Journal of Genetics and Genomics

Prenatal Genotyping of Four Common Oculocutaneous Albinism Genes in 51 Chinese Families

Ai-Hua Wei, Dong-Jie Zang, Zhao Zhang, Xiu-Min Yang, Wei Li

2015, 42(6): 279-286. doi: 10.1016/j.jgg.2015.05.001

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Abstract:
Oculocutaneous albinism (OCA) is an autosomal recessive disorder characterized by hypopigmentation in eyes, hair and skin, accompanied with vision loss. Currently, six genes have been identified as causative genes for non-syndromic OCA (OCA-1∼4, 6, 7), and ten genes for syndromic OCA (HPS-1–9, CHS-1). Genetic counseling of 51 Chinese OCA families (39 OCA-1 with mutations in theTYR gene, 6 OCA-2 with mutations in the OCA2 gene, 4 OCA-4 with mutations in the SLC45A2 gene, 1 HPS-1 (Hermansky–Pudlak syndrome-1) with mutation in the HPS1 gene, and 1 mixed OCA-1 and OCA-4) led us to perform the prenatal genetic testing of OCA using amniotic fluid cells through the implementation of our optimized strategy. In our cohort, eleven previously unidentified alleles (PUAs) (5 in TYR, 2 in OCA2, and 4 in SLC45A2) were found. Three missense PUAs (p.C112R, p.H363R and p.G379V of TYR) and one in-frame deletional PUA (p.S222del of SLC24A5) led to fetuses with OCA when co-inherited with other disease causative alleles. Three PUAs (p.P152H and p.W272X of TYR, p.A486T of SLC24A5) identified in the OCA probands did not co-transmit with known pathological alleles and thus gave rise to unaffected fetuses. Four PUAs (p.Q83X and p.A658T of TYR, p.G161R and p.G366R of SLC24A5) did not transmit to the unaffected fetuses. In addition, the in vitro transfection assays showed that the p.S192Y variant of TYR produced less pigment compared to the wild-type allele. A fetus with a digenic carrier of OCA-1 and OCA-4 was unaffected. In combination with functional assays, the family inheritance pattern is useful for the evaluation of pathogenicity of PUAs and genetic counseling of OCA.

Assembly of IMPDH2-Based, CTPS-Based, and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction

Gerson Dierley Keppeke, S. John Calise, Edward K.L. Chan, Luis Eduardo C. Andrade

2015, 42(6): 287-299. doi: 10.1016/j.jgg.2015.04.002

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Abstract:
Inhibition of guanosine triphosphate (GTP) and cytidine triphosphate (CTP) biosynthetic pathways induces cells to assemble rod/ring (RR) structures, also named cytoophidia, which consist of the enzymes cytidine triphosphate synthase (CTPS) and inosine-5′-monophosphate dehydrogenase 2 (IMPDH2). We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures. HeLa and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine (DON), ribavirin, or mycophenolic acid (MPA). Over 90% of DON-treated cells presented RR structures. In HeLa cells, 35% of the RR structures were positive for IMPDH2 alone, 26% were CTPS alone, and 31% were IMPDH2/CTPS mixed, while in COS-7 cells, 42% of RR were IMPDH2 alone, 41% were CTPS alone, and 10% were IMPDH2/CTPS mixed. Ribavirin and MPA treatments induced only IMPDH2-based RR. Cells were also transfected with an N-terminal hemagglutinin (NHA)-tagged CTPS1 construct. Over 95% of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100% presented CTPS1-based RR; when treated with ribavirin, over 94% of transfected cells presented IMPDH2-based RR and 37% presented CTPS1-based RR, whereas 2% of untreated transfected cells presented IMPDH2-based RR and 28% presented CTPS1-based RR. These results may help in understanding the relationship between CTP and GTP biosynthetic pathways, especially concerning the formation of filamentous RR structures.

A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

Andrew R. Bassett, Lesheng Kong, Ji-Long Liu

2015, 42(6): 301-309. doi: 10.1016/j.jgg.2015.03.011

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Abstract:
The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies.

