Molecular evolution of paclitaxel biosynthetic genes <Emphasis Type="Italic">TS</Emphasis> and <Emphasis Type="Italic">DBAT</Emphasis> of <Emphasis Type="Italic">Taxus</Emphasis> species

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.     

Overexpression of a novel imprinted gene,<Emphasis Type="Italic">PEG10</Emphasis>, in human hepatocellular carcinoma and in regenerating mouse livers

Many of the promising applications of the microarray technology are pertinent to identifying abnormalities in gene expression that contribute to malignant progression. We developed a bioinformatics tool to identify differentially expressed genes in human hepatocellular carcinoma (HCC). This involved the construction of a liver EST database (http://lestdb.nhri.org.tw) and in silico verification of differentially expressed genes with a human hepatoma microarray database. The stringency of the search was reinforced with a statistical analysis. A novel imprinted gene,Paternally Expressed 10 (PEG10) was identified as having an elevated level of expression in the majority of the HCC samples and was also induced to express during G₂/M phase of regenerating mouse liver. Ectopic expression ofPEG10 in 293T cells affects cell cycle progression. PEG10 is distributed in the cytosol and associates with the nuclear membrane. This is the first time that an imprinted gene has been found to reexpress in both human HCC and in the regenerating mouse liver. This result indicates that the induction of the paternally imprinted gene may play an important role during liver regeneration or carcinogenesis of the human hepatocyte. Understanding the molecular basis of the abnormal imprinting ofPEG10 will shed new light on the process that leads to liver disease.     

Characterization of Tn10d-Cam: a transposition-defective Tn10 specifying chloramphenicol resistance

Summary We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.     

Developmental expression of the amphioxus <Emphasis Type="Italic">Tbx1</Emphasis>/<Emphasis Type="Italic">10</Emphasis> gene illuminates the evolution of vertebrate branchial arches and sclerotome

We have isolated an amphioxus T-box gene that is orthologous to the two vertebrate genes, Tbx1 and Tbx10, and examined its expression pattern during embryonic and early larval development. AmphiTbx1/10 is first expressed in branchial arch endoderm and mesoderm of developing neurulae, and in a bilateral, segmented pattern in the ventral half of newly formed somites. Branchial expression is restricted to the first three branchial arches, and disappears completely by 4 days post fertilization. Ventral somitic expression is restricted to the first 10–12 somites, and is not observed in early larvae except in the most ventral mesoderm of the first three branchial arches. No expression can be detected by 4 days post fertilization. Integrating functional, phylogenetic and expression data from amphioxus and a variety of vertebrate model organisms, we have reconstructed the early evolutionary history of the Tbx1/10 subfamily of genes within the chordate lineage. We conclude that Tbx1/10-mediated branchial arch endoderm and mesoderm patterning functions predated the origin of neural crest, and that ventral somite specification functions predated the origin of vertebrate sclerotome, but that Tbx1 was later co-opted during the evolution of developmental programs regulating branchial neural crest and sclerotome migration.Edited by M. Akam     

穗醋栗MYB10基因克隆结构分析及功能验证

为探究花色苷合成相关转录因子MYB10在不同颜色穗醋栗果实着色差异的分子机理,通过cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)法从果实花青素含量有较大差异的黑穗醋栗(Ribes nigrum L.)、红穗醋栗(Ribes rubrum L.)和白穗醋栗(Ribes album L.)中分别克隆出MYB10基因,分别命名为RnMYB10 (KY786107)、RrMYB10 (KY786108)和RaMYB10(MW660848)。系统发育分析表明,RnMYB10和RrMYB10在进化上具有同源性。实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)结果表明:黑穗醋栗各时期果实中MYB10表达量均高于红穗醋栗且远远高于白穗醋栗。随着果实直径加大颜色加深,RnMYB10和RrMYB10表达量呈现先上升后下降的趋势(在果实转色程度75%时达到最大值),RaMYB10表达量极低,几乎无表达。过表达RnMYB10和RrMYB10的拟南芥呈现紫色叶柄和叶片,过表达RaMYB10的拟南芥无明显变化。说明...     

