基因捕捉及其在植物基因分离和功能基因组学上的应用

基因捕捉是一种报告基因的随机整合技术。基因捕捉系统已成为分离基因、鉴定基因功能的重要手段。基因捕捉(gene traps)包括增强子捕捉(enhancer trap)、启动子捕捉(promoter trap)和基因捕捉(gene trap),通称为基因捕捉(gcne traps)。在增强子捕捉中,报告基因与一个基本启动子融合,这个启动子不能使报告基因表达,但可被临近的增强子激活。在启动子捕捉和基因捕捉中,报告基因的启动子被去除,融合基因只有以正确的方向插入到转录单元内才能表达。对基因捕捉系统的结构特征、构建方法、应用范围、研究现状和应用前景等作了系统论述,并对有关问题进行了讨论。     

Construction of a synthetic variant of the bacteriophage f1 gene V by assembling oligodeoxy-nucleotides corresponding to only one strand of DNA

A simple and widely applicable procedure for constructing synthetic variants of a gene, involving the synthesis of only one strand of DNA, has been developed. The method is suited for cases in which a cloned DNA with a sequence related to the gene to be constructed is available. First, a heteroduplex DNA which is single-stranded throughout the region of interest is made. This single-stranded region is then used as a template to correctly align and allow ligation of synthetic oligos corresponding to the entire gene. To favor the replication of the strand encoding the synthetic gene, a template strand containing some substitutions of deoxyuridine for deoxythymidine is used. This procedure was used to construct a synthetic bacteriophage f1 gene V which differs from the wild-type (wt) gene at 45 positions out of 298. The synthetic gene was designed to include nine restriction sites without altering the sequence of the encoded DNA-binding protein. The gene construction was found to be very efficient, and about 40 % of the resulting plasmids contained the desired synthetic gene. The synthetic gene was found to be fully active and could substitute for the wt gene in bacteriophage f1.     

Preservation of a pseudogene by gene conversion and diversifying selection

Interlocus gene conversion is considered a crucial mechanism for generating novel combinations of polymorphisms in duplicated genes. The importance of gene conversion between duplicated genes has been recognized in the major histocompatibility complex and self-incompatibility genes, which are likely subject to diversifying selection. To theoretically understand the potential role of gene conversion in such situations, forward simulations are performed in various two-locus models. The results show that gene conversion could significantly increase the number of haplotypes when diversifying selection works on both loci. We find that the tract length of gene conversion is an important factor to determine the efficacy of gene conversion: shorter tract lengths can more effectively generate novel haplotypes given the gene conversion rate per site is the same. Similar results are also obtained when one of the duplicated genes is assumed to be a pseudogene. It is suggested that a duplicated gene, even after being silenced, will contribute to increasing the variability in the other locus through gene conversion. Consequently, the fixation probability and longevity of duplicated genes increase under the presence of gene conversion. On the basis of these findings, we propose a new scenario for the preservation of a duplicated gene: when the original donor gene is under diversifying selection, a duplicated copy can be preserved by gene conversion even after it is pseudogenized.     

Plasmid-specified FemABX-like immunity factor in Staphylococcus sciuri DD 4747

A plasmid from Staphylococcus sciuri DD 4747 had three open reading frames: a replication gene, an N-acetylmuramyl-l-alanine amidase-like gene, and a gene similar to the lysostaphin endopeptidase resistance gene (epr/lif). The epr-like gene was introduced into S. aureus RN4220; the recombinant strain was more resistant to lysostaphin endopeptidase and its cell wall peptidoglycan contained more serines and fewer glycines than the parental strain with the shuttle vector alone. Based on both its function and its similarity to femAB, this gene is a member of the femABX-like immunity gene family. Furthermore, this is the first example of a femABX-like immunity gene that is not linked to the gene for the bacteriolytic enzyme against which it specifies immunity.     

