Expression pattern of Cubitus interruptus from the mulberry silkworm Bombyx mori in late developmental stages

A partial genomic clone of Bombyx mori homologue of the segment polarity gene Cubitus interruptus (BmCi), encoding the conserved zinc finger domain and harbouring two introns, has been characterized. BmCi was expressed in the silkglands of B. mori from embryonic to the late larval stages(3rd, 4th and 5th intermoults). The expression was confined to the anterior region of the middle silkglands, overlapping with the domain of sericin-2 expression and excluding the domains of Bm invected expression, namely the middle and posterior regions of the middle silkglands. In the wing discs, the expression was restricted to the anterior compartment, which increased from 4th to 5th larval intermoults and declined later in the pupal wing buds. In gonadal tissues (both ovaries and testes) BmCi was expressed from the larval to pupal stages. The transcripts were localized to the sperm tubes containing spermatogonia in the testis of Bombyx larvae. BmCi expression, however, was not detected in any of these tissues during the moulting stages. Expression of Ci in the wing discs and gonads is evolutionarily conserved, while the silkgland represents a novel domain. Our results imply that BmCi is involved in the specification and maintenance of micro-compartment identity within the middle silkglands.

Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris.

Pattern formation during Caenorhabditis Elegans vulval development

Pattern formation during animal development involves at least three processes: establishment of the competence of precursor cells to respond to intercellular signals, formation of a pattern of different cell fates adopted by precursor cells, and execution of the cell fate by generating a pattern of distinct descendants from precursor cells. I have analyzed the fundamental mechanisms of pattern formation by studying the development of Caenorhabditis elegans vulva.

In C. elegans, six multipotential vulval precursor cells (VPCs) are competent to respond to an inductive signal LIN-3 (EGF) mediated by LET- 23 (RTK) and a lateral signal via LIN-12 (Notch) to form a fixed pattern of 3°-3°-2°-1°-2°-3°. Results from expressing LIN-3 as a function of time in animals lacking endogenous LIN-3 indicate that both VPCs and VPC daughters are competent to respond to LIN-3. Although the daughters of VPCs specified to be 2° or 3° can be redirected to adopt the 1°fate, the decision to adopt the 1° fate is irreversible. Coupling of VPC competence to cell cycle progression reveals that VPC competence may be periodic during each cell cycle and involve LIN-39 (HOM-C). These mechanisms are essential to ensure a bias towards the 1° fate, while preventing an excessive response.

After adopting the 1° fate, the VPC executes its fate by dividing three rounds to form a fixed pattern of four inner vulF and four outer vulE descendants. These two types of descendants can be distinguished by a molecular marker zmp-1::GFP. A short-range signal from the anchor cell (AC), along with signaling between the inner and outer 1° VPC descendants and intrinsic polarity of 1° VPC daughters, patterns the 1° lineage. The Ras and the Wnt signaling pathways may be involved in these mechanisms.

The temporal expression pattern of egl-17::GFP, another marker ofthe 1° fate, correlates with three different steps of 1° fate execution: the commitment to the 1° fate, as well as later steps before and after establishment of the uterine-vulval connection. Six transcription factors, including LIN-1(ETS), LIN-39 (HOM-C), LIN-11(LIM), LIN-29 (zinc finger), COG-1 (homeobox) and EGL-38 (PAX2/5/8), are involved in different steps during 1° fate execution.

