Micropthalmia transcription factor a new prognostic marker

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用生物信息学挖掘玉米中的microRNAs及其靶基因

。近年来 , miRNA 的研究已经成为分子子 RNA 发展到在不同物种中大量发现 miRNA, 而随着研究的不断深入以及相关理论和实验技术的完善 , 发现在植物中存在一些序
本研究由国家自然科学基金项目 (30900901)和教育部高等学校博士学科点专项科研基金项目 (20095103120002)资助。
microRNAs (miRNA)是一大类内源性的、 19~24 碱基长度的小分子非编码 RNA, 它可以通过与靶 mRNA 分子完全或部分匹配 , 指导 mRNA 剪切或者抑止翻译等方式调控动植物的生命过程 [1], 从而在多细胞生物的基因表达调节和控制中扮演十分重要的角色 , 是以 RNA 为基础的基因表达调控系统的关键成员
用生物信息学挖掘玉米中的 microRNAs 及其靶基因
张志明
摘
宋锐
彭华
罗茂
沈亚欧
刘丽
赵茂俊
潘光堂*
四川农业大学玉米研究所 , 四川雅安 625014
要 : microRNA (miRNA)是一类内源性的、 19~24 碱基长度的小分子非编码 RNA, 通过碱基互补调控靶基因的表
达 , 在多细胞生物的基因表达调控过程中扮演着十分重要的角色。植物中的 miRNAs 具有高度的保守性 , 这为通过同源比对发现保守的 miRNAs 提供了思路和途径。本研究通过对拟南芥、水稻等植物已知的 miRNAs 与玉米 EST 和 GSS 数据库的比对 , 并设置一系列严格的筛选标准 , 共筛选到 23 条新的玉米 miRNAs; 利用 WMD 3 在线植物 miRNAs 靶基因预测软件 , 对新发现的玉米 miRNAs 进行靶基因预测 , 总共预测到 89 个靶基因 , 进一步功能分析发现 , 这些靶基因参与玉米的生长发育、信号转导、转录调节、新陈代谢及逆境胁迫响应等调控过程。关键词 : microRNA (miRNA); 生物信息学 ; 预测 ; 靶基因 ; 玉米

分子生物学复习题及答案附带模拟考卷

分子生物学复习思考题1．写出分子生物学广义的与狭义的定义，现代分子生物学研究的主要内容，以及5个分子生物学发展的主要大事纪（年代、发明者、简要内容）。

广义上:分子生物学包括对蛋白质和核酸等生物大分子结构与功能的研究、以及从分子水平上阐明生命的现象和生物学规律。

狭义概念:既将分子生物学的范畴偏重于核酸（基因）的分子生物学，主要研究基因或DNA结构与功能、复制、转录、表达和调节控制等过程。

其中也涉及到与这些过程相关的蛋白质和酶的结构与功能的研究。

现代分子生物学研究的主要内容有：基因与基因组的结构与功能，DNA的复制、转录和翻译，基因表达调控的研究，DNA重组技术，结构分子生物学等。

5个分子生物学发展的主要大事纪（年代、发明者、简要内容）:1.1944年,著名微生物学家Avery 等人在对肺炎双球菌的转化实验中证实了DNA是生物的遗传物质。

这一重大发现打破了长期以来，许多生物学家认为的只有象蛋白质那样的大分子才能作为细胞遗传物质的观点，在遗传学上树立了DNA是遗传信息载体的理论。

2. 2.1953年，是开创生命科学新时代具有里程碑意义的一年，Watson和Crick发表了“脱氧核糖核酸的结构”的著名论文，他们在Franklin和Wilkins X-射线衍射研究结果的基础上，推导出DNA双螺旋结构模型，为人类充分揭示遗传信息的传递规律奠定了坚实的理论基础。

同年，Sanger历经8年，完成了第一个蛋白质——胰岛素的氨基酸全序列分析。

3. 1954年Gamnow从理论上研究了遗传密码的编码规律, Crick在前人研究工作基础上，提出了中心法则理论，对正在兴起的分子生物学研究起了重要的推动作用。

4. 1956年Volkin和Astrachan发现了mRNA(当时尚未用此名)。

5. 1985年,Saiki等发明了聚合酶链式反应(PCR)；Sinsheimer首先提出人类基因组图谱制作计划设想；Smith等报导了DNA测序中应用荧光标记取代同位素标记的方法；Miller等发现DNA结合蛋白的锌指结构。

Translation-翻译

• Cap binding protein, eIF4E, binds to cap
• The N-terminus of eIF4G binds eIF4E and the Cterminus binds eIF4A
• Th20e21/44/40S subunit binds to eIF4G via eIF3
AAAAAA
1
A “Simple” Eukaryotic Gene
Transcription Start Site
5’ UTR
Introns
3’ UTR
5’
Exon 1 Int. 1 Exon 2 Int. 2 Exon 3
3’
Promoter/ Control Region
Exons
Transcription
• 70-80 nt long
• 3’ end has the 5’- CCA sequence to which aa are linked
• The opposite end contains the anticodon loop
• Contains modified
ba2s02e1/4s/4
7
2021/4/4
4
Model of eukaryotic ribosome
• rRNAs are believed to play a catalytic role in protein synthesis.
• After removal of 95% of the ribosomal proteins, the 60S subunit can catalyze formation of peptide bonds.

