Real-time detection of gene promoter activity: quantitation of toxin gene transcription.

We have developed a new method for quantification of promoter activity in cell lines transfected with recombinant plasmids containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) by real-time PCR. As the efficiency of transfection has a direct influence on the total mRNA produced, we have used the neomycin-resistance gene present within the same vector DNA to normalize the measurement of mRNA levels. Three promoters from genes encoding toxins (pre-synaptic neurotoxin phospholipase A(2), post-synaptic alpha neurotoxin and cardiotoxin), believed to have evolved from the same ancestor but exhibiting different promoter activities, have been employed in this study to demonstrate the feasibility and accuracy of the method in CAT gene reporter analysis.

Regulatory properties of the pyruvate dehydrogenase complex of Pseudomonas aeruginosa.

The pyruvate dehydrogenase multienzyme complex of Pseudomonas aeruginosa was subjected to a steady-state kinetic analysis using the exponential model for a regulatory enzyme and a sensitive statistical fitting procedure. This showed that all the substrates, pyruvate, CoA and NAD+, exhibit cooperative kinetics towards the native multienzyme complex.

Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs.

Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.

Cloning, characterization and expression of a cDNA clone encoding rabbit ubiquitin-conjugating enzyme, E2(32k).

A cDNA clone encoding rabbit E2(32k) was obtained by library screening and PCR. The cDNA contains an open reading frame coding for 238 amino acids which shows an overall identity of 81% to human CDC34, the cell cycle-related ubiquitin-conjugating enzyme. A 50% homology to yeast CDC34 within the conserved core domain was also observed. Northern blot analysis indicated that three transcripts existed in all six rabbit tissues examined but their expression levels varied over a wide range. The putative cDNA coding region was highly expressed in Escherichia coli as a his-tagged protein which was purified to homogeneity. The ability of this expressed protein to form a thiolester bond with ubiquitin showed that it was functionally active. The ability of this protein to catalyze the conjugation of ubiquitin to histone H2A and H2B was also examined.

Cloning and expression analysis of a cytochrome P-450(11 beta) cDNA in sheep.

A full length ovine steroid 11 beta-hydroxylase (cytochrome P-450(11 beta)) cDNA clone from a sheep adrenal cortex cDNA library was isolated. Sequence analysis indicates that this cDNA clone resembles bovine P-450(11 beta) cDNA (95% nucleotide sequence homology) more closely than rat P-450(11 beta) cDNA (69% nucleotide sequence homology). Although the levels of nucleotide sequence homology of this cDNA clone to the rat P-450(11 beta) cDNA and the rat P-450aldo cDNA are similar, the putative amino acid sequence shows a closer resemblance to rat P-450aldo protein. Northern blot analysis shows that there are three sizes of transcript and they are expressed throughout the adrenal cortex.

Sputa nerve growth factor forms a preferable substitute to mouse 7S-beta nerve growth factor.

The NGF (nerve growth factor) from Naja sputatrix has been purified by gel filtration followed by reversed-phase HPLC. The protein showed a very high ability to induce neurite formation in PC12 cells relative to the mouse NGF. Two cDNAs encoding isoforms of NGF have been cloned and an active recombinant NGF, sputa NGF, has been produced in Escherichia coli as a His-tagged fusion protein. Sputa NGF has been found to be non-toxic under both in vivo and in vitro conditions. The induction of neurite outgrowth by this NGF has been found to involve the high-affinity trkA-p75NTR complex of receptors. The pro-survival mechanism of p75NTR has been mediated by the activation of nuclear factor kappaB gene by a corresponding down-regulation of inhibitory kappaB gene. Real-time PCR and protein profiling (by surface-enhanced laser-desorption-ionization time-of-flight) have confirmed that sputa NGF up-regulates the expression of the endogenous NGF in PC12 cells. Preliminary microarray analysis has also shown that sputa NGF is capable of promoting additional beneficial effects such as the up-regulation of arginine vasopressin receptor 1A, voltage-dependent T-type calcium channel. Hence, sputa NGF forms a new and useful NGF.

Regulation of erythropoietin gene expression in fetal sheep by glucocorticoids.

