scholarly journals Molecular characterization of the BRO beta-lactamase of Moraxella (Branhamella) catarrhalis.

1996 ◽  
Vol 40 (4) ◽  
pp. 966-972 ◽  
Author(s):  
H J Bootsma ◽  
H van Dijk ◽  
J Verhoef ◽  
A Fleer ◽  
F R Mooi
Keyword(s):  
Amino Acid ◽  
Active Sites ◽  
Signal Sequence ◽  
Amino Acid Difference ◽  
Beta Lactamase ◽  
Amino Acid Residues ◽  
Beta Lactam ◽  
Insertional Inactivation ◽  
Membrane Compartment ◽  
Branhamella Catarrhalis

A rapid increase in the prevalence of beta-lactamase-producing Moraxella (Branhamella) catarrhalis strains has been noticed during the last decades. Today, more than 80% of strains isolated worldwide produce beta-lactamase. To investigate beta-lactamase(s) of M. catarrhalis at the molecular level, the BRO-1 beta-lactamase gene (bla) was isolated as part of a 4,223-bp HindIII fragment. Sequence analysis indicated that bla encodes a polypeptide of 314 amino acid residues. Insertional inactivation of bla in M. catarrhalis resulted in complete abrogation of beta-lactamase production and ampicillin resistance, demonstrating that bla is solely responsible for beta-lactam resistance. Comparison with other beta-lactamases suggested that M. catarrhalis beta-lactamase is a unique enzyme with conserved residues at the active sites. The presence of a signal sequence for lipoproteins suggested that it is lipid modified at its N terminus. In keeping with this assumption was the observation that 10% of beta-lactamase activity was found in the membrane compartment of M. catarrhalis. M. catarrhalis strains produce two types of beta-lactamase, BRO-1 and BRO-2, which differ in their isoelectric points. The BRO-1 and BRO-2 genes from two ATCC strains of M. catarrhalis were sequenced, and only one amino acid difference was found between the predicted products. However, there was a 21-bp deletion in the promoter region of the BRO-2 gene, possibly explaining the lower level of production of BRO-2. The G + C content of bla (31%) was significantly lower than those of the flanking genes (47 and 50%), and the overall G + C content of the M. catarrhalis genome (41%). These results indicate that bla was acquired by horizontal gene transfer from another, still unknown species.

scholarly journals Rat liver β-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells

1988 ◽  
Vol 250 (2) ◽  
pp. 547-555 ◽  
Author(s):  
P P Powell ◽  
J W Kyle ◽  
R D Miller ◽  
J Pantano ◽  
J H Grubb ◽  
...  
Keyword(s):  
Amino Acid ◽  
Liver Enzyme ◽  
Rat Liver ◽  
Signal Sequence ◽  
Proteolytic Processing ◽  
Amino Acid Difference ◽  
Amino Acid Residues ◽  
Sequence Comparisons ◽  
Chimeric Clone ◽  
Cos Cells

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3′ untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.

scholarly journals A Two-Amino Acid Difference in the Coat Protein of Satellite panicum mosaic virus Isolates Is Responsible for Differential Synergistic Interactions with Panicum mosaic virus

2019 ◽  
Vol 32 (4) ◽  
pp. 479-490 ◽  
Author(s):  
R. V. Chowda-Reddy ◽  
Nathan Palmer ◽  
Serge Edme ◽  
Gautam Sarath ◽  
Frank Kovacs ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Coat Protein ◽  
Mosaic Virus ◽  
Severe Disease ◽  
Amino Acid Difference ◽  
Amino Acid Residues ◽  
Genomic Rna ◽  
Proso Millet ◽  
Synergistic Interactions

