New approaches to increase the expression and stability of cloned foreign genes in Escherichia coli.

A family of expression plasmid vectors were constructed by fusing the strong P2 promoter of the rrnB gene of Escherichia coli (coding for ribosomal RNA) to the lac operator, thereby eliminating regulatory sequences from the rrnB gene and placing the expression under lac repressor control. This promoter proved to be stronger in vivo than the well-known consensus tac promoter, and its strength could be further increased by converting the sequence to consensus. The stability of the recombinant proteins could be increased by fusion to various lengths of the N-terminal end of beta-galactosidase, or by inserting a synthetic oligonucleotide, coding for heptathreonine. A new method was developed for the stabilization of recombinant plasmids without antibiotic selection, based on the presence of an essential gene on the plasmid and its absence from the chromosome. The application of this method is illustrated by the example of a plasmid expressing human proinsulin.     

Bacteriophage T7 RNA polymerase-directed, inducible and tissue-specific over-expression of foreign genes in transgenic plants

A widely applicable bacteriophage T7 RNA polymerase-directed, tissue-specific and inducible over-expression of foreign genes in transgenic plants was developed. This was achieved through the simultaneous transformation of a modified T7 RNA polymerase to specifically transcribe the foreign gene placed under the control of T7 expression signals. The T7 RNA polymerase recognized the chimeric uidA gene integrated randomly into tobacco and rice genomes. Results from the use of six different promoters with different tissue specificities indicated that the recombinant protein was expressed at a several-fold (3-10-fold) higher level when compared with transgenes expressed directly under the control of these tissue-specific promoters. An important feature of the T7 system in plants was the near-uniform expression in the independently transformed plants, in contrast with the large variations observed in transgene expression under the direct control of plant promoters. In addition, our results demonstrated the application of the T7 system in the regulation of transgene expression through chemically inducible mechanisms. This versatility of controlled and regulated expression offers a powerful tool that could be used in various programmes in plant biotechnology and genomic studies.     

Restricting expression prolongs expression of foreign genes introduced into animals by retroviruses

If foreign genes are ubiquitously expressed in mice using a viral vector, expression is abrogated by CD8(+) cells in 2 to 4 weeks. However, if the expression of the genes is confined to skeletal muscle cells, the CD8(+) T-cell response is much weaker and expression is maintained for more than 6 weeks. These data show that restricting the expression of foreign genes to skeletal muscle cells and presumably to other cells that are inefficient at antigen presentation can prolong the expression of a foreign gene product.     

Myb genes from Hordeum vulgare: tissue-specific expression of chimeric Myb promoter/Gus genes in transgenic tobacco

The structures of the three Myb -related genes Hv1 , Hv5 and Hv33 from barley were determined. They contain a single intron located in the second repeat unit of the Myb -related domain. By analogy to the animal MYB oncoproteins this conserved region of the gene product was shown by filter-binding experiments to exhibit nucleic acid-binding activity. Tobacco plants transgenic for chimeric Myb promoter/ Gus genes express the enzyme in a developmentally controlled and tissue-specific manner. During germination and early stages of plant growth, GUS activity is seen in the root cap and adjacent meristematic tissue. At later stages of plant development, GUS activity is predominantly observed in the shoot apical meristem, the roots and the nodal regions of the stem. Within the stem at stages of secondary growth, Myb promoters are active in defined cell types. In the internode low GUS activity is displayed by the innermost cell layer of the cortex, the starch sheath, that surrounds the vascular cylinder of secondary xylem and phloem tissue, as well as in pith rays originating from vascular cambium initials. In the nodal region Myb promoter-controlled Gus expression is mainly confined to the abaxial starch sheath of the leaf trace, to the branch traces and to internal strands of primary phloem. It is suggested that in addition to their activity in meristematically active plant tissues Myb genes are expressed in conductive tissues that are closely associated with vascular bundles.     

The methods to generate transgenic animals and to control transgene expression

Transgenic animals have been used for years to study gene function and to create models for the study of human diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white. Improvement of animal production by transgenesis is still in infancy.Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA. The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes.All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.     

A promoter directing high level expression in pistils of transgenic plants

The promoter of the potato (Solanum tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase, was cloned. Various fragments of the SK2-promoter, from 1 kb down to 0.23 kb in length, were fused to the GUS reporter gene. Chimaeric SK2 promoter-GUS fusion constructs were transformed into potato by Agrobacterium tumefaciens-mediated transformation. The SK2-GUS transgenic potato plants exhibited a highly specific GUS activity in the pistil. Expression in the pistil was shown to be developmentally regulated. In addition to the GUS activity in pistils, transgenic plants also showed a much weaker ectopic expression in anthers. In other tissues no systematic expression was detectable. All SK2 promoter fragments analysed conferred pistil-specific expression without significant qualitative or quantitative differences, demonstrating that the regulatory elements mediating this expression pattern are located within a 230 bp SK2 promoter fragment. The SK2 promoter may be used to engineer high levels of expression in pistils of transgenic plants.     

