The expression of chicken NOV, a member of the CCN gene family, in early stage development

The nephroblastoma overexpressed gene, NOV, is a member of the CCN gene family. We investigated the NOV gene expression pattern in the chicken during early stage embryogenesis. Several embryonic structures showed a distinct expression pattern. The initial expression was detected in Hensen's node (Hamburger and Hamilton stage (HH) 5). The expression was noted in the presumptive notochord and floor plate forming cells. The expression on the left side was more elongated posteriorly, a type of left-right asymmetry. Chicken NOV gene expression in the forming notochord and floor plate was observed until HH 18. The expression was also detected in the ventral area of the mesencephalon and isthmus at HH 14-16.     

Oligonucleotide-based strategies to reduce gene expression

Research on embryonic development and differentiation provides a sensitive, but challenging opportunity to use a variety of techniques designed to modulate gene expression. Changes in the expression of a single gene can alter levels of other genes and provide information on developmentally regulated gene expression pathways. The morphological consequences of altered gene expression can link gene expression to developmental fate. Oligonucleotide-based approaches offer a variety of means to potentially disrupt normal gene expression. The basis for some of these approaches is presented in this review.     

毕赤酵母蛋白表达系统研究进展

选择合适的蛋白表达系统是外源基因能否成功表达的关键.毕赤酵母(Pichia pastoris)蛋白表达系统是近些年来发展起来的一种真核表达系统,与其他表达系统相比,该系统所具有的诸多优势使其研究价值和应用价值越来越广泛,已经成功表达了多种蛋白质.简要综述其特点、表达宿主菌、表达载体以及其元件、外源蛋白的表达及其影响因素等方面的基础研究和最新进展.     

A hormone response element in the human apolipoprotein CIII (ApoCIII) enhancer is essential for intestinal expression of the ApoA-I and ApoCIII genes and contributes to the hepatic expression of the two linked genes in transgenic mice

We have generated transgenic mice carrying wild-type promoters of the human apolipoprotein A-I (apoA-I)-apoCIII gene cluster or promoters mutated in their hormone response elements. The wild-type cluster directed high levels of apoA-I gene expression in liver and intestine, moderate expression in kidney, and low to minimal expression in other tissues. It also directed high levels of chloramphenicol acetyltransferase (CAT) expression (used as a reporter for the apoCIII gene) in liver, low levels in intestine and kidney, and no expression in other tissues. Mutations in the apoCIII promoter and enhancer abolished the intestinal and renal expression of the apoA-I gene, reduced hepatic apoA-I expression by 80%, and abolished CAT expression in all tissues. A similar pattern of expression was obtained by mutations in the apoCIII enhancer alone. Mutations in the proximal apoA-I promoter reduced by 85% hepatic and intestinal apoA-I expression and did not affect CAT expression. The findings suggest that a hormone response element within the apoCIII enhancer is essential for intestinal and renal expression of apoA-I and apoCIII genes and also enhances hepatic expression. The hormone response elements of the proximal apoA-I promoter or the apoCIII enhancer can promote independently low levels of hepatic and intestinal expression of the apoA-I gene in vivo.     

A suite of parallel vectors for baculovirus expression

The expression of proteins using recombinant baculoviruses is a mature and widely used technology. However, some aspects of the technology continue to detract from high throughput use and the basis of the final observed expression level is poorly understood. Here, we describe the design and use of a set of vectors developed around a unified cloning strategy that allow parallel expression of target proteins in the baculovirus system as N-terminal or C-terminal fusions. Using several protein kinases as tests we found that amino-terminal fusion to maltose binding protein rescued expression of the poorly expressed human kinase Cot but had only a marginal effect on expression of a well-expressed kinase IKK-2. In addition, MBP fusion proteins were found to be secreted from the expressing cell. Use of a carboxyl-terminal GFP tagging vector showed that fluorescence measurement paralleled expression level and was a convenient readout in the context of insect cell expression, an observation that was further supported with additional non-kinase targets. The expression of the target proteins using the same vectors in vitro showed that differences in expression level were wholly dependent on the environment of the expressing cell and an investigation of the time course of expression showed it could affect substantially the observed expression level for poorly but not well-expressed proteins. Our vector suite approach shows that rapid expression survey can be achieved within the baculovirus system and in addition, goes some way to identifying the underlying basis of the expression level obtained.     

