Expression of mammalian liver glycolytic/ gluconeogenic enzymes in Escherichia coli: Recovery of active enzyme is strain and temperature dependent

A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions betweenAulonium ruficorne [Coleoptera: Colydiidae] and other natural enemies of bark beetles [Coleoptera: Scolytidae]

The within tree distribution of some common natural enemies of bark beetles in pine plantations in Israel and some aspects of their feeding habits were studied with special emphasis on the potential impact on the predatorAulonium ruficorne Olivier. A total of 12 predators and 2 parasite species were found associated withA. ruficorne in the natural enemy complex of bark beetles on pines. No secondary parasites were detected. The anthocoridScoloposcelis pulchella (Zetterstedt) and the dipteranMedetera striata Parent were observed feeding on immature stages ofA. ruficorne in the absence of scolytids. The associated Coleoptea:Nemosema elongatum F.,Rhizophagus bipustulatus L.,Corticeus spp.,Plastysoma spp. andPlegaderus discisus Erickson are thought to compete withA. ruficorne on larvae and pupae of bark beetles when the latter are in limited quantities (especially in the lower section of the tree). The parasites, mainlyMetacolus unifasciatuss Forster andDendrosoter caenopachoides Ruschka are assumed to compete withA. ruficorne during the larval period in the smooth bark section of the stem. Competition might occur mainly during spring and fall. Deutonymphs of the miteIpiduropoda sellnicki were detected on the abdomen ofA. ruficorne adults. Larvae of the predator were rarely infected in the field by the bacteriaSerratia sp. while laboratory cultures suffered high rate of mortality caused by this pathogen.      

Identification of consensus patterns in unaligned DNA sequences known to be functionally related

We have developed a method for identifying consensus patternsin a set of unaligned DNA sequences known to bind a common proteinor to have some other common biochemical function. The methodis based on a tnatrix representation of binding site patterns.Each row of the matrix represents one of the four possible bases,each column represents one of the positions of the binding siteand each element is determined by the frequency the indicatedbase occurs at the indicated position. The goal of the methodis to find the most significant matrix-i.e. the one with thelowest probability of occurring by chance-out of all the matricesthat can be formed from the set of related sequences. The reliabilityof the method improves with the number of sequences, while thetime required increases only linearly with the number of sequences.To test this method, we analysed 11 DNA sequences containingpromoters regulated by the Escherichia coli LexA protein. Thematrices we' found were consistent with the known consensussequence, and could distinguish the generally accepted LexAbinding sites from other DNA sequences. Received on November 6, 1989; accepted on December 20, 1989     

A statistical power analysis of the 'internal reference' technique for comparing growth and growth depensation of tilapia strains

Experiments were conducted to compare the growth and growth compensation of three strains of juvenile Oreochromis niloticus . Ten full sib families (10 replicates) per strain were split and grown under crowded and uncrowded conditions for 3 weeks (the treatment). Both treatments were then grown an additional 12 weeks under less crowded conditions (the compensation). Standard length measurements were made at the end of crowding and the end of compensation. Each replicate included a size-matched control population of a fourth (red) reference strain. ANCOVA with the reference strain used as a cofactor revealed significant strain effects on specific growth throughout the experiment. The reference strain removed most of the random among-replicate error variance as shown by an increase of r 2 from 0.06 to 0.91 when it was included in the statistical models. If the reference fish had not been used, approximately 450 replicate families would have been needed to achieve the sensitivity of the present experiment (a difference of 7% among strains significant at P =0.05). We conclude that the CLSU strain grows significantly more slowly than the Israel and NIFI strains under the experimental conditions, that the crowding effect was essentially eliminated after 12 weeks of compensation, and that the reference strain greatly improved the resolution of the strain-testing experiment.     

A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles.

A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures. In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases. Immunogold labeling of thin sections of P. aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm. It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled. Since division is due to cell constriction in this P. aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled. Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm. Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide.     

Growth suppression in early-stationary-phase nutrient broth cultures of Salmonella typhimurium and Escherichia coli is genus specific and not regulated by sigma S.

We have studied the growth suppression seen in early-stationary-phase LB broth cultures of Salmonella typhimurium. Multiplication of small numbers of an antibiotic-resistant S. typhimurium mutant was prevented when the mutant was added to 24-h cultures of the antibiotic-sensitive parent strain, whereas an antibiotic-resistant mutant of an Escherichia coli strain added to the same culture grew well. A 24-h E. coli culture produced a similar specific bacteriostatic inhibition against E. coli. In older cultures, a specific bactericidal effect similar to that observed by M. M. Zambrano and R. Kolter (J. Bacteriol. 175:5642-5647, 1993) was also observed. Whether incubated statically or shaken, sufficient nutrients were present in the filtered supernatants of 24-h cultures for small inocula of the same strain to multiply to ca. 10(9) CFU/ml after reincubation. Introduction of the rpoS mutation had no effect on the specific bacteriostatic inhibition. Similar specific inhibition was also observed in strains of Citrobacter freundii, Klebsiella pneumoniae, Enterobacter agglomerans, and Shigella spp. Experiments in which the 24-h culture was physically separated from the antibiotic-resistant mutant by using a dialysis membrane were carried out. These results indicated that the inhibition might be mediated by a diffusible but labile chemical mediator.     

Regulated, stable expression and nuclear presence of retrovirus double-stranded RNA-binding protein sigma3 in HeLa cells.

