scholarly journals Characterization of an Ebosin derivative produced by heterologous gene replacement in

2014 ◽  
Vol 13 (1) ◽  
pp. 103
Author(s):  
Yang Zhang ◽  
Junjie Shan ◽  
Yonggang Bao ◽  
Liping Bai ◽  
Rong Jiang ◽  
...  
Keyword(s):  
Gene Replacement ◽  
Heterologous Gene

scholarly journals Characterization of an Ebosin derivative produced by heterologous gene replacement in Streptomyces sp. 139

2014 ◽  
Vol 13 (1) ◽  
Author(s):  
Yang Zhang ◽  
Junjie Shan ◽  
Yonggang Bao ◽  
Liping Bai ◽  
Rong Jiang ◽  
...  
Keyword(s):  
Gene Replacement ◽  
Heterologous Gene ◽  
Streptomyces Sp

Cloning and Characterization of a Prolinase Gene (pepR) from Lactobacillus rhamnosus

1998 ◽  
Vol 64 (5) ◽  
pp. 1831-1836 ◽  
Author(s):  
Pekka Varmanen ◽  
Terhi Rantanen ◽  
Airi Palva ◽  
Soile Tynkkynen
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Gene Replacement ◽  
Lactobacillus Helveticus ◽  
Open Reading Frame ◽  
Transcriptional Unit ◽  
Wild Type ◽  
Reading Frame ◽  
Gene Expressing ◽  
Produce Acid

ABSTRACT A peptidase gene expressingl-proline-β-naphthylamide-hydrolyzing activity was cloned from a gene library of Lactobacillus rhamnosus 1/6 isolated from cheese. Peptidase-expressing activity was localized in a 1.5-kbSacI fragment. A sequence analysis of the SacI fragment revealed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long. The ORF1-encoded 34.2-kDa protein exhibited 68% identity with the PepR protein from Lactobacillus helveticus. Additional sequencing revealed the presence of another open reading frame (ORF2) following pepR; this open reading frame was 459 bp long. Northern (RNA) and primer extension analyses indicated that pepR is expressed both as a monocistronic transcriptional unit and as a dicistronic transcriptional unit with ORF2. Gene replacement was used to construct a PepR-negative strain of L. rhamnosus. PepR was shown to be the primary enzyme capable of hydrolyzing Pro-Leu in L. rhamnosus. However, the PepR-negative mutant did not differ from the wild type in its ability to grow and produce acid in milk. The clonedpepR expressed activity against dipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, and Leu-Gly-Gly and the chromogenic substrates l-leucine-β-naphthylamide andl-phenylalanine-β-naphthylamide were hydrolyzed by the PepR of L. rhamnosus.

scholarly journals Functional Characterization of Ubiquitin-Like Core Autophagy Protein ATG12 in Dictyostelium discoideum

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 72 ◽  
Author(s):  
Sarah Fischer ◽  
Ramesh Rijal ◽  
Peter Frommolt ◽  
Prerana Wagle ◽  
Roman Konertz ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Functional Characterization ◽  
Gene Replacement ◽  
Cellular Functions ◽  
Fruiting Body Formation ◽  
Spore Viability ◽  
Additional Function ◽  
Independent Functions ◽  
Mutant Strains

Autophagy is a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagy protein ATG12 plays, together with ATG5 and ATG16, an essential role in the expansion of the autophagosomal membrane. In this study we analyzed gene replacement mutants of atg12 in Dictyostelium discoideum AX2 wild-type and ATG16‾ cells. RNAseq analysis revealed a strong enrichment of, firstly, autophagy genes among the up-regulated genes and, secondly, genes implicated in cell motility and phagocytosis among the down-regulated genes in the generated ATG12‾, ATG16‾ and ATG12‾/16‾ cells. The mutant strains showed similar defects in fruiting body formation, autolysosome maturation, and cellular viability, implying that ATG12 and ATG16 act as a functional unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12 and ATG16 resulted in slightly more severe defects in axenic growth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional function in these processes. Phagocytosis of yeast, spore viability, and maximal cell density were much more affected in ATG12‾/16‾ cells, indicating that both proteins also have cellular functions independent of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.

scholarly journals Cloning and Characterization of the Biosynthetic Gene Cluster for Tomaymycin, an SJG-136 Monomeric Analog

2009 ◽  
Vol 75 (9) ◽  
pp. 2958-2963 ◽  
Author(s):  
Wei Li ◽  
ShenChieh Chou ◽  
Ankush Khullar ◽  
Barbara Gerratana
Keyword(s):  
Cluster Analysis ◽  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Biosynthetic Gene Cluster ◽  
Gene Replacement ◽  
Biosynthetic Gene

ABSTRACT Tomaymycin produced by Streptomyces achromogenes is a naturally produced pyrrolobenzodiazepine (PBD). The biosynthetic gene cluster for tomaymycin was identified and sequenced. The gene cluster analysis reveals a novel biosynthetic pathway for the anthranilate moiety of PBDs. Gene replacement and chemical complementation studies were used to confirm the proposed biosynthetic pathway.

