are distinct patterns at single position 21 with glycine, leucine and isoleucine corresponding to subtype C, B and all possible recombination forms chimeric with CRF01_AE, which also indicate distinct geographic features. Our method widens the scope of inference of signature from geographic, genetic, and genomic viewpoints. These findings may provide a valuable reference for epidemiological research or vaccine design.

Cloning and Sequence Analysis of Glycoprotein D Gene of Bovine Herpesvirus-1 Strain Luojing

By means of PCR,the gene encoding gD of bovine herpesvirus-1 (BHV-1) strain Luojing was amplified,cloned and sequenced.The nucleotide sequence of this gD gene was (1 251 bp,)encoding 417 amino acids.Comparied with the published P8-2 strain,the homology of the necleotide sequence is 99.92%,and that of the deduced amino acid sequence is 100%.The results indicated that gD of BHV-1 was highly conservative.

Study on Focal Mechanism Changing Characteristics of the 2013 Zogang-Markam Ms6. 1 Earthquake Sequence

The M_S6. 1 earthquake was a foreshock-mainshock-aftershock type which occurred in the boundary region between Zogang and Markam counties on August 12,2013. Within 9hours before the main shock seven earthquakes of greater than M_L2. 0 occurred,with a maximum of M_L4. 7. In this paper,the earthquake focal mechanism changing process of the Zogang-Markam M_S6. 1 earthquake sequence is studied by calculating the correlation coefficient of body wave spectral amplitudes,and the result shows that the correlation coefficients of spectral amplitude of foreshocks present high value fluctuation with an average value of 0. 86,which shows that the focal mechanism of foreshocks are similar;and the correlation coefficients of spectral amplitude of aftershocks present low value,which shows that the possibility of a large earthquake is not high after a time.

The genome of herpes simplex virus type 1 is prone to form short repeat sequences

Herein, we report a very high content of simple sequence repeats (SSRs) covering 66.12% of the herpes simplex virus type 1 (HSV-1) genome when a low threshold is adopted to define SSRs, indicating that repeat sequence is a very important character of the HSV-1 genome. The repeats with two iterations account for 68.33% of the total repeats. In reality, the genome of HSV-1 is prone to form shorter repeat sequences. For mono-, di- and trinucleotide repeats, the repeat numbers decreased with the increase of repeats iterations, implicating that the formation tendency of SSRs might be from low iterations to high iterations. The high iterations SSRs might have subjected to strong selected pressure and survived to perform different functions. The analysis suggested that the repeats formation may be an essential evolutionary driving force for the HSV-1 genome, and the results might be helpful for studying the genome structure, repeats genesis and genome evolution of HSV-1.

Role of host immune responses in sequence variability of HIV-1 Vpu

Viral protein U （Vpu） is an accessory protein associated with two main functions important in human immu-nodeficiency virus type 1 （HIV-1） replication and dis-semination; these are down-regulation of CD4 receptor through mediating its proteasomal degradation and en-hancement of virion release by antagonizing tetherin/BST2. It is also well established that Vpu is one of the most highly variable proteins in the HIV-1 proteome. However it is still unclear what drives Vpu sequence variability, whether Vpu acquires polymorphisms as a means of immune escape, functional advantage, or otherwise. It is assumed that the host-pathogen inter-action is a cause of polymorphic phenotype of Vpu and that the resulting functional heterogeneity of Vpu may have critical significance in vivo . In order to compre-hensively understand Vpu variability, it is important to integrate at the population level the genetic association approaches to identify specifc amino acid residues and the immune escape kinetics which may impose Vpu functional constraints in vivo . This review will focus on HIV-1 accessory protein Vpu in the context of its sequence variability at population level and also bring forward evidence on the role of the host immune re-sponses in driving Vpu sequence variability; we will also highlight the recent findings that illustrate Vpu func-tional implication in HIV-1 pathogenesis.

Cloning, Sequence Analysis, and Prokaryotic Expression of the Porcine DECR1 Gene

2,4-dienoyl-CoA reductase 1 （ DECR1 ） is the key rate-limiting enzyme in the metabolism of polyunsaturated fatty acids. Although this protein has been studied in a variety of mammals, its role in por- cine is yet to be fully elucidated. However, it is a candidate determinant/indicator of meat quality, growth traits, and carcass quality. Here, we employed RT-PCR and rapid amplification of cDNA ends （RACE） analysis to amplify the full-length cDNA of DECR1 from Mashen pig liver, and cloned it into the expression vector pET-32a＋. After confirmation by sequencing and restriction analysis, the recombinant plasmid was transformed into E. coli BL21 cells. The cDNA of pig DECR1 contained 2,352 nucleotides, including a 987 bp open reading frame flanked by a 53 bp 5＇-untranslated region （UTR） and a 1,312 bp 3＇-UTR. The pig DECR1 coding sequence encoded 328 amino acid residues, which shared 99%, 88%, 87%, 87%, 87%, 87%, and 83% identity with those of Sus scrofa （predicted）, Bos taurus, Homo sapiens, Macaca mulatta, Pan troglodytes, Equus caballus, Canis, and Mus musculus, respectively. SDS-PAGE analysis revealed that the recombinant protein was expressed and that the expression level reached its highest level after 4 h induction. Western blot analysis indicated that the molecular weight of the expressed protein was the same as that predicted, ap- proximately 35 kDa. Collectively these data provide the basis for further studies into the physiological functions and molecular mechanisms of the pig DE- CR1 gene.

