Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda.

Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein. Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site. Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed. Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells. The different applications of these plasmids for expression of cloned genes are discussed.     

Sucrose-diet feeding induces gene expression of heat shock protein in rat brain under stress

Stress-induced hyperphagia is enhanced in the presence of sweets, particularly sucrose, which may act to attenuate stress. Recently, it was also reported that heat shock protein (HSP) may be involved in the defense against stress. To explore whether sucrose alters gene expression of HSP under stress, we determined the HSP mRNA levels in the hypothalamus, cerebellum, and cerebral cortex after restraint stress in sucrose-diet-fed rats. Competitive RT-PCR revealed that gene expressions of HSP27 in the cerebral cortex and cerebellum and of HSP70 in the cerebral cortex, hypothalamus, and cerebellum were induced by restraint stress under a sucrose-diet-fed condition. However, restraint stress by itself or sucrose diet alone did not induce expression of HSP27 or HSP70 mRNA in any of the three anatomical parts. It is suggested that sucrose facilitates the gene expression of HSP27 and HSP70 in brain after restraint stress, which may attenuate stress.     

Renaturation of denatured lambda repressor requires heat shock proteins

The temperature-sensitive bacteriophage lambda cI857 repressor protein rapidly renatures after thermal inactivation. E. coli mutants in the heat shock protein genes dnaK, dnaJ, and grpE do not efficiently reactivate heat-denatured repressor. Our results suggest that protein refolding is promoted by heat shock proteins and that such a process is the basis of the homeostatic role played by these proteins in the heat shock response.     

sn-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (htpR)

Overexpression of the Escherichia coli sn-glycerol-3-phosphate (glycerol-P) acyltransferase, an integral membrane protein, causes formation of ordered arrays of the enzyme in vitro. The formation of these tubular structures did not occur in an E. coli strain bearing a mutation in the htpR gene, the regulatory gene for the heat shock response. The htpR165 mutation was shown by genetic analysis to be the lesion responsible for blockage of tubule formation. Similar amounts of glycerol-P acyltransferase were produced in isogenic htpR+ and htpR165 strains, ruling out an effect of htpR165 on expression of glycerol-P acyltransferase. Further, phospholipid metabolism was not altered in either strain after induction of glycerol-P acyltransferase synthesis. Increased glycerol-P acyltransferase synthesis caused a partial induction of the heat shock response which was dependent upon a wild type htpR gene. The heat shock proteins induced were identified as the groEL and dnaK gene products on two-dimensional gels. These two proteins have been implicated in the assembly of bacteriophage coats. These heat shock proteins appear essential for tubule formation.     

Cloning of the replication gene O of E. coli bacteriophage lambda and its expression under the control of the lac promoter

The expression of the replication gene O of bacteriophage lambda was put under the control of the lac promoter-operator region integrated into the pBR322 cloning vehicle. The new plasmid pKK104 was introduced into minicells and the O gene induced by isopropyl-beta-thiogalactoside (IPTG). The O protein could be identified as a major component in extracts from these cells, in association with the cell membrane fractions. The molecular weight of the O protein in SDS gels is about 33 000, and it is metabolically unstable but apparently stable upon isolation as a membrane-associated fraction.     

In vivo gene delivery and expression by bacteriophage lambda vectors

AIMS: Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. METHODS AND RESULTS: Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. CONCLUSIONS: Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.     

Flavonoids inhibit the expression of heat shock proteins

Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetin and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DM cells. Quercetin inhibited the induction of hsp70 at the level of mRNA accumulation. This is the first report to describe the inhibition of hsp expression by reagents.     

Heat shock induces variably the major heat shock proteins of CV1 clones

CV1 cells have been subcloned several times. Five of these clones were studied for the induction of the major heat shock proteins. These CV1 clones exhibit morphological differences as well as differences in SDS-PAGE protein profiles. These clones responded to heat shock variably as judged from the induction of the major heat shock proteins, 70, 72 and 92 kDa. Variable expression of the heat shock proteins suggests that the selective pressure for isolation of cell clones may affect gene expression differently.     

Cloning and expression in Escherichia coli of the TL-DNA gene 4 of Agrobacterium tumefaciens under the control of the PR promoter of bacteriophage lambda

A plasmid was constructed that directs expression of the TL-DNA gene 4 protein in E. coli. The different steps of the construction were as follows: i) a region of gene 4 encoding the amino-terminal portion of the protein was fused in frame to DNA encoding an enzymatically active carboxy-terminal fragment of beta-galactosidase. The hybrid gene was poorly expressed from the upstream lambda PL promoter carried by the vector. ii) in order to generate an efficient procaryotic ribosome binding site, a DNA fragment carrying the lambda PR promoter with the nearby Shine-Dalgarno (SD) sequence of gene cro was placed in front of the gene 4-lacZ fusion. A recombinant plasmid, termed pGV793, that expressed efficiently a fused protein 4-beta-galactosidase was identified among the Lac+ clones. DNA sequencing analysis showed that pGV793 carried a hybrid ribosome binding site composed of the cro SD sequence, a five bp sequence and the ATG codon of gene 4. Plasmid pGV793 directed the synthesis of three polypeptides of molecular weight 132 Kd, 126 Kd and 122 Kd that carried beta-galactosidase antigenic determinants. The largest polypeptide had the expected size for the hybrid protein. The fusion proteins which accounted for about 0.5% of the total cellular proteins were purified by immunoadsorption using anti-beta-galactosidase antiserum. iii) the complete gene 4 coding sequence was reconstituted, with the lambda PR promoter in place. The resulting pGV822 plasmid expressed a polypeptide whose molecular weight 27 Kd corresponded to the expected size for the gene 4 product. The pI was about 7.     

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Summary Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a prophage. Without amplified photolyase, the prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and is even more effective if cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.