Understanding Agarose Gel Electrophoresis, PCR, and DNA Sequencing


Added on  2019-09-23

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Running Head: VirologyVirology
Understanding Agarose Gel Electrophoresis, PCR, and DNA Sequencing_1

Virology1The ProhibitinProtein pair of prohibitin 1 and prohibitin 2 is universally expressed as the prohibitin proteins where prohibitin 2 or PHB2 is also referred as REA (receptor estrogen activity) repressor or BAP-37 (B-cell receptor associate protein). Both these proteins belong to flotilin, stomatin, HflK/C, and prohibitin superfamily (Mishra, Murphy & Murphy, 2017). It is designated as “prohibitin” because originally PHB1 cDNA transfection led to arrest of cell cycle (Mishra,Murphy & Murphy, 2006), ("Isolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver - ScienceDirect", 2006). It has been showed that PHB1 and PHB2 are present in cytosol, mitochondria, nucleus, and incertain receptors of cell membrane ("Prohibitin Ligands in Cell Death and Survival: Mode of Action and TherapeuticPotential - ScienceDirect", 1989). At present, PHBs are considered as the best examples that illustrate distinctive andclear functions which depend on intercellular localization. PHB1 and PHB2 inside mitochondria make an alternating ring-like heterodimeric complex helpful in mitochondrial stability. On the contrary, both the independent PHBs inside the nucleus helps in transcriptionally suppressing the target genes (Mishra, Murphy & Murphy, 2006), ("Prohibitin Ligands in Cell Death and Survival: Mode of Action and Therapeutic Potential - ScienceDirect", 1989). Although PHBs in the nucleus influence cell cycle and many transcription factors, the prohibitin loss of any type andmost of the observed cellular effects are attributed to their mitochondrial function (Merkwirth et al., 2008). Level of ROS (reactive oxygen species), inflammation, and PHBs’ expressions is correlated; hence, inflammatory component diseases such as diabetes, cancer, and degenerative disorders of neuromuscles are more likely to be present with localization and/or expression of altered PHB1/2. In fact, experiments on altered expression of PHB in dissimilar model systems copies numerous pathologies of inflammation (Merkwirth et al., 2012), (Supale et al., 2013), (Theiss et al., 2007), (Kasashima, Ohta, Kagawa & Endo, 2006). While majority of researches till date on the PHBs has beencollected from PHB1 studies, this study focuses on recent advancements made in the field of PHB2 which implicatesintercellular communication nature of PHBs between mitochondria and the nucleus (Bavelloni, Piazzi, Raffini, Faenza & Blalock, 2015).Figure 1: Gene structure of PHB1 and PHB2, their coding region, and full-length protein as result. PHB1 and PHB2 introns and exons (boxes) are also included in the gene structure.Shaded boxes point out coding sequences of exon protein while unshaded boxes point out not coded exon sequence for protein. Resulting full-length proteins, major domainsand start and stop of approximate amino acid are also shown(Bavelloni, Piazzi, Raffini, Faenza & Blalock, 2015).Figure 2: Modification sites of post-translational PHB2 are shown. Post-translational modification (PTMs) sites gathered from PhosphositePlus database are sectioned into tyrosine phosphorylation, threonine/serine phosphorylation, and lysine modifications (ubiquitylation/acetylation). Bold red sites indicate validated modification through biochemical evidence at the site. Bold black sites indicate the existence of specific and significant evidence (e.g. targeted mass spectrometry) of site modification without any biochemical validation. Small black sites indicate identified sites through high-throughput techniques without any particular manner and existence of biochemical evidence (Bavelloni, Piazzi, Raffini, Faenza & Blalock, 2015).Figure 3: PHB2 mediated Signal transduction. (A) Diagram of the PHB2 playing canonical role in transcription induced by regulating estrogen receptor (ER)-α and in maintaining mitochondrial protection. (B) The transcriptional regulating role of PHB2 in myogenic differentiation and regulation through phosphorylation dependent on CaMK IV
Understanding Agarose Gel Electrophoresis, PCR, and DNA Sequencing_2

Virology2and AKT2 binding at S91. (C) The anti-apoptotic and transcriptional regulating role of PHB2 in differentiating myeloid. Erythroid differentiation shown on left panel through E3 ligase RNF2 PHB2 binding. The role of nuclear PHB2 phosphorylation dependent on hierarchical AKT shown on right panel during promyelocytic leukemia cell differentiation mediated through all-trans retinoic acid (Bavelloni, Piazzi, Raffini, Faenza & Blalock, 2015).Gene Structure and ExpressionThe chromosome 12p13.31 mapped phb2 gene, encodes 10 exons, expressed in all tested tissues virtually, and covers5.47 kb (Fig. 1), (Bavelloni, Piazzi, Raffini, Faenza & Blalock, 2015). Since the gene is conserved highly by evolution, the coding sequence of human phb2 maintains 69%, 91%, and 58% homology with fruit fly, phb2 in mouse, and yeast, respectively. While phb2 transcriptive regulation is understood poorly, it is predicted that phb2 promoters roughly contain 130 varied binding sites for transcription factors, including Fork head box protein (FoxO),GATA-1,-2,-3, CAAT-enhancer binding protein (C/EBP)-a, activator of transcription (STAT)-1, -3, and -5 and signal transducer, peroxisome proliferating-activated receptor (PPAR)-a/-c, nuclear factor (NF)-Sp1, jB, and homeobox (HOX) factors (The Search Portal for Champion ChiP Transcription Factor). PHB2 basal expressions are majorly influenced by Sp1 and other such constitutive transcription factors. Tight linkage of PHB2 with inflammation and metabolic tissues can be explained by presence of many other transcription factors. Interestingly, all the PHB2 negatively regulated transcription factors such as MyoD, ERa, and MEF-2, have binding sites of consensus inside the phb2 promoter, signifying a PHB2 expression involved feedback loop. Phb2 transcription leads to the expression of an mRNA of ~1,505 bp and encoding of a 299-amino acid protein (Fig. 1), (Bavelloni, Piazzi, Raffini, Faenza & Blalock, 2015). Furthermore, out of 13 documented splice variants, 6 do not result in expression ofprotein, and 7 transcripts code either for identified or for putative PHB2 isoforms (Bavelloni, Piazzi, Raffini, Faenza & Blalock, 2015).PHB2 Protein and Post-translational ModificationWith 33.3 kDa molecular weight full-length PHB2 has a sequence similarity of amino acid between human and mouse, yeast, or fruit fly at 100%, 56%, and 71% respectively (Mishra, Murphy & Murphy, 2006). While the sequence similarity of amino acid in PHB1 and PHB2 is 54% only, their PHB domains are identical by 74% (Mishra,Murphy & Murphy, 2006). Just like PHB1, for mitochondrial localization amino acid 1–36 (transmembrane domain) and amino acid 36–201 (central prohibitin domain) followed by an amino acid 188–264 (overlapping coiled-coil domain) is also contained in PHB2 (Fig. 1), (Bavelloni, Piazzi, Raffini, Faenza & Blalock, 2015). Moreover, PHB2 also have an ER-binding domain inside the prohibitin domain. Contrasting to PHB1 that contains a nuclear exclusionputative sequence, PHB2 contains a nuclear import putative sequence (NIS) that is situated inside ER-binding domain, explaining post-translational modifications (PTMs) or connection with other proteins directs sub-cellular localization of PHB2 (Mishra, Murphy & Murphy, 2006), (Theiss et al., 2011) (Fig. 1).
Understanding Agarose Gel Electrophoresis, PCR, and DNA Sequencing_3

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