乳糖操纵子介导的半乳糖苷酶在转基因动物中的应用前景

细菌的乳糖操纵子可以在哺乳动物中调控基因的表达,修饰阻抑物基因和操纵基因可调控阻抑物诱导能力及其对操纵基因的亲和力,更好的适应高等动物的内环境。半乳糖苷酶对乳糖操纵子系统有正向调控作用,利用乳糖操纵子在转基因动物中可诱导性调控半乳糖苷酶基因的表达,能有效的提高转基因动物利用半乳糖苷、吸收营养物质的能力。以下从乳糖操纵子的结构功能、乳糖阻抑物功能活性的基因调控、操纵基因的功能以及其在哺乳动物的应用现状四个方面,结合半乳糖苷酶的生理功能和其在转基因动物中应用两个方面进行综述,对乳糖操纵子介导的半乳糖苷酶在转基因动物中的应用效果和前景进行了分析探讨。     

Salmonella typhimurium has two homologous but different umuDC operons: cloning of a new umuDC-like operon (samAB) present in a 60-megadalton cryptic plasmid of S. typhimurium.

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The DNA which can restore UV mutability to a umuD44 strain and to a umuC122::Tn5 strain of E. coli has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the cloned DNA potentially encoded proteins with calculated molecular weights of 15,523 and 47,726 and was an analog of the E. coli umuDC operon. We have termed this cloned DNA the samAB (for Salmonella mutagenesis) operon and tentatively referred to the umuDC operon of S. typhimurium LT2 (C. M. Smith, W. H. Koch, S. B. Franklin, P. L. Foster, T. A. Cebula, and E. Eisenstadt, J. Bacteriol. 172:4964-4978, 1990; S. M. Thomas, H. M. Crowne, S. C. Pidsley, and S. G. Sedgwick, J. Bacteriol. 172:4979-4987, 1990) as the umuDCST operon. The samAB operon is 40% diverged from the umuDCST operon at the nucleotide level. Among five umuDC-like operons so far sequenced, i.e., the samAB, umuDCST, mucAB, impAB, and E. coli umuDC operons, the samAB operon shows the highest similarity to the impAB operon of TP110 plasmid while the umuDCST operon shows the highest similarity to the E. coli umuDC operon. Southern hybridization experiments indicated that (i) S. typhimurium LT2 and TA1538 had both the samAB and the umuDCST operons and (ii) the samAB operon was located in a 60-MDa cryptic plasmid. The umuDCST operon is present in the chromosome. The presence of the two homologous but different umuDC operons may be involved in the poor mutability of S. typhimurium by UV and chemical mutagens.     

Lateral transfer of the lux gene cluster

The lux operon is an uncommon gene cluster. To find the pathway through which the operon has been transferred, we sequenced the operon and both flanking regions in four typical luminous species. In Vibrio cholerae NCIMB 41, a five-gene cluster, most genes of which were highly similar to orthologues present in Gram-positive bacteria, along with the lux operon, is inserted between VC1560 and VC1563, on chromosome 1. Because this entire five-gene cluster is present in Photorhabdus luminescens TT01, about 1.5 Mbp upstream of the operon, we deduced that the operon and the gene cluster were transferred from V. cholerae to an ancestor of Pr. luminescens. Because in both V. fischeri and Shewanella hanedai, luxR and luxI were found just upstream of the operon, we concluded that the operon was transferred from either species to the other. Because most of the genes flanking the operon were highly similar to orthologues present on chromosome 2 of vibrios, we speculated that the operon of most species is located on this chromosome. The undigested genomic DNAs of five luminous species were analysed by pulsed-field gel electrophoresis and Southern hybridization. In all the species except V. cholerae, the operons are located on chromosome 2.     

Cloning and characterisation of a novel ompB operon from Vibrio cholerae 569B

The ompB operon of Vibrio cholerae 569B has been cloned and fully sequenced. The operon encodes two proteins, OmpR and EnvZ, which share sequence identity with the OmpR and EnvZ proteins of a variety of other bacteria. Although the order of the ompR and envZ genes of V. cholerae is similar to that of the ompB operon of E. coli, S. typhimurium and X. nematophilus, the Vibrio operon exhibits a number of novel features. The structural organisation and features of the V. cholerae ompB operon are described.     