A comprehensive analysis of the developmental and tissue-specific expression of the isoactin multigene family in the rat.

The present study represents the first comprehensive analysis of isoactin gene expression in the developing rat. Our results clearly demonstrate that the developmental and tissue-specific expression of the actin multigene family is a highly integrated and complex process involving a variety of regulatory paradigms. The distinct temporal patterns of expression reported in this study indicate that there are three key phases in the regulation of expression of the actin multigene family during development. These include early embryonic development, late fetal development, and early postnatal development. The specific spatial patterns of expression observed in this study demonstrate that the expression of the actin multigene family is much more permissive than previously reported. This permissive expression includes a wide range of "ectopic" expression of the striated muscle isoactins as well as an extended expression of the alpha-smooth muscle isoactin. These findings expand our current understanding of the expression of the actin multigene family in development and provide a fundamental basis for future studies directed at investigating these processes.     

The patterns of gene expression in the tomato shoot apical meristem.

In this paper, we describe the synthesis of a cDNA library from the vegetative shoot apical meristem and the analysis of clones selected from it. Using in situ hybridization, we characterized the patterns of expression of these genes in the tomato shoot apical meristem, as well as the patterns obtained from other sources. The results from the analysis of 15 cDNAs indicated the following six main patterns of gene expression in the shoot apical meristem: overall expression, zero expression, expression limited to the epidermis, expression excluded from the epidermis, punctate expression, and expression elevated in the flanks of the meristem. The patterns observed and the nature and number of the genes showing these patterns necessitate a reinterpretation of the models of meristem structure and function. In particular, the data suggest a compartmentation within the shoot apical meristem based on the spatial expression of particular subsets of genes. This paper also reports on the specific and precise criteria essential for the correct identification of meristem-specific gene expression. The data give new insight into the molecular organization of the shoot apical meristem and provide the framework for a detailed dissection of the factors controlling this organization.     

Sequence-based factors influencing the expression of heterologous genes in the yeast Pichia pastoris--A comparative view on 79 human genes

High yield expression of heterologous proteins is usually a matter of "trial and error". In the search of parameters with a major impact on expression, we have applied a comparative analysis to 79 different human cDNAs expressed in Pichia pastoris. The cDNAs were cloned in an expression vector for intracellular expression and recombinant protein expression was monitored in a standardized procedure and classified with respect to the expression level. Of all sequence-based parameters with a possible influence on the expression level, more than 10 were analysed. Three of those factors proved to have a statistically significant association with the expression level. Low abundance of AT-rich regions in the cDNA associates with a high expression level. A comparatively high isoelectric point of the recombinant protein associates with failure of expression and, finally, the occurrence of a protein homologue in yeast is associated with detectable protein expression. Interestingly, some often discussed factors like codon usage or GC content did not show a significant impact on protein yield. These results could provide a basis for a knowledge-oriented optimisation of gene sequences both to increase protein yields and to help target selection and the design of high-throughput expression approaches.     

Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression.

We report the identification of four additional genes of the Autographa californica nuclear polyhedrosis virus involved in expression from a late baculovirus promoter in transient expression assays. Three of these genes, p35, 39K, and p47, have been previously described. The role of the p35 gene product in late gene expression may be related to its ability to block apoptosis, since two other baculovirus genes also known to block apoptosis, Cp-iap and Op-iap, were able to functionally replace p35 in the transient expression assay. The requirement for p47 in this assay confirms its role in late gene expression, a role previously established by characterization of a temperature-sensitive mutant of p47, while the requirement for 39K may be related to its known association with the virogenic stroma. The fourth gene identified as a late expression factor gene, lef-11, was located immediately upstream of 39K and is predicted to encode a 13-kDa polypeptide. When plasmids containing these 4 genes were cotransfected with plasmids containing the 14 genes previously identified as late gene expression factors, the level of expression from the late capsid promoter was similar to that observed for a library of clones representing the entire viral genome. The genes provided by these 18 plasmids thus represent the viral genes necessary and sufficient to support expression from a late viral promoter in this transient expression assay.     

