Kilodalton Protein Research Articles

Overexpression, purification, and late transcription factor activity of the 17-kilodalton protein encoded by the vaccinia virus A1L gene

The A1L, A2L, and G8R open reading frames (ORFs) were previously shown by transfection assays to encode transactivators of late gene expression. We now present evidence that the 17-kDa protein product of the A1L gene can function in vitro as a transcription factor. Simultaneous overexpression of the transactivators was achieved by coinfecting HeLa cells with one recombinant vaccinia virus that encodes the bacteriophage T7 RNA polymerase and three recombinant vaccinia viruses that contain copies of A1L, A2L, and G8R ORFs regulated by T7 promoters. Extracts from the recombinant virus-infected cells exhibited greatly enhanced late in vitro transcription activity and served as a source of factors. The 17-kDa product of the A1L ORF represented approximately 8% of the ammonium sulfate-precipitated cell protein and copurified with a late transcription factor activity. The transcription factor activity could be specifically immunodepleted with immobilized antibody to the bacterially expressed A1L-encoded protein, providing additional evidence for its identity and role. A sequence encoding six consecutive histidines was added to the A1L ORF, which was then incorporated into the genome of a baculovirus expression vector. The 17-kDa protein, synthesized in insect cells and purified by binding to an Ni(2+)-chelating affinity column, could replace the vaccinia virus-overexpressed 17-kDa protein in transcription assays. In addition to the 17-kDa product of the A1L gene, which was named vaccinia virus late transcription factor 2, the proteins that stimulate specific transcription of late promoter-regulated templates included the viral multisubunit RNA polymerase, vaccinia virus late transcription factor 1 (the product of the G8R ORF), and at least one other partially purified protein.

Detection in an enzyme immunoassay of an immune response to a recombinant fragment of the 128 kilodalton protein (CagA) ofHelicobacter pylori

The possibility of using a recombinant fragment of the CagA (128 kDa protein) for the diagnosis of Helicobacter pylori infection was evaluated. Following cloning of the gene coding for the CagA, a recombinant fragment of it was expressed in Escherichia coli, purified and used in Western blot and an EIA to screen sera from 82 patients with gastroduodenal disease who underwent endoscopic examination. In Western blot, good correlation was found between the serological data obtained with the recombinant antigen and those obtained using non-purified extracts of Helicobacter pylori. The EIA using the antigen showed a sensitivity of 96.2% and a specificity of 96.6% compared with Western blot. These data indicate that the recombinant protein is a reliable antigen for detection of infections with Helicobacter pylori strains that are associated with disease. The EIA assay described may be used in follow-up of the progression of the illness and the results of therapy.

Growth factor modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cells.

Insulin-like growth factors (IGFs) stimulate growth and differentiation of osteoblasts in culture, and these biological functions can be modulated by their binding proteins (IGFBPs). Previous studies have shown that IGFBP-2 is the major IGFBP synthesized by fetal rat osteoblast-like (ROB) cells, which also secret a minor 24-kilodalton IGFBP, presumably IGFBP-4. In this study we examined the modulation of the abundance of IGFBPs by various growth factors. By immunoprecipitation and ligand blotting, we have proved that the 24 kilodalton protein, which appeared at 25 kilodalton in the present study, is IGFBP-4, whose level was strongly enhanced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB homodimer. Induction of IGFBP-4 by these factors was detected at 1 nM (10- to 30-fold) and increased to 50- to 70-fold of control at 10 nM. The abundance of IGFBP-4 was also increased but to a lesser degree by transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF). As opposed to the actions of other growth factors, TGF-beta 1 substantially lowered the levels of IGFBP-2 and IGFBP-4. PTH and PTH-related peptide did not induce IGFBPs in ROB cells. This is in contrast to the findings from a malignant rat osteoblast-like cell line, UMR 106-01. Since the level of IGFBP-4 after bFGF treatment remained elevated in the presence of hydroxyurea, the induction was likely to be independent of the stimulatory effects of these factors on mitogenesis. To further examine the mechanisms by which IGFBPs were regulated, cultures were treated with actinomycin D and cycloheximide with and without bFGF. Although the synthesis of IGFBP-2 and IGFBP-4 were both inhibited by cycloheximide, actinomycin D blocked the synthesis of basal and bFGF-induced IGFBP-4 but not IGFBP-2. Total RNA extracted from ROB cells and hybridized with specific rat complementary DNAs for IGFBP-2 and IGFBP-4 showed single transcripts of 1.3 and 2.2 kilobases, respectively. Regardless of the changes at the protein level, the abundance of IGFBP-4 transcripts was not different from the vehicle-treated controls at 2, 8, and 24 h after bFGF treatment. Similarly, the levels of messenger RNA for IGFBP-2 and IGFBP-4 did not change during the same time course in the TGF-beta 1 treatment. These data demonstrate that in ROB cells, the abundance of IGFBPs is differentially regulated by various growth factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of glucose transporter isoforms in the adult and developing human eye.

