Effects of STI571 and p27 gene clone on proliferation and apoptosis of K562 cells

AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line.p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot.MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index.RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined.The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58,P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell).CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.

Diversity of Microflora in Colonic Mucus from Severe Ulcerative Colitis Patients Analyzed by Terminal Restriction Fragment Length Polymorphism and Clone Libraries of Bacterial 16S rRNA Gene Sequences

Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.

1B Specific LMW-GS Primers Cloned a 1D Located Gene from Wheat Cultivar Xiaoyan 22

In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.

Defect in an immune regulator gene BrSRFR1 leads to premature leaf senescence in Chinese cabbage

Leaf senescence is the final stage of leaf development, where the nutrients and energy of senescent leaves are redistributed to developing tissues or organs for plant growth, reproduction, and defense. Outer leaves are photosynthetic organs that usually senesce at the late heading stage in Chinese cabbage, and premature leaf senescence often reduces leafy head yield and quality. In this study, 11 premature leaf senescence mutants were screened from an ethyl methanesulfonate-mutagenized population of the double haploid line ‘FT' in Chinese cabbage. At the early heading stage, the mutants exhibited edge yellowing within its outer leaves, and at the mature stage, its leafy head weight decreased significantly. Genetic analysis revealed that the mutated trait of all 11 mutants corresponds to single gene recessive inheritance. Semi-diallel cross tests showed that 5 of the 11 were allelic mutants. MutMap and Kompetitive Allele Specific PCR genotyping revealed that BraA01g001400.3C was the candidate gene, which is orthologous of Arabidopsis SUPPRESSOR OF rps4-RLD 1, encoding an immune regulator, so we named it as BrSRFR1. All the BrSRFR1 in the five allelic mutants exhibited single nucleotide polymorphisms at different positions on their exons and led to premature translation termination, which confirmed that defect in BrSRFR1 led to premature leaf senescence. These results verify the role of Br SRFR1 on leaf senescence and provide a new insight into the mechanisms of leaf senescence in Chinese cabbage, which reveals a novel function of SRFR1 in plant development.

Molecular Cloning and Bioinformatics Analysis of msrA Gene from Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to understand the structure and function of MsrA protein.[Methods]With Vibrio alginolyticus HY9901 as the object of study,primers were designed to amplify the full-length gene of msrA,and its bioinformatics analysis was carried out.[Results]The full length of msrA gene was 639 bp,encoding 212 amino acids,and its theoretical molecular weight was about 23729.60 Da.The protein had a stable structure,and it was hydrophobic overall.The structure of signal peptides at the N terminal of the amino acid sequence was predicted,and it was found that there was no signal peptide cleavage site and no transmembrane region.The amino acid sequence of MsrA contained multiple signal binding sites.Protein subcellular localization showed that MsrA protein was most likely located in the cytoplasm.Homology analysis showed that MsrA of V.alginolyticus had high homology with other Vibrio species,and the highest homology with V.alginolyticus.In the prediction of functional domains,MsrA had the function of methionine sulfoxide reduction.In secondary structure prediction,MsrA contained random coils at a proportion of 46.70%,which was the highest.The similarity between the tertiary structure model of MsrA and template Q87SW6.1.A was 89.15%.PTM analysis showed that MsrA protein had many PTM modification sites such as phosphorylation and glycosylation sites.[Conclusions]This study provides some reference value for further study on the role of MsrA in bacterial antioxidant stress.

Molecular Cloning of sodB Gene from Vibrio alginolyticus HY9901 and Its Bioinformatics Analysis

Vibrio alginolyticus is a zoonotic bacterium.A pair of specific primers was designed using the sodB gene sequence of Vibrio alginolyticus HY9901 in order to amplify the full length of the gene by PCR.The results indicated that the total length of the sodB gene was 585 bp and that it could encode 194 amino acids.The predicted amino acid sequence derivation indicated that the molecular weight of the protein was approximately 21.56 kDa,with an isoelectric point of 4.95.Upon prediction of the N-terminal signal peptide structure of the protein,no significant signal peptide cleavage site was observed,indicating that the protein lacked both a signal peptide and a transmembrane region.The amino acid sequence contained an N-glycosylation site,a casein kinase II phosphorylation site,a microsomal C-terminal target signal site,and a manganese and iron superoxide dismutase signal site.The probability of intracytoplasmic localization of the SodB protein was 56.5%,which was analyzed according to the subcellular localization of the protein.The amino acid sequence of the sodB gene of V.alginolyticus exhibited 98%-100%homology to other Vibrio species,clustering into the same subfamily with V.parahaem,indicating a relatively close relationship between them.In the prediction of protein structure,the proportions ofα-helix,random coil,β-sheet,and extended strand were 48.45%,30.41%,5.67%,and 15.46%,respectively.The similarity to template 1dt0.1.A reached 71.58%.A PTM site analysis revealed the presence of phosphorylation,glycosylation,ubiquitination,sumoylation,acetylation,and methylation modification sites,as well as the absence of lactylation modification sites.

