Purification and properties of allelic lactate dehydrogenase isozymes at the B2 locus in rainbow trout,Salmo gairdneri

• 1.1. The homotetrameric lactate dehydrogenase (LDH) isozymes, B₄^2′ and B₄^2″, from homozygous rainbow trout livers have been purified to homogeneity by affinity chromatography using a Sepharose-linked oxamate ligand.
• 2.2. The B₄^2″ isozyme has been found: (a) to have increased K_m (pyr) and K_i (lac) values, (b) to bind pyruvate strongly in the absence of coenzymes with a binding constant of 4.02 × 10⁴ M⁻¹ under conditions where other LDH isozymes showed no binding, and (c) to give a nonlinear noncompetitive lactate product inhibition pattern compared to a normal linear noncompetitive inhibition for the B₄^2′ isozyme.
• 3.3. Different kinetic mechanisms have been proposed for the two isozymes and an adaptive functional significance has been discussed.

     

Retina-specific LDH isozyme in blueback herring, Alosa aestivalis (Mitchell), and alewife, Aiosa pseudoharengus (Wilson)

The retina of 390 Alosa aestivalis and 410 Alosa pseudoharengus have been examined by means of starch-gel electrophoresis. The retina-specific E₄ isozyme has been found to occur in all the fish examined. This study demonstrates for the first time that the E₄ isozyme occurs in A. aestivalis. Because the E₄ isozyme is not polymorphic and has an identical mobility in A. pseudoharengus and in A. aestivalis it is neither suitable for use as a species identification characteristic nor a population marker. Alosa aestivalis and Alosa pseudoharengus are two commercially important ana-dromous species of fish in New Brunswick, Canada. These two species may occur together in the same spawning runs but w^rhile A. pseudoharengus has a wide distribution along the East coast of North America (Leim & Scott, 1966) A. aestivalis occurs in a very limited area in Canada where it is at the northern limit of its range and because of increasing threat of pollution has been listed by McAllister (1970) as one of 17 endangered species. These two species of fish are morphologically very similar and can only be separated by the colour of the abdominal peritoneum (Leim & Scott, 1966). McKenzie (1973) has compared these two species by means of protein electrophoresis and found that while the muscle myogen patterns were species specific, the LDH patterns were the same in both species. He described these two species as five isozyme fish showing the LDH isozymes A₄, A₃B, A₂B₂, AB₃ and B₄. Since Horowitz & Whitt (1972) have reported the presence of the E₄ isozyme in the retina of some teleosts including. A. pseudoharengus but not including A. aestivalis, I considered it worth-while to re-examine A. aestivalis and A. pseudoharengus to find out whether A. aestivalis possessed this isozyme and, if so, whether the mobility of the isozyme could be used as a species identification characteristic and as a population marker. The fish used in this study were collected from five different locations during the 1971 spring migration period and held deep frozen for eight months before they were examined. They were identical to the specimens used for the study reported by McKenzie (1973) where the collection dates, numbers of fish and geographic locations are given. One eyeball from each of 390 A. aestivalis and 410 A. pseudoharengus was removed. Each eyeball was homogenized in 10 drops of distilled water and allowed to stand for one hour at 4^oC. The samples were then centrifuged for 10 min at 12 000 g. The supernatants were used immediately for vertical starch-gel electrophoresis. The apparatus used was that described by Boyer & Hiner (1963). The conditions of electrophoresis were the same as used by Saunders & McKenzie (1971). The LDH bands were stained by the deposition of blue formazan dyes in the regions of LDH activity. The stain formula and details of methods are given in Whitt (1970). The LDH isozyme patterns of all the fish examined were identical. The location of the isozymes A₄, A₃B, A₂B₂, AB₃ and B₄ are indicated in Fig. 1. A₄ is abundant in muscle while B₄ is abundant in heart. Because the LDH subunits assemble preferentially into homodimeric pairs before forming tetramers (Markert & Ursprung, 1971). A₄. A₂B₂ and B₄ show up in electropherograms as strong bands while A₃B and AB₃ show up as weak bands. The retina-specific isozyme E₄ is shown between B₄ and AB₃. Whitt (1970) has already demonstrated that A. pseudoharengus is a five isozyme fish. This has been confirmed by McKenzie (1973) who also compared both A. pseudoharengus and A. aestivalis and found these five isozymes had identical mobilities in both species of fish. The present study demonstrates for the first time that the retina-specific E₄ occurs in A. aestivalis where it has the same electro-phoretic mobility as that of A. pseudoharengus. The patterns are the same and do not appear to vary with geographic origin of the fish. The reason why the presence of the E₄ isozyme was demonstrated in the present study and not in the previous one (McKenzie, 1973) is because of the method used. In that study the migration length was not long enough to sufficiently separate the enzymes. In the present study vertical starch-gel electrophoresis which allows for long migration distances was used. As has already been shown for the A-B isozymes (McKenzie, 1973), the E₄ isozyme is not polymorphic in these two species of fish. It therefore has no use as apopulation marker. Because the E₄ isozyme has an identical mobility in both species of fish, it cannot be used as a species identification characteristic.     

