scholarly journals Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide

1994 ◽  
Vol 299 (2) ◽  
pp. 381-387 ◽  
Author(s):  
G W Black ◽  
G P Hazlewood ◽  
G P Xue ◽  
C G Orpin ◽  
H J Gilbert
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Northern Blot Analysis ◽  
Catalytic Domain ◽  
Catalytic Properties ◽  
Single Gene ◽  
Reading Frame ◽  
Catalytic Domains ◽  
Neocallimastix Patriciarum ◽  
Derivatives Of

A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N. patriciarum xynA cDNA encoding xylanase A. A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated. Northern-blot analysis of mRNA from Avicel-grown N. patriciarum showed that xynB hybridized to a 3.4 kb mRNA species. The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066. The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases. The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times. Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain. To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography. The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose. The XYLB fusion did not cleave any cellulosic substrates. The data presented in this report suggest that the multiple xylanases of N. patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus. The possible origin of the xynB gene is discussed.

scholarly journals Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase

1994 ◽  
Vol 297 (2) ◽  
pp. 359-364 ◽  
Author(s):  
L Zhou ◽  
G P Xue ◽  
C G Orpin ◽  
G W Black ◽  
H J Gilbert ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Catalytic Domain ◽  
Crystalline Cellulose ◽  
Anaerobic Fungus ◽  
Beta Glucan ◽  
Reading Frame ◽  
Neocallimastix Patriciarum ◽  
Rumen Fungus ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

The cDNA designated celB from the anaerobic rumen fungus Neocallimastix patriciarum contained a single open reading frame of 1422 bp coding for a protein (CelB) of M(r) 53,070. CelB expressed by Escherichia coli harbouring the full-length gene hydrolysed carboxymethylcellulose in the manner of an endoglucanase, but was most active against barley beta-glucan. It also released reducing sugar from xylan and lichenan, but was inactive against crystalline cellulose, laminarin, mannan, galactan and arabinan. The rate of hydrolysis of cellulo-oligosaccharides by CelB increased with increasing chain length from cellotriose to cellopentaose. The predicted structure of CelB contained features indicative of modular structure. The first 360 residues of CelB constituted a fully functional catalytic domain that was homologous with bacterial endoglucanases belonging to cellulase family A, including five which originate from three different species of anaerobic rumen bacteria. Downstream from this domain, and linked to it by a serine/threonine-rich hinge, was a non-catalytic domain containing short tandem repeats, homologous to the C-terminal repeats contained in xylanase A from the same anaerobic fungus. Unlike previous fungal cellulases, genomic celB was devoid of introns. This lack of introns and the homology of its encoded product with rumen bacterial endoglucanases suggest that acquisition of celB by the fungus may at some stage have involved horizontal gene transfer from a prokaryote to N. particiarum.

scholarly journals Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase

1995 ◽  
Vol 308 (2) ◽  
pp. 425-432 ◽  
Author(s):  
A Charest ◽  
J Wagner ◽  
S H Shen ◽  
M L Tremblay
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Northern Blot Analysis ◽  
Catalytic Domain ◽  
Tyrosine Phosphatase ◽  
Glutathione S Transferase ◽  
Epitope Tag ◽  
Reading Frame ◽  
Protein Tyrosine ◽  
Cytosolic Protein

We have isolated the murine cDNA homologue of the human protein tyrosine phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic kidney library. The cDNA isolated has a single open reading frame predicting a protein of 775 amino acids. When expressed in vitro as a glutathione S-transferase fusion protein, the catalytic domain (residues 1-453) shows intrinsic phosphatase activity. Reverse transcriptase PCR and Northern-blot analysis show that MPTP-PEST mRNA is expressed throughout murine development. Indirect immunofluorescence in COS-1 cells against a heterologous epitope tag attached to the N-terminus of MPTP-PEST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cytosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in proline, glutamate, serine and threonine, otherwise known as PEST sequences. These are characteristic of proteins that display very short intracellular half-lives. Despite the presence of these motifs, pulse-chase labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.

scholarly journals A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulose-binding domain

1993 ◽  
Vol 294 (2) ◽  
pp. 349-355 ◽  
Author(s):  
L M Ferreira ◽  
T M Wood ◽  
G Williamson ◽  
C Faulds ◽  
G P Hazlewood ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Methoxycinnamic Acid ◽  
Cellulose Binding ◽  
Derivatives Of

