scholarly journals Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Erma Sulistyaningsih ◽  
Sukarti Moeljopawiro ◽  
Jarot Subandono ◽  
Wayan T. Artama
Keyword(s):  
Toxoplasma Gondii ◽  
Dna Sequences ◽  
Messenger Rna ◽  
Vaccine Candidate ◽  
Dna Encoding ◽  
Gene Encoding ◽  
Local Isolate ◽  
Ecori Restriction ◽  
Encoding Gene

Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA

scholarly journals Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

2015 ◽  
Vol 10 (2) ◽  
Author(s):  
Wayan T. Artama ◽  
Yulia Sari ◽  
Didik Tulus Subekti ◽  
Soenarwan Hery Poerwanto ◽  
Jarot Subandono
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Plasmid ◽  
Messenger Rna ◽  
Secretory Protein ◽  
Gene Encoding ◽  
Isolation System ◽  
Complementary Dna ◽  
E Coli ◽  
Local Isolate ◽  
Cell Target

Rhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the clonedgene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate.Keywords: Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP2

The skipping of constitutive exons in vivo induced by nonsense mutations

Science ◽  
1993 ◽  
Vol 259 (5095) ◽  
pp. 680-683 ◽  
Author(s):  
HC Dietz ◽  
D Valle ◽  
CA Francomano ◽  
RJ Kendzior ◽  
RE Pyeritz ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Constitutive Exon ◽  
Gyrate Atrophy ◽  
Splice Site Selection ◽  
Gene Encoding ◽  
Nonsense Mutations ◽  
Severe Reduction

Nonsense mutations create a premature signal for the termination of translation of messenger RNA. Such mutations have been observed to cause a severe reduction in the amount of mutant allele transcript or to generate a peptide truncated at the carboxyl end. Analysis of fibrillin transcript from a patient with Marfan syndrome revealed the skipping of a constitutive exon containing a nonsense mutation. Similar results were observed for two nonsense mutations in the gene encoding ornithine delta-aminotransferase from patients with gyrate atrophy. All genomic DNA sequences flanking these exons that are known to influence RNA splicing were unaltered, which suggests that nonsense mutations can alter splice site selection in vivo.

scholarly journals A unique serine-rich repeat protein (Srr-2) and novel surface antigen (ε) associated with a virulent lineage of serotype III Streptococcus agalactiae

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 1029-1040 ◽  
Author(s):  
Kyle N. Seifert ◽  
Elisabeth E. Adderson ◽  
April A. Whiting ◽  
John F. Bohnsack ◽  
Paula J. Crowley ◽  
...  
Keyword(s):  
Surface Antigen ◽  
Invasive Disease ◽  
Group B Streptococci ◽  
Gene Encoding ◽  
Repeat Protein ◽  
Encoding Gene ◽  
Group B ◽  
Mouse Model Of Infection ◽  
Highly Virulent

Group B streptococci (GBS) are pathogens of both neonates and adults, with serotype III strains in particular being associated with invasive disease and meningitis. In this study, a novel GBS surface antigen, ε, was found to be co-expressed with the previously reported δ antigen on an identical subset of serotype III GBS. Expression of δ/ε on the surface of serotype III GBS was shown to distinguish the restriction digest pattern (RDP) III-3 and multilocus sequence typing (ST)-17 lineage. ε-Specific antibodies were reactive with a unique, high-molecular-mass, serine-rich repeat protein (Srr-2) found exclusively in RDP III-3 strains. The gene encoding Srr-2 was located within a putative accessory secretory locus that included secY2 and secA2 homologues and had a genetic organization similar to that of the secY2/A2 locus of staphylococci. In contrast, serotype III δ/ε-negative strains and strains representative of serotypes Ia, Ib, Ic and II shared a common Srr-encoding gene, srr-1, and an organization of the secY2/A2 locus similar to that of previously reported serotype Ic, δ/ε-negative serotype III and serotype V GBS strains. Representative serotype III δ/ε-positive strains had LD90 values 3–4 logs less than those of serotype III δ/ε-negative strains in a neonatal mouse model of infection. These results indicate that the RDP III-3/ST-17 lineage expresses Srr-2 and is highly virulent in an in vivo model of neonatal sepsis.

scholarly journals Four new species of Trichoderma in the Harzianum clade from northern China

