Identification of the human papilloma virus-1a E4 gene products.

Antibodies prepared against a human papilloma virus-1 (HPV-1) E4/beta-galactosidase fusion protein identified several polypeptides in HPV-1, but not HPV-2 or 4, induced papillomas. The major E4 protein, that represented up to 30% of total cellular protein, was a 16/17-K doublet which was purified by column chromatography and analysed for amino acid content. A peptide derived by chymotryptic digestion was purified by h.p.l.c. and subjected to amino acid sequencing. The unique sequence obtained, Gly-His-Pro-Asp-Leu-Ser-Leu, identified the 16/17-K doublet as a product of the HPV-1 E4 gene region. Antibodies to both the E4/beta-galactosidase fusion protein and the 16/17-K doublet identified two smaller polypeptides (10/11-K) which may represent spliced products of E4. We propose that the products of the HPV-1 E4 gene region are not classical DNA tumor virus early proteins and suggest that they play a role in virus maturation.     

An assay for high-sensitivity detection of thrombin activity and determination of proteases activating or inactivating protease-activated receptors

This paper describes the development of galactosidase protease-activated receptor (GPAR) as a recombinant protein obtained by fusion of beta-galactosidase, the extracellular domains of protease-activated receptors (PARs), and a biotin acceptor domain. Used as an immobilized substrate, this protein allows the detection of thrombin in the sub-picomolar range. A comparative analysis for proteolytic cleavage of murine PAR1, PAR2, and PAR3 and human PAR4 was performed, involving mutated and nonmutated GPAR fusion proteins. Thrombin cleaved GPAR1 (2.6 mol(beta-galactosidase)/(mol(thrombin) * min)), GPAR3 (410 mmol(beta-galactosidase)/(mol(thrombin) * min)), and GPAR4 (4.3 mmol(beta-galactosidase)/(mol(thrombin) * min)) specifically at the proteolytic activation site. A second possible cleavage site for thrombin is present in murine PAR1 and PAR3. Trypsin and plasmin cleaved all receptor fusion proteins with little specificity for the activation site, except for a marked preference of trypsin for cleavage at the activation site of GPAR2. Chymotrypsin cleaves GPAR1 at a rate (58 mmol(beta-galactosidase)/(mol(thrombin) * min)) that suggests the possibility of chymotryptic inactivation of PAR1. Elastase may inactivate PAR1 and PAR3, but probably not PAR2 and PAR4. Neither activated protein C nor the plasminogen activators cleave any GPAR fusion protein at considerable rates.     

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.     

Turnover of beta-galactosidase in fibroblasts from patients with genetically different types of beta-galactosidase deficiency.

The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phenyltriazene (beta-Gal-MNT) and from the subsequent restoration of enzyme activity in cell cultures turnover times were calculated. By using [3H]beta-Gal-MNT, the hydrolytic activity per molecule of beta-galactosidase was determined. 3H-labelled beta-D-galactopyranosylmethylamine, the precursor of [3H]beta-gal-MNT, was obtained by Raney-nickel-catalysed exchange with 3H2O. The rate of synthesis of beta-galactosidase in normal and all mutant cells tested was found to be 0.4-0.5 pmol/day per mg of cellular protein. The GM1-gangliosidosis cells tested contain the normal amount of 0.5 pmol of beta-galactosidase/mg of protein with a normal turnover time of about 10 days, but only 10% of beta-galactosidase activity per enzyme molecule. Cells with combined beta-galactosidase and neuraminidase deficiency contain only 0.3 pmol of beta-galactosidase/mg of protein with a decreased turnover time of 1 day and normal hydrolytic properties (200 nmol of 4-methylumbelliferyl galactoside/h pmol of beta-galactosidase).     

Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli.

The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity. A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion. This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion. prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE. The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins. When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold. Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.     

A bifunctional chimeric protein consisting of MutS and beta-galactosidase

A bifunctional protein consisting of MutS, a mismatch binding protein and a beta-galactosidase reporter domain has been constructed. The fusion of beta-galactosidase to the MutS C-terminus was obtained by cloning the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. Milligram amounts of this huge chimeric protein (217 kDa monomer) were purified from 1l of overexpressing E. coli cells using metal-chelate affinity chromatography. The mismatch binding properties of the fusion protein were confirmed by DNA mobility shift assay in polyacrylamide gels. Binding to biotinylated mismatched DNA immobilized on streptavidin microplates followed by colorimetric reaction with X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), demonstrated both mismatch recognition and beta-galactosidase activity of the chimeric protein. The activity of beta-galactosidase domain of the fusion was similar to that of the native enzyme. A colorimetric assay for beta-galactosidase activity using X-Gal supplemented with NBT (nitro blue tetrazolium) allowed detection of 50 and 500 fmol of the chimeric protein with naked eye in 45 microl volumes after 120 and 15 min incubation, respectively.     