水稻小G蛋白Arf GTPase激活蛋白OsAGAP功能研究

　　锌指蛋白在植物生长发育中具有重要功能，它们可以识别并结合特定的DNA序列进行转录调控，还能够参与蛋白之间相互作用的调节。我们根据锌指蛋白等转录因子特征结构域的序列特点，从来自10 K水稻芯片的EST数据库中筛选出编码58个EST序列。通过对器官表达特异性的比较分析，从中选出七个只在单一器官表达的基因，并对这七个基因的功能进行研究。对其转基因水稻的表型分析发现，C1基因调节水稻的株高和穗的发育;LIM 家族的F9影响小花的形态，主要体现在雌蕊与雄蕊的发育;锌指蛋白S34调控叶倾角的变化;F14基因编码一个核定位的TFIIIA类锌指蛋白，具体功能尚不清楚;锌指蛋白F35转基因水稻主根缩短，侧根数目显著减少。它编码一个推测的ArfGAP (Arf GTPase activating protein)，据此我们将其命名为OsAGAP，并对其进行深入研究。 　　OsAGAP的cDNA全长为1328bp，编码的蛋白由320个氨基酸组成，含有两个保守结构域：锌指结构域和C2 结构域。其中锌指结构域属于CX2CX16CX2C类，即ArfGAP domain的特征结构。GTP酶活性测定试验表明，OsAGAP蛋白能够激活水稻Arf的GTP酶活性，另外，OsAGAP还能够恢复酵母ArfGAP缺失突变体的表型。说明OsAGAP编码的蛋白是水稻中的一个ArfGAP。 　　OsAGAP在水稻各器官中均有表达，但强弱有所不同。RNA原位杂交结果显示，它在茎尖分生组织与侧生原基及侧根部位表达强烈;它在根尖主要分布于中央维管组织、分生区、皮层细胞，最有趣的是恰好与生长素在根尖极性运输路径相吻合。在亚细胞水平，OsAGAP广泛分布于细胞膜、细胞质、细胞核。 　　OsAGAP超表达水稻主根、不定根长度缩短，侧根数目显著减少表现出类似于生长素极性运输突变体的表型。其主根伸长对TIBA的抑制作用不敏感，这暗示OsAGAP超表达水稻的生长素极性运输被破坏;另外，其对各种生长素的作用敏感性也发生变化，对IAA、2,4-D的不敏感，而对NAA的反应与野生型一致，根据各类生长素进出细胞机制不同，可以推测超表达水稻的输入能力存在缺陷。极性运输实验结果表明，超表达水稻极性运输能力被破坏;对生长素输入能力的测定进一步表明，超表达水稻根载体的介导的生长素输入能力显著下降。另外，NAA处理能够恢复超表达水稻中侧根发育受抑的表型缺陷。由此可见，OsAGAP在水稻中超表达破坏了生长素极性运输的输入能力。 　　FM1-43是一类特异标记囊泡运输的荧光染料。经其染色标记后，OsAGAP超表达水稻细胞内囊泡成片聚集，形成“BFA区间”，表现出囊泡运输被破坏的典型特征。透射电镜观察发现，超表达水稻细胞内有大量的小液泡，其中积累了电子密度很高的颗粒物质。由此推测，可能由于细胞的囊泡运输被破坏，导致胞内的代谢物质不能被正常运送或分泌，而在液泡中暂时贮存以维持细胞环境的稳定。 　　在酵母和动物细胞中的研究表明， ArfGAP是调控囊泡运输的一个重要因子，然而目前还没有关于ArfGAP在植物细胞中生理作用的报道。我们的结果说明，OsAGAP作为的一个ArfGAP，它通过调控水稻中的囊泡运输，而影响了生长素的极性运输，具体表现在对生长素输入能力的调控。由此，我们推测ArfGAP可能在生长素的极性运输中也起着重要的调控作用。 　　但OsAGAP在拟南芥中却通过调控植株生长素的水平，而影响了转基因拟南芥根的发育。每种生物都有多个ArfGAP，它们之间的分工存在联系，但各不相同。OsAGAP是拟南芥的外源基因，它在拟南芥中可能以不同于水稻的机制起作用。