Erythropoietin (Epo) gene expression in ovine fetal liver and kidneys was measured by competitive RT-PCR in situations in which fetal glucocorticoid status was altered. Bilateral adrenalectomy at 120 +/- 0.3 days gestation (term is 145-150 days) caused a significant (P < 0.05) 5-fold increase in renal Epo messenger RNA (mRNA) levels at 145 +/- 1 days compared to those in age-matched controls. With cortisol replacement in adrenalectomized fetuses, renal Epo mRNA levels dropped to 17% of this values (P < 0.05). Cortisol infusion (230 micrograms/h for 48 h) at 108 +/- 1 day decreased renal Epo gene expression significantly (P < 0.01) to 23% of the control value; dexamethasone treatment of the ewe at midgestation (0.76 mg/h for 48 h) also decreased fetal, but not adult, renal Epo mRNA levels (to 12% of control value; P < 0.01). Fetal and maternal liver Epo mRNA levels were unaffected by glucocorticoid status at any stage of pregnancy. Thus, glucocorticoids can influence fetal renal, but not maternal, Epo gene expression. In the presence of high concentrations of fetal glucocorticoids, plasma Epo values were consistently 4-5 mU/ml, close to the sensitivity of the assay, whereas in seven adrenalectomized fetuses, the plasma Epo value was 9.1 +/- 1.4 mU/ml.

Rabbit ubiquitin-activating enzyme E1: cDNA cloning, sequence and expression.

A cDNA clone encoding ubiquitin-activating enzyme E1 has been isolated from a rabbit heart cDNA library and sequenced. The 3.485 kb cDNA contains an open reading frame of 1058 amino acid residues which predicts a protein of approx. 118 kDa. The deduced protein sequence exhibits a very high homology to other ubiquitin-activating enzymes identified in a variety of organisms. Northern blot analysis reveals a single transcript of approx. 3.5 kb in all the rabbit tissues examined. The entire coding region of the rabbit E1 cDNA has been expressed as a his-tagged protein. The recombinant protein has been verified by its ability to cross-react with anti-human E1 antibodies. Ubiquitin thiolester assay shows that the recombinant rabbit E1 protein is functional.

Molecular cloning, characterization and evolution of the gene encoding a new group of short-chain alpha-neurotoxins in an Australian elapid, Pseudonaja textilis.

The structure and organization of five genes responsible for the synthesis of six isoforms of short-chain alpha-neurotoxins in Pseudonaja textilis venom are reported in this paper. This also forms the first report which describes the synthesis of two neurotoxin mRNA variants from one of these genes (Pt-sntx1) as a result of alternative splicing. Each gene consists of three exons which are separated by two introns and each has a functional promoter. The promoter activity was confirmed by both CAT assay and Real-Time PCR. A transcription initiation site, two putative TATA boxes, one CCAAT box and the transcription factor binding consensus sites for AP-1, GATA-2, c/EBPb were identified in the 5' non-coding region of each gene. Phylogenetic analysis showed that these five genes from P. textilis constituted a distinct group which has evolved by gene duplication followed by accelerated evolution from an ancestral gene.

Genomic structure of a potassium channel toxin from Heteractis magnifica.

We provide information on the gene encoding the K+ channel toxin, HmK, of the sea anemone Heteractis magnifica. A series of DNA amplifications by PCR, which included the amplification of the 5'-untranslated region of the gene, showed that an intron of 402 nucleotides separated the sequence that encodes the matured toxin from the signal peptide sequence. A second 264 nucleotide intron interrupted the 5'-untranslated region of the previously reported HmK cDNA. Two possible transcription-initiation sites were identified by primer extension analysis. Corresponding TATA-box consensus sequences, characteristic of a promoter region, were also located from PCR products of uncloned libraries of adaptor-ligated genomic DNA fragments. The coding region for matured HmK is intronless. The same is also true for other sea anemone toxins reported thus far. More notably, a similar intron-exon organization is present in other ion channel-blocking toxins from scorpions implying that molecules having similar functions share a similar organization at the genomic level suggesting a common path of evolution.

Structure and organization of the cardiotoxin genes in Naja naja sputatrix.

We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding cardiotoxins (CTX) of Naja naja sputatrix. The cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP-2, NFIL-6/C/EBP, NF-kappaB and PuF have been identified in the 5'-region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three-finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.

In situ hybridization and immunohistochemical analysis of the expression of cardiotoxin and neurotoxin genes in Naja naja sputatrix.