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B₄) but no trace of LDH-5 (A₄) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29—Embryonale Entwicklung and Differenzierung.     

The inheritance of tissue-specific lactate dehydrogenase isozymes in interspecific bass (Micropterus) hybrids

A combination of hybridization (in vivo and in vitro), immunochemical, and electrophoretic analyses reveals that both smallmouth bass, Micropterus dolomieui (Lacépède), and largemouth bass, M. salmoides (Lacépède), possess three homopolymeric lactate dehydrogenase (LDH) isozymes, A₄, B₄, and E₄. The retinal-specific E₄ isozymes of these two parental species possess different electrophoretic mobilities. The two bass species were hybridized to produce the interspecific F₁ hybrids. In addition, F₂ and F₃ hybrid generations were produced. The genetic data from these crosses indicate that the retinal-specific LDH isozyme is the product of a distinct nuclear gene (E locus) on an autosomal chromosome. This E gene appears to segregate independently of the gene for supernatant MDH. The LDH E gene is highly active in the bass neural retina and less active in other neural tissues. However, unlike in most teleosts, the bass LDH E gene also functions in such nonneural tissues as the heart and kidney.This research was supported by NSF grant GB 16425 to G. S. Whitt and by funds provided by the Illinois Natural History Survey to W. F. Childers.     

A complete cDNA coding for the sequence of glycinin A2B1a subunit precursor

Analysis of the A₂B_1a subunit precursor, one of the A₂-subunit family of glycinin, the main storage protein of soybean, revealed that it is composed of a signal peptide segment (18 amino acids), the A₂ acidic polypeptide (282 amino acids), followed by the B_1a basic polypeptide (185 amino acids). There was overall 63% homology between this subunit complex and pea legumin, which is an analogous protein to glycinin. As this degree of homology is rather higher than that in the A₃B₄ subunit, one of the A₃ subunit family, it seems that the genes encoding the A₂ subunit family are phylogenetically more strongly related to the legumin genes.     

Synthesis and pharmacological evaluation of novel 1,3,8- and 1,3,7,8-substituted xanthines as adenosine receptor antagonists

A number of novel xanthines bearing a variety of substituents at positions 1, 3, 7 and 8 were prepared and evaluated for their binding affinity to the human adenosine receptor A₁, A_2A, A_2B and A₃ subtypes. Several of the 1,3,8- and 1,3,7,8-substituted xanthines showed moderate-to-high affinity at human A_2B and A₁ receptors, with the most active compound (14q) having a pK_i of 7.57 nM for hA_2B receptors and a selectivity over hA_2A receptors of 8.1-fold and hA₁ receptors of 3.7-fold.     

REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridisation

The regenerating islet-derived (REG) gene family encodes a group of proteins highly expressed in several human pathologies, many of which are associated with epithelial inflammation. All human family members, namely REG1A, REG1B, REG3A and REG4, are closely related in genomic sequence and all are part of the c-type lectin superfamily. REGs are highly expressed during inflammatory bowel disease (IBD)-related colonic inflammation and the in vivo expression pattern of REG1A and REG4 has been localised by using immunohistochemistry. However, the function of the encoded proteins is largely unknown and the cellular localisation of REG expression during colonic inflammation has not been described. Therefore, we have used in situ hybridisation to demonstrate the cellular localisation of REG expression in healthy and diseased colonic mucosa. Samples drawn from an IBD cohort including both inflamed and un-inflamed colonic mucosa are described, as are samples from non-IBD inflammation and healthy controls. Immunohistochemistry against known cell-type markers on serial sections has localised the expression of REGs to metaplastic Paneth cells (REG1A, REG1B and REG3A) and enteroendocrine cells (REG4), with a marked expansion of expression during inflammation. The group of REGs can, based on gene expression patterns, be divided into at least two groups; REG1A, REG1B and REG3A with their expression focused in the crypt base spreading from Paneth cells and REG4 being more highly expressed towards the luminal face. This exploration of expression pattern forms provides the background for further exploration of REG function in the intestine.     