scholarly journals Characterization of the Human Artemis Promoter by Heterologous Gene Expression In Vitro and In Vivo

2011 ◽  
Vol 30 (10) ◽  
pp. 751-761 ◽  
Author(s):  
Megan M. Multhaup ◽  
Sweta Gurram ◽  
Kelly M. Podetz-Pedersen ◽  
Andrea D. Karlen ◽  
Debra L. Swanson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Heterologous Gene

The inborn errors of metabolism: General aspects

2020 ◽  
pp. 1929-1941
Author(s):  
Timothy M. Cox ◽  
Richard W.E. Watts
Keyword(s):  
Inborn Errors Of Metabolism ◽  
Quaternary Structure ◽  
Correct Diagnosis ◽  
Gene Replacement ◽  
Inborn Errors ◽  
Irreversible Damage ◽  
Presymptomatic Stage ◽  
General Aspects ◽  
High Level

The inborn errors of metabolism are those inherited diseases in which the phenotype includes a characteristic constellation of biochemical abnormalities related to an alteration in the catalytic activity of a single specific enzyme, activator, or transport protein. Mechanism of diseases—mutations in the proteins giving rise to the inborn errors of metabolism affect primary, secondary, tertiary, or quaternary structure. This can lead to an enormous variety of consequences. Clinical presentation—the manifestations of metabolic disease are protean and may seem nondescript, especially in adults, hence a high level of suspicion may be required to make a correct diagnosis. Prevention and screening—there is a strong case for mass population screening for some inborn errors of metabolism at the presymptomatic stage to allow early detection and introduction of proven treatment before irreversible damage occurs. Management—definitive cure of the underlying abnormality is available for a few disorders, but precise characterization of the biochemical disturbance often permits rational treatment to be organized and provides the basis for further therapeutic endeavours. General approaches include (1) restriction of a substrate that cannot be metabolized including molecules derived from the diet; (2) replacement of a missing metabolic product; (3) removal of poisonous metabolites or rebalancing overproduction of toxic intermediates; (4) administering pharmacological doses of a cofactor, sometimes a vitamin, that may also stabilize a mutant enzyme; (5) replacement of a missing gene product, usually by enzymatic augmentation therapy or pharmacological chaperones, to prevent premature aggregation and denaturation; (6) repression of an overproduced protein or metabolite by stable RNA inhibition; (7) transplantation of cells or organs as a ‘gene replacement therapy’; and (8) activation of a poorly functioning protein.

scholarly journals Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

2011 ◽  
Vol 286 (12) ◽  
pp. 10419-10428 ◽  
Author(s):  
Rasmus J. N. Frandsen ◽  
Claes Schütt ◽  
Birgitte W. Lund ◽  
Dan Staerk ◽  
John Nielsen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusarium Graminearum ◽  
Polyketide Synthase ◽  
Functional Characterization ◽  
Extracellular Enzyme ◽  
Gene Replacement ◽  
Orphan Genes ◽  
Pigment Biosynthesis ◽  
Targeted Gene Replacement

Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated γ-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to- aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.

scholarly journals Development of Genetic Tools forLactobacillus sakei: Disruption of the β-Galactosidase Gene and Use of lacZ as a Reporter Gene To Study Regulation of the Putative Copper ATPase, AtkB

2000 ◽  
Vol 66 (10) ◽  
pp. 4272-4278 ◽  
Author(s):  
Régis Stentz ◽  
Christophe Loizel ◽  
Christine Malleret ◽  
Monique Zagorec
Keyword(s):  
Escherichia Coli ◽  
Reporter Gene ◽  
Sequence Similarity ◽  
Gene Replacement ◽  
Negative Regulator ◽  
Lactobacillus Sakei ◽  
Enterococcus Hirae ◽  
Genetic Tools ◽  
P Type

ABSTRACT Downstream from the ptsHI operon of Lactobacillus sakei, the genes atkY and atkB, organized in an operon, were observed. The two putative proteins, AtkB and AtkY, show sequence similarity to the Enterococcus hirae copper P-type ATPase, responsible for copper efflux, and its negative regulator. Characterization of AtkB as a copper P-type ATPase could not be demonstrated since an atkB mutant did not show any phenotype. Thus, another strategy was followed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L. sakei. A plasmid was constructed, the use of which allowed gene replacement at the lacLM locus in L. sakei by two successive crossovers. A strain deleted of the lacLM operon encoding the β-galactosidase of L. sakei was constructed by this method, and the Escherichia coli lacZ gene could then be used as a reporter gene to investigate the regulation ofatkYB. Results show that the atkYB operon is induced by small concentrations of CuSO4 (30 to 40 μM) but not when CuSO4 is omitted or added at higher concentrations.

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum

Extremophiles ◽  
2021 ◽  
Author(s):  
Ilma Fauziah Ma’ruf ◽  
Yuka Sasaki ◽  
Anastasia Kerbs ◽  
Jochen Nießer ◽  
Yu Sato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Heterologous Gene ◽  
Thermoplasma Acidophilum
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