Evolutionary significance of gene expression divergence

Recent large-scale studies of evolutionary changes in gene expression among mammalian species have led to the proposal that gene expression divergence may be neutral with respect to organismic fitness. Here, we employ a comparative analysis of mammalian gene sequence divergence and gene expression divergence to test the hypothesis that the evolution of gene expression is predominantly neutral. Two models of neutral gene expression evolution are considered: 1-purely neutral evolution (i.e., no selective constraint) of gene expression levels and patterns and 2-neutral evolution accompanied by selective constraint. With respect to purely neutral evolution, levels of change in gene expression between human-mouse orthologs are correlated with levels of gene sequence divergence that are determined largely by purifying selection. In contrast, evolutionary changes of tissue-specific gene expression profiles do not show such a correlation with sequence divergence. However, divergence of both gene expression levels and profiles are significantly lower for orthologous human-mouse gene pairs than for pairs of randomly chosen human and mouse genes. These data clearly point to the action of selective constraint on gene expression divergence and are inconsistent with the purely neutral model; however, there is likely to be a neutral component in evolution of gene expression, particularly, in tissues where the expression of a given gene is low and functionally irrelevant. The model of neutral evolution with selective constraint predicts a regular, clock-like accumulation of gene expression divergence. However, relative rate tests of the divergence among human-mouse-rat orthologous gene sets reveal clock-like evolution for gene sequence divergence, and to a lesser extent for gene expression level divergence, but not for the divergence of tissue-specific gene expression profiles. Taken together, these results indicate that gene expression divergence is subject to the effects of purifying selective constraint and suggest that it might also be substantially influenced by positive Darwinian selection.     

From gene trees to species trees II: species tree inference by minimizing deep coalescence events

When gene copies are sampled from various species, the resulting gene tree might disagree with the containing species tree. The primary causes of gene tree and species tree discord include incomplete lineage sorting, horizontal gene transfer, and gene duplication and loss. Each of these events yields a different parsimony criterion for inferring the (containing) species tree from gene trees. With incomplete lineage sorting, species tree inference is to find the tree minimizing extra gene lineages that had to coexist along species lineages; with gene duplication, it becomes to find the tree minimizing gene duplications and/or losses. In this paper, we present the following results: 1) The deep coalescence cost is equal to the number of gene losses minus two times the gene duplication cost in the reconciliation of a uniquely leaf labeled gene tree and a species tree. The deep coalescence cost can be computed in linear time for any arbitrary gene tree and species tree. 2) The deep coalescence cost is always not less than the gene duplication cost in the reconciliation of an arbitrary gene tree and a species tree. 3) Species tree inference by minimizing deep coalescence events is NP-hard.     

Problems and paradigms: Induction mechanism of a single gene molecule: Stochastic or deterministic?

A new field of gene expression regulation research is emerging that has previously been overlooked. This new area is concerned with distinguishing the expression of a single gene from the averaged expression of many gene copies within the cell population. This paper reviews research focused on individual genes in inducible gene expression systems. The main experimental strategy is to measure the gene expression level of a single cell containing a single reporter gene molecule. In contrast to the commonly held belief, gene induction is found to be stochastic under certain conditions. The possible mechanisms and implications are discussed.     

Insertional gene synthesis, a novel method of assembling consecutive DNA sequences within specific sites in plasmids. Construction of the HIV-1 tat gene.

The construction of the HIV-1 tat gene using a novel method termed insertional gene synthesis (IGS) is described. IGS is used to assemble a gene or any DNA sequence in a stepwise manner within a plasmid containing a single stranded DNA phage origin of replication. The IGS method is based upon consecutive targeted insertions of long DNA oligonucleotides (greater than 100 bases) within the plasmid by oligonucleotide-directed mutagenesis. IGS therefore involves synthesis of only a few oligonucleotides corresponding to one strand of a gene. Furthermore, the gene is synthesized directly adjacent to bacterial gene regulatory sequences for direct expression. Using this approach, the 261 bp tat gene was assembled in three successive cycles adjacent to the lac promoter in the pEMBL-derivative, pKH125. The 15 kD tat protein was produced from this synthetic gene in E. coli upon IPTG induction. However, it was necessary to tightly control the expression of tat by including the lac I gene directly within the tat expression vector.     

