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。     

How Escherichia coli sets different basal levels in SOS operons

The recA and sfiA genes of Escherichia coli are SOS operons regulated negatively by the LexA repressor. The steady state level of expression of recA is 10-fold higher than that of sfiA, as measured by means of recA::lac and sfiA::lac operon fusions. To study the molecular basis of this difference, we have compared the expression of these two operons in strains in which the concentration of LexA repressor was normal (lexA+), zero (spr amber mutation) or higher than normal (plasmid pJL45, carrying the lexA gene linked to the lac promoter). The results indicate (i) that the recA promoter is about 4 times stronger than the sfiA promoter (as measured in the spr strains), (ii) that neither operon has a physiologically significant level of lexA-independent expression (pJL45 strains), and (iii) that the recA operator has about 2.5 times lower affinity than the sfiA operator for LexA repressor (comparison of lex+ and spr strains). Considering our previous results that the sfiA operon (high operator affinity of LexA) is derepressed very rapidly after inducing treatments and that the recA operon (low operator affinity) is repressed very rapidly when induction is stopped, we conclude that differences in operator affinity do not affect inducibility but serve only to set the basal levels of the different SOS functions.     

Translocations and a balanced polymorphism in a Drosophila population

A Drosophila population cage initiated with equal numbers of two viable II–III translocation homozygotes rapidly evolved into a balanced polymorphism with the two translocations maintained throughout 25 generations at which time the experiment was terminated. The fertility of this population averaged 26%; a control population averaged 90%. The establishment of the polymorphism was interpreted with reference to the reduced viability of the two homozygotes such that their net fitness was considerably less than that of partially sterile double heterozygote. By the incorporation of specific values for the relative fitness of the three genotypes in a computer programme it was possible to simulate the polymorphism.     

Adaptive evolution of the lactose utilization network in experimentally evolved populations of Escherichia coli

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network.     

Fusions of the lac and trp Regions of the Escherichia coli Chromosome

Two classes of strains were studied in which the lac operon is transposed to a chromosomal site close to the tonB and trp loci. The two classes differ in the orientation of the lac region on the chromosome. In both types of strains, tonB mutants were selected in which deletions removing the tonB locus also caused a fusion of the lac and trp regions. The study of the properties of such fusion strains provides information on the control of both the lac and trp operons.     

Contribution of horizontal gene transfer and deletion events to development of distinctive patterns of fimbrial operons during evolution of Salmonella serotypes.

Only certain serotypes of Salmonella represent 99% of all human clinical isolates. We determined whether the phylogenetic distribution of fimbrial operons would account for the host adaptations observed for Salmonella serotypes. We found that three fimbrial operons, fim, lpf, and agf, were present in a lineage ancestral to Salmonella. While the fim and agf fimbrial operons were highly conserved among all Salmonella serotypes, sequence analysis suggested that the lpf operon was lost from many distantly related lineages. As a consequence, the distribution of the lpf operon cannot be explained easily and may be a consequence of positive and negative selection in different hosts for the presence of these genes. Two other fimbrial operons, sef and pef, each entered two distantly related Salmonella lineages and each is present only in a small number of serotypes. These results show that horizontal gene transfer and deletion events have created unique combinations of fimbrial operons among Salmonella serotypes. The presence of sef and pef correlated with serotypes frequently isolated from common domesticated animals.     

Lag in adaptation to lactose as a probe to the timing of permease incorporation into the cell membrane.

If bacteria are incapable of forming and incorporating proteins into the cytoplasmic membranes in all phases of the cell cycle, then not all cells from an asynchronous culture should be capable of growth when switched to a new carbon and energy source whose metabolism requires new membrane function. The transfer of an inducible culture to low lactose provides such a situation since the cells cannot grow unless galactoside permease can function to concentrate the lactose internally. From such experiments, it was concluded that the Y gene product of the lac operon is synthesized, incorporated, and can start functioning in active transport, at any time throughout the bulk of the cell cycle. Not only were the lags before growth re-ensued much shorter than would be expected if the membrane transport capability could only be developed in a small portion of the cycle, but brief pulses of a gratuitous inducer shortened the lags much further. Three types of Escherichia coli ML 30 culture were studied: cells that had exhausted the limiting glucose; cells taken directly from glucose-limited chemostats; and a washed suspension of highly catabolite repressed cells from cultures grown in high levels of glucose and gluconate. The growth studies reported here were performed on-line with a minicomputer. They represent at least an order of magnitude increase in accuracy in estimating growth parameters over previous instrumentation.     

