The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

Hot spot for Tn1000 insertions in cloned repressor gene of the L phage

Insertions of Tn1000 into a cloned fragment of the L-phage genome comprising the repressor gene were prepared. Repressor activities produced by plasmids with insertions were assayed in vivo. As a result, the repressor gene was localized within 0.5 kb near one end of the cloned fragment. Transposon insertions were nonrandomly clustered within the repressor gene and in its close vicinity. An analysis of supercoiled plasmid DNA with the nuclease S1 revealed no distortion of the secondary structure of DNA in this region.     

A site-targeted recombinant nuclease probe of DNA structure

A genetically engineered lac repressor/T7 endonuclease hybrid protein shows repressor-like binding toward restriction fragments carrying the lac operator. In addition, fragments carrying the operator near a particular pBR322 sequence are cleaved into specific products. Cleavage occurs at precise positions within that sequence, is independent of the orientation of the operator, is inhibited by isopropyl-1-thio-beta-D-galactopyranoside, and is observed when the target is separated from the operator by at least as few as 150 and as many as 240 base pairs. This evidence indicates that the hybrid protein is a site-directed nuclease that requires the following two structural elements for activity: the lac operator and a target. Repressor-like binding directs the enzyme to the operator and nearby single-stranded DNA targets. The discovery of an unusual target in a well-studied DNA sequence is evidence of the power of this approach for probing unusual structures in non-supercoiled duplex DNA.     

Two-hybrid system for characterization of protein-protein interactions in E. coli

The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeric protein containing the 434 repressor DNA binding domain. In the second hybrid, a second test protein, the prey, is fused downstream of a chimeric protein with the DNA binding specificity of the lambda repressor. Reporters were designed to express cat and lacZ under the control of a low-affinity lambda operator. At low expression levels, lambda repressor hybrids weakly repress the reporter genes. A high-affinity operator recognized by 434 repressor was placed nearby, in a position that does not yield repression by 434 repressor alone. If the test proteins interact, the 434 hybrid bound to the 434 operator stabilizes the binding of the lambda repressor hybrid to the lambda operator, causing increased repression of the reporter genes. Reconstruction experiments with the fos and jun leucine zippers detected protein-protein interactions between either homodimeric or heterodimeric leucine zippers.     

人工锌指核酸酶介导的基因组定点修饰技术

锌指核酸酶(ZFN)由锌指蛋白(ZFP)结构域和Fok I核酸内切酶的切割结构域人工融合而成,是近年来发展起来的一种可用于基因组定点改造的分子工具。ZFN可识别并结合特定的DNA序列,并通过切割这一序列的特定位点造成DNA的双链断裂(DSB)。在此基础上,人们可以对基因组的特定位点进行各种遗传操作,包括基因打靶、基因定点插入、基因修复等,从而能够方便快捷地对基因组实现靶向遗传修饰。这种新的基因组定点修饰方法的突出优势是适用性好,对物种没有选择性,并且可以在细胞和个体水平进行遗传操作。文章综述了ZFN技术的研究进展及应用前景,重点介绍ZFN的结构与作用机制、现有的靶点评估及锌指蛋白库的构建与筛选方法、基因组定点修饰的策略,以及目前利用这一技术已成功实现突变的物种及内源基因,为开展这一领域的研究工作提供参考。     

Hot spot for Tn1000 insertions in cloned reprossor gene of the L phage

Insertions of Tn1000 into a cloned fragment of the L-phage genome comprising the repressor gene were prepared. Repressor activities produced by plasmids with insertions were assayed in vivo. As a result, the repressor gene was localized within 0.5 kb near one end of the cloned fragment. Transposon insertions were nonrandomly clustered within the repressor gene and in its close vicinity. An analysis of supercoiled plasmid DNA with the nuclease S1 revealed no distortion of the secondary structure of DNA in this region.     

Interplay of Protein and DNA Structure Revealed in Simulations of the lac Operon

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.     

Electron microscopic study of the repressor of bacteriophage λ and its interaction with operator DNA

The structure of purified phage λ repressor has been examined by high resolution electron microscopy. The repressor molecule appears predominantly as a tetramer of about 95 Å × 120 Å. We have proposed a model to account for the variety of aspects seen on the electron micrographs. Spreading DNA without protein film and use of uranyl formate staining allowed the simultaneous visualization of the DNA and the structure of the repressor molecule bound to it. Mapping the positions of λ repressor bound to whole λ DNA shows preferential binding to the region containing the operators. At high resolution multiple binding of repressor to the operator can be demonstrated. Depending on the amount of repressor present, rows of one to four repressor tetramers are seen on the DNA, confirming the model of the operator containing four binding sites for repressor. The bound repressor can consequently protect against nuclease digestion of operator pieces of approximately 30, 57, 87 and 111 base-pairs. The isolated operator appears in the electron microscope as short double-stranded DNA fragments which can be shown to rebind repressor.     

In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain.

Both the amino-terminal and the carboxy-terminal domain of the LexA repressor have been purified using the LexA protein autodigestion reaction at alkaline pH, which leads to the same specific products as the physiological RecA-catalyzed proteolysis of repressor. We show by circular dichroism (c.d) that, upon non-specific binding to DNA, the purified amino-terminal domain induces a very similar if not identical conformational change of the DNA as does the entire repressor. The positive c.d. signal increases approximately 3-fold if the DNA lattice is fully saturated with protein. Further, the amino-terminal domain of the LexA protein binds specifically to the operator of the recA gene, producing qualitatively the same effects on the methylation pattern of the guanine bases by dimethylsulfate as the entire repressor, consisting of a methylation inhibition effect at four distal operator guanines and a slight enhancement at the central bases. The spacing between these contacts suggests that LexA does not bind to the operator along the same face of the DNA helix. As shown by c.d. studies the amino-terminal domain harbours a substantial amount of residues in alpha-helical conformation, a prerequisite for DNA recognition via a helix--turn--helix structural motif as proposed for many other regulatory proteins.     