团头鲂生长相关基因在不同发育时期生长轴组织的表达分析

为了探讨生长相关基因对团头鲂生长发育的调控, 研究采用Real-time PCR的方法定量分析了团头鲂6个生长相关基因在其不同生长发育阶段(3、6、12月龄)相关组织(脑、肝脏、肌肉)的表达情况, 并比较了这些基因在生长快和慢两个群体的表达差异. 结果显示: GHRs基因在肝脏与肌肉中的表达量高于脑, 在6月龄表达量高于3月龄与12月龄, 生长快群体中的表达量高于生长慢群体(P0.05); IGFs基因在三个组织中均有表达, 肝脏表达量最高, 生长快群体中的表达量高于生长慢群体(P0.05). MSTN a与MSTN b基因在组织中表达模式存在差异, MSTN a在肌肉中高表达, MSTN b主要在脑与肝脏中表达. HCL聚类结果表明: 除了MSTN a基因外, 其他5个基因在生长差异的两个群体中表达量均分别聚为一支. 不同时期组织表达聚类结果表明, 除了3月龄肝脏与12月龄肌肉组织, 6个生长相关基因在不同时期的同一组织中的表达模式存在相似性. Pearson相关分析显示: GHRs与IGFs呈正相关, MSTN a基因与GHR 2、IGFs基因呈负相关, 相同基因在两个群体中呈极显著相关(P0.01).       

An intron control region differentially regulates expression of thyroid hormone receptor beta2 in the cochlea, pituitary, and cone photoreceptors

The Thrb gene, encoding thyroid hormone receptor beta (TRbeta), serves key roles in endocrine regulation and the development of the senses of hearing and color vision. The versatile functions of this gene depend upon its expression of distinct receptor isoforms by differential promoter activation. The TRbeta2 isoform has a particularly specialized distribution including in the anterior pituitary and cochlea. TRbeta2 is also found in immature cone photoreceptors where it has a unique role in programming the expression pattern of opsin photopigments that mediate color vision. Given the importance of precise, tissue-specific expression for the function of TRbeta2, we investigated the genomic control elements that direct this expression in vivo using lacZ reporter transgenes in mice. The TRbeta2 promoter region is sufficient for cochlear expression, whereas a complex intron control region is necessary for pituitary and retinal expression. In the retina, the intron region directs peak expression in the embryo in postmitotic, immature cones. The retinal control region is further subdivided into domains that specify and amplify expression, respectively, indicating that timely, cone-specific expression reflects an integrated response to complex signals. The mammalian Thrb gene has therefore incorporated several mechanisms into a multifunctional intron control region that regulates developmental induction of the distant promoter. This specialized genomic organization underlies the unique expression pattern and functions of TRbeta2.     

Expression of complement components coincides with early patterning and organogenesis in Xenopus laevis