水稻冷诱导基因OSCOIN的功能分析

对于双子叶模式植物拟南芥在逆境应答中的机理研究已取得了很大的进展，但在单子叶植物中的相关研究相对滞后。在单子叶植物水稻中仅仅报道了一些转录因子类基因以及与代谢有关的酶类基因，但与低温有关的分子伴侣、离子通道和载体等类基因的研究少见报道。 受低温诱导的水稻基因OsCOIN（AK104280）来自水稻10K cDNA芯片，它的cDNA全长有1593bp，开放阅读框内为1089bp，编码363个氨基酸，蛋白质的计算分子量42kDa，等电点5.25。在基因组序列中有7个外显子，6个内含子。生物信息分析显示，OsCOIN在第72—106氨基酸间形成一个指环结构域。OsCOIN蛋白没有跨膜区，定位于细胞质和细胞核。通过酵母双杂交实验证明了指环蛋白OsCOIN没有转录活性，故不是转录因子。RT-PCR结果表明，OsCOIN在所选的11种水稻组织中都有不同程度的表达，4C处理0.5h时OsCOIN基因开始较强地表达，持续较强地表达到4C处理48小时，72h时OsCOIN的表达量下降到起始的水平。另外,该基因表达还受ABA和盐诱导。 利用农杆菌介导的转化手段，得到三个OsCOIN超表达株系和六个RNAi株系。RNAi株系分蘖增多、植株矮化。为了分析OsCOIN与逆境胁迫的关系，实验中分析了超表达植株对低温等胁迫的耐性。结果表明，4C处理60h、72h、84h后，所有植株都出现萎蔫，恢复生长两周后超表达植株的存活率(分别为76.2%、71.4％和50％)明显高于野生型的存活率(分别为52.4%、22.2%和14.8%)。超表达OsCOIN水稻中，OsLti6b、OsNAC6和OsP5CS的表达量明显增加，而OsDhn1和OsDREB1a的表达量明显地降低，OsCDPK7和OsLti6a表达水平未受影响。 上述结果表明OsCOIN基因的过量表达抑制了OsDhn1和OsDREB1a的表达，促进了OsLti6b、OsNAC6和OsP5CS的表达。OsCOIN基因参与的低温响应途径与ABA相关，OsCOIN超表达植株不仅能耐低温，而且对盐和干旱胁迫有一定的抗性。

Xenopus RCOR2 (REST corepressor 2) interacts with ZMYND8, which is involved in neural differentiation

Regulation of neuronal gene expression is critical to nervous system development. REST (RE1-silencing transcription factor) regulates neuronal gene expression through interacting with a group of corepressor proteins including REST corepressors (RCOR). Here we show that Xenopus RCOR2 is predominantly expressed in the developing nervous system. Through a yeast two-hybrid screen, we isolated Xenopus ZMYND8 (Zinc finger and MYND domain containing 8) as an XRCOR2 interacting factor. XRCOR2 and XZMYND8 bind each other in co-immunoprecipitation assays and both of them can function as transcriptional repressors. XZMYND8 is co-expressed with XRCOR2 in the nervous system and overexpression of XZMYND8 inhibits neural differentiation in Xenopus embryos. These data reveal a RCOR2/ZMYND8 complex which might be involved in the regulation of neural differentiation. (C) 2010 Elsevier Inc. All rights reserved.

Sox and Zfx genes in giant panda

By using PCR cloning techniques, the DNA sequences of the HMG box regions of six Sox genes (pSox) and the zinc finger domains of two Zfx genes (pZfx) in the giant panda were identified. The giant panda Sox genes fell into two subfamilies, SOX-S1 and SOX-S2. The pSox and pZfx genes of the giant panda were highly homologous to the corresponding genes in mammals and revealed close substitution rates to those in the primates.

Transcriptome of embryonic and neonatal mouse cortex by high-throughput RNA sequencing

Brain structure and function experience dramatic changes from embryonic to postnatal development. Microarray analyses have detected differential gene expression at different stages and in disease models, but gene expression information during early brain development is limited. We have generated >27 million reads to identify mRNAs from the mouse cortex for>16,000 genes at either embryonic day 18 (E18) or postnatal day 7 (P7), a period of significant synapto-genesis for neural circuit formation. In addition, we devised strategies to detect alternative splice forms and uncovered more splice variants. We observed differential expression of 3,758 genes between the 2 stages, many with known functions or predicted to be important for neural development. Neurogenesis-related genes, such as those encoding Sox4, Sox11, and zinc-finger proteins, were more highly expressed at E18 than at P7. In contrast, the genes encoding synaptic proteins such as synaptotagmin, complexin 2, and syntaxin were up-regulated from E18 to P7. We also found that several neurological disorder-related genes were highly expressed at E18. Our transcriptome analysis may serve as a blueprint for gene expression pattern and provide functional clues of previously unknown genes and disease-related genes during early brain development.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Characterization and expression analysis of TNFR-associated factor 1 (TRAF1) in grass carp Ctenopharyngodon idella

