电化学发光在基因检测中的应用

电化学发光基因检测是把电化学发光的高灵敏性和传统分子生物学方法的稳定性结合于一体的一种新型的基因检测技术。与传统的基因检测方法相比,它具有无放射性危害、高灵敏度、操作简便等优点。近年被广泛地应用于核酸序列分析,基因突变分析,遗传病、转基因物种、病毒、微生物等的检测。本文概述了电化学发光的基本原理以及传统的基因检测技术,详细地介绍了电化学发光在当前基因检测中的应用现状,并对其前景作了展望。     

酵母基因中断技术研究进展

综述酵母基因中断技术（gene disruption）自创立以来的不断改进与发展。随着分子生物学技术的更新,酵母基因中断技术已经实现了目标基因的精确缺失、多重中断、无痕中断等,已成为功能基因组学研究和酵母分子育种研究的重要手段之一。     

The organization of the dihydrofolate reductase gene of baby hamster kidney fibroblasts.

Using cloned DNA complementary to mouse dihydrofolate reductase (DHFR) mRNA, the organization of the hamster DHFR gene has been determined in two baby hamster kidney (BHK) cell lines, A5 and B1. A5 cells are highly methotrexate-resistant, containing 200-fold more copies of the DHFR gene than do the parental B1 cells. The DHFR gene has the same organization in A5 and B1 cells, suggesting that it has not been altered by the amplification process. The BHK DHFR gene spans a maximum of 10.7 kb and contains at least three introns. Thus the BHK DHFR gene is much smaller than the mouse DHFR gene, which has a minimum size of 42 kb and at least five introns. This striking size difference is probably due to much smaller introns in the BHK DHFR gene.     

The cloning and expression of the RNAse structural gene of Bacillus intermedius]

The structure gene of extracellular alkaline ribonuclease Bacillus intermedius (binase) has been cloned in E. coli cells in composition of pMT 316 plasmid carrying the inhibitor gene (barstar of barnase--binase structure homologue. The possibility to use such vector has been proved during the barstar action on binase catalytic activity. Using biochemical immunochemical analysis the expression of binase gene in E. coli cells has been confirmed. The recombinant clone E. coli which contains both plasmids simultaneously--carrying gene for barster and for benase has been produced. The given vector is suggested to be used for cloning of inhibitor gene to obtain a viable producer of alkaline intracellular ribonuclease.     

Non-viral gene therapy for Duchenne muscular dystrophy: progress and challenges

Duchenne muscular dystrophy (DMD) is one of the most common lethal, hereditary diseases of childhood. Since the identification of the genetic basis of this disorder, there has been the hope that a cure would be developed in the form of gene therapy. This has yet to be realized, but many different gene therapy approaches have seen dramatic advances in recent years. Although viral-mediated gene therapy has been at the forefront of the field, several non-viral gene therapy approaches have been applied to animal and cellular models of DMD. These include plasmid-mediated gene delivery, antisense-mediated exon skipping, and oligonucleotide-mediated gene editing. In the past several years, non-viral gene therapy has moved from the laboratory to the clinic. Advances in vector design, formulation, and delivery are likely to lead to even more rapid advances in the coming decade. Given the relative simplicity, safety, and cost-effectiveness of these methodologies, non-viral gene therapy continues to have great promise for future gene therapy approaches to the treatment of DMD.     

Zfa is an expressed retroposon derived from an alternative transcript of the Zfx gene.

ZFY, a gene on the Y chromosome encoding a zinc finger protein, has been proposed as a candidate for the human testis determining gene. Sequences related to ZFY, called ZFX, are present on the X chromosome of a wide range of placental mammals. Unlike most mammals the mouse has four genes homologous to ZFY; two on the Y chromosome, Zfy-1 and Zfy-2, an X-linked gene, Zfx, and an autosomal gene, Zfa. We show here that Zfa has arisen recently by retroposition of one of at least three alternatively spliced mRNAs transcribed from the Zfx gene. Zfa is an unusual retroposon in that it has retained an open reading frame and is expressed, although its function may be limited or altered by the presence of a potentially inactivating mutation in the third of its zinc fingers. This mutation must have occurred at the same time or soon after the retroposition event as it is also present in the Zfa gene of Mus spretus. Interestingly the third finger of the M. musculus musculus Zfy-2 gene has also sustained a mutation suggesting that this gene family may be rapidly evolving in mice.     