The complement system is the central component of innate immunity and an important player in the adaptive immunity of vertebrates. We analyzed the expression patterns of several key members of the complement cascade during Xenopus development. We found extensive expression of these molecules already during gastrula/early neurula stage. Remarkably, several genes also showed an organ-specific expression pattern during early organogenesis. Early expression is notable for two different expression patterns in the neuroectoderm. In one group, there is early strong neural plate and neural precursor expression. This is the case of properdin, C1qA, C3 and C9. The second pattern, seen with C1qR and C6, is noteworthy for its expression at the periphery of the neural plate, in the presumptive neural crest. Two genes stand out for their predominantly mesodermal expression. C3aR, the message for the cognate receptor for C3 in the complement cascade, is expressed at the same time as C3, but in a complementary, reciprocal pattern in the mesoderm. C1qA expression also predominates in somites, pronephros, visceral mesoderm and ventral blood islands. Finally, several genes are characterized by later expression in developing organs. C1qR displays a reticular pattern consistent with expression in the developing vasculature. The late expression of C1qA and C3bC4b is strongest in the pronephros. Finally, the expression of properdin in the hindbrain and in the developing lens are novel findings. The expression patterns of these molecules suggest that these components of the complement system may have in Xenopus a so far undefined developmental role.     

Multi-Host Expression System for Recombinant Production of Challenging Proteins

Recombinant production of complex eukaryotic proteins for structural analyses typically requires a profound screening process to identify suitable constructs for the expression of ample amounts of properly folded protein. Furthermore, the evaluation of an optimal expression host has a major impact on protein yield and quality as well as on actual cost of the production process. Here we present a novel fast expression system for multiple hosts based on a single donor vector termed pFlp-Bac-to-Mam. The range of applications of pFlp-Bac-to-Mam comprises highly efficient transient transfection of HEK293-6E in serum-free suspension culture and subsequent large-scale production of challenging proteins expressing in mg per Liter level using either the baculoviral expression vector system or stable CHO production cell lines generated by Flp-mediated cassette exchange. The success of the multi-host expression vector to identify the optimal expression strategy for efficient production of high quality protein is demonstrated in a comparative expression study of three model proteins representing different protein classes: intracellular expression using a fluorescent protein, secretion of a single-chain-Fv-hIgG1Fc fusion construct and production of a large amount of highly homogeneous protein sample of the extracellular domain of a Toll-like receptor. The evaluation of the production efficiency shows that the pFlp-Bac-to-Mam system allows a fast and individual optimization of the expression strategy for each protein class.     

Large-scale analysis of gene expression profiles

The accumulation of DNA microarray data has now made it possible to use gene expression profiles to analyse expression data. A gene expression profile contains the expression data for a given gene over various samples, and can be contrasted with an expression signature, which contains the expression data for a single sample. Gene expression profiles are most revealing when samples are grouped appropriately, either by standard clinical or pathological categories or by categories discovered through cluster analysis techniques. Expression profiles can exist at various levels of abstraction, yielding information across various tissues or across diseases within a particular tissue. Hypothesis tests may be applied to expression profiles on a large scale to identify candidate genes of interest.     

T7 RNA聚合酶基因表达系统在鱼腥藻7120中构建及hG-CSF的表达

外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7 RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7 RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7 RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Western blotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7 RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系统使hG-CSF基因的表达量提高2倍。该表达系统将为蓝藻基因工程的应用提供更优的工具,将促进蓝藻作为底盘细胞在合成生物学等领域的发展。     

Wrestling with pleiotropy: genomic and topological analysis of the yeast gene expression network

The vast majority (>95%) of single-gene mutations in yeast affect not only the expression of the mutant gene, but also the expression of many other genes. These data suggest the presence of a previously uncharacterized "gene expression network"--a set of interactions between genes which dictate gene expression in the native cell environment. Here, we quantitatively analyze the gene expression network revealed by microarray expression data from 273 different yeast gene deletion mutants.(1) We find that gene expression interactions form a robust, error-tolerant "scale-free" network, similar to metabolic pathways(2) and artificial networks such as power grids and the internet.(3-5) Because the connectivity between genes in the gene expression network is unevenly distributed, a scale-free organization helps make organisms resistant to the deleterious effects of mutation, and is thus highly adaptive. The existence of a gene expression network poses practical considerations for the study of gene function, since most mutant phenotypes are the result of changes in the expression of many genes. Using principles of scale-free network topology, we propose that fragmenting the gene expression network via "genome-engineering" may be a viable and practical approach to isolating gene function.