Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Five aspartate aminotransferase (EC 2.6.1.1; AAT) isozymes were identified in soybean seedling extracts and designated AAT1 to AAT5 based on their rate of migration on non-denaturing electrophoretic gels. AAT1 was detected only in extracts of cotyledons from dark-grown seedlings. AAT3 and AAT4 were detected in crude extracts of leaves and in cotyledons of seedlings grown in the light. AAT2 and AAT5 were detected in all tissues examined. A soybean leaf cDNA clone, pSAT17, was identified by hybridization to a carrot AAT cDNA clone at low stringency. pSAT17 had an open reading frame which could encode a 50,581 Da protein. Alignment of the deduced amino acid sequence from the pSAT17 open reading frame with mature AAT protein sequences from rat disclosed a 60 amino acid N-terminal extension in the pSAT17 protein. This extension had characteristics of a plastid transit peptide. A plasmid, pEXAT17, was constructed which encoded the mature protein lacking the putative chloroplast transit polypeptide. Transformed Escherichia coli expressed a functional soybean AAT isozyme, which comigrated with the soybean AAT5 isozyme during agarose gel electrophoresis. Differential sucrose gradient sedimentation of soybean extracts indicated that AAT5 specifically cofractionated with chloroplasts. Antibodies raised against the pEXAT17-encoded AAT protein specifically reacted with the AAT5 isozyme of soybean and not with any of the other isozymes, indicating that the soybean cDNA clone, pSAT17, encodes the chloroplast isozyme, AAT5.
Plant Mol Biol 1993 Mar
PMID:Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase. 768 17

A soybean leaf cDNA clone, pSAT2, was isolated by hybridization to a carrot aspartate aminotransferase (EC 2.6.1.1.; AAT) cDNA clone at low stringency. pSAT2 contained an open reading frame encoding a 47640 Da protein. The protein encoded by pSAT2 showed significant sequence similarity to AAT proteins from both plants and animals. It was most similar to two Panicum mitochondrial AATs, 81.5% and 82.0% identity. Alignment of the pSAT2-encoded protein with other mature AAT enzymes revealed a 25 amino acid N-terminal extension with characteristics of a mitochondrial transit peptide. A plasmid, pEXAT2, was constructed to encode the mature pSAT2 protein lacking the putative mitochondrial transit peptide. Escherichia coli containing the plasmid expressed a functional AAT isozyme which comigrated with the soybean AAT4 isozyme during agarose gel electrophoresis. Equilibrium sucrose gradient sedimentation of soybean extracts demonstrated that AAT4 specifically cofractionated with mitochondria. Antibodies raised against the pEXAT2-encoded AAT protein reacted with AAT4 of soybean and not with other AAT isozymes detected in soybean tissues, providing further evidence that clone pSAT2 encodes the soybean mitochondrial isozyme AAT4.
Plant Mol Biol 1995 Mar
PMID:Characterization of a soybean cDNA clone encoding the mitochondrial isozyme of aspartate aminotransferase, AAT4. 776 91

A clone encoding aspartate aminotransferase (AAT, EC 2.6.1.1) was isolated from an Arabidopsis thaliana leaf cDNA library. This clone contains a 1365 bp open reading frame encoding a polypeptide of 49.8 kDa, designated Ataat1. The clone was shown to contain a chloroplastic isoenzyme as an in organellar protein import assay demonstrated that a radiolabelled transcription/translation product of 49.8 kDa was imported into viable pea chloroplasts and was subsequently processed to yield a mature protein of 45 kDa. The open reading frame corresponding to the predicted mature AAT was manipulated into an expression construct (pEC14). Transformed Escherichia coli cells containing pEC14 expressed up to 16 times more AAT activity than vector only controls, thus demonstrating conclusively that the clone encoded AAT.
Plant Mol Biol 1995 Mar
PMID:Isolation, characterisation and expression of a cDNA clone encoding plastid aspartate aminotransferase from Arabidopsis thaliana. 776 5

We have placed two different penicillin structural genes from Aspergillus nidulans, ipnA (encoding isopenicillin N synthetase, IPNS) and acyA (encoding acyl-CoA:6-aminopenicillanic acid acyltransferase, AAT), under the control of the strong alcA promoter [alcA(p)]. Single copies of these transcriptional fusions were targeted to the same chromosomal location and conditions have been worked out which simultaneously allow induction of the alcA(p) and support penicillin biosynthesis. Transcriptional induction of the chimeric genes alcA(p)::ipnA or alcA(p)::acyA(cdna) in the relevant recombinant strains results in 10-fold higher levels of the ipnA or acyA transcripts than those resulting from transcription of the corresponding endogenous genes. This increase causes a 40-fold rise in IPNS activity or a 8-fold rise in AAT activity. Despite this rise in enzyme levels, forced expression of the ipnA gene results in only a modest increase in levels of exported penicillin, whereas forced expression of the acyA gene reduces penicillin production, showing that neither of these enzymes is rate-limiting for penicillin biosynthesis in A. nidulans. A genomic version of the alcA(p)::acyA fusion in which the acyA gene is interrupted by three small introns, is inducible by threonine to a lesser extent (as determined by both acyA mRNA levels and AAT enzyme levels) than the corresponding cDNA version, suggesting that processing of the introns present in the primary transcript may limit acyA expression.
Mol Gen Genet 1995 Jan 06
PMID:Overexpression of two penicillin structural genes in Aspergillus nidulans. 782 6

