scholarly journals Isolation of Metallothionein (MT) Gene from Phytoremediation Agent, Eleusine indica

2021 ◽  
Vol 934 (1) ◽  
pp. 012027
Author(s):  
M Mardalisa ◽  
U M Batubara
Keyword(s):  
Heavy Metals ◽  
Gene Isolation ◽  
Sequencing Analysis ◽  
Amino Acid Residues ◽  
Eleusine Indica ◽  
Band Size ◽  
Pcr Product ◽  
Visualization Process ◽  
Poaceae Family ◽  
Pcr Technology

Abstract Belulang grass (Eleusine indica) is a plant in the Poaceae family that is commonly found in the coastal area of Dumai, Riau Province. Eleusine indica is characterized by narrow leaves, concave stems that can reach up to 95 cm high and strong roots. E. indica is known to be very tolerant of its environment, including the environment contaminated with heavy metals. The ability of E. indica as a phytoremediation agent in absorbing heavy metals has been widely known as the role of metallothionein (MT) protein. MT is believed to have a function in the metal metabolism and detoxification process through the metal chelating interaction between the cysteine amino acid residues. This unique function prompted the interest to isolate the MT gene from E. indica. This method involves the isolation of genomic DNA from E. indica followed by the process of amplification of the MT gene using specific primers, namely MTFS and MTRS by polymerase chain reaction (PCR) technology. The success of the MT gene isolation process from E. indica was evidenced by the presence of a single band size of around 172 bp via the visualization process on 1% agarose gel. Furthermore, the results of the PCR product are purified for the purpose of sequencing activity. The results of sequencing analysis of the 172 bp fragment showed 99.31% identical similarity with the complete metallothionein gene from E. indica (DQ082855.1) by using the BLASTN tool, NCBI website.

EVALUATION OF PTYCHOBOTHRIDEAN CESTODES USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS FROM FRESH WATER FISHES IN GODAVARI BASIN (M.S.) INDIA

2017 ◽  
Vol 23 (2) ◽  
Author(s):  
SUNITA BORDE ◽  
ASAWARI FARTADE ◽  
AMOL THOSAR ◽  
RAHUL KHAWAL
Keyword(s):  
Fresh Water ◽  
Rapd Markers ◽  
Random Amplified Polymorphic Dna ◽  
Band Pattern ◽  
Genomic Variation ◽  
Polymorphic Band ◽  
Rapd Profile ◽  
Band Size ◽  
Pcr Product ◽  
The Common

Ptychobothridean genera like Senga and Circumoncobothrium are the common parasites of fresh water fishes. The genotypic study of these parasites was taken by RAPD. The RAPD profile of these two parasites were not similar to each other as depicted by the band pattern in picture. These results suggest the presence of inter-specific polymorphism among cestode parasites of two different genera for RAPD analysis. The present study demonstrated that genetic differentiation of cestode parasites could be accomplished on the basis of genomic variation with polymorphic band pattern using RAPD. All the detected bands (PCR product) were polymorphic and band size ranged from 500-5000 bp in length. The RAPD of profiles using GBO-31, GBO-32, GBO-33, GBO-34, GBO-35 and GBO-36. Primers were able to characterize inter-specific polymorphism among the two genus ( Senga and Circumoncobothrium ). Genetic analysis suggests that Senga and Circumoncobothrium show genetic diversity with respect to RAPD patterns using all the six primers used for the present study. The genetic distance between the analyzed genuses ranged from 0.14 to 0.80. The differentiation of the two parasites on the basis of genetic markers could greatly facilitate study on the biology of these parasites.

scholarly journals Gene Cloning and Molecular Characterization of Lysine Decarboxylase from Selenomonas ruminantiumDelineate Its Evolutionary Relationship to Ornithine Decarboxylases from Eukaryotes

2000 ◽  
Vol 182 (23) ◽  
pp. 6732-6741 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Yoshihiro Yamaguchi ◽  
Minenobu Ono ◽  
Yoshiyuki Kamio
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Pyridoxal Phosphate ◽  
Evolutionary Relationship ◽  
Binding Domain ◽  
Sequencing Analysis ◽  
Lysine Decarboxylase ◽  
Amino Acid Residues ◽  
Phosphate Binding ◽  
Link Type

ABSTRACT Lysine decarboxylase (LDC; EC 4.1.1.18 ) from Selenomonas ruminantium comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both l-lysine andl-ornithine with similar Km andVmax values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063–1069, 1999). Here, the LDC-encoding gene (ldc) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of S. ruminantium LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC 4.1.1.17 ), including the mouse,Saccharomyces cerevisiae, Neurospora crassa,Trypanosoma brucei, and Caenorhabditis elegansenzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved inS. ruminantium LDC. Computer analysis of the putative secondary structure of S. ruminantium LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward bothl-lysine and l-ornithine with a substrate specificity ratio of 0.83 (defined as thek cat/Km ratio obtained with l-ornithine relative to that obtained withl-lysine). We have succeeded in converting S. ruminantium LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in S. ruminantium LDC.