MycoKeys ◽  
2020 ◽  
Vol 73 ◽  
pp. 109-132
Author(s):  
Xin Gu ◽  
Rui Wang ◽  
Quan Sun ◽  
Bing Wu ◽  
Jing-Zu Sun
Keyword(s):  
New Species ◽  
Dna Sequences ◽  
Elongation Factor ◽  
Northern China ◽  
Gene Encoding ◽  
Translation Elongation ◽  
Nuclear Rna ◽  
Encoding Gene ◽  
Elongation Factor 1 ◽  
Nuclear Rna Polymerase

The Harzianum clade of Trichoderma comprises many species, which are associated with a wide variety of substrates. In this study, four new species of Trichoderma, namely T. lentinulae, T. vermifimicola, T. xixiacum, and T. zelobreve, were encountered from a fruiting body and compost of Lentinula, soil, and vermicompost. Their colony and mycelial morphology, including features of asexual states, were described. For each species, their DNA sequences were obtained from three loci, the internal transcribed spacer (ITS) regions of the ribosomal DNA, the gene encoding the second largest nuclear RNA polymerase subunit (RPB2), the translation elongation factor 1-α encoding gene (TEF1-α). The analysis combining sequences of the three gene regions distinguished four new species in the Harzianum clade of Trichoderma. Among them, T. lentinulae and T. xixiacum clustered with T. lixii, from which these new species differ in having shorter phialides and smaller conidia. Additionally, T. lentinulae differs from T. xixiacum in forming phialides with inequilateral to a strongly-curved apex, cultural characteristics, and slow growth on PDA. Trichoderma vermifimicola is closely related to T. simmonsii, but it differs from the latter by producing phialides in verticillate whorls and smaller conidia. Trichoderma zelobreve is the sister species of T. breve but is distinguished from T. breve by producing shorter and narrower phialides, smaller conidia, and by forming concentric zones on agar plates. This study updates our knowledge of species diversity of Trichoderma.

scholarly journals Three new species of soil-inhabiting Trichoderma from southwest China

MycoKeys ◽  
2018 ◽  
Vol 44 ◽  
pp. 63-80 ◽  
Author(s):  
Min Qiao ◽  
Xing Du ◽  
Zhe Zhang ◽  
JianPing Xu ◽  
ZenFen Yu
Keyword(s):  
New Species ◽  
Dna Sequences ◽  
Elongation Factor ◽  
Gene Encoding ◽  
Translation Elongation ◽  
Closely Related Species ◽  
Encoding Gene ◽  
Three New Species ◽  
Elongation Factor 1 ◽  
Nuclear Rna Polymerase

Fungi in the genus Trichoderma are widely distributed in China, including in Yunnan province. In this study, we report three new soil-inhabiting species in Trichoderma, named as T.kunmingense, T.speciosum and T.zeloharzianum. Their colony and mycelial morphology, including features of asexual states, were described. For each species, their DNA sequences were obtained from three loci, the internal transcribed spacer (ITS) regions of the ribosomal DNA, the translation elongation factor 1-α encoding gene (tef1) and the gene encoding the second largest nuclear RNA polymerase subunit (rpb2). Our analyses indicated that the three new species showed consistent divergence amongst each other and from other known and closely related species. Amongst the three, T.speciosum and T.kunmingense belong to the Viride Clade. Specifically, T.speciosum is related to three species – T.hispanicum, T.samuelsii and T.junci and is characterised by tree-like conidiophores, generally paired branches, curved terminal branches, spindly to fusiform phialides and subglobose to globose conidia. In contrast, T.kunmingense morphologically resembles T.asperellum and T.yunnanense and is distinguished by its pyramidal conidiophores, ampulliform to tapered phialides, discrete branches and ovoidal, occasionally ellipsoid, smooth-walled conidia. The third new species, T.zeloharzianum, is a new member of the Harzianum Clade and is closely associated with T.harzianum, T.lixii and T.simmonsii but distinguished from them by having smaller, subglobose to globose, thin-walled conidia.

The Role of DNA Polymerase ζ in Benzo[a]pyrene-induced Mutagenesis in the Mouse Lung

Mutagenesis ◽  
2021 ◽  
Author(s):  
Yuji Ishii ◽  
Shinji Takasu ◽  
Petr Grúz ◽  
Kenichi Masumura ◽  
Kumiko Ogawa ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Mouse Lung ◽  
Gene Encoding ◽  
Protective Mechanisms ◽  
Induced Mutagenesis ◽  
Gpt Delta