Panicum mosaic virus (PMV) (genus Panicovirus, family Tombusviridae) and its molecular parasite, Satellite panicum mosaic virus (SPMV), synergistically interact in coinfected proso and pearl millet (Panicum miliaceum L.) plants resulting in a severe symptom phenotype. In this study, we examined synergistic interactions between the isolates of PMV and SPMV by using PMV-NE, PMV85, SPMV-KS, and SPMV-Type as interacting partner viruses in different combinations. Coinfection of proso millet plants by PMV-NE and SPMV-KS elicited severe mosaic, chlorosis, stunting, and eventual plant death compared with moderate mosaic, chlorotic streaks, and stunting by PMV85 and SPMV-Type. In reciprocal combinations, coinfection of proso millet by either isolate of PMV with SPMV-KS but not with SPMV-Type elicited severe disease synergism, suggesting that SPMV-KS was the main contributor for efficient synergistic interaction with PMV isolates. Coinfection of proso millet plants by either isolate of PMV and SPMV-KS or SPMV-Type caused increased accumulation of coat protein (CP) and genomic RNA copies of PMV, compared with infections by individual PMV isolates. Additionally, CP and genomic RNA copies of SPMV-KS accumulated at substantially higher levels, compared with SMPV-Type in coinfected proso millet plants with either isolate of PMV. Hybrid viruses between SPMV-KS and SPMV-Type revealed that SPMV isolates harboring a CP fragment with four differing amino acids at positions 18, 35, 59, and 98 were responsible for differential synergistic interactions with PMV in proso millet plants. Mutation of amino acid residues at these positions in different combinations in SPMV-KS, similar to those as in SPMV-Type or vice-versa, revealed that A35 and R98 in SPMV-KS CP play critical roles in enhanced synergistic interactions with PMV isolates. Taken together, these data suggest that the two distinct amino acids at positions 35 and 98 in the CP of SPMV-KS and SPMV-Type are involved in the differential synergistic interactions with the helper viruses.

scholarly journals Isolation and Expression in Escherichia coli ofcslA and cslB, Genes Coding for the Chondroitin Sulfate-Degrading Enzymes Chondroitinase AC and Chondroitinase B, Respectively, from Flavobacterium heparinum

2000 ◽  
Vol 66 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Ana Lydia Tkalec ◽  
Dominique Fink ◽  
Françoise Blain ◽  
Guiyi Zhang-Sun ◽  
Maryse Laliberte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Chondroitin Sulfate ◽  
Dna Sequences ◽  
Dermatan Sulfate ◽  
Signal Sequence ◽  
Open Reading Frames ◽  
Amino Acid Residues ◽  
Chondroitin Ac Lyase ◽  
Flavobacterium Heparinum

ABSTRACT In medium supplemented with chondroitin sulfate,Flavobacterium heparinum synthesizes and exports two chondroitinases, chondroitinase AC (chondroitin AC lyase; EC 4.2.2.5 ) and chondroitinase B (chondroitin B lyase; no EC number), into its periplasmic space. Chondroitinase AC preferentially depolymerizes chondroitin sulfates A and C, whereas chondroitinase B degrades only dermatan sulfate (chondroitin sulfate B). The genes coding for both enzymes were isolated from F. heparinum and designated cslA (chondroitinase AC) and cslB(chondroitinase B). They were found to be separated by 5.5 kb on the chromosome of F. heparinum, transcribed in the same orientation, but not linked to any of the heparinase genes. In addition, the synthesis of both enzymes appeared to be coregulated. The cslA and cslB DNA sequences revealed open reading frames of 2,103 and 1,521 bp coding for peptides of 700 and 506 amino acid residues, respectively. Chondroitinase AC has a signal sequence of 22 residues, while chondroitinase B is composed of 25 residues. The mature forms of chondroitinases AC and B are comprised of 678 and 481 amino acid residues and have calculated molecular masses of 77,169 and 53,563 Da, respectively. TruncatedcslA and cslB genes have been used to produce active, mature chondroitinases in the cytoplasm of Escherichia coli. Partially purified recombinant chondroitinases AC and B exhibit specific activities similar to those of chondroitinases AC and B from F. heparinum.

scholarly journals Identification of betacellulin as a major peptide growth factor in milk: purification, characterization and molecular cloning of bovine betacellulin

1999 ◽  
Vol 344 (3) ◽  
pp. 713-721 ◽  
Author(s):  
Andrew J. DUNBAR ◽  
Ilka K. PRIEBE ◽  
David A. BELFORD ◽  
Chris GODDARD
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Bovine Milk ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Wide Range ◽  
Rapid Amplification

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family of peptide growth factors, was purified from a growth-factor-enriched whey fraction of bovine milk by a combination of ion-exchange chromatography, gel-filtration chromatography, affinity chromatography and reverse-phase HPLC. Bovine BTC (bBTC) had an apparent molecular mass of 21-22 kDa on SDS/PAGE and exists in a glycosylated form. The cDNA encoding bBTC was obtained by a combination of 5ʹ and 3ʹ rapid amplification of cDNA ends (‘RACE’). The primary translation product consists of 178 amino acid residues containing a putative signal sequence, a transmembrane domain, the mature BTC domain and a cytoplasmic domain containing a highly hydrophilic Arg-Lys-rich region similar to that of mouse BTC and human BTC. The amino acid sequence of the bBTC precursor was 88% identical with human BTC and 79% identical with mouse BTC. The bBTC gene was found to be expressed in a wide range of tissues, including the mammary gland. The identification of BTC in milk raises the possibility that it has a major role in the growth and development of the neonatal gastrointestinal tract.