High Throughput SNP Genotyping with Two Mini-sequencing Assays

2004 ◽  
Vol 36 (6) ◽  
pp. 379-384 ◽  
Author(s):  
Chunqing Luo ◽  
Libin Deng ◽  
Changqing Zeng ◽  
You-Xin Jin
Keyword(s):  
High Throughput ◽  
Snp Genotyping ◽  
Success Rates ◽  
Ionization Time ◽  
Data Process ◽  
Pcr Product ◽  
Human Chromosome 3 ◽  
Pcr Technology ◽  
The Cost

Abstract Two mini-sequencing methods, FP-TDI (template-directed dye-terminator incorporation with fluorescence-polarization) and MassArray (matrix assisted laser desorption ionization time of flight detection mass spectrometry), were optimized. A numeric standard was introduced to evaluate the SNP scoring quality of FP-TDI assay, thus made the optimization work easier. At the same time, using multi-PCR technology, 8-plex genotyping of MassArray assay was successfully carried out, some softwares were developed and the data process of MassArray was highly automated. Then these two methods were applied to high throughput SNP genotyping, the accuracy, efficiency and robustness were compared. The result shows FP-TDI is more sensitive to the concentration of SNPprimer and PCR product, as well as extension cycles, the SNPprimer length of FP-TDI should be 24–30 bp long, whereas MassArray assay prefers to be as short as only 16 bp. Altogether 6440 SNP sites of human chromosome 3 were genotyped in a sample of 90 individuals, 4792 sites by FP-TDI assay and 1648 sites by MassArray assay, the success rates of FP-TDI and MassArray were 67.7% and 93.6% respectively. The throughput of MassArray was higher than FP-TDI, and the cost of MassArray was lower, MassArray was more suitable for high throughput SNP genotyping.

scholarly journals Utilization of Parupuk Plants (Phragmites Karka) To Reduce Mercury Levels In Waters Former Of Diamond And Golden Mining

2019 ◽  
Vol 280 ◽  
pp. 05012
Author(s):  
Lailan Ni’mah ◽  
Muhammad Adzhari Anshari ◽  
Hari Apriyan Saputra ◽  
Adi Rahmadi ◽  
Ulfa Fitriati
Keyword(s):  
Waste Water ◽  
Heavy Metals ◽  
Mass Variation ◽  
Poaceae Family ◽  
Phragmites Karka ◽  
Effective Condition ◽  
Waste Degradation

The waters in Cempaka Sub-district have been contaminated by heavy metals that exceed the quality of waste water standard. The process that can be used to reduce waste pollution is by phytoremediation method. Phytoremediation is a method of waste degradation using plant capability. One of the plants is the parupuk plant (phragmites karka). Parupuk plant (Phragmites karka) has a big ability to degradation the waste. Parupuk plant was included in poaceae family. The purpose of this study is to analyze the influence of mass variations and the duration of immersion of parupuk plants in absorbing heavy metals mercury. In this study begins with static phytoremediation was performed by plant mass variation of 0.5 kg, 1 kg and 1.5 kg. The Soaking period of 8 hours, 80 hours, 152 hours and 224 hours. From the result of research known, the effective condition for the parapuk plant to absorbs the heavy metals mercury is at the 8th hour of phytoremediation and 1.5 kg parupuk plant can absorb the heavy metals mercury by 90,74%.

A New Human CD20 Spliced mRNA as a Potential Molecular Marker for the Follow-up of B-Cell Malignancies.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1479-1479
Author(s):  
Marina Deschamps ◽  
Carole Henry ◽  
Eric Robinet ◽  
Francine Garnache-Ottou ◽  
Jean-Paul Remy-Martin ◽  
...  
Keyword(s):  
B Cells ◽  
Molecular Marker ◽  
B Cell ◽  
Cell Lines ◽  
Specific Marker ◽  
Molecular Response ◽  
Sequencing Analysis ◽  
Pcr Product ◽  
Donor And Acceptor