Regulation of the expression of lactate dehydrogenase isozymes in human lymphocytes

Mitogen activation of human peripheral lymphocytes leads to a switch in the isozymes of LDH; resting cells contain low activities of only the B₄ and B₃A forms, whereas activated cells contain high activities of the A₄ and A₃B forms. B₄ LDH is not altered in activated cells. In this study we show that the appearance of the A subunits occurs concomitantly with a several fold increase in the steady state levels of LDH-A mRNA. Responses in LDH-A mRNA are observed within 12 hrs of activation, and are, thus, associated with the G0/G1 transition or with early G1 (Marjanovicet al. Exp. Cell Res. (1991) 193: 425–431). Maximal expression of LDH-A mRNA requires both phorbol ester and concanavalin A, implying a complex regulatory pathway involving cascade systems activated through both the antigen receptor (TR) and protein kinase C.     

Resolution and reconstitution of complex V of the mitochondrial oxidative phosphorylation system: properties and composition of the membrane sector

The antigenic properties of purified glycinin subunits were studied using antibodies prepared against them. Antisera against native glycinin did not react with the isolated subunits, and antibodies prepared against the purified subunits were not active against native glycinin. When native glycinin -was denatured, the antiglycinin immunoglobulins lost their ability to react with it, although the denatured complex was then recognized by antibodies against the purified subunits. Substantial structural rearrangement apparently occurred when the native complex was denatured and disaggregated. Acidic polypeptides A_1a, A_1b, and A₂ had similar determinants as judged by their reactions against A_1a and A_1a antisera. The reaction of the A₃ polypeptides with these antibodies was of lower intensity and in each case clear spurs of cross-reactivity were visible. No cross-reaction was detected between polypeptide A₄ and either anti-A_1a or A₂. Anti-A₃ antibodies reacted with each of the acidic polypeptides of glycinin, and distinct spurs of cross-reactivity were observed between A₃ vs A_1a, A₃ vs A₂, and A₃ vs A₄. B₁ Antisera developed a reaction of identity between basic polypeptides B₁ and B₂, but reacted very weakly with B₃ and B₄. The acidic and basic polypeptides of glycinin were immunologically unrelated. The results demonstrated that immunological tests would successfully differentiate some members of the family of acidic subunits, and other immunoglobulins would discriminate between members of the family of basic subunits.     

Crystallographic and enzymatic insights into the mechanisms of Mg-ADP inhibition in the A1 complex of the A1AO ATP synthase

F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A₁A_O ATP synthases (A₃B₃CDE₂FG₂ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A₃B₃D- and A₃B₃DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments.     

Subunit Structure of Soybean 11S Globulin

The acidic and the basic subunits were shown to be present in equimolar amounts in the 11S globulin molecule by the densitometric scanning of the SDS gel and the molecular weight consideration. The four acidic subunits (A₁, A₂, A₃ and A₄) were found to be present in the approximate molar ratio of 1:1:2:2. Four basic subunits separated and designated as B₁, B₂, B₃ and B₄ based on the relative mobilities in the acidic gel in 7 m urea were found to be present in the approximate molar ratio of 1:1:2:2. The four basic subunits were fractionated in approximately same amounts into three different peaks, peak I (B₁ and B₂), peak II (B₃) and peak III (B₄) by CM-Sephadex C–50 column chromatography in the presence of 6 m urea. Three kinds of intermediary subunits of 11S globulin were fractionated with DEAE-Sephadex A–50 in the absence of reducing agents in 6 m urea, and disulfide bonds appeared to participate in the binding between the acidic and the basic subunits in the molar ratio of 1: 1 with the following combinations; A₁ and A₂ combined with B₃, A₃ with B₁ and B₂, and A₄ with B₄. In view of the above results and molecular weight consideration, a new model of subunit structure was proposed for 11S globulin.     

Interaction of the Thermoplasma acidophilum A1A0-ATP synthase peripheral stalk with the catalytic domain

The peripheral stalk of the archaeal ATP synthase (A₁A₀)-ATP synthase is formed by the heterodimeric EH complex and is part of the stator domain, which counteracts the torque of rotational catalysis. Here we used nuclear magnetic resonance spectroscopy to probe the interaction of the C-terminal domain of the EH heterodimer (E_CT1H_CT) with the N-terminal 23 residues of the B subunit (B_NT). The data show a specific interaction of B_NT peptide with 26 residues of the E_CT1H_CT domain, thereby providing a molecular picture of how the peripheral stalk is anchored to the A₃B₃ catalytic domain in A₁A₀.