Isolation and Complete Nucleotide Sequence of the Measles Virus IMB-1 Strain in China

The complete nucleotide sequence of the measles virus strain IMB-1,which was isolated in China,was determined.As in other measles viruses,its genome is 15,894 nucleotides in length and encodes six proteins.The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%-5% at the nucleotide sequence level.This isolate has amino acid variations over the full genome,including in the hemagglutinin and fusion genes.This report is the first to describe the full-length genome of a genotype H1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.

Analyses of the Sequence and Structure of the BnNIP7;1 Gene in Brassica napus

Rapeseed,the largest oil crop in China,is a boron-loving plant.Boron has an important influence on the growth and development of rapeseed.In this study,four different boron-efficient Brassica napus varieties were used as the material to analyze the structure and sequence of BnNIP7;1 gene,aiming to provide a basis for studying the role of BnNIP7;1 in the absorption and transport of boric acid in rapeseed.The results showed that BnNIP7;1 exists in multiple copies of the Brassica napus genome,consisting of five exons and four introns.There are base insertions,base deletions and more base substitutions in introns.However,there is no base insertion or base deletion in the exon,only a small number of base substitution mutations are present,bringing about three amino acid changes in the encoded protein of BnNIP7;1-ZS9b in Zhongshuang 9.The BnNIP7;1-HY7b of Huayou 7 has an ochre mutation that will lead to a protein of only 47 amino acids,thus losing its function.Bioinformatics indicates that BnNIP7;1 protein is a membrane protein with six transmembrane regions,indicating that it is involved in the absorption and transport of boric acid.

Isolation and sequence analysis of a Dof protein gene(PtDof1) in Populus

Both cDNA and DNA clones of PtDof1 （GenBank Accession No. FJ402844 and FJ402845） were isolated from plants grown in tissue culture ofPopulus tornentosa. The DNA sequence is 1597 bp including two exons and one intron. The cDNA is 969 bp in length with a 765 bp open reading frame which is capable of encoding 255 amino acids. The deduced amino acids sequence of the PtDofl protein shares 65%, 56% and 55% identity with Vitis vinifera （CAO48618）, Nicotiana tabacum （CAA08755） and Glycine max （ABI 16022） Dof protein by blast analysis in GenBank. Phylogenic analysis suggests PtDof1 gene could belong to the Dofgene family. PtDofl protein contains an unusual conserved single zinc finger with the pattern of C-X2-C-X21-C-X2-C, which may play a functional role in tissue-specific expression and possibly the auxin response of endogenous plant genes.

Characterizations of Chinese isolates of Coxiella burnetii in the com1 gene sequence

Objective: To know some genetical characterizations of Coxiella burnetii Chinese isolates by comparing the coml gene sequence. Methods: com1 gene sequences of Chinese isolates were amplified, se-quenced, and analyzed by comparing our result and the previous published data. Results: Three different com1 sequences were identified in 7 Chinese isolates. Sequence comparison indicated that the isolates harboring the QpRS plasmid could be defined as a new group and, in addition, the isolates carrying the same plas-mid type showed similar com1 gene sequence. Conclusion: Study suggests that the classification of the group based on the coml gene sequence is highly associated with the plasmid type of the isolates and, however, little related to disease forms and geographical origins of the isolates.

ANALYSIS OF MOTIF IN TCR Vβ HV4 SEQUENCES BINDING SUPERANTIGEN TSST-1

首先对４１种人和小鼠的Ｔ细胞受体β链可变基因编码肽段（Ｖβ）的氨基酸序列进行多序列对准，就Ｖβ之第四高变区（ＨＶ４）片段进行比较，分析与超抗原毒素休克综合征毒素－１（ＴＳＳＴ－１）结合的四种Ｖβ（小鼠Ｖβ３、Ｖβ１５、Ｖβ１７和人Ｖβ２）之ＨＶ４序列内是否存在特定的氨基酸残基排列模式。结果发现：小鼠Ｖβ３和Ｖβ１７的ＨＶ４具有特异的ＲＦＳＡＸＣＸＳＮＳ模式，而小鼠Ｖβ１５和人Ｖβ２的ＨＶ４则含独特的ＫＦＸＩＸＨ模式。提示：与ＴＳＳＴ－１结合的四种Ｖβ所对应的Ｔ细胞识别表位可能不止一个。

Sequence determination of exp-1 Gene of Plasmodium Falciparum Isolate FCC1 /HN

目的 测定恶性疟原虫FCCl／HN株exp-1基因序列。方法 根据exp-1基因已知序列设计合成1对引物，用PCR技术从FCCl／HN株基因组DNA中扩增exp-1基因；将exp-1基因克隆入pMD-18T载体，转化大肠杆菌JMl09感受态细胞，铺x—gal LB平板；挑取阳性菌落，用酶切，PCR扩增进行鉴定。以正确的重组质粒为模板，用双脱氧链末端终止法测定exp-1基因序列。结果 从恶性疟原虫FCCl／HN株基因组DNA中获取exp-1基因，成功克隆入pMD-18T载体；测序表明FCCl／HN株exp-1基因全长937bp，编码162个氨基酸。结论 克隆了恶性疟原虫FCCl／HN株exp-1基因，并测定了其核苷酸序列，为进-步研究其功能奠定基础。