Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS2) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS1). A sequence identical to an N-terminal sequence determined from a purified preparation of the mature GS2 polypeptide (NH2-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46-61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS2 subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS2-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS2 mRNA, and an anti-GS antiserum to assay for GS2 protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS2 mRNA; class II, which had normal or increased levels of GS2 mRNA but very little GS2 protein; and class III, which had significant amounts of GS2 protein but little or no GS2 activity.
Plant Mol Biol 1990 Mar
PMID:Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase. 198 86

The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.
J Mol Biol 1991 Feb 05
PMID:Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin. All six modules are independently folded. 199 38

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.
Mol Cell Biol 1991 May
PMID:A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin. 201 64

A series of tripeptides that satisfy the -Asn-Xaa-Thr/Ser- primary sequence requirement [Marshall, R. D. (1972) Annu. Rev. Biochem. 41, 673-702] for N-glycosylation have been synthesized and examined as potential acceptors in an oligosaccharyltransferase assay. Of these, six (Ac-Asn-Ala-Thr-NH2, Ac-Asn-Leu-Thr-NH2, Ac-Asn-Asp-Thr-NH2, Ac-Asn-D-Ala-Thr-NH2, Ac-Asn-Pro-Thr-NH2, and Ac-Asn-AIB-Thr-NH2) were examined for solution conformational properties in dimethyl sulfoxide with use of amide proton temperature coefficients, 3JHN alpha analysis [Pardi, A., et al. (1984) J. Mol. Biol. 180, 741-751], and 2-D ROESY experiments [Bothner-By, A. A., et al. (1984) J. Am. Chem. Soc. 106, 811-813]. The analysis reveals that the peptides that serve as acceptors in the transferase assay demonstrate similar conformational properties in solution. These are highlighted by a secondary structural motif that involves the interaction between the asparagine side-chain carboxamide and the backbone amide of the threonine. The peptides that show very poor acceptor, or even nonacceptor, properties in the oligosaccharyltransferase assay demonstrate different conformational features in solution. These observations may explain the distinct biological activity observed for these peptides.
 ...
PMID:Differences between Asn-Xaa-Thr-containing peptides: a comparison of solution conformation and substrate behavior with oligosaccharyltransferase. 202 29

The Phanerochaete chryososporium trpC gene has been isolated by complementation of an Escherichia coli trpC mutant. The full extent of the fungal gene, determined by sequence analysis, was found to be 2414bp. This includes a single intron of 50bp, the presence of which was confirmed by RNA-primed polymerase chain reaction analysis. This features makes the P. chrysosporium gene unique when compared to equivalent genes from other filamentous fungi. The P. chrysosporium trpC gene encodes a single protein containing three enzyme activities involved in tryptophan biosynthesis arranged in the order: NH2-GAT-IGPS-PRAI-COOH. This order is conserved in all filamentous fungi so far examined and, indeed, is the gene order within the E. coli trp operon.
Mol Microbiol 1991 Feb
PMID:The trpC gene of Phanerochaete chrysosporium is unique in containing an intron but nevertheless maintains the order of functional domains seen in other fungi. 204 79

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.
Mol Cell Biol 1991 Jul
PMID:Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone. 204 70

