Isolation and Characterization of Avirulence Genes in Magnaporthe oryzae

Jee, Mui Sie; Lim, Leonard Whye Kit; Dirum, Martina Azelin; Hashim, Sara Ilia Che; Masri, Muhammad Shafiq; Tan, Hui Ying; Lai, Lee San; Yeo, Freddy Kuok San; Chung, Hung Hui

doi:10.33736/bjrst.389.2017

Cited by 9 publications

(2 citation statements)

References 53 publications

(59 reference statements)

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“…The inactivate mixture was analysed on 1% agarose gel electrophoresis at 90 V for 40 minutes. The cloning process was conducted in accordance to that portrayed by Jee et al (2017).…”

Section: /8mentioning

confidence: 99%

Mutagenesis Analysis of ABCB8 Gene Promoter of Danio rerio

Lai

Lim

Chung

2021

TUR

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The ABCB8 is one of the members under the ABCB subfamily of ATP-Binding Cassette (ABC) transporter which possess the ability in regulating the intracellular iron and heme transport. The loss of function mutation of ABCB8 gene leads to iron and heme accumulation in the cell which is highly toxic to human. However, the information regarding the expression regulation of this gene remains scarce. Hence, the objectives of this project are to determine the transcription factors binding site (TFBS) of ABCB8 and to identify the transcriptional roles of the cis-elements through mutagenesis analysis. To examine this, total genomic DNA was extracted from Danio rerio and the promoter sequence was isolated by using specific pair of primers through polymerase chain reaction (PCR). The sample was sent for DNA sequencing and the result showed 98% similarities to the zebrafish DNA sequence from clone DKEYP-87A6 in linkage group 24. Besides, the TFBS was studied in aspect of TFBS abundance, TFBS composition and TFBS distribution. The two most abundant TFBSs based on liver-specific profile were HNF-3β and C/EBPβ, with 38 and 39 binding sites, respectively. The sequence of ABCB8 promoter gene was mutated through substitution of the AP-1 binding site at location 535 with other nucleotides by using a pair of mutagenic primers (forward primer: 5’-TGGGGGTTTAGATATTGAAAC-3’; reverse primer: 5’-AACTCGC ATACATTTCAGTCATC-3’). This result may benefit the development of new diagnostics and therapeutics for iron-associated disorder.

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“…The inactivate mixture was analysed on 1% agarose gel electrophoresis at 90 V for 40 minutes. The cloning process was conducted in accordance to that portrayed by Jee et al (2017).…”

Section: /8mentioning

confidence: 99%

Mutagenesis Analysis of ABCB8 Gene Promoter of Danio rerio

Lai

Lim

Chung

2021

TUR

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“…Gradient PCR targeting the ABCG2 gene promoter was conducted as described by Lim, Chung, and Hasnain (2020) with adjustment on the extension time to 1.5 min. The cloning procedures were mirrored from that of Jee et al (2017). The plasmid used to house the promoter gene insert is the pGL3.0 (Promega, USA).…”

Section: Dna Extraction Pcr and Cloningmentioning

confidence: 99%

Mutagenesis Analysis of ABCG2 Gene Promoter of Zebrafish (Danio Rerio)

Yusni

Lim

Chung

2020

TUR

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Breast cancer is the commonest cancer among women worldwide and the probability of a woman dying from breast cancer is high (about 1 in 38 of total human population (2.6%)).The main factor for mortality is due to the resistance of this particular disease to chemotherapeutic agents. One of the most well-known proteins to be found to correlate significantly with breast cancer resistance to chemotherapeutic agent is the ATP-binding cassette super-family G member 2 (ABCG2). Knowledge on ABCG2 gene regulation is still lacking in terms of how the increased cytotoxic levels are closely related to induce a hype in gene transcript levels and ultimately cause of the reduction in chemotherapeutic agents. The approach taken in this study is through mutational analysis of selected transcription factor governing the expression of ABCG2. In order to achieve this, a previously cloned ABCG2 promoter which has been isolated (around 1500 bp in size) from Danio rerio and inserted into pGL3.0 plasmid, was subjected to site-directed mutagenesis. Selected transcription factor which is AP-1 was successfully mutated by deletion of 5'- TGACGCG -3' sequence at position 1113 bp from TSS+1 where it would bind in order to define their role in ABCG2 physiological function. Sequencing result after site-directed mutagenesis shows high similarities about 98% with ABCG2 gene of Danio rerio. Upon validation, it was found that the intended AP-1 binding site has been mutated. In future work, the mutated clone here will be subjected to transfection analysis where dual-luciferase assay will be conducted to verify the loss of activity from the ABCG2 promoter upon mutation of the targeted AP-1 site. Hence, the mutagenesis analysis of ABCG2 promoter are able to provide information on the involvement of AP-1 transcription factor in multidrug resistance mechanism of breast cancer and thus will be a potential target for chemotherapeutic agent.

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