Structured summary

MINT-7260681: Hct (refseq:NP_393485), Ect1 (uniprotkb:Q9HM68) and Bnt (uniprotkb:Q9HM64) physically interact (MI:0915) by nuclear magnetic resonance (MI:0077)     

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A1 and A2B adenosine receptor subtypes

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N¹ (1,2) or N¹⁰ (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A₁ and A_2B AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A₁ and A_2B ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs.Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A₁ AR and A_2B AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A₁ and A_2B ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.     

4-Substituted-7-N-alkyl-N-acetyl 2-aminobenzothiazole amides: Drug-like and non-xanthine based A2B adenosine receptor antagonists

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A_2B receptor antagonists because of its slight selectivity against A₁ and A_2A receptors and modest A_2B potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A_2B potency, modest selectivity versus A_2A and A₁ receptors, and good rodent PK properties.     

The Influence of the 1-(3-Trifluoromethyl-Benzyl)-1H-Pyrazole-4-yl Moiety on the Adenosine Receptors Affinity Profile of Pyrazolo[4,3-e][1,2,4]Triazolo[1,5-c]Pyrimidine Derivatives

A new series of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) derivatives has been developed in order to explore their affinity and selectivity profile at the four adenosine receptor subtypes. In particular, the PTP scaffold was conjugated at the C2 position with the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole, a group believed to confer potency and selectivity toward the human (h) A_2B adenosine receptor (AR) to the xanthine ligand 8-(1-(3-(trifluoromethyl)benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (CVT 6975). Interestingly, the synthesized compounds turned out to be inactive at the hA_2B AR but they displayed affinity at the hA₃ AR in the nanomolar range. The best compound of the series (6) shows both high affinity (hA₃ AR K_i = 11 nM) and selectivity (A₁/A₃ and A_2A/A₃ > 9090; A_2B/A₃ > 909) at the hA₃ AR. To better rationalize these results, a molecular docking study on the four AR subtypes was performed for all the synthesized compounds. In addition, CTV 6975 and two close analogues have been subjected to the same molecular docking protocol to investigate the role of the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole on the binding at the four ARs.     

Secretion of Hexosaminidase Isozymes by Tetrahymena*

SYNOPSIS. Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of β-N-acetylhexosaminidase (2-acetamido-2-deoxy-β-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A₁ and B₁, and 2 minor forms, A₂ and B₂, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B₁ has a molecular weight of ?93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-β-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A₁ has a molecular weight of ?170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-β-D-galactosaminide than the B forms. Neither hexosaminidases A₁ or B₁ has any endo-β-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete ?20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxy-benzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

Malate dehydrogenase in subtropical fish belonging to the orders characiformes,siluriformes and perciformes I. Duplicate gene expression and polymorphism

• 1.1. Electrophoretic analysis of the soluble malate dehydrogenase (sMDH) from 22 subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes, collected in 10 reservoirs of São Paulo State and in two lakes of Minas Gerais State, Brazil, indicates that at least two sMDH loci, MDH-A¹ and MDH-B¹, are active. In addition to this latter locus, in Hoplias malabaricus (Erythrinidae, Characiformes), a MDH-A 1,3¹ isoloci is proposed in order to explain the six-banded pattern detected in all the individuals screened.
• 2.2. In attempting to explain the multiplicity of compounds detected in 87% of the Geophagus brasiliensis (Cichlidae, Perciformes) specimens analyzed, three hypotheses are proposed: the event of duplication in processing the presence of three loci with a null allele within the MDH-B¹, and overdominance.
• 3.3. In 87% of the species here studied, a bidirectionally divergent pattern of expression of the sMDH loci was observed, in which the least anodal isozyme A₂ predominated in liver, and the most anodal isozyme B₂ predominated in skeletal muscle. In two siluriform species, Pimelodela gracilis and Hypostomus regani, and in one perciform, Tilapia rendalli, a unidirectionally divergent pattern, in which the isozyme A₂ predominated in every tissue analyzed, was observed.
• 4.4. Polymorphism in at least one of the sMDH loci was detected in 9% of the species studied here: Leporinus friderici (Characiformes) at the MDH-A¹ and P. gracilis at both sMDH loci. In L. friderici and Pimelodus maculatus (Siluriformes), rare alleles at the MDH-B¹ locus were detected. Polymorphism at the mitochondrial locus was detected in Tilapia rendalli.

     

Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments

This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.