New Tool for Metabolic Pathway Engineering in Escherichia coli: One-Step Method To Modulate Expression of Chromosomal Genes

A simple and highly efficient method was developed to produce a library of Escherichia coli clones that express a particular chromosomal gene at a wide range of expression levels. The basic strategy was to replace all or part of the upstream region of a coding sequence containing the elements involved in its expression (promoter, operator, gene coding for a regulator, ribosome binding site, and start codon) with a PCR-generated library of expression cassettes.     

Cloning of the Atlantic salmon (Salmo salar) estrogen receptor-alpha gene

A cDNA clone encoding most of an Atlantic salmon (Salmo salar) estrogen receptor (ER) was obtained from a liver cDNA library and the remainder of the coding sequence from the gene was isolated from a genomic library. Sequence comparisons showed that the cloned gene represents ER-alpha. Expression of the ER-alpha gene in male and female salmon parr was analysed by RT-PCR. Highest expression was found in brain and liver, with lower levels of ER-alpha mRNA present in all other tissues tested. There was little difference in expression of ER-alpha between male and female.     

Molecular cloning, primary structure and disruption of the structural gene of aldolase from Saccharomyces cerevisiae

A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.     

Coding sequence and growth regulation of the human vimentin gene.

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.     

Complete characterization of the gene coding for the Epstein-Barr virus major membrane antigen gp 220/340 and selective expression of a secreted form of gp 220

The gene which encodes the Epstein-Barr gp 220/340 was inserted into a eukaryotic expression vector. A cDNA clone corresponding to the mature mRNA coding for gp 220 was isolated from an Epstein-Barr virus cDNA library and inserted in the same expression vector, enabling us to identify the precise location of the intron within the gp 220/340 coding sequence. The recombinant plasmids direct the expression of membrane proteins detected by immunofluorescence experiments using an anti-gp 220/340 monoclonal antibody in transfected human cells. The region of the gp 220/340 gene encoding the domain for membrane anchorage was removed from the two recombinant plasmids and the sequence containing the intron produced secreted forms of both truncated gp 220 and gp 340 whereas only the former was obtained with the intronless sequence.     

Characterization of a barley gene coding for an α-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.     

Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity

Genomic DNA from the silk moth Antheraea pernyi bearing the gene of a pheromone binding protein has been isolated from a partial genomic library using specific cDNA probes. The DNA spans 3.5 kilobases, contains three exons and two intervening sequences that interrupt the protein coding region of the gene. A DNA fragment of a second gene was isolated and the complete primary structure of a corresponding cDNA clone was unravelled. The expression of two different genes, giving rise to different pheromone binding proteins, implies a more specific function of these proteins than was hitherto assumed.     

Identification of regulatory regions of the putative tumor suppressor gene DMBT1

The lack of expression of the putative tumor suppressor gene DMBT1 has been described in gliomas, lung carcinomas, and other malignancies. To investigate the mechanisms regulating DMBT1 expression we have screened a human genomic library with a 5' cDNA probe and identified a fragment of approximately 3 kb upstream of the proposed coding sequence of DMBT1. This fragment contains subregions that may up- or down-regulate the expression of a reporter gene. These findings may help to elucidate the mechanisms regulating the alternative splicing and the tissue specific expression of DMBT1.     

A study of the cloned human erythropoietin gene expression in mammalian cells in vitro

The human gene coding for the principal factor of erythroid cells differentiation, erythropoietin, has been isolated from the genomic phage library using an oligonucleotide probe for the gene. The construction of series of plasmids carrying the erythropoietin gene under the control of various regulatory elements is reported. Efficiency of the erythropoietin gene expression was estimated by testing of the biologically active erythropoietin in conditioned media 48 h after transient transfection of COS 1 and CHO cell lines.     

Macronuclear structure of the G surface antigen gene of Paramecium primaurelia and direct expression of its repeated epitopes in Escherichia coli.