Abstract DNA polymerase zeta (Polζ) is a heterotetramer composed of the catalytic subunit Rev3l, Rev7 and two subunits of Polδ (PolD2/Pol31 and PolD3/Pol32), and this polymerase exerts translesion DNA synthesis (TLS) in yeast. Because Rev3l knockout results in embryonic lethality in mice, the functions of Polζ need further investigation in vivo. Then, we noted the two facts that substitution of leucine 979 of yeast Rev3l with methionine reduces Polζ replication fidelity and that reporter gene transgenic rodents are able to provide the detailed mutation status. Here, we established gpt delta mouse knocked in the constructed gene encoding methionine instead of leucine at residue 2610 of Rev3l (Rev3l L2610M gpt delta mice), to clarify the role of Polζ in TLS of chemical-induced bulky DNA adducts in vivo. Eight-week-old gpt delta mice and Rev3l L2610M gpt delta mice were treated with benzo[a]pyrene (BaP) at 0, 40, 80, or 160 mg/kg via single intraperitoneal injection. At necropsy 31 days after treatment, lungs were collected for reporter gene mutation assays. Although the gpt mutant frequency (MF) was significantly increased by BaP in both mouse genotypes, it was three times higher in Rev3l L2610M gpt delta than gpt delta mice after treatment with 160 mg/kg BaP. The frequencies of G:C base substitutions and characteristic complex mutations were significantly increased in Rev3l L2610M gpt delta mice compared with gpt delta mice. The BaP dose–response relationship suggested that Polζ plays a central role in TLS when protective mechanisms against BaP mutagenesis, such as error-free TLS, are saturated. Overall, Polζ may incorporate incorrect nucleotides at the sites opposite to BaP-modified guanines and extend short DNA sequences from the resultant terminal mismatches only when DNA is heavily damaged.

scholarly journals MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome

2015 ◽  
Vol 112 (17) ◽  
pp. 5509-5514 ◽  
Author(s):  
Lin Chen ◽  
Kaifu Chen ◽  
Laura A. Lavery ◽  
Steven Andrew Baker ◽  
Chad A. Shaw ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Rett Syndrome ◽  
Dna Sequences ◽  
Neurodevelopmental Disorder ◽  
Adult Brain ◽  
Gene Encoding ◽  
Dna Elements ◽  
Timing Of Onset

Epigenetic mechanisms, such as DNA methylation, regulate transcriptional programs to afford the genome flexibility in responding to developmental and environmental cues in health and disease. A prime example involving epigenetic dysfunction is the postnatal neurodevelopmental disorder Rett syndrome (RTT), which is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2). Despite decades of research, it remains unclear how MeCP2 regulates transcription or why RTT features appear 6–18 months after birth. Here we report integrated analyses of genomic binding of MeCP2, gene-expression data, and patterns of DNA methylation. In addition to the expected high-affinity binding to methylated cytosine in the CG context (mCG), we find a distinct epigenetic pattern of substantial MeCP2 binding to methylated cytosine in the non-CG context (mCH, where H = A, C, or T) in the adult brain. Unexpectedly, we discovered that genes that acquire elevated mCH after birth become preferentially misregulated in mouse models of MeCP2 disorders, suggesting that MeCP2 binding at mCH loci is key for regulating neuronal gene expression in vivo. This pattern is unique to the maturing and adult nervous system, as it requires the increase in mCH after birth to guide differential MeCP2 binding among mCG, mCH, and nonmethylated DNA elements. Notably, MeCP2 binds mCH with higher affinity than nonmethylated identical DNA sequences to influence the level of Bdnf, a gene implicated in the pathophysiology of RTT. This study thus provides insight into the molecular mechanism governing MeCP2 targeting and sheds light on the delayed onset of RTT symptoms.

scholarly journals SUBKLONING DAN ISOLASI GEN PENYANDI MIKRONEMA 3 (MIC-3) Toxoplasma gondii ISOLAT LOKAL

Molekul ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. 10
Author(s):  
Diana Indrasanti ◽  
Aris Haryanto ◽  
Wayan T. Artama
Keyword(s):  
Toxoplasma Gondii ◽  
Expression Vector ◽  
Recombinant Dna ◽  
Cell Infection ◽  
Recombinant Dna Technology ◽  
Specific Primers ◽  
Gene Encoding ◽  
E Coli ◽  
Local Isolate ◽  
Microneme Protein