Clonal analysis of the lac operons from Klebsiella M5al and the Lac plasmid (pRE2) from Klebsiella V9A

The chromosomal lac region of the coliform bacterium Klebsiella M5al was cloned into the multicopy plasmid pBR322 to give pHE7 and pHE8. pHE8 contains 12.6 kb of M5al DNA, including its complete lac operon, and pHE7 contains 2.5 kb of M5al DNA and includes the complete lac Y gene and a small segment of lacZ. The M5al operon has the same gene order as the Escherichia coli lac operon. The lac genes of the Lac plasmid of Klebsiella V9A were cloned into pBR322 to give pHE1 and pHE2, of approximately 39 and 43 kb. Both plasmids were unstable in an E. coli RecA-strain, in contrast to the stability of pHE8. Polyacrylamide gel electrophoresis tests suggested that the M5al beta-galactosidase monomer is about 5% longer, i.e. has about 50 more amino acids, than that of the E. coli Z gene. Tests made on the enzymes coded by the lac operons of M5al, another Klebsiella strain (V9A) and its resident Lac plasmid, and several Lac+ Enterobacteria, led to the conclusion that only Escherichia coli among the Enterobacteria contains an active lacA gene.     

Studies on beta-galactoside transport in a Proteus mirabilis merodiploid carrying an Escherichia coli lactose operon

Merodiploid derivatives bearing an F-linked lac operon (i(+), o(+), z(+), y(+), a(+)) from Escherichia coli were prepared from a Proteus mirabilis strain unable to utilize lactose and from a lac deletion strain of E. coli. A suitable growth medium was found in which the episomal element in the P. mirabilis derivative was sufficiently stable to allow induction of the episome-borne lac operon and thus to permit a comparison of the activities and properties of E. coli lac products in the intracellular environments of P. mirabilis and E. coli. In both derivatives the episomal lac operon was shown to be repressed in the absence of inducer. Kinetics of induction with gratuitous inducer (isopropyl-1-thio-beta-d-galactoside) were similar for both beta-galactosidase activity (beta-d-galactoside galactohydrolase, EC 3.4.1.23) and beta-galactoside transport activity in both derivatives, although the ratio of galactoside transport to beta-galactosidase activity was approximately 1.6-fold higher in the E. coli derivative. Comparison of beta-galactosidase and M-protein (lac y gene product)-specific activities indicated coordinate expression of the induced lac operon in both derivatives. Quantitatively, the maximal beta-galactosidase specific activity was two or three times higher for the E. coli derivative. A significant sodium azide inhibition (65% inhibition by 10 mM sodium azide) of lactose permease-mediated transport of o-nitrophenyl-beta-galactoside from an outside region of high concentration to an inside region of very low concentration ("downhill transport") was observed for the P. mirabilis derivative. Identical conditions for the E. coli derivative yielded only about 15% inhibition. Active transport of thiomethyl-beta-galactoside was similar for both derivatives, the major difference being that active transport was more sensitive to azide poisoning in the P. mirabilis derivative. Preliminary examination of the thiomethyl-beta-galactoside derivatives following active transport did not demonstrate the accumulation of a phosphorylated product in either strain but did reveal an unidentified derivative present in the P. mirabilis merodiploid extract which was not detectable in the E. coli merodiploid.     

A global analysis of adaptive evolution of operons in cyanobacteria

Operons are an important feature of prokaryotic genomes. Evolution of operons is hypothesized to be adaptive and has contributed significantly towards coordinated optimization of functions. Two conflicting theories, based on (i) in situ formation to achieve co-regulation and (ii) horizontal gene transfer of functionally linked gene clusters, are generally considered to explain why and how operons have evolved. Furthermore, effects of operon evolution on genomic traits such as intergenic spacing, operon size and co-regulation are relatively less explored. Based on the conservation level in a set of diverse prokaryotes, we categorize the operonic gene pair associations and in turn the operons as ancient and recently formed. This allowed us to perform a detailed analysis of operonic structure in cyanobacteria, a morphologically and physiologically diverse group of photoautotrophs. Clustering based on operon conservation showed significant similarity with the 16S rRNA-based phylogeny, which groups the cyanobacterial strains into three clades. Clade C, dominated by strains that are believed to have undergone genome reduction, shows a larger fraction of operonic genes that are tightly packed in larger sized operons. Ancient operons are in general larger, more tightly packed, better optimized for co-regulation and part of key cellular processes. A sub-clade within Clade B, which includes Synechocystis sp. PCC 6803, shows a reverse trend in intergenic spacing. Our results suggest that while in situ formation and vertical descent may be a dominant mechanism of operon evolution in cyanobacteria, optimization of intergenic spacing and co-regulation are part of an ongoing process in the life-cycle of operons.