The gene encoding the G surface antigen of Paramecium primaurelia was cloned from a macronuclear DNA library by a screening procedure involving differential hybridization with cDNA probes synthesized from polyadenylated RNAs of cells expressing one of two alternate antigens. S1 mapping experiments and sequencing of the cloned DNA and the mRNA showed that the cloned gene corresponded to the high-molecular-weight mRNA that had been indirectly identified as that of the G surface antigen. Because the genetic code of Paramecium spp. is different from the "universal" code, this mRNA cannot be correctly translated in vitro; direct proof that it encoded the antigenic determinants of this protein was therefore obtained through expression of fragments of the coding sequence in Escherichia coli by using the expression vector lambda gt11. Studies on the structure of this gene revealed that the central part of the coding sequence contained at least five tandem repeats of 222 base pairs, encoding immunogenic domains of the protein. We also showed that, like other surface antigen genes of trypanosomes and paramecia, this gene lay next to a chromosome end and that no rearrangement of its immediate genomic environment was associated with its expression.     

Localization of the gene encoding steroid hydroxylase cytochrome P-450 from Rhizopus nigricans inside a HindIII fragment of genomic DNA

The gene encoding steroid inducible cytochrome P450 of Rhizopus nigricans ATCC 6227b has been found inside a HindIII fragment of the genomic DNA by hybridization with a partial length cDNA probe. The latter was isolated by immunoscreening a cDNA library prepared in the lambda gt11 expression system and identified on the basis of inducibility and sequence analysis. The nucleotide sequence of the cDNA probe revealed a coding sequence for the heme binding segment characteristic of the P450 gene family.     

Assignment of the human 8.5 H gene to chromosome 5, region 5q35

The human 8.5 H probe was isolated from a human cerebellum cDNA library with a probe corresponding to the coding region of the murine 8.5 M cDNA. This cDNA isolated from a murine cDNA library constructed from newborn cerebral hemispheres was selected because of its strong expression in embryonic neurons. Consequently the corresponding human gene could be a candidate for hereditary neurodegenerative diseases. The human 8.5 H gene was assigned by somatic hybrid analysis to chromosome 5; this chromosome contains the gene(s) for spinal muscular atrophy (SMA), a group of heritable degenerative diseases that selectively affect the anterior horn motor neuron of the spinal cord. The localization by in situ hybridation of 8.5 H on 5q35 excluded the possibility that this gene is identical to SMA. The SMA gene(s) was (were) known, from linkage analysis, to be in a region (5q11.2-q13.3) very distant from 5q35.     

Regional assignment of the gene coding for a human Graves' disease autoantigen to 10q21.3–q22.1

A cDNA coding for a human Graves' disease autoantigen (hGT) has been isolated from a thyroid expression library. Using this cDNA as a probe, the gene for hGT, previously assigned to chromosome 10, has been further localized to 10q21.3–q22.1 by non-isotopic in situ hybridization.     

A plasmid system for optimization of Fab' production in Escherichia coli: importance of balance of heavy chain and light chain synthesis

We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene. A plasmid system that enables random combination of these variant SP coding regions is used to construct optimized Fab' expression plasmids. These small plasmid libraries facilitated selection of optimal Fab' expression plasmids and resulted in increases of periplasmic yield, up to 580 mgL(-1) from E. coli fermentations and will enable rapid variable region subcloning and selection of future Fab(') expression plasmids.     

人EYA基因家族的克隆、表达及转录活性分析

采用PCR方法从人组织或细胞cDNA文库中扩增Eya基因家族的完整编码序列,将所扩增的基因克隆到带FLAG标签的真核表达载体上,转染人胚肾293T细胞,Western blot鉴定Eya基因家族的表达。检测Eya基因及其与Six基因共转染对MEF3-Luc转录活性的影响。经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确,通过Western blot实验证明Eya基因家族真核表达载体构建成功。荧光素酶活性测定显示Eya能协同Six激活肌细胞生成素的表达。本研究进一步证实Eya是Six的转录共激活因子,能提高肌细胞增强因子的活性。