Microneme protein (MIC) is one of proteins that belongs to excretory-secretory antigens (ESAs) of Toxoplasma gondii. Microneme 3 protein (MIC-3) is the protein that plays an important role in the invasion proccess during cell infection as a mediator attachment parasite to the host cell. The aim of this research is to clone mic3 (gene encoding for MIC-3) of T. gondii from local isolate using recombinant DNA technology by cloning mic3 in an expression vector. Deoxyribonucleic acid (DNA) from T. gondii tachyzoites was amplified by PuRe Taq RTG-PCR Beads using mic3 specific primers. Amplified DNA was double digested using EcoRV and HindIII restriction endonucleases and then purified using EZ-10 spin coloumn purification kit. The mic3 DNA was ligated into pET-32a(+) expression vector and transformated into Escherichia coli BL21. The results showed that recombinant mic3gene 4.2 kDa has been successfully performed by cloning gene encoding for MIC-3 protein of T. gondii local isolate into pET-32a(+) and transformed to E. coli BL21.

scholarly journals In vitro selection implicates ROP1 as a resistance gene for an experimental therapeutic benzoquinone acyl hydrazone in Toxoplasma gondii

2020 ◽  
Author(s):  
Matthew C. Martens ◽  
Madalyn M. Won ◽  
Harim I. Won ◽  
Thomas T. Schulze ◽  
Abigail K. Judge ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Resistance Gene ◽  
In Vitro Selection ◽  
Acute Infection ◽  
Chemical Mutagenesis ◽  
Gene Encoding ◽  
Acyl Hydrazone ◽  
Globally Distributed

Toxoplasma gondii is a globally distributed apicomplexan parasite and the causative agent of toxoplasmosis in humans. While pharmaceuticals exist to combat acute infection, they can produce serious adverse reactions, demonstrating a need for enhanced therapies. KG8 is a benzoquinone acyl hydrazone chemotype identified from a previous chemical screen for which we previously showed in vitro and in vivo efficacy against T. gondii. However, the genetic target and mechanism of action of KG8 remain unknown. To investigate potential targets, we generated resistant T. gondii lines by chemical mutagenesis followed by in vitro selection. Whole genome sequencing of resistant clones revealed a P207S mutation in the gene encoding rhoptry organelle protein 1 (ROP1), in addition to two lesser resistance-conferring mutations in the genes for rhoptry organelle protein 8 (ROP8) and a putative ADP/ATP carrier protein (TGGT1_237700). Expressing ROP1P207S in parental parasites was sufficient to confer significant (10.3-fold increased EC50) KG8 resistance. After generating a library of mutants carrying hypermutated rop1 alleles followed by KG8 pressure, we sequenced the most resistant clonal isolate (>16.9-fold increased EC50) and found independent recapitulation of the P207S mutation, along with three additional mutations in the same region. We also demonstrate that a rop1 knockout strain is insensitive to KG8. These data implicate ROP1 as a putative resistance gene of KG8. This work further identifies a compound which can be used in future studies to better understand ROP1 function and highlights this novel chemotype as a potential scaffold for the development of improved T. gondii therapeutics.

scholarly journals Cloning Gene Encoding Micronema 3 (Mic3) Protein of Tachyzoite Toxoplasma Gondii Local Isolate

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Wayan T. Artama ◽  
Ni Nyoman Ayu Dewi ◽  
Didik Tulus Subekti
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Dna ◽  
Computer Software ◽  
Gene Encoding ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Local Isolate ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Toxoplasma Gondii Infection

Microneme 3 (MIC3) protein tachyzoites Toxoplasma gondii is one of protein which plays an important roleduring cell host invasion. Gene encoding MIC3 protein has been studied and it was suggested a potent vaccinecandidate against Toxoplasma gondii infection. The aim of this research is to clone and sequence the gene encodingMIC3 protein of tachyzoites Toxoplasma gondii local isolate by amplification using polymerase chain reaction withspecific primers. The amplified DNA fragment was cloned into pGEM-T and transformed into E. coli XL-1 Blue byheat shock method. Recombinant plasmids were isolated using alkali lysis method and analyzed by digestionusing restriction endonuclease enzymes PstI, HindIII, NcoI and EcoRV. The recombinant plasmids then sequencedto find out the nucleotide sequence of insert gene by ABIPRISM 377 DNA Sequencer. The DNA sequence thenwere analyzed by computer software for alignment. The result showed that transformation in E. coli XL-1 Blue bypGEM-T produced one clone that was encoding MIC3 protein. Analysis of 489 bp from 5’ and 447 from 3’ of genesequence showed 97-98% homology with gene encoding for MIC3 protein of RH isolate.Keywords: MIC3 protein, Toxoplasma gondii, tachyzoite, recombinant DNA

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