Abstract The human CD20 molecule (hCD20) is a B-cell lineage specific marker expressed by normal and leukemic B cells from the pre-B to the plasma-cell stages and a target for Rituximab (Rx) immunotherapy. During the course of a CD20 RTPCR on B-cell line cDNA, we unexpectedly obtained a shorter PCR product (ΔCD20) in addition to the expected 894bp PCR product (wtCD20). Sequencing analysis revealed that this additional fragment was identical to Genbank published CD20 sequence, but lacked 501bp. In silico analysis of the wtCD20 sequence, using Genesplicer and NetGene 2 softwares, showed donor and acceptor sites (nt112 & nt611 respectively, from ATG codon) matching the newly identified spliced DCD20 form and a branched site located in nt595 exon 3 of the gene respectively. The CD20 spliced mRNA form links part of the end of exon 3 to that of exon 7. The truncated sequence is on the reading frame and can code a putative protein of 130 amino-acids (~15KD) including the intracellular C- terminal domain with part of the transmembrane 1 (TM1) domain and the end of the N-terminal intracellular domain. The extracellular domain and large parts of the 4 TM segments are deleted suggesting that the putative ΔCD20 may be a non-anchored membrane protein. Using CD20 PCR assays amplifying either both wtCD20 and ΔCD20 forms or ΔCD20 alone, we detected the truncated mRNA DCD20 form in different B cell lines (n=12) but not in different T-cell lines (n=4). With a QPCR assay allowing for the specific quantification of either wtCD20 or truncated DCD20 mRNA we also detected the ΔCD20 spliced form [expressed as relative % of ΔCD20: R = (ΔCD20/wtCD20+ΔCD20) × 100] in in-vitro EBV-transformed B-cell lines (2.9 ± 4.51%, n=6); as well as in CD19+ cell sorted cells from tonsillectomy samples (9 ± 2.2%, n=7) and in-vitro B blast cells (14 ± 7.8%, n=5). Interestingly, screening of a panel of B-cell hematologic malignancies showed that the spliced form is detectable at various levels. We found a mean of 3.6 ± 5.1% in B-ALL (n=27); 3.9 ± 5.3% in follicular lymphomas (n=5); 2.9 ± 4.5% in mantle lymphomas (n=6); 3.2 ± 2.2% in high grade lymphomas (n=5); and 0.1 ± 0.2% in B-CLL (n=8). However, this spliced form was not detected in peripheral blood mononuclear cells or in immunomagnetically-sorted CD19+ or CD20+ blood cells from healthy donors. To explore clinical relevance, molecular monitoring of ΔCD20 in the marrow was performed in 2 patients. A 1rst patient with mantle lymphoma, showed an R of 4.2% at diagnosis (diag). After chemotherapy (VAD + chloraminophen) + Rx, R was 2.2% at +12 mo (×1.9 decrease from diag) and 1.8% at +18mo (×1.3 decrease/+12mo). This was in accordance with the cytological (absence of lymphoma cells) and the molecular response (absence of cyclinD1 overexpression). A second patient, with a Phi+ B-ALL, showed an ×13.7 R-decrease compared to diag (from 4.1% to 0.3%) following treatment according to the molecular response. Interestingly, R increased up to 2.6% at +8 months (×8.6/6mo) and 3% (×1.1/8mo) at +12 months after treatment, while molecular and cytological relapse was evidenced only 12 mo after treatment. In conclusion, we report evidence of a novel alternatively spliced transcript of the hCD20 gene, specifically expressed at detectable levels in leukemic, lymphoma, activated or EBV-transformed B cells, but not in normal resting B cells. Further experiments will determine whether the ΔCD20 mRNA spliced form modulates wtCD20 expression and thus influences the response to Rx treatment in hematologic B diseases. However, our initial results suggest that DCD20 quantification may be an indicator of minimal residual disease, as a potential predictive marker of relapse, especially in patients with no other molecular marker.

Molecular Cloning of TSARG6 Gene Related to Apoptosis in Human Spermatogenic Cells

2004 ◽  
Vol 36 (2) ◽  
pp. 93-98 ◽  
Author(s):  
Gang Liu ◽  
Guang-Xiu Lu ◽  
Xiao-Wei Xing
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Critical Role ◽  
Human Testis ◽  
Amino Acid Residues ◽  
Spermatogenic Cells ◽  
Protein Protein Interactions ◽  
New Gene ◽  
Pcr Technology ◽  
Dnak Protein

Abstract Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accession No. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan program and PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns is located in human chromosome 11q13.3. Its protein containing 316 amino acid residues is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6 protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result of RT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and the transcript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene which probably inhibited human testis spermatogenesis apoptosis.

scholarly journals Isolation and Genetic Analysis of Bovine Viral Diarrhea Virus from Infected Cattle in Indiana