scholarly journals Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY

2007 ◽  
Vol 190 (4) ◽  
pp. 1172-1183 ◽  
Author(s):  
Josephine R. Chandler ◽  
Gary M. Dunny
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Constitutive Expression ◽  
Direct Interaction ◽  
Proteolytic Processing ◽  
Sequence Specificity ◽  
Amino Acid Residues ◽  
And Control ◽  
Specificity Determinants

ABSTRACT Conjugative transfer of the Enterococcus faecalis plasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently.

Chemical Probes of Amino Acid Residues Affect the Active Sites of the Glucocorticoid Receptor*

Endocrinology ◽  
1980 ◽  
Vol 106 (3) ◽  
pp. 922-929 ◽  
Author(s):  
DENNIS M. DiSORBO ◽  
DAVID S. PHELPS ◽  
GERALD LITWACK
Keyword(s):  
Amino Acid ◽  
Glucocorticoid Receptor ◽  
Active Sites ◽  
Amino Acid Residues ◽  
Chemical Probes

scholarly journals Analysis of Amino Acid Residues Involved in Catalysis of Polyethylene Glycol Dehydrogenase from Sphingopyxis terrae, Using Three-Dimensional Molecular Modeling-Based Kinetic Characterization of Mutants

2006 ◽  
Vol 72 (6) ◽  
pp. 4388-4396 ◽  
Author(s):  
Takeshi Ohta ◽  
Takeshi Kawabata ◽  
Ken Nishikawa ◽  
Akio Tani ◽  
Kazuhide Kimbara ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Amino Acid ◽  
Molecular Modeling ◽  
Binding Site ◽  
Active Sites ◽  
Substrate Binding ◽  
Three Dimensional ◽  
Kinetic Characterization ◽  
Amino Acid Residues

ABSTRACT Polyethylene glycol dehydrogenase (PEGDH) from Sphingopyxis terrae (formerly Sphingomonas terrae) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from Aspergillus niger and Penicillium amagasakiense. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q10 was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.

scholarly journals A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

2019 ◽  
Vol 48 (1) ◽  
pp. 316-331 ◽  
Author(s):  
Lisa Kesselring ◽  
Csaba Miskey ◽  
Cecilia Zuliani ◽  
Irma Querques ◽  
Vladimir Kapitonov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Phylogenetic Analyses ◽  
Sleeping Beauty ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Dna Transposition ◽  
Dead End ◽  
Single Amino Acid Change ◽  
Induced Pluripotent

Abstract The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int− transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int− transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.

scholarly journals Molecular Cloning and Bioinformatics Analysis of DQA Gene from Mink (Neovison vison)

2019 ◽  
Vol 20 (5) ◽  
pp. 1037
Author(s):  
Zhaobin Fan ◽  
Houfeng Zhang ◽  
Min Rong ◽  
Dongmei Meng ◽  
Zhenxing Yu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Signal Sequence ◽  
Amino Acid Sequences ◽  
Neovison Vison ◽  
Random Coil ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Full Length Sequence

In the present study, we cloned, sequenced, and explored the structural and functional characteristics of the major histocompatibility complex (MHC)-DQA gene from mink (Neovison vison) for the first time. The full-length sequence of DQA gene was 1147-bp-long, contained a coding region of 768-bp, which was predicted to encoding 255 amino acid residues. The comparison between DQA from mink (Neovison vison) and other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of the mink DQA gene exhibited high similarity with the ferret (Mustela pulourius furo). Phylogenetic analysis revealed that mink (Neovison vison) DQA is grouped with that of ferret (Mustela pulourius furo). The cloned sequence contained a 23-amino acid NH2-terminal signal sequence with the signal peptide cutting site located in amino acids 23–24, and had three Asn-Xaa-Ser/Thr sequons. Three cysteine residues were also identified (Cys-85, Cys-121, and Cys-138). The 218 to 240 amino acids were predicted to be the transmembrane domains. The prediction of the secondary structure revealed three α-helixes and fourteen β-sheets in Neovison vison DQA protein, while random coil was a major pattern. In this study, the whole CDS sequence of Neovison vison DQA gene was successfully cloned, which was valuable for exploring the function and antiviral molecular mechanisms underlying the molecule. The findings of the present study have laid the foundation for the disease resistance and breeding of mink.

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