Functions of the duplicated hik31 operons in central metabolism and responses to light, dark, and carbon sources in Synechocystis sp. strain PCC 6803

There are two closely related hik31 operons involved in signal transduction on the chromosome and the pSYSX plasmid in the cyanobacterium Synechocystis sp. strain PCC 6803. We studied the growth, cell morphology, and gene expression in operon and hik mutants for both copies, under different growth conditions, to examine whether the duplicated copies have the same or different functions and gene targets and whether they are similarly regulated. Phenotype analysis suggested that both operons regulated common and separate targets in the light and the dark. The chromosomal operon was involved in the negative control of autotrophic events, whereas the plasmid operon was involved in the positive control of heterotrophic events. Both the plasmid and double operon mutant cells were larger and had division defects. The growth data also showed a regulatory role for the chromosomal hik gene under high-CO(2) conditions and the plasmid operon under low-O(2) conditions. Metal stress experiments indicated a role for the chromosomal hik gene and operon in mediating Zn and Cd tolerance, the plasmid operon in Co tolerance, and the chromosomal operon and plasmid hik gene in Ni tolerance. We conclude that both operons are differentially and temporally regulated. We suggest that the chromosomal operon is the primarily expressed copy and the plasmid operon acts as a backup to maintain appropriate gene dosages. Both operons share an integrated regulatory relationship and are induced in high light, in glucose, and in active cell growth. Additionally, the plasmid operon is induced in the dark with or without glucose.     

The gene yjfQ encodes the repressor of the yjfR-X regulon (ula), which is involved in L-ascorbate metabolism in Escherichia coli

Mutations in yjfQ allowed us to identify this gene as the regulator of the operon yjfS-X (ula operon), reported to be involved in L-ascorbate metabolism. Inactivation of this gene renders constitutive the expression of the ula operon, indicating that YjfQ acts as a repressor. We also demonstrate that this repressor regulates the nearby yjfR gene, which in this way constitutes a regulon with the ula operon.     

In silico evolved lac operons exhibit bistability for artificial inducers, but not for lactose

Bistability in the lac operon of Escherichia coli has been widely studied, both experimentally and theoretically. Experimentally, bistability has been observed when E. coli is induced by an artificial, nonmetabolizable, inducer. However, if the lac operon is induced with lactose, the natural inducer, bistability has not been demonstrated. We derive an analytical expression that can predict the occurrence of bistability both for artificial inducers and lactose. We find very different conditions for bistability in the two cases. Indeed, for artificial inducers bistability is predicted, but for lactose the condition for bistability is much more difficult to satisfy. Moreover, we demonstrate that in silico evolution of the lac operon generates an operon that avoids bistability with respect to lactose, but does exhibit bistability with respect to artificial inducers. The activity of this evolved operon strikingly resembles the experimentally observed activity of the operon. Thus our computational experiments suggest that the wild-type lac operon, which regulates lactose metabolism, is not a bistable switch. Nevertheless, for engineering purposes, this operon can be used as a bistable switch with artificial inducers.     

Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl-tRNA synthetase operon.

Among mutants of Escherichia coli resistant to p-fluorophenylalanine (PFP) were some with constitutive expression of the phenylalanine biosynthetic operon (the pheA operon). This operon is repressed in the wild type by phenylalanine. The mutation in three of these mutants mapped in the aroH-aroD region of the E. coli chromosome at 37 min. A plasmid bearing wild-type DNA from this region restored p-fluorophenylalanine sensitivity and wild-type repression of the pheA operon. Analysis of subclones of this plasmid and comparison of its restriction map with published maps indicated that the mutations affecting regulation of the pheA operon lie in the structural genes for phenylalanyl-tRNA synthetase, pheST, probably in pheS. Thus, the pheST operon has a role in the regulation of phenylalanine biosynthesis, the most likely being that wild-type phenylalanyl-tRNA synthetase maintains a sufficient intracellular concentration of Phe-tRNA(Phe) for attenuation of the pheA operon in the presence of phenylalanine. A revised gene order for the 37-min region of the chromosome is reported. Read clockwise, the order is aroD, aroH, pheT, and pheS.     

Vulnerabilities in Yersinia pestis caf operon are unveiled by a Salmonella vector

During infection, Yersinia pestis uses its F1 capsule to enhance survival and cause virulence to mammalian host. Since F1 is produced in large quantities and secreted into the host tissues, it also serves as a major immune target. To hold this detrimental effect under proper control, Y. pestis expresses the caf operon (encoding the F1 capsule) in a temperature-dependent manner. However, additional properties of the caf operon limit its expression. By overexpressing the caf operon in wild-type Salmonella enterica serovar Typhimurium under a potent promoter, virulence of Salmonella was greatly attenuated both in vitro and in vivo. In contrast, expression of the caf operon under the regulation of its native promoter exhibited negligible impairment of Salmonellae virulence. In-depth investigation revealed all individual genes in the caf operon attenuated Salmonella when overexpressed. The deleterious effects of caf operon and the caf individual genes were further confirmed when they were overexpressed in Y. pestis KIM6+. This study suggests that by using a weak inducible promoter, the detrimental effects of the caf operon are minimally manifested in Y. pestis. Thus, through tight regulation of the caf operon, Y. pestis precisely balances its capsular anti-phagocytic properties with the detrimental effects of caf during interaction with mammalian host.