In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner

Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.     

Induction of bphA, encoding biphenyl dioxygenase, in two polychlorinated biphenyl-degrading bacteria, psychrotolerant Pseudomonas strain Cam-1 and mesophilic Burkholderia strain LB400

We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gm(r) fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30 degrees C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7 degrees C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15 degrees C was approximately five times less than the level in LB400-1 grown at 30 degrees C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.     

Characterization,expression and regulation of a third gene encoding glutathione S-transferase from the fission yeast

A third gene encoding glutathione S-transferase (GSTIII) was cloned from the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined was found to contain 2110 base pairs including an open reading frame of 242 amino acids that would encode a protein of a molecular mass of 26,620 Da. The cloned GSTIII gene could be expressed in S. pombe, S. cerevisiae and Escherichia coli cells which gave 1.4-, 2.1-, and 3.0-fold higher GST activity in an assay using 1-chloro-2,4-dinitrobenzene as a substrate, respectively. The cloned GSTIII gene caused higher survivals of S. pombe cells on solid media with cadmium chloride or mercuric chloride. The GSTIII protein has 16% and 18% homologies with the GSTI and GSTII proteins, respectively. To independently monitor the regulation of the GSTIII gene, its 1168 bp upstream region and N-terminal 33 amino acid-coding region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357. The synthesis of beta-galactosidase from the fusion plasmid pGY357 was greatly enhanced by cadmium chloride (50 microM), cupric chloride (10 microM), aluminum chloride (5 mM, 10 mM), mercuric chloride (1 microM), and zinc chloride (10 mM). However, the synthesis of beta-galactosidase from the fusion plasmid pGY357 was not affected by superoxide-generating menadione, and o-dinitrobenzene, whereas they could significantly induce the expression of the GSTI and GSTII genes of S. pombe. The overproduced Pap1 inhibited the induction of beta-galactosidase synthesis from the fusion plasmid pGY357 by cadmium chloride, which is opposite to the previously known role of Pap1 in the response to oxidative stress. Our results collectively indicate that the three GST genes of S. pombe are subjected to different regulatory mechanisms. The major role of the GSTIII protein in S. pombe may be the detoxification of various metals.     

Expression of hepatitis B virus surface antigen in adult rat liver. Co-introduction of DNA and nuclear protein by a simplified liposome method.

We established a simple and efficient method for gene transfer in vitro (to cultured cells) and in vivo (to an adult organ) using liposomes. Plasmid DNA and proteins were efficiently co-encapsulated in liposomes by agitation and sonication, and were co-introduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion. Introduction of the Escherichia coli beta-galactosidase gene with non-histone chromosomal protein high mobility group 1 (HMG1) into LLCMK2 cells resulted in about 3 times higher beta-galactosidase activity than that on introduction of the gene alone. Two days after injection of HVJ-liposomes containing the beta-galactosidase gene and HMG1 under the perisplanchnic membrane of adult rat liver, hepatic cells near the injection site were found by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to have beta-galactosidase activity. After similar injection of HVJ-liposomes containing the hepatitis B virus surface antigen (HBsAg) gene and HMG1, HBsAg was detected in the serum for 9 days with a maximum of 25-45 ng/ml on day 2 after the injection.     

Pap1-dependent regulation of the GSTII gene from the fission yeast

The genomic DNA encoding a second glutathione S-transferase (GSTII) was previously isolated from the fission yeast Schizosaccharomyces pombe. Its expression was shown to be induced by menadione, mercuric chloride, o-dinitrobenzene, and NO-generating S-nitroso-N-acetylpenicillamine using the GSTII-lacZ fusion harboring the 910 bp upstream region from the translational initiation point. In this study, the additional fusion plasmids pGST50-590 and pGST50-6R-590 were constructed to carry the 590 bp upstream region in the vectors YEp357 and YEp367R, respectively. The synthesis of beta-galactosidase from the fusion plasmid pGST50-590 was about 3-fold higher than that from the fusion plasmid pGST50-F, indicating the presence of negatively activating sequence in the -910 to approximately -590 region. It was also enhanced by the same agents, which induced the synthesis of beta-galactosidase from the fusion plasmid pGST50-F. The synthesis of beta-galactosidase from both fusion plasmids pGST50-F and pGST50-590 was enhanced by the overexpressed Pap1 protein. The synthesis of beta-galactosidase from the two YEp367R derivatives pGST50-6R-F and pGST50-6R-590 was greatly decreased in the Pap1-negative strain TP108-3C. These results propose the Pap1-dependent regulation of the GSTII gene from the fission yeast.     