The 5′ regions of genes xynB and xynC, coding for a xylanase and arabinofuranosidase respectively, are identical and are reiterated four times within the Pseudomonas fluorescens subsp. cellulosa genome. To isolate further copies of the reiterated xynB/C 5′ region, a genomic library of Ps. fluorescens subsp. cellulosa DNA was screened with a probe constructed from the conserved region of xynB. DNA from one phage which hybridized to the probe, but not to sequences upstream or downstream of the reiterated xynB/C locus, was subcloned into pMTL22p to construct pFG1. The recombinant plasmid expressed a protein in Escherichia coli, designated esterase XYLD, of M(r) 58,500 which bound to cellulose but not to xylan. XYLD hydrolysed aryl esters, released acetate groups from acetylxylan and liberated 4-hydroxy-3-methoxycinnamic acid from destarched wheat bran. The nucleotide sequence of the XYLD-encoding gene, xynD, revealed an open reading frame of 1752 bp which directed the synthesis of a protein of M(r) 60,589. The 5′ 817 bp of xynD and the amino acid sequence between residues 37 and 311 of XYLD were almost identical with the corresponding regions of xynB and xynC and their encoded proteins XYLB and XYLC. Truncated derivatives of XYLD lacking the N-terminal conserved sequence retained the capacity to hydrolyse ester linkages, but did not bind cellulose. Expression of truncated derivatives of xynD, comprising the 5′ 817 bp sequence, encoded a non-catalytic polypeptide that bound cellulose. These data indicate that XYLD has a modular structure comprising of a N-terminal cellulose-binding domain and a C-terminal catalytic domain.

scholarly journals Characterization of the Plasmid Encoded Virulence Region pat-1 of Phytopathogenic Clavibacter michiganensis subsp. michiganensis

1997 ◽  
Vol 10 (2) ◽  
pp. 195-206 ◽  
Author(s):  
Jens Dreier ◽  
Dietmar Meletzus ◽  
Rudolf Eichenlaub
Keyword(s):  
Structural Gene ◽  
Dna Sequences ◽  
Repetitive Sequence ◽  
Transcription Initiation ◽  
Serine Proteases ◽  
Tandem Repeats ◽  
Sequence Motif ◽  
Clavibacter Michiganensis ◽  
Reading Frame ◽  
Clavibacter Michiganensis Subsp

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, causing bacterial wilt and canker, harbors two plasmids, pCM1 (27.5 kb) and pCM2 (72 kb), carrying genes involved in virulence. The region of plasmid pCM2 encoding the pathogenicity locus pat-1 was mapped by deletion analysis and complementation studies to a 1.5-kb BglII/SmaI DNA fragment. Introduction of the pat-1 region into endophytic, plasmid-free isolates of C. michiganensis subsp. michiganensis converted these bacteria into virulent pathogens. Based on the nucleotide sequence of the pat-1 region, an open reading frame (ORF1) can be predicted, coding for a protein of 280 amino acids and 29.7 kDa with homology to serine proteases. Introduction of a frame-shift mutation in ORF1 leads to a loss of the pathogenic phenotype. Northern (RNA) hybridizations identified an 1.5-knt transcript of the pat-1 structural gene. The site of transcription initiation was mapped by primer extension and a typical -10/-35 region was located with significant homology to the consensus Escherichia coli σ70 and Bacillus subtilis σ43 promoters. Downstream of the pat-1 structural gene, a peculiar repetitive sequence motif (pat-1rep) is located, consisting of 20 direct tandem repeats preceded by a run of 14 guanosine residues. DNA sequences homologous to pat-1rep were isolated and characterized from four virulent C. michiganensis subsp. mich-iganensis strains exhibiting a high extent of structural conservation. The deletion of this repetitive sequence reduced virulence significantly but did not lead to a complete loss of the virulence phenotype.

scholarly journals Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

2002 ◽  
Vol 184 (20) ◽  
pp. 5753-5761 ◽  
Author(s):  
Sophie Bozonnet ◽  
Marguerite Dols-Laffargue ◽  
Emeline Fabre ◽  
Sandra Pizzut ◽  
Magali Remaud-Simeon ◽  
...  
Keyword(s):  
Leuconostoc Mesenteroides ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Carboxy Terminus ◽  
Reading Frame ◽  
Average Mass ◽  
Catalytic Domains ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of α-1,6 and α-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and α-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.

The reaction mechanism of FokI excludes the possibility of targeting zinc finger nucleases to unique DNA sites

2011 ◽  
Vol 39 (2) ◽  
pp. 584-588 ◽  
Author(s):  
Stephen E. Halford ◽  
Lucy E. Catto ◽  
Christian Pernstich ◽  
David A. Rusling ◽  
Kelly L. Sanders
Keyword(s):  
Zinc Finger ◽  
Dna Sequences ◽  
Catalytic Domain ◽  
Dna Recognition ◽  
Zinc Finger Nucleases ◽  
Target Sequence ◽  
Base Pairs ◽  
Catalytic Domains ◽  
Zinc Finger Domains ◽  
Better Than

The FokI endonuclease is a monomeric protein with discrete DNA-recognition and catalytic domains. The latter has only one active site so, to cut both strands, the catalytic domains from two monomers associate to form a dimer. The dimer involving a monomer at the recognition site and another from free solution is less stable than that from two proteins tethered to the same DNA. FokI thus cleaves DNA with two sites better than one-site DNA. The two sites can be immediately adjacent, but they can alternatively be many hundreds of base pairs apart, in either inverted or repeated orientations. The catalytic domain of FokI is often a component of zinc finger nucleases. Typically, the zinc finger domains of two such nucleases are designed to recognize two neighbouring DNA sequences, with the objective of cutting the DNA exclusively between the target sequences. However, this strategy fails to take account of the fact that the catalytic domains of FokI can dimerize across distant sites or even at a solitary site. Additional copies of either target sequence elsewhere in the chromosome must elicit off-target cleavages.