Mutational analysis of the -10 region from the Mycobacterium tuberculosis lipF promoter

The promoter driving expression of the Mycobacterium tuberculosis gene lipF (Rv3487c) had previously been identified as being upregulated by acidic stress. Subsequently a 59 base pair (bp) acid inducible minimal promoter region was isolated in which a putative -10 region was identified. In this study we use mutational analysis to investigate the -10 region of the lipF promoter. Mutations within this region lead to a dramatic decrease of promoter activity, while a mutation outside of this region does not affect promoter activity.     

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1 μM of l-phenylalanine (6.2 g L⁻¹). Addition of 1 μM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7 μg L⁻¹), which was not detected in the untreated control culture. Supplementation with 100 μM of l-phenylalanine together with 100 μM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3 μg L⁻¹ (control untreated culture) to 568.2 μg L⁻¹, the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8 μg L⁻¹) was observed when the medium was supplemented with 100 μM of the precursor in combination with methyl jasmonate.Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.     

Taxane Production in Suspension Culture of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">Media</Emphasis> var. <Emphasis Type="Italic">Hicksii</Emphasis> Carried Out in Flasks and Bioreactor

Paclitaxel and 10-deacetylbaccatin III (10-DAB III) were produced in suspension cultures of Taxus × media var. Hicksii grown in shake-flasks and in a 7-l bioreactor reaching, in the bioreactor, 4.4 mg l⁻¹ (on day 14) and 37.5 mg l⁻¹ (on day 11). In shake-flasks the highest total content of paclitaxel and 10-DAB III was 7.3 mg l⁻¹ (on day 4) and 8.8 mg l⁻¹ (on day 18). Phenylalanine, at 0.05 mM, increased paclitaxel accumulation in cells cultivated in bioreactor and flasks 30-fold and 9-fold (from 0.02 mg l⁻¹ to 0.6 mg l⁻¹ and to 0.2 mg l⁻¹, respectively). The 10-DAB III content in cells from flasks was increased from 0.4 mg l⁻¹ to 1.6 mg l⁻¹.     

An Investigation on the Expression Level of Interleukin 10 (IL-10) in the Healthy and Mastitic Holstein Cows and the Bioinformatics Analysis of Nucleosome Profile

Cytokines are immune regulators that play an essential role in regulating immune response against various infections. The present study focused on the possible association between the expression level of Interleukin 10 (IL-10) in blood and milk samples of 25 healthy and 25 mastitic cows in Fars province, Iran, using a quantitative real-time PCR assay. The experimental groups were categorized according to the number of calvings. The expression level of IL-10 was significantly higher in the blood and milk samples of mastitic cows compared to the healthy ones. Concomitant to increasing the number of calving, a numerical elevation in the expression of IL-10 in blood was observed (P?IL-10 gene revealed the promoter, exon-intron regions, and nucleosome profile. The nucleosome occupancy site was finally predicted using NUPOP software. Our result indicated that the promoter was not exactly placed in the nucleosome region, which was finally aimed to predict the position and expression of IL-10 gene in the mastitic cows.     

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q₁₀ (CoQ₁₀), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ₁₀ precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ₁₀ was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ₁₀-producing E. coli strain resulted in an increase in CoQ₁₀ content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ₁₀ content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.     

Increased taxane accumulation in callus cultures of Taxus cuspidata and Taxus × media by some elicitors and precursors

In Taxus cuspidata callus, vanadyl sulfate (10 mg l^–1) induced a high (146 g g^–1 dry wt) production of 10-deacetylbaccatin III in comparison to 7 g g^–1 dry wt of the control. The content of paclitaxel in this species increased from 16 g g^–1 to 74 g g^–1 dry wt when 20 mg phenylalanine l^–1 was used. In T. media, p-aminobenzoic acid induced the highest content of 10-deacetylbaccatin III (481 g g^–1 dry wt) versus 181 g g^–1 in the control. Paclitaxel increased from 89 to 139 g g^–1 dry wt after adding chitosan (20 mg l^–1) to the cultures.     