Reovirus genome segment S4 codes for polypeptide sigma3, a major outer capsid component of virions and a double-stranded RNA (dsRNA)-binding protein implicated in viral cytopathogenesis. We have constructed a stable HeLa cell line (S4tTA) that produces functional sigma3 under tetracycline transactivator control. In the absence of tetracycline, S4tTA cells synthesized stable dsRNA-binding sigma3 that accumulated in the nucleus as well as in the cytoplasm. However, in induced S4tTA cells also expressing reovirus outer shell polypeptide mu1/mu1C, migration of sigma3 into the nucleus was blocked, probably as a result of formation of a complex with mu1/mu1C which was exclusively in the cytoplasm. Mutant analyses indicated a correlation between dsRNA-binding activity and nuclear entry of sigma3, suggesting an additional role(s) for this capsid protein in virus-cell interactions.     

Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

A major impediment to the effective use of adenovirus vectors for gene therapy is a lack of knowledge of how these vectors interact with diverse cell types in vivo. Adenovirus attachment to most human cell types is mediated by the fiber protein, which binds to an as yet unidentified cell receptor. In contrast to this, we report that adenovirus type 2 (Ad2) attachment to hematopoietic cells is facilitated by interaction of the penton base protein with members of the beta2 integrin family. Adenovirus particles were capable of binding to human monocytic cells, which lack fiber receptors, and virus binding could be blocked by a soluble penton base or by a function-blocking monoclonal antibody to integrin alphaMbeta2. To confirm the role of alphaMbeta2 integrins in Ad2 binding to hematopoietic cells, we analyzed virus attachment and gene delivery to CHO cells expressing recombinant beta2 integrins. alphaMbeta2-expressing CHO cells supported 3- to 5-fold-higher levels of Ad2 binding and 5- to 10-fold-larger amounts of gene delivery than did nontransfected CHO cells, indicating that alphaMbeta2 facilitates adenovirus attachment to and infection of hematopoietic cells. While beta2 integrins promote Ad2 attachment to hematopoietic cells, further studies demonstrated that alphav integrins were required for the next step in infection, virus internalization into cell endosomes. These studies reveal a novel pathway of Ad2 infection of hematopoietic cells mediated by distinct integrins which facilitate separate events in virus entry. They also suggest a possible strategy for selective adenovirus-mediated gene delivery to hematopoietic cells.     

Overexpression of the herpes simplex virus type 1 immediate-early regulatory protein, ICP27, is responsible for the aberrant localization of ICP0 and mutant forms of ICP4 in ICP4 mutant virus-infected cells.

ICP0 and ICP4 are immediate-early regulatory proteins of herpes simplex virus type 1. Previous studies by Knipe and Smith demonstrated that these two proteins are characteristically observed in the nuclei of wild-type virus-infected cells but predominantly in the cytoplasms of cells infected with several ICP4 temperature-sensitive (ts) mutant viruses at the nonpermissive temperature (NPT) (D. M. Knipe and J. L. Smith, Mol. Cell. Biol. 6:2371-2381, 1986). Consistent with this observation, it has been shown previously that ICP0 is present predominantly in the cytoplasms of cells infected with an ICP4 null mutant virus (n12) at high multiplicities of infection and that the level of ICP27, a third viral regulatory protein, plays an important role in determining the intracellular localization of ICP0 (Z. Zhu, W. Cai, and P. A. Schaffer, J. Virol. 68:3027-3040, 1994). To address whether the cytoplasmic localization of ICP0 is a common feature of cells infected with all ICP4 mutant viruses or whether mutant ICP4 polypeptides, together with ICP27, determine the intracellular localization of ICP0, we used double-staining immunofluorescence tests to examine the intracellular staining patterns of ICP0 and ICP4 in cells infected with an extensive series of ICP4 mutant viruses. In these tests, compared with the localization pattern of ICP0 in wild-type virus-infected cells, more ICP0 was detected in the cytoplasms of cells infected with all ICP4 mutants tested at high multiplicities of infection. Each of the mutant forms of ICP4 exhibiting predominantly cytoplasmic staining contains both the nuclear localization signal and the previously mapped ICP27-responsive region (Z. Zhu and P. A. Schaffer, J. Virol. 69:49-59, 1995). No correlation between the intracellular staining patterns of ICP0 and mutant forms of ICP4 was demonstrated, suggesting that mutant ICP4 polypeptides per se are not responsible for retention of ICP0 in the cytoplasm. This observation was confirmed in studies of cells cotransfected with plasmids expressing ICP0 and mutant forms of ICP4, in which the staining pattern of ICP0 was not changed in the presence of mutant ICP4 proteins. Studies of cells infected at low multiplicities with a variety of ICP4 ts mutant viruses at the NPT showed that both ICP0 and ts forms of ICP4 were localized predominantly within the nucleus. These observations are a further indication that the aberrant localization of the ts forms of ICP4 at the NPT is not a direct result of specific mutations in the ICP4 gene. In the final series of tests, the localization of ICP0 in cells infected with a double-mutant virus unable to express either ICP4 or ICP27 was examined. In these tests, ICP0 was detected exclusively in the nuclei of Vero cells but in both the nuclei and the cytoplasms of ICP27-expressing cells infected with the double mutant. These results demonstrate that ICP27, rather than the absence of functional ICP4, is responsible for the cytoplasmic localization of ICP0 in ICP4 mutant virus-infected cells. Taken together, these findings demonstrate that the aberrant localization of ICP0 and certain mutant forms of ICP4 in cells infected with ICP4 mutant viruses is mediated by high levels of ICP27 resulting from the inability of mutant forms of ICP4 to repress the expression of ICP27.