Despite the extensive search for disease causing mutations in exons 28-36 of the neurofibromatosis type 1 (NF1) gene, the NF1 specific mutations so far documented account for only a small proportion of all NF1 cases. In this study, we have used 8 sets of new primers to amplify sequences throughout the NF1 gene, including 10 different exons and their flanking intron sequences. The derived PCR products from 150 independent NF1 patients and 50 normal controls were examined by heteroduplex analysis on Hydrolink gels. Three novel mutations were identified and characterised. Two of these mutations include the same 3-bp deletion (AAT) within exon 17 with a silent codon change from ACA (threonine) to ACG (threonine) and a loss of the codon ATG (methionine). The third mutation is a 10-bp deletion (TTCTCTTGGA) within exon 44 resulting in the formation of an inappropriate stop codon. These results should be useful for the further elucidation of the molecular basis of NF1.
Hum Mol Genet 1993 Nov
PMID:Neurofibromatosis type 1 (NF1): the search for mutations by PCR-heteroduplex analysis on Hydrolink gels. 790 9

The effects of a 7-day intraperitoneal infusion with VIP (0.03 nmol.kg-1.min-1) and its antagonist [4-Cl-D-Phe6,Leu17]-VIP (VIP-A; 3 nmol.kg-1.min-1) were studied in sham and bilaterally adrenalectomized rats bearing ACTH and angiotensin II (ANG-II)-responsive adrenocortical autotransplants. VIP significantly increased plasma aldosterone (ALDO) concentration (PAC) and lowered plasma renin activity (PRA) in both groups of animals, without affecting plasma levels of ACTH and corticosterone. This treatment caused a marked hypertrophy of adrenal zona glomerulosa (ZG) and its parenchymal cells (without inducing any significant change in the zona-fasciculata morphology), as well as of ZG-like cells of autotransplants. Isolated ZG cells and autotransplant quarters obtained from VIP-infused rats evidenced a notable increase in both their basal and maximally ACTH- or ANG-II-stimulated ALDO secretion. The simultaneous infusion of rats with VIP-A completely reversed all these effects of VIP. The infusion with VIP-A alone caused, in sham-operated rats, a net decrease in PAC, coupled with a rise in PRA, and a marked atrophy of ZG and ZG cells; basal and maximally stimulated ALDO secretion of dispersed ZG cells was also significantly lowered. Conversely, VIP-A did not evoke any appreciable effect in autotransplanted rats. These findings suggest that endogenous VIP is specifically involved in the maintenance of the growth and secretory capacity of rat adrenal ZG. Since regenerated adrenocortical autotransplants, which are responsive to VIP but not to VIP-A infusion, are completely deprived of chromaffin cells, the hypothesis is advanced that adrenal medulla may be the source of endogenous VIP regulating ZG function.
J Steroid Biochem Mol Biol 1994 Oct
PMID:Evidence that endogenous vasoactive intestinal peptide (VIP) plays a role in the maintenance of the growth and steroidogenic capacity of rat adrenal zona glomerulosa. 794 54

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.
Mol Cell Biol 1994 Sep
PMID:C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7. 806 49

Mutations in the p53 tumor suppressor gene are detected in approximately half of non-melanoma skin cancers. The type of base-pair changes observed strongly suggests solar radiation as the causative mutagen. Mutations are distributed nonrandomly and form moderate hotspots. We studied the capacity of ultraviolet B light (UVB, 280-320 nm) to induce base-pair changes into the p53 exon 7 sequence extending from nt 14067 to 14075 in human skin fibroblasts. This sequence contains hotspot codon 248. UVB induced mostly C-->A and G-->T transversions. The base-pair change with the highest relative abundance was C-->A in the first position of codon 250 (CCC-->ACC), followed by (in diminishing relative abundance) G-->T in the third position of codon 249 (AGG-->AGT), C-->A in the first position of codon 248 (CGG-->AGG), and C-->A in the third position of codon 247 (AAC-->AAA). The C-->T transition in the third position of codon 247 (AAC-->AAT) occurred with moderate efficiency. These base-pair changes are compatible with pyrimidine photodimers as premutagenic lesions, but they could also form opposite 8-hydroxyguanine, which is the major oxidation product of guanine. No evidence was obtained for the presence of tandem double CC-->TT transitions in the untranscribed strand at codons 247/248 and 250. The relative abundance of mutations induced by UVB in the p53 sequence extending from codon 247 to 250 in human fibroblasts does not correlate with mutations observed in the DNA from non-melanoma skin cancer. This lack of correlation suggests that the mutability of this p53 sequence at the DNA level plays only a minor role in the pathogenesis of non-melanoma skin cancer in humans.
Mol Carcinog 1994 Aug
PMID:Ultraviolet B light-induced mutagenesis of p53 hotspot codons 248 and 249 in human skin fibroblasts. 806 78