The expression of glucose transporter isoforms (Glut 1, Glut 3, Glut 4, and Glut 5) in the human eye was investigated at various ages ranging between 8 weeks gestation (first trimester) and adult using Western blot and immunohistochemical analyses. Glut 1 and Glut 3 expression and cellular localization patterns were similar to those of human brain. Glut 1 (50-kilodalton protein) was expressed by epithelial cells (retinal pigmented epithelium, choroidal, iridial, and pars planus), which form the blood-eye barrier, retinal Mueller cells, the lens fiber cells, iridial microvascular endothelial cells, and to a lesser extent by the outer segments of the photoreceptor cells in the adult eye. This pattern was conserved throughout development and was evident as early as 8 weeks gestation. In addition, the endothelial cells of vitreous hyaloid vessels expressed Glut 1 at 8 weeks gestation. Glut 3 (50 to 55-kilodalton protein) immunoreactivity was observed only in the adult inner synaptic layer of the retina. Neither Glut 4 nor Glut 5 was expressed in any occular tissue at any age examined. These results suggest that Glut 1 is the main glucose transporter of the human eye and that it is ontogenically conserved. In contrast, Glut 3 is associated with selective neuronal processes, and its expression is developmentally altered.

A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis

The effect of a 20-kDa protein on cell viability and CytA crystal production in its natural host, Bacillus thuringiensis, was studied by expressing the cytA gene in the absence or presence of this protein. In the absence of the 20-kDa protein, B. thuringiensis cells either were killed during sporulation (strain cryB) or produced very small CytA crystals (strain 4Q7). Expression of cytA in the presence of the 20-kDa protein, however, preserved cell viability, especially in strain cryB, and in both strains yielded bipyramidal crystals of the CytA protein that were larger than those of wild-type B. thuringiensis. These results suggest that the 20-kDa protein promotes crystal formation, perhaps by chaperoning CytA molecules during synthesis and crystallization, concomitantly preventing the CytA protein from interacting lethally with the bacterial host cell.

The cDNA sequence of an abundant Entamoeba histolytica 20-kilodalton protein containing four repetitive domains

The cDNA sequence of an abundant Entamoeba histolytica 20-kilodalton protein containing four repetitive domains

Parastatin (porcine chromogranin A347-419), a novel chromogranin A-derived peptide, inhibits parathyroid cell secretion.