The conversion of 3 beta-hydroxy-5-ene steroids by the enzyme complex 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues. To develop a model more closely related to the human, we have isolated and characterized cDNA clones encoding macaque 3 beta-HSD by screening a rhesus monkey ovary lambda gt11 cDNA library using a human 3 beta-HSD cDNA probe. Nucleotide sequence of 1629 bp from overlapping cDNA clones predicts a protein of 372 amino acids with a calculated molecular mass of 41,874 (excluding the first Met). The deduced amino acid sequence of macaque 3 beta-HSD displays 79.4% and 93.9% similarity with that of bovine and human 3 beta-HSD, respectively. RNA blot analysis performed under high stringency conditions of macaque poly(A)+ RNA samples using full-length 32P-labeled macaque 3 beta-HSD cDNA revealed the presence of an approximately 1.7 kb mRNA species in classical steroidogenic tissues, namely the ovary, testis and adrenal glands as well as in several peripheral tissues including the liver, kidney and epididymis. Computer analysis of the deduced macaque 3 beta-HSD protein sequence predicts the presence of an NH2-terminal membrane-associated segment as well as four additional membrane-spanning segments, thus suggesting that 3 beta-HSD is an integral protein. The availability of macaque cDNA should permit detailed studies concerning the tissue-specific expression as well as the hormonal regulation of 3 beta-HSD mRNA in classical steroidogenic glands as well as in peripheral tissues which are an important site of steroidogenesis in primates.
Mol Cell Endocrinol 1991 Feb
PMID:Characterization of macaque 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase: structure and expression in steroidogenic and peripheral tissues in primate. 205 Feb 70

The nucleotide sequence of a cDNA that codes for the major phenobarbital (PB)-inducible male beagle dog hepatic cytochrome P450 has been determined. Using a rabbit P450IIB cDNA probe (R. Gasser, M. Negishi, and R. M. Philpot, 1988, Mol. Pharmacol, 32, 22-30), a cDNA clone with a 2.6-kilobase pair insert was isolated from a lambda gt11 library prepared from hepatic mRNA from a PB-treated dog. The cloned insert was sequenced and found to contain an open reading frame coding for a polypeptide of 494 amino acids (Mr 56,183). The encoded protein can be assigned to the P450IIB subfamily on the basis of homology to cytochromes P450 from other species. The deduced amino acid sequence is 79% identical to that reported for rabbit P450 BO (P450IIB4) and 75% identical to that for rat P450b (P450IIB1). The sequence identity decreases to less than 52% when the dog sequence is compared with other P450II subfamilies. The deduced NH2-terminal 30 amino acids encoded by the dog cDNA are identical to those determined by sequence analysis of purified dog cytochrome P450 PBD-2, and the amino acid composition concurs with that determined for the PBD-2 protein (D. B. Duignan, I. G. Sipes, T. B. Leonard, and J. R. Halpert, 1987, Arch. Biochem. Biophys. 255, 290-303). Northern blots revealed two mRNA species of approximately 1.9 and 2.9 kilobases in length, which hybridized to the coding region of the dog P450IIB cDNA. The level of total hybridizable mRNA was increased approximately sixfold in livers from PB-treated dogs compared with that in untreated animals. This increase correlates well with the reported nearly sixfold increase in the level of PBD-2 protein and the fivefold increase in the rate of hepatic metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl following PB treatment. The two mRNA species may result from the use of different polyadenylation signals located in the 3'-noncoding region or from transcription of more than one gene for PBD-2. Southern blot analysis indicated that the dog P450IIB subfamily contains at least two closely related genes.
 ...
PMID:cDNA and deduced amino acid sequences of a dog hepatic cytochrome P450IIB responsible for the metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl. 211 65

The primary structure of a human mu heavy chain (DAG) protein is described. The native protein is a circular decamer with a molecular weight (Mr) of 500 kDa, each decamer being constituted of the constant domains C mu 2, C mu 3 and C mu4 and interlinked by 15 disulfide bridges. At its NH2-terminal each monomeric chain starts with an "extra sequence". The amino acid sequence of this segment is Arg-Gln-Ser-Asp-Asp-Pro-Val-Leu-Arg-Gly-Thr-Thr-Val-Pro-Val-Thr-Glu and its reinitiation point is located at Val223 (Gal numbering), at the beginning of C mu 2. This sequence has no homology with any other protein included in the present databases.
Mol Immunol 1990 Aug
PMID:A new extra sequence at the amino terminal of a mu heavy chain disease protein (DAG). 211 80

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.
Mol Plant Microbe Interact
PMID:Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco. 213 Oct 96


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