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Roman M. Pogranichniy ◽  
Megan E. Schnur ◽  
Eran A. Raizman ◽  
Duane A. Murphy ◽  
Maria Negron ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Clinical Signs ◽  
Bovine Viral Diarrhea ◽  
Sequencing Analysis ◽  
Diagnostic Laboratory ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Mucosal Disease ◽  
Pcr Product ◽  
Viral Diarrhea Virus

Species and biotype distribution was determined in 44 bovine viral diarrhea virus- (BVDV-) positive samples submitted to the Animal Disease Diagnostic Laboratory (ADDL) in Indiana during 2006–2008. BVDV RNA was detected in the 5′-untranslated region and Nproregion using reverse transcriptase PCR followed by sequencing analysis of the PCR product. Additionally, cases were classified into one of six categories according to history and/or lesions: acute symptomatic, hemorrhagic, respiratory distress, reproductive, persistent infection (PI), and mucosal disease (MD). Of 44 BVDV-positive samples, 33 were noncytopathic (ncp), 10 were cytopathic (cp), and one presented both ncp and cp biotypes. Sequencing analysis demonstrated that all samples belonged to BVDV-1a, BVDV-1b, or BVDV-2. The most common isolate was ncp BVDV-1b, (44%) followed by ncp BVDV-2a (24%). Among the six categories, respiratory clinical signs were the most common (36%) followed by PI (25%) and MD (16%).

scholarly journals Keanekaragaman dan Kemelimpahan Jenis Tumbuhan Invasif di Hutan Wisata Penggaron Kabupaten Semarang Jawa Tengah

2018 ◽  
Vol 20 (2) ◽  
pp. 100
Author(s):  
Sri Utami ◽  
M Murningsih
Keyword(s):  
Genetic Resources ◽  
Invasive Plants ◽  
Cynodon Dactylon ◽  
Pennisetum Purpureum ◽  
Eleusine Indica ◽  
Central Java ◽  
Forest Genetic ◽  
Poaceae Family ◽  
Forest Genetic Resources ◽  
Synedrella Nodiflora

Forests are ecosystems that have very potential natural resources, including storing high genetic resources. One of the things that threatens the decline of genetic resources in the forest is the presence of invasive species. This study aims to determine the species of invasive plants and their abundance in the Penggaron tourism forest of Ungaran Regency, Central Java. The research method was carried out by exploring the entire forest area through the path. The results of the study showed that 13 species of invasive plants were included in 7 families. The most number of invasive plants from the Poaceae family include 5 species : Axonopus Compressus, Cynodon dactylon, Pennisetum purpureum, Paspalum conjugatum and Eleusine indica. The highest relative abundance was Eleusine indica and was followed by Synedrella nodiflora, Elephantopus scaber and Paspalum conjugatum. The species of invasive plants, especially the abundant ones, need to be controlled by the population so as not to threaten native plants and cause environmental degradation in Penggaron tourism forests. Key word : Invansive plant, Penggaron tourism forest, genetic resources.

Cloning of the obese gene (ob) of adipose tissue, and differences in ob gene expression with age of piglets

2005 ◽  
Vol 2 (3) ◽  
pp. 207-212
Author(s):  
Wang Yi-Zhen ◽  
Chu Xiao-Na ◽  
Huang Hai-Qing ◽  
Han Fei-Fei ◽  
Liu Jian-Xin
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Amino Acid ◽  
Internal Control ◽  
Mrna Levels ◽  
Amino Acid Residues ◽  
Rt Pcr ◽  
Amino Acid Homology ◽  
Pcr Product ◽  
The Difference

AbstractGenomic RNA was extracted from the subcutaneous adipose tissue of piglets (Duroc×Landrace×Tai-hu) at 1, 14, 28, 42 and 56 days of age, and obese gene (ob) mRNA was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR). A DNA fragment of about 504 bp was obtained and the PCR product was cloned into a pGEM-T vector. The ob gene was isolated and sequenced from the positive clones screened. Sequence analysis suggested that this fragment was a partial sequence of ob cDNA, coding 167 amino acid residues, which constituted the major part of leptin mature protein. The gene homology of the fragment obtained in this study compared to the reported ob cDNA sequence in adipocytes of pig was 99.405%, and amino acid homology was 98.94%. Based on the ob gene clone, we successfully constructed an optimal semi-quantitative RT-PCR method. Using β-actin as the internal control, we investigated the difference of ob gene expression at different ages of piglets. Results showed that ob mRNA levels increased steadily at postnatal days 1–28 (preweaning), peaked at postnatal day 28, when piglets were weaned, and decreased from day 28 to 56.

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