Chromogranin A (CgA), previously referred to as secretory protein-I, is a 50-kilodalton protein present in secretory granules of many endocrine and neuroendocrine cells. In the parathyroid it is present and cosecreted with PTH in response to hypocalcemia. CgA appears to be a precursor of bioactive peptides including pancreastatin, beta-granin, vasostatin, and chromostatin. The presence of several highly conserved pairs of basic amino acids, putative cleavage sites, in the CgA molecule suggests that other yet unidentified bioactive peptides might exist within the molecule. We tested this speculation by subjecting porcine parathyroid CgA to digestion by endoproteinase Lys-C. Resulting CgA-derived peptides were isolated by reverse-phase C18 HPLC and tested for their ability to affect low-Ca2+ stimulated secretion by porcine parathyroid cells. We characterized one peptide, which we named parastatin, that inhibited secretion of both PTH and CgA in a dose-dependent fashion over the range of 0.2-0.6 microM. Parastatin migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent mol wt of 11,000. Edman degradation yielded the sequence L-S-F-R-A-P-A-Y-G-F-R-G-P-G-L corresponding to residues 347-361 of porcine CgA. Amino acid analysis of endoproteinase Lys-C and endoproteinase Asp-N-generated fragments indicated that parastatin corresponds to residues 347-419 of CgA. A synthetic NH2-terminal fragment of rat parastatin corresponding to residues 1-19 was an inhibitory as intact porcine parastatin on parathyroid gland secretion. These results extend the concept that CgA is a precursor of biologically active peptides.

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

Paraneoplasia and Autoimmunologic Injury of the Nervous System: The Anti‐Hu Syndrome

In recent years, antineuronal autoantibodies of varying antigenic specificity have come to be associated with a number of paraneoplastic neurologic disorders. Anti-Hu is a polyclonal complement-fixing IgG directed against a 35 to 40 kilodalton protein concentrated in the nuclei of neurons throughout the central and peripheral neuraxes. Its elaboration at high titer in serum and cerebrospinal fluid is invariably associated with a neurologic syndrome characterized chiefly by subacutely evolving sensory neuropathy and an array of central disturbances that include bulbar and cerebellar dysfunction, limbic encephalitis and motor neuron disease. The manufacture of anti-Hu IgG is triggered in a great majority of cases by underlying small cell carcinomas of pulmonary origin, typically limited in stage and otherwise silent, that aberrantly express the native neuronal antigen or an antigenically indistinguishable epitope. Both neoplastic and diseased neural tissues contain lymphocytes of B and T lineage specifically cognizant of the Hu antigen as well as concentrated anti-Hu IgG bound to tumor cells and neurons, respectively. These observations suggest that an immune response serving initially to limit the growth and spread of its inciting neoplasm comes subsequently to be misdirected against the nervous system of the host, resulting in autoimmunologically-mediated neurologic injury. Clinical, neuropathologic and immunologic data derived from a series of 71 sero-confirmed cases of the anti-Hu-associated paraneoplastic sensory neuronopathy/encephalomyelitis complex are reviewed.

Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome.

Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome.

Cloning and expression of a cDNA encoding epitopes shared by 15- and 60-kilodalton proteins of Cryptosporidium parvum sporozoites.

A cDNA (CP15/60) encoding epitopes of Cryptosporidium parvum 15- and 60-kDa sporozoite proteins was isolated and expressed in Escherichia coli toward the goal of developing an immunogen for producing high-titer anticryptosporidial colostrum. Antisera prepared in rats to native C. parvum 15-kDa protein and used to identify the CP15/60 bacteriophage clone recognized both 15- and 60-kDa in vitro translation products derived from sporozoite RNA. Antisera specific for recombinant CP15/60 antigen recognized native 15- and 60-kDa C. parvum sporozoite proteins by immunoblotting and identified both surface and internal antigens on C. parvum sporozoites by immunofluorescence staining. Northern (RNA) and Southern blot hybridization experiments using sporozoite RNA and DNA indicated that CP15/60 DNA is transcribed as a single 1.4-kb RNA species from a single-copy gene. Recombinant CP15/60 antigen was recognized by hyperimmune colostrum from cows immunized with C. parvum oocyst-sporozoite protein and by convalescent-phase sera from C. parvum-infected calves.