The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production

The balhimycin biosynthetic gene cluster of the glycopeptide producer Amycolatopsis balhimycina includes a gene (orf1) with unknown function. orf1 shows high similarity to the mbtH gene from Mycobacterium tuberculosis. In almost all nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters, we could identify a small mbtH-like gene whose function in peptide biosynthesis is not known. The mbtH-like gene is always colocalized with the NRPS genes; however, it does not have a specific position in the gene cluster. In all glycopeptide biosynthetic gene clusters the orf1-like gene is always located downstream of the gene encoding the last module of the NRPS. We inactivated the orf1 gene in A. balhimycina by generating a deletion mutant. The balhimycin production is not affected in the orf1-deletion mutant and is indistinguishable from that of the wild type. For the first time, we show that the inactivation of an mbtH-like gene does not impair the biosynthesis of a nonribosomal peptide.     

The evolution of the novel Sdic gene cluster in Drosophila melanogaster

The origin of new genes and of new functions for existing genes are fundamental processes in molecular evolution. Sdic is a newly evolved gene that arose recently in the D. melanogaster lineage. The gene encodes a novel sperm motility protein. It is a chimeric gene formed by duplication of two other genes followed by multiple deletions and other sequence rearrangements. The Sdic gene exists in several copies in the X chromosome, and is presumed to have undergone several duplications to form a tandemly arrayed gene cluster. Given the very recent origin of the gene and the gene cluster, the analysis of the composition of this gene cluster represents an excellent opportunity to study the origin and evolution of new gene functions and the fate of gene duplications. We have analyzed the nucleotide sequence of this region and reconstructed the evolutionary history of this gene cluster. We found that the cluster is composed by four tandem copies of Sdic; these duplicates are very similar but can be distinguished by the unique pattern of insertions, deletions, and point mutations in each copy. The oldest gene copy in the array has a 3' exon that has undergone accelerated diversification, and also shows divergent regulatory sequences. Moreover, there is evidence that this might be the only gene copy in the tandem array that is transcribed at a significant level, expressing a novel sperm-specific protein. There is also a retrotransposon located at the 3' end of each Sdic gene copy. We argue that this gene cluster was formed in the last two million years by at least three tandem duplications and one retrotransposition event.     

转基因植物中外源基因的沉默及应对策略

随着转基因技术在作物育种领域的应用,转基因植物中外源基因表达量低的现象较为普遍。导致外源基因表达量低的主要原因是基因沉默。外源基因沉默可分为转录水平的基因沉默和转录后水平的基因沉默。如何应对基因沉默,提高外源基因的表达量,是转基因技术发展亟待解决的问题。     

Ethnic variation of genetic disease: roles of drift for recessive lethal genes.

Using Wright's distribution of gene frequencies for a recessive lethal gene, a method is given to analyze the probability that any particular gene frequency is greater than a given threshold gene frequency. The method is introduced to analyze the plausibility of drift for explaining observed data.     

Use of beta-glucuronidase reporter gene for gene expression analysis in turfgrasses

The beta-glucuronidase (GUS) gene has been successfully used as a reporter gene in innumerable number of plant species. The functional GUS gene produces blue coloration in plants upon integration into the plant genome. Because of the ease it provides to analyze the gene expression (as no expensive equipment is needed), GUS gene is surely plant biotechnologist's first choice as a reporter gene. The turfgrass family contains the world's most economically important horticultural crops. There is a world-wide drive for genetic modification of grasses due to its huge economic importance. GUS gene can be transiently or stably expressed in grasses for the purpose of promoter analysis and to study tissue-specific and developmental gene expression. This paper summarizes the use of GUS gene for transient and stable expression studies in various turfgrass species.