Comparison of the structures of operator DNA free and in complex with lambda repressor

The structures of operator DNA unbound and in complex with lambda repressor protein are compared. The conformation of the left 10 base pairs of a lambda right regulatory operator DNA sequence has been previously determined in solution using nuclear magnetic resonance techniques and the structure of a homologous left regulatory operator DNA bound to lambda repressor N-terminal domain had been previously solved using X-ray crystallography. The DNA adopts an overall linear B-form DNA both in the absence and presence of lambda repressor. Superimpositioning of the DNA structures reveals small differences between them that are due to the binding of protein and not to the different techniques used for their determination.     

DNA-induced conformational changes in bacteriophage 434 repressor

Although bacteriophage 434 repressor binds to its specific DNA sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator DNA. Our results suggest that both specific and non-specific DNA induce conformational changes in repressor that lead to formation of repressor dimers. The repressor conformational changes induced by DNA occur at concentrations much lower than those needed for binding of repressor, suggesting that the alternative conformations of repressor persist even if the protein is not in direct contact with DNA. Hence, DNA acts in a "catalytic" fashion to induce a steady-state amount of an alternative repressor conformation that has an enhanced affinity for its specific binding site. These findings suggest that the repressor conformer induced by non-specific DNA is the form of the repressor that is optimized for searching for DNA binding sites along non-specific DNA. Upon finding a binding site, the repressor protein undergoes an additional conformational change that allows it to "lock-on" to its specific site.     

在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100%不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。     

在杂交作物分子育种中利用普通核雄性不育的几个可能途径

由一对隐性基因控制的普通核雄性不育性遗传方式能够满足对植物最佳雄性不育系选育的要求,是水稻等作物杂种优势利用的极好遗传工具。如果能解决其不育系繁殖问题,将优于现有的其他杂种优势利用方式。克隆出普通核雄性不育性的可育基因,通过叶绿体转化,将核雄性不育性可育基因向普通核雄性不育株细胞质转移,创造普通核雄性不育株的保持系;通过种子成熟后表达的启动子;和以位点特异性重组技术为基础的基因开关以及化学诱导启动子的利用,都可能繁殖出100％不育株率的普通核雄性不育系,创造普通核雄性不育性利用的新途径,对植物杂种优势利用产业有十分重要的意义。     

Nuclease sensitivity of the mouse HPRT gene promoter region: differential sensitivity on the active and inactive X chromosomes.

We investigated the conformation of the X-linked mouse hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) promoter region both in chromatin from the active and inactive X chromosomes with DNase I and in naked supercoiled DNA with S1 nuclease. A direct comparison of the chromatin structures of the active and inactive mouse HPRT promoter regions was performed by simultaneous DNase I treatment of the active and inactive X chromosomes in the nucleus of interspecies hybrid cells from Mus musculus and Mus caroli. Using a restriction fragment length polymorphism to distinguish between the active and inactive HPRT promoters, we found a small but very distinct difference in the DNase I sensitivity of active versus inactive chromatin. We also observed a single DNase I-hypersensitive site in the immediate area of the promoter which was present only on the active X chromosome. Analysis of the promoter region by S1 nuclease digestion of supercoiled plasmid DNA showed an S1-sensitive site which maps adjacent to or within the DNase I-hypersensitive site found in chromatin but upstream of the region minimally required for normal HPRT gene expression.     

Price of disorder in the lac repressor hinge helix

The Lac system of genes has been pivotal in understanding gene regulation. When the lac repressor protein binds to the correct DNA sequence, the hinge region of the protein goes through a disorder to order transition. The structure of this region of the protein is well understood when it is in this bound conformation, but less so when it is not. Structural studies show that this region is flexible. Our simulations show this region is extremely flexible in solution; however, a high concentration of salt can help kinetically trap the hinge helix. Thermodynamically, disorder is more favorable without the DNA present.     

The solution structure and dynamics of an Arc repressor mutant reveal premelting conformational changes related to DNA binding.

The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Arc repressor. A backbone rmsd versus the average of 0.37 A was obtained for the well-defined core region. For both Arc-MYL and the wild-type Arc repressor, evidence for a fast equilibrium between a packed ("in") conformation and an extended ("out") conformation of the side chain of Phe 10 was found. In the MYL mutant, the "out" conformation is more highly populated than in the wild-type Arc repressor. The Phe 10 is situated in the DNA-binding beta-sheet of the Arc dimer. While its "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Arc, where the Phe 10 ring contacts the phosphate backbone [Raumann, B. E., et al. (1994) Nature 367, 754-757]. As well as DNA binding, denaturation by urea and high temperatures induces the functionally active "out" conformation. With a repacking of the hydrophobic core, this characterizes a premelting transition of the Arc repressor. The dynamical properties of the Arc-MYL and the wild-type Arc repressor were further characterized by 15N relaxation and hydrogen-deuterium exchange experiments. The increased main chain mobility at the DNA binding site compared to that of the core of the protein as well as the reorientation of the side chain of Phe 10 is suggested to play an important role in specific DNA binding.