Genetic transformation of Leishmania has relied upon two exogenous selectable markers, neo and hyg, encoding resistance to G418 and hygromycin B respectively. There is a need for multiple independent selectable markers, since Leishmania is diploid and experimental sexual crosses are not currently feasible. Here we report on the development of two additional markers: pac, conferring resistance to the glycopeptide antibiotic puromycin, and phleo, conferring resistance to the DNA-binding drug phleomycin. We constructed a set of four analogous shuttle vectors with these four markers, using DNA segments flanking the Leishmania major H region hmtxr gene to provide information required for expression. These constructs (pHM-NEO, pHM-HYG, pHM-PAC and pHM-PHLEO) were successfully transfected into L. major, mostly with efficiencies comparable to those observed with previous DHFR-TS-based neo and hyg-containing constructs. The exception was pHM-PHLEO, which transfected 30-fold less efficiently; this may be related to the nonenzymatic mechanism of resistance encoded by phleo. All four constructs were shown to replicate extra-chromosomally. Stable transfectants bearing all paired combinations of pHM constructs were obtained by a second round of transfection. These data show that the four markers are functionally independent and in conjunction with the Leishmania N-acetylglucosaminyl transferase gene, brings the number of selectable markers available in Leishmania to five.
Mol Biochem Parasitol 1993 Nov
PMID:Two more independent selectable markers for stable transfection of Leishmania. 811 24

Phosphorothioate pesticides, such as parathion (O,O-diethyl-O-4-nitrophenyl phosphorothioate), undergo enzymic oxidation to the active insecticidal agents that are the analogous organophosphorus compounds. In hepatic microsomal fractions, the NADPH-mediated conversion of parathion to paraoxon occurs with concomitant loss of cytochrome P450 (P450) and associated activities. In this study, the capacity of parathion to inactivate specific P450 enzymes was studied in rat hepatic microsomes. Parathion was a potent inhibitor of P450 3A2- and 2C11-mediated androst-4-ene-3,17-dione (androstenedione) 6 beta- and 16 alpha-hydroxylation (Ki values of 13 +/- 2 and 2.3 +/- 0.1 microM, respectively, and Km/Ki ratios of 1.4 +/- 0.2 and 11 +/- 1, respectively). After a 10-min preincubation between parathion and NADPH-supplemented microsomes, to inactivate P450 before androstenedione hydroxylation was carried out, the corresponding Km/Ki ratios were increased to 3.5 +/- 0.4 and 35 +/- 6, reflecting 2.5- and 3.2-fold enhancement of inhibition of P450 3A2- and 2C11-dependent activities. In contrast to these findings, P450 2A1/2-mediated androstenedione 7 alpha-hydroxylation was refractory to inhibition and P450 2C6-mediated progesterone 21-hydroxylation was inhibited but not inactivated by the pesticide. Further studies established that androstenedione 6 beta- and 16 alpha-hydroxylation pathways were inactivated with maximal half-times of 2.59 min and 1.72 min, respectively. Although the incubation of parathion (50 microM) with rat liver microsomes for 10 min led to a 16% decrease in P450 estimated spectrophotometrically, immunoblot analysis revealed no change in the microsomal content of P450 2C11 apoprotein. Finally, NADPH-mediated metabolism of parathion to paraoxon (by desulfuration) and 4-nitrophenol (by oxidative cleavage of the phosphorothioate ester) occurred efficiently in microsomes (4.32 and 4.35 nmol/min/mg of protein, respectively). P450 loss was estimated under the same incubation conditions and, thus, 210 parathion molecules were oxidized for each molecule of holo-P450 lost. These findings establish that parathion is a potent inhibitor and inactivator of the principal constitutive P450s, 3A2 and 2C11, in rat liver, whereas the P450s 2A1 and 2A2 are refractory to either inhibition or inactivation. Another major constitutive enzyme, P450 2C6, is inhibited effectively by parathion but does not appear to be subject to inactivation.
Mol Pharmacol 1993 Jun
PMID:Inhibition and inactivation of constitutive cytochromes P450 in rat liver by parathion. 831 22


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