Molecular methods for environmental monitoring and containment of genetically engineered microorganisms

Plans to introduce genetically engineered microorganisms into the environment has led to concerns over safety and has raised questions about how to detect and to contain such microorganisms. Specific gene sequences, such as lacZ, have been inserted into genetically engineered microorganisms to permit their phenotypic detection. Molecular methods have been developed based upon recovery of DNA from environmental samples and gene probe hybridization to specific diagnostic gene sequences for the specific detection of genetically engineered microorganisms. DNA amplification using the polymerase chain reaction has been applied to enhance detection sensitivity so that single gene targets can be detected. Detection of messenger RNA has permitted the monitoring of gene expression in the environment. The use of reporter genes, such as the lux gene for bioluminescence, likewise has permitted the observation of gene expression. Conditional lethal constructs have been developed as models for containment of genetically engineered microorganisms. Suicide vectors, based upon the hok gene have been developed as model containment systems.     

Characteristics of expression of the tetracycline resistance gene from the plasmid pBR322 in Bacillus subtilis cells

The expression of Tc resistance gene derived from plasmid pBR322 has been studied in Bacillus subtilis cells where this alien gene is not usually expressed. Fragments of Bacillus subtilis chromosome were inserted into the Tc resistance gene promoter region of the hybrid plasmid pGG20 and the expression of this gene was registered. Plasmid pGG20 confers a constitutive mode of Tc resistance in Escherichia coli cells. In contrast, the inducibility of Tc resistance gene expression in Bacillus subtilis cells has been reported. Optimal concentration for the highest inducibility of Tc resistance by the antibiotic has been determined.     

Evolutionary genetics: changed sex determination in honeybees

In several insects and fish, and probably some mammals, the gene controlling the male-female switch has changed during evolution. It now seems that this has also happened in honeybees, where the sex-determining gene has now been shown to be a duplicate of another Hymenopteran sex-determining gene.     

Study of growth hormone gene in non-familial complete somatotropin deficiency

Familial growth hormone deficiency has been often associated to homozygous gene deletions. In this work we have looked for the possible absence of this gene in patients with isolated GH deficiency. The patient genomic DNAs have been digested with two restriction enzymes and hybridized with a 32P labelled growth hormone cDNA. The presence of the growth hormone gene has been proved in the patients. This situation, in which the gene is present but not expressed, might be due to changes in gene regulation or to punctual gene deletions or mutations.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

An evolution revolution provides further revelation

The extent of copy-number variation (CNV) in the human genome has been appreciated only recently. Nevertheless, for almost four decades, gene duplication has been a prevailing hypothesis for evolutionary change. Recently, gene CNV spanning 60 million years of human and primate evolution has been determined enabling lineage-specific gene CNV to be identified. Primate lineage-specific gene CNV studies reveal that almost one third of all human genes exhibit a copy-number change in one or more primate species. Intriguingly, human lineage-specific gene amplification can be correlated to the emergence of human-specific traits such as cognition and endurance running.     

A cardiovascular EST repertoire: progress and promise for understanding cardiovascular disease

The application of expressed sequence tag (EST) technology has proven to be an effective tool for gene discovery and the generation of gene expression profiles. The generation of an EST resource for the cardiovascular system has revealed significant insights into the changes in gene expression that guide heart development and disease. Furthermore, an important genetic resource has been developed for cardiovascular biology that is valuable for data mining and disease gene discovery.     

The mammalian alphaD-globin gene lineage and a new model for the molecular evolution of alpha-globin gene clusters at the stem of the mammalian radiation

We have explored the evolution of the alpha-globin gene family by comparative sequence and phylogenetic analyses of mammalian alpha-globin genes. Our analyses reveal the existence of a new alpha-globin gene lineage in mammals that is related to the alpha(D)-globin genes of birds, squamates and turtles. The gene is located in the middle of the alpha-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the alpha(D)-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of alpha-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5'-zeta2-zeta1-alpha(D)-alpha3-alpha2-alpha1-theta-omega-3'), including seven alpha-like genes and one beta-like globin gene (omega-globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although omega-globin has been lost from the alpha-globin gene cluster of humans.