Secretory processes and their regulation have been extensively studied in mammalian salivary parotid glands. However, little is known regarding the secretory mechanism in the venom glands of snakes, which invariably produce one of the most complex of all animal secretions. The pharmacologically important and toxic components of the Malayan spitting cobra (Naja naja sputatrix) venom include postsynaptic neurotoxins (NTX), presynaptic neurotoxins (phospholipase A2, PLA2), and cardiotoxins (CTX) which, for convenience, have been collectively referred to as "toxins." We report here for the first time the mechanism of toxin gene expression by studying the accumulated mRNA level and protein synthesis rates for the three toxins over a period of 8 days after stimulation of venom synthesis by manual "milking" of the venom gland. Immunofluorescence and in situ hybridization were used to localize the toxins and their mRNAs in venom gland sections. The rate of protein synthesis, as determined by immunofluorescence and liquid chromatography-mass spectrometry (LC-MS) techniques, increased in parallel with the increase in the toxin mRNA content in the secretory epithelial cells, suggesting that transcriptional regulation of the toxin genes is involved. (J Histochem Cytochem 47:551-560, 1999)

Adrenergic regulation of RNA synthesis in the rat parotid gland.

Adrenergic regulation of RNA synthesis by in vivo stimulated parotid glands and dispersed parotid lobules was studied by a combination of in vivo and in vitro methods. Following a single intraperitoneal injection of isoproterenol, [3H]uridine incorporation into RNA was increased by 50% after the first hour. Amylase mRNA content was also elevated within 1 hr and was 2-3-fold higher than control values at 4 hr. An increase in the rate of total protein synthesis was detectable after 2 hr, and maximal rates were achieved 6 hr after isoproterenol administration. In dispersed parotid lobules, both isoproterenol and epinephrine stimulated [3H]uridine incorporation and at optimal concentrations increased incorporation by almost 200%. Phenylephrine (10 microM) caused a slight increase of about 20% whereas methoxamine (10 microM) had no effect. Stimulation by epinephrine was reversed by propranolol, but not by either phentolamine or prazosin. The increase in RNA synthesis induced by isoproterenol or epinephrine was dose dependent and half-maximal stimulation required 5.0 x 10(-8) M isoproterenol and 7.9 x 10(-7) M epinephrine. Dibutyryl cyclic AMP also stimulated [3H]uridine incorporation, whereas 8-bromo cyclic GMP, A23187 and phorbol myristate acetate had no effect. The importance of protein phosphorylation in mediating the observed stimulation was evaluated using protein kinase and phosphatase inhibitors. N-[2-(Methylamino)ethyl]-5-isoquinolinesulphonamide, an inhibitor of cyclic nucleotide-dependent protein kinases, substantially diminished the isoproterenol-induced stimulation. Okadaic acid treatment of lobules increased [3H]uridine incorporation. Furthermore, okadaic acid synergistically potentiated the stimulatory effect of a suboptimal concentration of isoproterenol. The results demonstrate that activation of the beta-adrenergic receptor induces the synthesis of certain RNA species in the parotid gland and that protein phosphorylation by a cyclic AMP-dependent protein kinase is a key event in the signal transduction pathway.

Ontogeny of aquaporins 1 and 3 in ovine placenta and fetal membranes.

A sensitive and highly reproducible method has been used to show that Aquaporin 3 (AQP(3)) mRNA is present in the ovine placenta and chorion from at least 60 days of gestation (term=145-150d) with levels increasing substantially (>16 fold) at 100 days, and remaining constant thereafter. By immuno- and hybridization histochemistry, the epithelial cells expressing AQP(3)were found to be the trophoblast cells. Some AQP(3)was expressed in fibroblasts of the amnion and allantois but none was expressed in the epithelia of these membranes. AQP(1)was expressed in endothelial cells of fetal and maternal blood vessels but not in any epithelial cell of the ovine placenta and fetal membranes. The level of AQP(3)expression is consistent with known ovine placental permeabilities to water, glycerol and urea.

Effect of hemorrhage and nephrectomy on erythropoietin gene expression in the ovine fetus.