Heart specific expression of mouse BMP-10 a novel member of the TGF-β superfamily

Here we report the cloning and expression of murine BMP-10, a novel member of the TGF-β superfamily. In the mouse embryo, BMP-10 expression begins at 9.0 d.p.c. and is restricted to the developing heart. Initially, BMP-10 expression localizes to the trabeculated part of the common ventricular chamber and to the bulbus cordis region. After 12.5 d.p.c., additional BMP-10 expression is seen in the atrial wall. The data presented here suggest that BMP-10 plays an important role in trabeculation of the embryonic heart.     

miR-10b对MCF10A细胞表达N-糖链 及O-糖链的影响

蛋白质糖基化是蛋白质翻译后修饰之一,对蛋白质功能有重要的调节作用,而异常糖基化在肿瘤的发生、发展以及癌细胞转移过程中起到关键作用.MiRNAs在癌症的发生发展过程中同样起到非常关键的作用,但其如何影响糖基化进而在肿瘤恶性转化过程中发挥生物学功能的研究甚少.本文将miR-10b在人正常乳腺上皮细胞MCF10A中过表达,利用糖类相关基因芯片系统筛选了发生显著变化的糖基转移酶;随后利用本实验室建立的N-糖链及O-糖链测定方法,分析糖链水平的表达差异;最后对关键糖基转移酶基因Fut8、MGAT3及OGT通过荧光定量PCR、蛋白质免疫印迹和凝集素免疫印迹进行了验证,为研究miR-10b在乳腺癌中的作用提供更多糖组学方面的理论基础.     

Up‐regulation of miR‐10b‐3p promotes the progression of hepatocellular carcinoma cells via targeting CMTM5

In this study, we investigated how miR‐10b‐3p regulated the proliferation, migration, invasion in hepatocellular carcinoma (HCC) at both in vitro and in vivo levels. CMTM5 was among the differentially expressed genes (data from TCGA). The expression of miR‐10b‐3p and CMTM5 was detected by qRT‐PCR and Western blot (WB). TargetScan was used to acquire the binding sites. Dual‐luciferase reporter gene assay was used to verify the direct target relationship between miR‐10b‐3p and CMTM5. WB analysis proved that miR‐10b‐3p suppressed CMTM5 expression. Furthermore, proliferation, invasion and migration of HCC cells were measured by MTT assay, colony formation assay, transwell assay and wound‐healing assay, respectively. Kaplan‐Meier plotter valued the overall survival of CMTM5. Finally, xenograft assay was also conducted to verify the effects of miR‐10b‐3p/CMTM5 axis in vivo. Up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were detected in HCC tissues and cell lines. CMTM5 was verified as a target gene of miR‐10b‐3p. The overexpression of CMTM5 contributed to the suppression of the proliferative, migratory and invasive abilities of HCC cells. Moreover, the up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were observed to be associated with worse overall survival. Lastly, we have confirmed the carcinogenesis‐related roles of miR‐10b‐3p and CMTM5 in vivo. We concluded that the up‐regulation of miR‐10b‐3p promoted the progression of HCC cells via targeting CMTM5.     

KCTD10 interacts with proliferating cell nuclear antigen and its down‐regulation could inhibit cell proliferation

A novel gene (GenBank accession No. AF113208) named KCTD10 (potassium channel tetramerisation domain‐containing 10) was cloned from our 5300 EST database of human aorta cDNA library. Computational analysis showed that KCTD10 cDNA is 2,638 bp long, encoding 313 amino acids with a proliferating cell nuclear antigen binding motif, mapped to chromosome 12q24.11 with 7 exons, ubiquitously expressed in all 12 tested normal tissues and 7 of 8 tested tumor cell lines from MTN membranes by Northern blot. Nuclear localization of KCTD10 was observed in A549 cells. Yeast two‐hybrid analysis and immunoprecipitation assay showed that KCTD10 can interact with PCNA. In A549 cells, KCTD10 down‐regulation could inhibit cell proliferation, but its over‐expression could not influence cell proliferation. The results suggest that KCTD10 may be associated with DNA synthesis and cell proliferation. J. Cell. Biochem. 106: 409–413, 2009. © 2009 Wiley‐Liss, Inc.