Sang Chul Nam

A full-length cDNA for the mature, mitochondrial form of human mitochondrial RNA polymerase was cloned and expressed under the control of T5 or tac promoter in Escherichia coli. The cDNA was efficiently expressed at 37 degrees C, but virtually all the polymerase produced was insoluble, and renaturation of the inclusion bodies was unsuccessful. When the cells were grown at 25 degrees C, however, a portion of approximately 10% was soluble and active. The protein was purified 100-fold from the soluble lysates to homogeneity by two-step chromatography using Ni-nitrilotriacetic acid-Sepharose and heparin-agarose columns, as an N-terminal histidine tag attached and as the tag cleaved away. The purified polymerases with and without the histidine tag were both active in RNA polymerization in vitro as measured with poly(dA-dT) template, and specific activity was 140,000 units/mg. The purified enzyme has the same biochemical properties as the polymerase fraction partially purified from the human mitochondria, except for the promoter-specific activity that was not observed with the purified polymerase in the presence of mitochondrial transcription factor A. Additional factor(s) and/or mammalian-specific or regulatory modification(s) of the polymerase should be necessary for promoter-specific transcription.

Cell polarity genes provide positional cues for elongating photoreceptors during photoreceptor morphogenesis. Coordinated interactions between cytoskeleton and cell polarity proteins are involved in many polarized cellular processes. It has recently been found that stable microtubules in Drosophila photoreceptors, a non-ciliary rhabdomeric eye, were linked to photoreceptor cell polarity. Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental cell biological processes in which dynamic cellular polarity need to be established and maintained. Since Spectraplakin, an actin-microtubule cross-linker, is able to bind microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of photoreceptor cell polarity. The Spectraplakin in developing pupal photoreceptors specifically localizes at rhabdomere terminal web, a sub-rhabdomeric fibrous actin meshwork, which was recently proposed to be involved in a secretory trafficking. Photoreceptors lacking the Spectraplakin showed dramatic misplacements of apical membrane domains, adherens junctions, and the stable microtubules. This role of Spectraplakin in photoreceptor cell polarity was further supported by gain-of-function studies. Spectraplakin overexpression in photoreceptors caused an apical-basal cell polarity defect. These data suggest that Spectraplakin, an actin-microtubule cross-linker, at rhabdomere terminal web is essential in photoreceptor cell polarity.

Transcription termination of the human mitochondrial genome requires specific binding to termination factor mTERF. In this study, mTERF was produced in E. coli and purified by two-step chromatography. mTERF-binding DNA sequences were isolated from a pool of randomized sequences by the repeated selection of bound sequences by gel-mobility shift assay and polymerase chain reaction. Sequencing and comparison of the 23 isolated clones revealed a 16-bp consensus sequence of 5\'-GTGTGGCA GANCCNGG-3\' in the light-strand (underlined residues were absolutely conserved), which nicely matched the genomic 13-bp terminator sequence 5\'-TGGCAGAGCCC GG-3\'. Moreover, mTERF binding assays of heteroduplex and single-stranded DNAs showed mTERF recognized the light strand in preference to the heavy strand. The preferential binding of mTERF with the light-strand may explain its distinct orientation-dependent termination activity.

Traditional developmental biology laboratory classes have utilized a number of different model organisms to allow students to be exposed to diverse biological phenomena in developing organisms. This traditional approach has mainly focused on the diverse morphological and anatomical descriptions of the developing organisms. However, modern developmental biology is focusing more on conserved genetic networks which are responsible for generating conserved body patterns in developing organisms. Therefore, it is necessary to develop a new pedagogical tool to educate undergraduate biology students in the laboratory class of developmental biology with the genetic principles which are responsible for generating and controlling the developing body patterns. A new undergraduate laboratory class for developmental biology was developed in order to offer students the opportunity to explore a wide range of experimental procedures, also incorporating the instructor's on-going research. Thereby the course can serve as a bridge between research and education by combining both into a single theme. The course design involves a sequence of exercises which can be easily adapted to the faculty's ongoing research. This style of laboratory coursework could be a transitional form between a regular laboratory course and a discovery-based laboratory course. (c) 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):141-150, 2018.

Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Here, Tau, a microtubule associated protein, was analyzed to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins' localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. It is found that Tau has a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation.

Background: Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes' role in photoreceptor morphogenesis.
Methodology/Principal Findings: Here, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn's gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs).
Conclusions/Significance: These results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor.

Photoreceptor morphogenesis requires specific and coordinated localization of junctional markers at different stages of development. Here, we provide evidence that Drosophila Klp64D, a homolog of Kif3A motor subunit of the heterotrimeric Kinesin II complex, is essential for viability of developing photoreceptors and localization of junctional proteins. Genetic analysis of mutant clones shows that absence of Klp64D protein in early larval eye disc does not affect initial differentiation, but results in abnormal nuclear position in differentiating photoreceptors. These cells eventually die in the pupal stage, indicating klp64D's role in cell viability. The function of Klp64D protein is cell type specific because the p35 cell death inhibitor can rescue cell death in cone cells but not photoreceptors. In contrast to early induction of mutant clones, late induction during third instar larval stage just prior to pupation allows survival of single- or few-celled clones of klp64D mutant cells. Analysis of these lately induced clones shows that Klp64D function is essential for Bazooka (Par-3 homolog) and Armadillo localization to the adherens junction (AJ) in pupal photoreceptors. These findings suggest that Kinesin II complex plays a cell type-specific function in the localization of AJ and cell polarity proteins in the developing retina, thereby contributing to photoreceptor morphogenesis. genesis 48:522-530, 2010. (C) 2010 Wiley-Liss, Inc.

Background: Mutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development.
Methodology/Principal Findings: The spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin's gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity.
Conclusions/Significance: These results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis.

Spectrins are major proteins in the cytoskeletal network of most cells. In Drosophila, beta(Heavy)-Spectrin encoded by the karst gene functions together with Crb during photoreceptor morphogenesis. However, the roles of two other Spectrins (alpha- and beta-Spectrins) in developing photoreceptor cells have not been studied. Here, we analyzed the effects of spectrin mutations on developing eyes to determine their roles in photoreceptor morphogenesis. We found that the Spectrins are dispensable for retinal differentiation in eye imaginal discs during larval stage. However, photoreceptors deficient in alpha- or beta-Spectrin display dramatic apical membrane expansions including Crb and show morphogenesis defects during pupal eye development, suggesting that alpha- and beta-Spectrins are specifically required for photoreceptor polarity during pupal eye development. Karst localizes apically, whereas beta-Spectrin is preferentially distributed in the basolateral region. We show that overexpression of beta-Spectrin causes a strong shrinkage of apical membrane domains, and loss of beta-Spectrin causes an expansion of apical domains, implying an antagonistic relationship between beta-Spectrin and Karst. These results indicate that Spectrins are required for controlling photoreceptor morphogenesis through the modulations of cell membrane domains. genesis 47:744-750, 2009. (C) 2009 Wiley-Liss, Inc.