Characterization of genes with tissue-specific differential expression patterns in Populus

Like many plants, Populus has an evolutionary history in which several, both recent and more ancient, genome duplication events have occurred and, therefore, constitutes an excellent model system for studying the functional evolution of genes. In the present study, we have focused on the properties of genes with tissue-specific differential expression patterns in poplar. We identified the genes by analyzing digital expression profiles derived by mapping 90,000+ expressed sequence tags (ESTs) from 18 sources to the predicted genes of Populus. Our sequence analysis suggests that tissue-specific differentially expressed genes have less diverged paralogs than average, indicating that gene duplication events is an important event in the pathway leading to this type of expression pattern. The functional analysis showed that genes coding for proteins involved in processes of functional importance for the specific tissue(s) in which they are expressed and genes coding for regulatory or responsive proteins are most common among the differentially expressed genes, demonstrating that the expression differentiation process is under strong selective pressure. Thus, our data supports a model where gene duplication followed by gene specialization or expansion of the regulatory and responsive networks leads to tissue-specific differential expression patterns. We have also searched for clustering of genes with similar expression pattern into gene-expression neighborhoods within the Populus genome. However, we could not detect any major clustering among the analyzed genes with highly specific expression patterns. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Dual promoter expression system with insulator ensures a stringent tissue-specific regulation of two reporter genes in the transgenic fish

The precise control of spatiotemporal expression of target genes is crucial when establishing transgenic animals, and the introduction of genes for fluorescent marker proteins is inevitable for accelerating research at molecular levels. To assist this, we constructed a novel dual promoter expression vector for two independent reporter genes, green fluorescent protein (GFP) and red fluorescent protein (mCherry). Their expression is designed under the control of two distinct tissue-specific promoters, e.g. zebrafish cardiac muscle-specific promoter (cmlc2) and medaka skeletal muscle-specific promoter (myl2) derived from the myosin light chain 2 genes, and they are placed in a head-to-head orientation. After microinjecting the dual promoter expression vector into fertilized eggs of medaka, the developing fish embryos and the resulting transgenic fish lines showed strong GFP signal in the whole body (skeletal muscle) and mCherry signal in the heart (cardiac muscle). However, weak GFP signal was observed in the heart, indicating a leakiness of the skeletal muscle promoter. To improve the stringency of dual promoter expression, we inserted two chicken-derived insulators, e.g. tandem copies of the core sequence (250 bp) of cHS4 (5′-hypersensitive site-4 chicken beta-globin insulator), in the boundary of two promoters. The dual promoter expression vector with insulator now ensured the stringent tissue-specific expression in the transgenic fish lines. Thus, our dual promoter expression system with insulator is compatible to the conventional IRES and fused reporter gene systems and will be an alternative method to produce the transgenic fishes.     

外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change

It is increasingly clear that alterations in Na+-K+-ATPase kinetics to fit the demands in specialized cell types is vital for the enzyme to execute its different physiological roles in diverse tissues. In addition to tissue-dependent expression of isoforms of the conventional subunits, alpha and beta, auxiliary FXYD proteins appear to be essential regulatory components. The present study identified genes belonging to this family in Atlantic salmon by analysis of expressed sequence tags. Based on the conserved domain of these small membrane proteins, eight expressed FXYD isoforms were identified. Phylogenetic analysis suggests that six isoforms are homologues to the previously identified FXYD2, FXYD5, FXYD6, FXYD7, FXYD8, and FXYD9, while two additional isoforms were found (FXYD11 and FXYD12). Using quantitative PCR, tissue-dependent expression of the different isoforms was analyzed in gill, kidney, intestine, heart, muscle, brain, and liver. Two isoforms were expressed in several tissues (FXYD5 and FXYD9), while six isoforms were distributed in a discrete manner. In excitable tissues, two isoforms were highly expressed in brain (FXYD6 and FXYD7) and one in skeletal muscle (FXYD8). In osmoregulatory tissues, one isoform was expressed predominantly in gill (FXYD11), one in kidney (FXYD2), and one equally in kidney and intestine (FXYD12). Expression of several FXYD genes in kidney and gill differed between fresh water and seawater salmon, suggesting significance during osmoregulatory adaptations. In addition to identifying novel FXYD isoforms, these studies are the first to show the tissue dependence in their expression and modulation by salinity in any teleosts.     

New type of polyubiquitin-like genes with intein-like autoprocessing domains

Genome analysis of ciliates identified a new type of polyubiquitin-like genes. These contain tandem repeats of ubiquitin-like domains interspersed with autocatalytic intein-like domains. Inteins and related protein domains post-translationally process their own precursor proteins by protein-splicing, cleavage and ligation reactions. The structure of these polyubiquitin-like genes suggests their precursor products undergo maturation and conjugation in cis. This novel gene structure also illustrates the genetic modularity of ubiquitin-like and intein-like domains. Our suggested autoprocessing of ubiquitin-like polyproteins is a new potential general way for controlling protein functions.