The RING Finger Protein NtRCP1 Is Involved in the Floral Transition in Tobacco (Nicotiana tabacum)

Hai-Yun Wang, Yi Yu, Yong-Duo Sun, Li-Bo Han, Xiao-Min Wu, Jia-He Wu, Gui-Xian Xia, Guo-Qin Liu

2015, 42(6): 311-317. doi: 10.1016/j.jgg.2015.03.010

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Abstract:
The transition from the vegetative phase to the reproductive phase is a major developmental process in flowering plants. The underlying mechanism controlling this cellular process remains a research focus in the field of plant molecular biology. In the present work, we identified a gene encoding the C3H2C3-type RING finger protein NtRCP1 from tobacco BY-2 cells. Enzymatic analysis demonstrated that NtRCP1 is a functional E3 ubiquitin ligase. In tobacco plants, expression level of NtRCP1 was higher in the reproductive shoot apices than in the vegetative ones. NtRCP1-overexpressing plants underwent a more rapid transition from the vegetative to the reproductive phase and flowered markedly earlier than the wild-type control. Histological analysis revealed that the shoot apical meristem of NtRCP1-overexpressing plants initiated inflorescence primordia precociously compared to the wild-type plant due to accelerated cell division. Overexpression of NtRCP1 in BY-2 suspension cells promoted cell division, which was a consequence of the shortened G2 phase in the cell cycle. Together, our data suggest that NtRCP1 may act as a regulator of the phase transition, possibly through its role in cell cycle regulation, during vegetative/reproductive development in tobacco plant.

Fast-Suppressor Screening for New Components in Protein Trafficking, Organelle Biogenesis and Silencing Pathway in Arabidopsis thaliana Using DEX-Inducible FREE1-RNAi Plants

Qiong Zhao, Caiji Gao, PoShing Lee, Lin Liu, Shaofang Li, Tangjin Hu, Jinbo Shen, Shuying Pan, Hao Ye, Yunru Chen, Wenhan Cao, Yong Cui, Peng Zeng, Sheng Yu, Yangbin Gao, Liang Chen, Beixin Mo, Xin Liu, Shi Xiao, Yunde Zhao, Silin Zhong, Xuemei Chen, Liwen Jiang

2015, 42(6): 319-330. doi: 10.1016/j.jgg.2015.03.012

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Abstract:
Membrane trafficking is essential for plant growth and responses to external signals. The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex (endosomal sorting complex required for transport). FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body (MVB), vacuolar protein transport and vacuole biogenesis, and autophagic degradation. FREE1 knockout plants show defective MVB formation, abnormal vacuolar transport, fragmented vacuoles, accumulated autophagosomes, and seedling lethality. To further uncover the underlying mechanisms of FREE1 function in plants, we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype ofFREE1-RNAi transgenic plants. The obtained mutants are termed as suppressors of free1 (sof). To date, 229 putative sof mutants have been identified. Barely detecting of FREE1 protein with M₃ plants further identified 84 FREE1-related suppressors. Also 145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants. Through next-generation sequencing (NGS) of bulked DNA from F₂ mapping population of two RNAi-related sof mutants, FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly. These FREE1- and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis, as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

OsCBL1 Modulates Lateral Root Elongation in Rice via Affecting Endogenous Indole-3-Acetic Acid Biosynthesis

Jing Yang, Xiaomu Zhang, Yunqing Huang, Yuqi Feng, Yangsheng Li

2015, 42(6): 331-334. doi: 10.1016/j.jgg.2015.03.004

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Transcriptomic Analysis of Thermoanaerobacter tengcongensis Grown at Different Temperatures by RNA Sequencing

Chuan Wang, Chunlei Jin, Jihui Zhang, Qiyu Bao, Bo Liu, Huarong Tan

2015, 42(6): 335-338. doi: 10.1016/j.jgg.2015.03.005

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The 3rd Symposium on Animal Models of Primates – The Application of Non-Human Primates to Basic Research and Translational Medicine

Yong-Gang Yao, Yong-Bin Chen, Bin Liang

2015, 42(6): 339-341. doi: 10.1016/j.jgg.2015.04.007

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2015 Vol. 42, No. 6