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.     

50 residues coded by exon 2 of chicken lysozyme carry residual catalytic activity

The generally accepted hypothesis that exons code for fundamental polypeptide structures was tested with a fusion protein consisting of almost the entire polypeptide coded by exon 2 of chicken lysozyme fused to the N-terminus of beta-galactosidase of E.coli. Exon 2 encodes residues 28-81 of lysozyme. It thus contains Glu 35 and Asp 52, which are essential for hydrolysis of glycosidic bonds. The exon 2-beta-galactosidase fusion protein hydrolysed the substrate 4-methylumbelliferyl-N,N',N'-triacetyl-chitotrioside with a reaction rate about 1/40,000 of that of native lysozyme. The low hydrolysis rate of exon 2-peptide is partially caused by its low affinity to its substrate.     

Induction of bphA, Encoding Biphenyl Dioxygenase, in Two Polychlorinated Biphenyl-Degrading Bacteria, Psychrotolerant Pseudomonas Strain Cam-1 and Mesophilic Burkholderia Strain LB400

We investigated induction of biphenyl dioxygenase in the psychrotolerant polychlorinated biphenyl (PCB) degrader Pseudomonas strain Cam-1 and in the mesophilic PCB degrader Burkholderia strain LB400. Using a counterselectable gene replacement vector, we inserted a lacZ-Gm^r fusion cassette between chromosomal genes encoding the large subunit (bphA) and small subunit (bphE) of biphenyl dioxygenase in Cam-1 and LB400, generating Cam-10 and LB400-1, respectively. Potential inducers of bphA were added to cell suspensions of Cam-10 and LB400-1 incubated at 30°C, and then beta-galactosidase activity was measured. Biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately six times greater than the basal level in cells incubated with pyruvate. In contrast, the beta-galactosidase activities in LB400-1 incubated with biphenyl and in LB400-1 incubated with pyruvate were indistinguishable. At a concentration of 1 mM, most of the 40 potential inducers tested were inhibitory to induction by biphenyl of beta-galactosidase activity in Cam-10. The exceptions were naphthalene, salicylate, 2-chlorobiphenyl, and 4-chlorobiphenyl, which induced beta-galactosidase activity in Cam-10, although at levels that were no more than 30% of the levels induced by biphenyl. After incubation for 24 h at 7°C, biphenyl induced beta-galactosidase activity in Cam-10 to a level approximately four times greater than the basal level in cells incubated with pyruvate. The constitutive level of beta-galactosidase activity in LB400-1 grown at 15°C was approximately five times less than the level in LB400-1 grown at 30°C. Thus, there are substantial differences in the effects of physical and chemical environmental conditions on genetic regulation of PCB degradation in different bacteria.     

The CRH family coding for cell wall glycosylphosphatidylinositol proteins with a predicted transglycosidase domain affects cell wall organization and virulence of Candida albicans

In Candida albicans UTR2 (CSF4), CRH11, and CRH12 are members of a gene family (the CRH family) that encode glycosylphosphatidylinositol-dependent cell wall proteins with putative transglycosidase activity. Deletion of genes of this family resulted in additive sensitivity to compounds interfering with normal cell wall formation (Congo red, calcofluor white, SDS, and high Ca(2+) concentrations), suggesting that these genes contribute to cell wall organization. A triple mutant lacking UTR2, CRH11, and CRH12 produced a defective cell wall, as inferred from increased sensitivity to cell wall-degrading enzymes, decreased ability of protoplasts to regenerate a new wall, constitutive activation of Mkc1p, the mitogen-activated protein kinase of the cell wall integrity pathway, and an increased chitin content of the cell wall. Importantly, this was accompanied by a decrease in alkali-insoluble 1,3-beta-glucan but not total glucan content, suggesting that formation of the linkage between 1,3-beta-glucan and chitin might be affected. In support of this idea, localization of a Utr2p-GFP fusion protein largely coincided with areas of chitin incorporation in C. albicans.As UTR2 and CRH11 expression is regulated by calcineurin, a serine/threonine protein phosphatase involved in tolerance to antifungal drugs, cell wall morphogenesis, and virulence, this points to a possible relationship between calcineurin and the CRH family. Deletion of UTR2, CRH11, and CRH12 resulted in only a partial overlap with calcineurin-dependent phenotypes, suggesting that calcineurin has additional targets. Interestingly, cells deleted for UTR2, CRH11, and CRH12 were, like a calcineurin mutant, avirulent in a mouse model of systemic infection but retained the capacity to colonize target organs (kidneys) as the wild type. In conclusion, this work establishes the role of UTR2, CRH11, and CRH12 in cell wall organization and integrity.