Rat duodenal iron-binding protein mobilferrin is a homologue of calreticulin

Background: Mobilferrin is a water soluble 56-kilodalton protein isolated from human and rat duodenal mucosa. It binds iron and other transitional metals in vivo and in vitro and is postulated to play a role in their absorption and intracellular metabolism. The purpose of this study was to characterize mobilferrin. Methods: Mobilferrin was characterized by identification of the N-terminal amino acid sequence, two-dimensional protein electrophoresis, and studies of mobilferrin and homologues using anti-mobilferrin antibody and competitive metal binding. Results: The N-terminal amino acid sequence of mobilferrin was Asp-Pro-Ala-Ile-Tyr-Phe-Lys-Glu-Gln-Phe-Leu-Asp-Gly-Asp-Ala-Ser-Thrand is a homologue of calreticulin (calregulin). The proteins had a similar molecular mass (56 kilodalton) and isoelectric point (4.7). Anti-mobilferrin antibodies react with calreticulin. Both proteins bind iron and calcium but have a greater affinity for iron. Conclusions: Mobilferrin and calreticulin are homologues that bind iron with greater affinity than calcium and other transitional metals. Competitive binding of metals by mobilferrin provides insight into the absorptive pathway shared by both essential and toxic transitional metals.

65-kDa protein binds to destabilizing sequences in the IFN-beta mRNA coding and 3' UTR.

The transient expression of human interferon (IFN-beta) gene in response to viral induction is regulated both at the transcriptional and posttranscriptional levels. The decrease in levels of IFN-beta mRNA, which requires protein synthesis, is due to transcriptional repression as well as a rapid turnover of beta mRNA. Previous studies have shown the presence of two destabilizing sequences, one in the 3' untranslated region (UTR) and the other in the coding region. We have shown in this study that the coding region destabilizing element resides in the 3' end of the coding region (+538 to +637) and that the degradation does not require the translation of IFN-beta mRNA through its coding region. In addition, we have identified three domains of 19, 20, and 29 nucleotides long that specifically bind a 65-kilodalton (kDa) cytoplasmic protein. One of the binding sites is in the 3' end of the coding region and the other two in the 3' UTR. All these regions are AU-rich and show considerable homology to each other. Interestingly, the levels of the 65-kDa protein was increased after poly rI.rC induction. We suggest that this 65-kDa protein is a component of the IFN-beta mRNA destabilizing complex or plays a role in the degradation of IFN-beta mRNA.

The immunodominant 90-kilodalton protein is localized on the terminal tip structure of Mycoplasma pneumoniae

Immunoblot analysis of convalescent-phase sera of experimentally infected chimpanzees or monoclonal antibodies (MAbs) specific to the 90- and 40-kDa proteins of Mycoplasma pneumoniae indicated that both proteins were present in cytadsorbing, pathogenic strains PI-1428, M129, and FH but absent in noncytadsorbing, nonpathogenic strain M129-B176. Adsorption of convalescent-phase chimpanzee sera with virulent strain PI-1428 removed reactivity, whereas adsorption with avirulent strain M129-B176 did not remove reactivity to these two proteins. By using proteolysis and specific MAbs, we demonstrated that the 90- and 40-kDa proteins were surface exposed. Immunoelectron microscopy employing specific MAbs showed that the 90-kDa protein is localized on the terminal tip attachment apparatus. However, the MAb specific for the 40-kDa protein failed to indicate a similar localization. Nevertheless, these data, taken together, indicate that the immunodominant 90- and 40-kDa proteins are surface exposed, are localized on the terminal tip apparatus, and might be involved in the attachment mechanism.

Use of PCR in routine diagnosis of treated and untreated pulmonary tuberculosis.

To assess the routine use of a polymerase chain reaction (PCR) assay for the direct detection of Mycobacterium tuberculosis in expectorated sputum specimens. A pair of primers (20-mer) were designed to amplify the 38 kilodalton protein of M tuberculosis. The specificity of the assay was evaluated in 31 M tuberculosis strains, 15 atypical mycobacterium species, and several commensal bacteria of the upper respiratory tract. The assay was subsequently applied to 519 sputum specimens from 85 inpatients of a chest hospital in Hong Kong. An amplified product of 239 base pairs was found in all M tuberculosis strains, standard strains of M bovis, and M africanum but not in the other bacterial strains tested. For the 51 patients with pulmonary radiographic lesions, the diagnosis of pulmonary tuberculosis was subsequently confirmed by both culture and PCR in 41 of them. Five patients who were treated before admission were positive by PCR alone. All but one patient in the control group (patients with acute exacerbation of chronic obstructive airway diseases) or those with atypical mycobacterial diseases were PCR negative. The PCR remained positive after four weeks of anti-tuberculosis treatment in 29 patients, 16 of whom had become culture negative. This PCR assay is a useful technique for the diagnosis of untreated and recently treated cases of pulmonary tuberculosis.