CAS Scientists Clone Dentinogenesis Gene

Scientists from Shanghai Institutes of Biological Science under theChinese Academy of Sciences (CAS) have cloned dentinogenesisgene, which is believed responsible for a genetic tooth disease.The disease, Dentinogenesis imperfecta 1, one of the most commongenetic tooth problems, causes brittle teeth for one out of every six toeight thousand humans in the world. There is no effective treatment

Study on the Cloning and Isolation of sus scrofa GPX2 Gene by RACE Method

[Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment analysis of GPX2 gene of human, rat, mouse, dog and cattle. A sus scrofa GPX2 gene sequence of 330 bp was obtained by RT-PCR application method. Primes were designed respectively according to the known sequence, sus scrofa GPX2 gene was isolated and cloned by 3-RACE and 5-RACE method and analyzed the gene sequence. [Result] A mRNA sequence of 924 bp was successfully cloned and isolated in this research. This sequence contained complete 3＇end and had higher sequence homology with human,mouse,cattle and dog GPX2 gene, and there was codon called TGA which encoding Sec on the position of No. 114-116 gene. [Conclusion] Sequence alignment analysis showed that the cloned gene was sus scrofa GPX2 gene （ NCBI GenBank database, the sequence number was D098982）.

Cloning and Bioinformatic Analysis of Cinnamoyl-CoA Reductase Gene (CCR) from Pennisetum purpureum

[Objective] The aim was to clone the cDNA and DNA sequences of the CCR （Cinnamoyl-CoA reductase） gene which involves in lignin biosynthesis, from Pennisetum purpureum, and to make comprehensive analysis on these sequences. [Method] CCR sequences were cloned from P. purpureum by using conventional RT-PCR and RACE （Rapid Amplification of cDNA Ends） methods; and the bioinformatic analyses of the CCR were conducted by means of NCBI, ProtParam ProtScale, TMHMM, TargetP, SignalP, Pfam20.0, Prosite, Swiss-Model, ClustalW2, DNAman, DNAstar and MEGA5. [Result] The cloned PpCCR （P. purpureum CCR） cDNA sequence was 1 316 bp, including a 1 110 bp ORF and 206 bp 3’-UTR. The cloned DNA sequence from PpCCR was 6 133 bp in full-length, containing five exons and four introns. Bioinformatic analysis indicated that PpCCR encoded a polypeptide of 369 amino acids, the secondary structure of which was primarily composed of random coil and α-helix, belonging to NAD-dependent epimerase/dehydratase family, and its co-factor binding sites and substrate binding sites were highly conserved. [Conclusion] DNA and cDNA sequences of CCR gene were obtained from P. purpureum, which had the typical characteristics of other homologous genes. The obtained bioinformatic data provided theoretical references for the further analysis of CCR and better application of P. purpureum in the future.

Cost-Effective Method of Gene Synthesis by Sequencing from Microchip-Derived Oligos for Droplet Cloning