scholarly journals Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joanna L. Fox ◽  
Michelle A. Hughes ◽  
Xin Meng ◽  
Nikola A. Sarnowska ◽  
Ian R. Powley ◽  
...  
Keyword(s):  
Cell Death ◽  
Structural Analysis ◽  
Cell Fate ◽  
Catalytic Domain ◽  
Caspase 8 ◽  
Type I ◽  
Signaling Complex ◽  
Catalytic Domains ◽  
Regulated Cell Death ◽  
Death Effector

AbstractRegulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

scholarly journals Islands of complex DNA are widespread in Drosophila centric heterochromatin.

Genetics ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 283-303
Author(s):  
M H Le ◽  
D Duricka ◽  
G H Karpen
Keyword(s):  
Dna Sequences ◽  
Structure And Function ◽  
Southern Analysis ◽  
Cloning And Sequencing ◽  
Pulsed Field ◽  
Drosophila Heterochromatin ◽  
And Function ◽  
Heterochromatin Structure ◽  
Derivatives Of ◽  
Eukaryotic Genomes

Abstract Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three "islands" of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed.

scholarly journals IIMLP: integrated information-entropy-based method for LncRNA prediction

2021 ◽  
Vol 22 (S3) ◽  
Author(s):  
Junyi Li ◽  
Huinian Li ◽  
Xiao Ye ◽  
Li Zhang ◽  
Qingzhe Xu ◽  
...  
Keyword(s):  
Machine Learning ◽  
Dna Sequences ◽  
Information Entropy ◽  
Area Under The Curve ◽  
Prediction Method ◽  
Machine Learning Algorithms ◽  
Reading Frame ◽  
Non Coding Rna ◽  
The One ◽  
Long Non Coding Rna

Abstract Background The prediction of long non-coding RNA (lncRNA) has attracted great attention from researchers, as more and more evidence indicate that various complex human diseases are closely related to lncRNAs. In the era of bio-med big data, in addition to the prediction of lncRNAs by biological experimental methods, many computational methods based on machine learning have been proposed to make better use of the sequence resources of lncRNAs. Results We developed the lncRNA prediction method by integrating information-entropy-based features and machine learning algorithms. We calculate generalized topological entropy and generate 6 novel features for lncRNA sequences. By employing these 6 features and other features such as open reading frame, we apply supporting vector machine, XGBoost and random forest algorithms to distinguish human lncRNAs. We compare our method with the one which has more K-mer features and results show that our method has higher area under the curve up to 99.7905%. Conclusions We develop an accurate and efficient method which has novel information entropy features to analyze and classify lncRNAs. Our method is also extendable for research on the other functional elements in DNA sequences.

scholarly journals Cosegregation of Single Genes Associated with Fertility Restoration and Transcript Processing of Sorghum Mitochondrial orf107 and urf209

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 383-391 ◽  
Author(s):  
Hoang V Tang ◽  
Ruying Chang ◽  
Daryl R Pring
Keyword(s):  
Fertility Restoration ◽  
Mitochondrial Gene ◽  
Pollen Viability ◽  
Single Gene ◽  
Dominant Gene ◽  
Nuclear Gene ◽  
Male Sterile ◽  
Reading Frame ◽  
Transcript Processing ◽  
Backcross Populations

Abstract Defective nuclear-cytoplasmic interactions leading to aberrant microgametogenesis in sorghum carrying the IS1112C male-sterile cytoplasm occur very late in pollen maturation. Amelioration of this condition, the restoration of pollen viability, involves a novel two-gene gametophytic system, wherein genes designated Rf3 and Rf4 are required for viability of individual gametes. Rf3 is tightly linked to, or represents, a single gene that regulates a transcript processing activity that cleaves transcriptsof orf107, a chimeric mitochondrial open reading frame specific to IS1112C. The mitochondrial gene urf 209 is also subject to nucleus-specific enhanced transcript processing, 5′ to the gene, conferred by a single dominant gene designated Mmt1. Examinations of transcript patterns in F2 and two backcross populations indicated cosegregation of the augmented orf107 and urf209 processing activities in IS1112C. Several sorghum lines that do not restore fertility or confer orf107 transcript processing do exhibit urf209 transcript processing, indicating that the activities are distinguishable. We conclude that the nuclear gene(s) conferring enhanced orf107 and urf209 processing activities are tightly linked in IS1112C. Alternatively, the similarity in apparent regulatory action of the genes may indicate allelic differences wherein the IS1112C Rf3 allele may differ from alleles of maintainer lines by the capability to regulate both orf107 and urf209 processing activities.

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