TNF receptor associated factor 1 (TRAF1) plays an important role in regulating the TNF signaling and protecting cells from apoptosis. In the present study, a TRAF1 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA is 2235 bp, including a 250 bp 5' UTR (untranslated region), a 1659 bp open reading frame, and a 326 bp 3'UTR. The polyadenylation signal (AATAAA, AATAA) and one mRNA instability motif (AUUUA) were found followed by a poly (A) tail in the 3'UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF1 (gcTRAF1). The putative amino acids of gcTRAF1 share 72% identity with the homologue in zebrafish. It is characterized by a zinc finger at the N-terminus and a TRAF domain (contains one TRAF-C and one TRAF-N) at the C-terminus. The identity of the TRAF domain among all the TRAF1 homologues in vertebrates varies from 52% to 58%, while the identities of TRAF-C were almost the same as 70%. The recombinant gcTRAF1 has been constructed successfully and expressed in Escherichia coli by using pET-32a expression vector. The polyclonal antibody for rabbit has been successfully obtained. The expression of gcTRAF1 in different organs was examined by real-time quantitative PCR and Western blotting, respectively. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of TRAF1 homologue molecule found in fish. (c) 2007 Elsevier B.V. All rights reserved.

Complete nucleotide sequence of the S10 genome segment of grass carp reovirus (GCRV)

Hemorrhagic disease, caused by the grass carp reovirus (GCRV), is one of the major diseases of grass carp in China. Little is known about the structure and function of the gene segments of this reovirus. The S10 genome segment of GCRV was cloned and the complete nucleotide sequence is reported here. The S10 is 909 nucleotides long and contains a large open reading frame (ORF) encoding a protein of 276 amino acids with a deduced molecular weight of approximately 29.7 kDa. Comparisons of the deduced amino acid sequence of GCRV S10 with those of other reoviruses revealed no significant homologies. However, GCRV S10 shared a putative zinc-finger sequence and a similar distribution of hydrophilic motifs with the outer capsid proteins encoded by Coho salmon aquareovirus (SCSV) S10, striped bass reovirus (SBRV) S10, and mammalian reovirus (MRV) S4. It was predicted that this segment gene encodes an outer capsid protein.

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.

Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools

Cystic Fibrosis (CF) is an autosomal recessive monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene with the ΔF508 mutation accounting for approximately 70% of all CF cases worldwide. This thesis investigates whether existing zinc finger nucleases designed in this lab and CRISPR/gRNAs designed in this thesis can mediate efficient homology-directed repair (HDR) with appropriate donor repair plasmids to correct CF-causing mutations in a CF cell line. Firstly, the most common mutation, ΔF508, was corrected using a pair of existing ZFNs, which cleave in intron 9, and the donor repair plasmid pITR-donor-XC, which contains the correct CTT sequence and two unique restriction sites. HDR was initially determined to be <1% but further analysis by next generation sequencing (NGS) revealed HDR occurred at a level of 2%. This relatively low level of repair was determined to be a consequence of distance from the cut site to the mutation and so rather than designing a new pair of ZFNs, the position of the existing intron 9 ZFNs was exploited and attempts made to correct >80% of CF-causing mutations. The ZFN cut site was used as the site for HDR of a mini-gene construct comprising exons 10-24 from CFTR cDNA (with appropriate splice acceptor and poly A sites) to allow production of full length corrected CFTR mRNA. Finally, the ability to cleave closer to the mutation and mediate repair of CFTR using the latest gene editing tool CRISPR/Cas9 was explored. Two CRISPR gRNAs were tested; CRISPR ex10 was shown to cleave at an efficiency of 15% and CRISPR in9 cleaved at 3%. Both CRISPR gRNAs mediated HDR with appropriate donor plasmids at a rate of ~1% as determined by NGS. This is the first evidence of CRISPR induced HDR in CF cell lines.

Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.