Gene synthesis has provided important contributions in various fields including genomics and medicine. Current genes are 7 - 30 cents depending on the assembly and sequencing methods performed. Demand for gene synthesis has been increasing for the past few decades, yet available methods remain expensive. A solution to this problem involves microchip-derived oligonucleotides (oligos), an oligo pool with a substantial number of oligo fragments. Microchips have been proposed as a tool for gene synthesis, but this approach has been criticized for its high error rate during sequencing. This study tests a possible cost-effective method for gene synthesis utilizing fragment assembly and golden gate assembly, which can be employed for quicker manufacturing and efficient execution of genes in the near future. The droplet method was tested in two trials to determine the viability of the method through the accuracy of the oligos sequenced. A preliminary research experiment was performed to determine the efficacy of oligo lengths ranging from two to four overlapping oligos through Gibson assembly. Of the three oligo lengths tested, only two fragment oligos were correctly sequenced. Two fragment oligos were used for the second experiment, which determined the efficacy of the droplet method in reducing gene synthesis cost and speed. The first trial utilized a high-fidelity polymerase and resulted in 3% correctly sequenced oligos, so the second trial utilized a non-high-fidelity polymerase, resulting in 8% correctly sequenced oligos. After calculating, the cost of gene synthesis lowers down to 0.8 cents/base. The final calculated cost of 0.8 cents/base is significantly cheaper than other manufacturing costs of 7 - 30 cents/base. Reducing the cost of gene synthesis provides new insight into the cost-effectiveness of present technologies and protocols and has the potential to benefit the fields of bioengineering and gene therapy.

Cloning of Rabbit Bone Morphogenetic Protein 15 and Its Expression During in vitro Maturation of Rabbit Oocytes

Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.

Identification of the Rice Vacuolar ATPase B Subunit Gene and Its Expression Pattern Analysis Under Phosphorus Deficiency

A vacuolar ATPase (V-ATPase.) B subunit gene has been cloned and characterized front a phosphorus starvation induced rice root subtractive cDNA library by suppression subtractive hybridization (SSH) method and RT-PCR amplification. This gene encodes a polypeptide of 487 amino acid residues, containing a conservative ATP binding site and with a molecular weight of 54.06 kD and an isoelectric point of 4.99, southern analysis of the. genomic DNA indicates that V-ATPase B subunit is encoded by a single gene in rice genome. The amino acid homologies of V-ATPase B subunits among different organisms range from 76% to 97% and reveals that the evolution of V-ATPase B subunit is accompanied with the biological evolution. Expression pattern analysis indicated that the maximal expression of V-ATPase B subunit gene occurred at an early stage (6 - 12 h) after phosphorus starvation in roots, and lately stage (24 - 48 It) in leaves. Under phosphorus deficiency, the up-regulated expression of V-ATPase gene was presumed to strengthen the proton transport and provide the required energy to maintain an electrochemical gradient across the tonoplast to facilitate Phosphorus transport.

Cloning and Prokaryotic Expression of FnBP Ligand Binding Gene of Staphylococcus aureus

[Objective] The study aimed to clone the FnBP ligand binding gene of Staphylococcus aureus and run prokaryotic expression by constructing a prokaryotic expression vector. [Method] The gene encoding FnBP ligand binding gene was amplified from S.aureus chromosomal DNA by PCR technique. After T-A cloning, plasmid pMD18- FnBP was constructed. pMD18- FnBP and pET28a(+)were digested by BamH Ⅰ and EcoR Ⅰ double enzymes, then the purified FnBP ligand binding gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-FnBP was thus constructed. The constructed plasmid pET28a-FnBP was transformed into Escherichia coli BL21(DE3) competent cells. The bacterium was induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blot. [Result] The gene fragment with the length of 370 bp was amplified by PCR approach. One approximately 30 kD exogenous protein was observed in SDS-PAGE analysis. Western blot analysis indicates the protein has antigenicity of S.aureus. [Conclusion] The FnBP ligand binding gene of S.aureus was successfully cloned and expressed in prokaryotic cells.

Cloning and Bioinformatics Analysis of P23 Gene from Theileria sergenti

[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.

Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C→T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences （PSQQQP）. The peptide fraction PF（Y）PP（Q）is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Dul seems to be more homologous with the genes on chromosome 6B.

枝状枝孢霉MD2的Unigene A03231的克隆及原核表达分析

为研究真菌紫杉醇合成相关基因的功能,从产紫杉醇真菌枝状枝孢霉MD2中克隆了Unigene A03231的DNA和c DNA全长序列(918 bp).结果表明:该基因不含内含子,其编码产物含305个氨基酸,与短链脱氢酶/还原酶一致性为64%,预测蛋白分子量为33071.5 Da,不存在信号肽,含有2个跨膜结构.Unigene A03231以单拷贝形式存在于基因组,在0~30 h内其表达水平随茉莉酸甲酯诱导表现为先升高后降低.构建了该基因的原核表达菌株,初步优化了其在大肠杆菌BL21中的诱导表达条件为:诱导温度28℃,IPTG浓度0.6 mmol/L,诱导时间8 h.为深入分析该基因的催化功能奠定了基础.