Antibody development against northern fowl mites (Acari: Macronyssidae) in chickens.

This study reports the development of an antibody against protein(s) from the tissue of the northern fowl mite, Ornithonyssus sylviarum (Canestrini & Fanzago). Northern fowl mite proteins were obtained by affinity chromatography and used for immunization. Western blot analysis identified proteins that were reactive with sera from birds immunized with the antigen; this indicated that serum antibodies against the northern fowl mite had been produced. Chickens that had been immunized or infested, or both, with the northern fowl mite produced sera that were reactive with a 100 kilodalton (kD) protein. The response was greater if the chicken had been immunized with the antigen and infested with the northern fowl mite. Experimentally immunized and infested chickens experienced limited decreases in the levels of northern fowl mite infestation. Survival of bloodfed mites after ingestion of the immune chicken blood was assessed in an in vitro feeding study using blood-filled parafilm sacs; minor differences in northern fowl mite feeding tendencies were noted. The chickens developed antibodies to the northern fowl mite proteins, but this immunity did not decrease the infestation level or in vitro feeding.

The E1B 19-kilodalton protein is not essential for transformation of rodent cells in vitro by adenovirus type 5

The newly constructed adenovirus type 5 mutant in1 carries a single AT base pair insertion immediately after nucleotide position 1715 in the E1B gene sequence which destroys the proximal AUG normally present in E1B messages and prevents production of intact E1B 19-kDa protein in infected cells. We have used in1, variants of in1 containing mutant alleles of viral genes known to enhance transformation frequency, and adenovirus type 5 mutant dl337 (S. Pilder, J. Logan, and T. Shenk, J. Virol. 52:664-671, 1984), in which the sequence between nucleotides 1770 and 1916 within the 19-kDa reading frame is deleted, to test the generally accepted hypothesis that this E1B protein is essential for the transformation of rodent cells and maintenance of the transformed phenotype. We find that these mutants transform rat embryo cells, rat kidney and mouse kidney primary cells, and cells of the 3Y1 rat line with decreased frequencies only when virus is added to these various cells at high input multiplicities of infection. In contrast, when lower doses of virus are used, the mutants transform with wild-type frequencies. Cells infected with higher doses of mutant virus show increased levels of DNA degradation and cell killing compared with those of cells infected with the same levels of wild-type virus, and these effects most likely contribute to the decreased transformation frequencies observed. On the basis of these results and the results of phenotypic analyses of numerous transformants, we propose that the E1B 19-kDa protein is not required for induction and/or maintenance of transformed-cell characteristics in rodent cells infected with adenovirus type 5.

High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes.

Interactions among the 20-kDa protein gene and the cytA and cryIVD genes located in a 9.4-kb HindIII fragment were studied. A series of plasmids containing a combination of these different genes was constructed by using the Escherichia coli/Bacillus thuringiensis shuttle vector pHT3101. The plasmids were then used to transform an acrystalliferous strain, cryB, derived from B. thuringiensis subsp. kurstaki. The results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses suggest that although the 20-kDa protein is required for the efficient CytA protein production in E. coli, it is not required in B. thuringiensis. With or without the truncated 20-kDa protein gene, the CtyA and/or CryIVD proteins are produced and form parasporal inclusions in B. thuringiensis cells. However, more-efficient expression is obtained when a second protein, probably acting as a chaperonin, is present. In addition, the time course studies show that the CytA and CryIVD proteins are coordinately produced. Both the crude B. thuringiensis culture and purified inclusions from each recombinant B. thuringiensis strain are toxic to Culex quinquefasciatus larvae. The parasporal inclusions formed in B. thuringiensis cells are mosquitocidal, with CytA synergizing CryIVD toxicity.