The purposes of this study were to determine whether (1) erythropoietin (Epo) gene expression could be stimulated, by severe hemorrhage, in both kidney and liver, at three stages of gestation (75-80, 106-112, and 140-142 days; term approximately equal to 150 days) in chronically-cannulated ovine fetuses and (2) whether the liver would compensate for the lack of kidneys at 110 days. Blood was removed (20% of estimated blood volume) at each of 0, 1, 19 h, and tissues collected at 24 h. In hemorrhaged fetuses (H) the liver mRNA levels were 2.7-, 5-, and 3-times that of control fetuses (C) at 78, 110 and 141 days, respectively. The kidney H:C ratios, for Epo mRNA, were 5, 6.4 and 43, respectively, at these three stages of gestation. In six fetuses at 108 days, nephrectomized 5-7 days before hemorrhage (HN) Epo mRNA increased 5-fold in the liver, and plasma Epo values were significantly lower (P < 0.05) than in intact (H). Thus hemorrhage stimulates increased Epo gene expression in both liver and kidney from mid-gestation, in the ovine fetus, but the liver does not compensate for the lack of kidneys at 110 days.

A genomic region encoding stonefish (Synanceja horrida) stonustoxin beta-subunit contains an intron.

The polymerase chain reaction (PCR) has been used to amplify a 1899 base pair fragment from stonefish genomic DNA. A comparison of the translated nucleotide sequence of this product with the separately determined N-terminal amino acid sequence of the beta-subunit reveals the presence of a 416 bp intron at Gly 18. The nucleotide sequence following this intron encodes 476 amino acids whose sequence showed no homology to other known toxins. This region, however, contained amino acid sequences identical to internal peptide sequences determined separately from the toxin's beta-subunit.

Molecular cloning of a cardiotoxin structural gene from Malayan spitting cobra (Naja naja sputatrix).

The structural gene and cDNA encoding cardiotoxin in Naja naja sputatrix have been cloned and characterized with a view to study the gene protein relationships and also to produce pure protein in large amounts. Using the polymerase chain reaction on the total RNA isolated from the venom glands, the structural gene (180 bp) has been synthesized and expressed in Escherichia coli to produce a fusion protein with beta-galactosidase. Immunoblotting using polyclonal antibodies raised against the total venom in rabbits demonstrated the presence of cross-reacting proteins in plaques produced by recombinant lambda gt11 phages.

Four new postsynaptic neurotoxins from Naja naja sputatrix venom: cDNA cloning, protein expression, and phylogenetic analysis.

cDNAs encoding 4 short chain alpha-neurotoxins from Malayan spitting cobra (Naja naja sputatrix) venom were cloned and expressed in Escherichia coli. The recombinant toxins possessed identical amino acid sequences to alpha-neurotoxins. This is the first report on cloning and expression of isoforms of neurotoxins from a species of spitting cobra. Two of these isoforms were also identified in the crude venom by reverse phase-high performance liquid chromatography (RP-HPLC), capillary electrophoresis followed by mass spectrometry and characterized by protein sequencing. Based on the variable amino acid residues, the neurotoxins in N. n. sputatrix could be assigned to 2 major groups, 10E11T and 10Q11A, which could be further subdivided into 10E11T28S: 10E11T28G and 10Q11A28S; 10Q11A28G respectively. These substitutions were also found to be unique to N. n. sputatrix neurotoxins. Phylogenetic analysis based on molecular properties of the toxins provided further support for the classification of N. n. sputatrix neurotoxin into 2 fundamental groups.

Cloning and characterization of cDNAs encoding three isoforms of phospholipase A2 in Malayan spitting cobra (Naja naja sputatrix) venom.

cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

Stimulation of RNA synthesis in rat parotid lobules by phorbol myristate acetate.

Incubation of parotid lobules with phorbol 12-myristate 13-acetate (PMA) transiently stimulated the incorporation of [3H]uridine into RNA. At 100 nM PMA, the rate of RNA synthesis during the first hour was 30% above control rates. The Ca2+ ionophore A23187 had no effect on either basal or PMA-stimulated RNA synthesis. Stimulation by 100 nM PMA was reversed by the protein kinase C inhibitor staurosporin (10 nM). When PMA was added together with isoproterenol or okadaic acid, both of which are potent activators of RNA synthesis, the increase in RNA synthesis was additive rather than synergistic. The results suggest that in the rat parotid gland, protein kinase C induces the rapid transcription of certain cellular genes by a mechanism that is independent of the beta-adrenergic receptor-activated pathway.