Cloning and Expression of Wheat Heat-shock Protein 60 (HSP60) Gene in E.coli

[Objective]The aim was to construct the wheat heat-shock protein 60 （HSP60） gene in prokaryotic expression vector and express HSP60 efficiently in E.coli. [Method]According to the wheat HSP60 gene sequence in GenBank,a pair of primers P1/P2 were designed and synthesized. The wheat HSP60 gene fragment was amplified from the wheat RNA by RT-PCR and inserted into bacterial expression vector of pGEX-4T-1. The construct of pGEX-4T-1-HSP60 was subsequently transformed into E.coli BL21. [Result]The construct of pGEX-4T-1-HSP60 was verified by restriction endonuclease digestion and sequenced. Compared with the sequences of wheat HSP60 genes in GenBank,homology accounted to 100%. Expression of the GST-HSP60 fusion protein was induced with IPTG. Its molecular weight was about 90 kD. The result was identified by electrophoresis of SDS-PAGE. Expression of the protein bands was consistent with the expected size. [Conclusion]The recombinant prokaryotic expression vector in pGEX-4T-1-HSP60 was constructed successfully and expressed stably in E.coli BL21. This will lay the foundation for further study on the functions of the protein and its mechanism.

Cloning and Sequence Analysis of a Novel Cold-Adapted Lipase Gene from Strain lip35 (Pseudomonas sp.)

A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.

Cloning and Characterization of karmoisin Homologue Gene(Nlka) in Two Brown Planthopper Strains with Different Eye Colors

The brown planthopper （BPH）, Nilaparvata lugens, is a destructive insect pest of rice throughout Asia. Different from brown-eye color wild type, BPH also has red-eye color mutation phenotype. As a visible genetic marker, the red-eye mutant in BPH is a valuable material. To reveal the eye color mutation mechanism, a karmoisin homologue gene （named as Nlka） was cloned from BPH. And karmoisin is always deemd as a xanthommatin-related gene in other insects, encoding phenoxazinone synthetase （PHS）. Nlka is consisted of 7 exons and encodes a protein with 502 amino acids （NIKA）. NIKA showed high amino acid identities with its insect homologues （48.8%-51.8%）. Nlka transcripts can be detected at all the developmental stages and in all tissues tested, including egg, nymph, adult, body wall, ovary, fat body, midgut and Malpighian tubule. However, no constant In/Del or non-synonymous mutation was observed between the mutant and the wild type strains. Quantitative real-time PCR experiment also showed that Nlka transcript level had no significant differences between them. These results indicated that Nlka is not the target gene causing the red-eye color mutation phenotype of BPH. Through the second structure and motif analysis, the present study also showed that all the proteins deduced from the karmoisin genes in insects may be members of monocarboxylate transporters （MCTs） rather than PHSs.

Phylogenetic diversity of planktonic bacteria in the Chukchi Borderland region in summer

Planktonic bacteria are abundant in the Chukchi Borderland region. However, little is known about their di- versity and the roles of various bacteria in the ocean. Seawater samples were collected from two stations K2S and K4S where sea ice was melting obviously. The analysis of water samples with fluorescence in situ hybridization （FISH） showed that DMSP-degrading bacteria accounted for 13% of the total bacteria at the station K2S. No aerobic anoxygenic phototrophic （AAP） bacteria were detected in both samples. The bacterial communities were characterized by two 16S rRNA gene clone libraries. Sequences fell into four major lineages of the domain Bacteria, including Proteobacteria （Alpha, Beta and Gamma subclasses）, Bac- teroidetes, Actinobacteria and Firmicutes. No significant difference was found between the two clone li- braries. SAR11 and Rhodobacteraceae clades of Alphaproteobacteria and Pseudoalteromonas of Gammapro- teobacteria constituted three dominant fractions in the clone libraries. A total of 191 heterotrophic bacterial strains were isolated and 76% showed extracellular proteolytic activity. Phylogenetic analysis reveals that the isolates fell into Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The most common genus in both the bacterial isolates and protease-producing bacteria was Pseudoalteromonas. UniFrac data showed suggestive differences in bacterial communities between the Chukchi Borderland and the northern Bering Sea.