动物园非洲野犬繁殖研究初探

2017年第12期[9]马亚南.miR-27a 在WM239细胞中的表达及对其生物学特性的影响[D].济南大学,2015[10]Dong C,et al.Coat color determination by miR-137mediateddown-regulation of microphthalmia-associated transcription factorin a mouse model[J].Rna-a Publication of the Rna Society,2012,18(9):1679-86.[11]Yan B,et al.microRNA regulation of skin pigmentation in fish[J].Journal of Cell Science,2013,126(15):3401-8.[12]Kennell J A,et al.The microRNA miR-8is a positive regulatorof pigmentation and eclosion in Drosophila[J].DevelopmentalDynamics,2012,241(1):161-168.[13]Zhang J,et al.Role of microRNA508-3p in melanogenesis bytargeting microphthalmia transcription factor in melanocytes of alpaca[J].Animal,2017,11(2):236-243.[14]Shi Q,et al.Oxidative stress-induced overexpression of miR-25:the mechanism underlying the degeneration of melanocytes in vitiligo[J].Cell Death &Differentiation,2016,23(3):496-508.[15]Yang S,et al.Identification of a novel microRNA importantfor melanogenesis in alpaca (Vicugna pacos)[J].Journal of AnimalScience,2015,93(4):1622-31.非洲野犬（Lycaon pictus ）是食肉目犬科非洲野犬属的唯一物种。

斑马鱼的mophalino

Targeted Inhibition of miRNA Maturationwith Morpholinos Reveals a Role for miR-375 in Pancreatic Islet DevelopmentWigard P.Kloosterman1,Anne gendijk1,Rene´F.Ketting1*,Jon D.Moulton2,Ronald H.A.Plasterk11Hubrecht Laboratory-KNAW,Utrecht,The Netherlands,2Gene Tools,Philomath,Oregon,United States of AmericaSeveral vertebrate microRNAs(miRNAs)have been implicated in cellular processes such as muscle differentiation, synapse function,and insulin secretion.In addition,analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis.Nonetheless,only a few loss-of-function phenotypes for individual miRNAs have been described to date.Here,we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development.Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs.Morpholinos can block processing of the primary miRNA(pri-miRNA)or the pre-miRNA,and they can inhibit the activity of the mature miRNA.We used this strategy to knock down13miRNAs conserved between zebrafish and mammals.For most miRNAs,this does not result in visible defects,but knockdown of miR-375causes defects in the morphology of the pancreatic islet.Although the islet is still intact at24hours postfertilization,in later stages the islet cells become scattered.This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375precursor,excluding off-target effects as cause of the phenotype.The aberrant formation of the endocrine pancreas,caused by miR-375knockdown,is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development.The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.Citation:Kloosterman WP,Lagendijk AK,Ketting RF,Moulton JD,Plasterk RHA(2007)Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375in pancreatic islet development.PLoS Biol5(8):e203.doi:10.1371/journal.pbio.0050203IntroductionMicroRNAs(miRNAs)have a profound impact on the development of multicellular organisms.Animals lacking the Dicer enzyme,which is responsible for the processing of the precursor miRNA into the mature form,cannot live[1–3]. MiRNA mutants have been described only for Caenorhabditis elegans and Drosophila,reviewed in[4].From these studies,it is clear that invertebrate miRNAs are involved in a variety of cellular processes,such as developmental timing[5,6], apoptosis[7,8],and muscle growth[9].Analysis of conditional Dicer null alleles in mouse has indicated a general role for miRNAs in morphogenesis of the limb,skin,lung epithelium, and hair follicles[10–13].Overexpression studies in mouse have implicated speciﬁc vertebrate miRNAs in cardiogenesis and limb development[14,15].In zebraﬁsh,embryos lacking both maternal and zygotic contribution of Dicer have severe brain defects[2].Strikingly,the brain phenotype of maternal-zygotic Dicer zebraﬁsh can be restored by injection of miR-430,the most abundant miRNA in early zebraﬁsh develop-ment.Despite all these studies describing functions for miRNAs in development,no vertebrate miRNA mutant has been described to date.Genetically,it is challenging to obtain mutant miRNA alleles in zebraﬁsh,because their small size makes them less prone to mutations by mutagens,and for many miRNAs,there are multiple alleles in the genome or they reside in families of related sequence.Temporal inhibition of miRNAs by antisense molecules provides another strategy to study miRNA function.29-O-methyl oligonucleotides have been successfully used in vitro and in vivo to knock down miRNAs[16–18].Morpholinos are widely applied to knock down genes in zebraﬁsh development [19]and have recently been used to target mature miR-214in zebraﬁsh[20].However,off-target phenotypes are often associated with the use of antisense inhibitors.Here,we show that morpholinos targeting the miRNA precursor can knock down miRNAs in the zebraﬁsh embryo. Several independent morpholinos can knock down the same miRNA,and these serve as positive controls toﬁlter out off-target effects.Morpholinos can block miRNA maturation at the step of Drosha or Dicer cleavage,and they can inhibit the activity of the mature miRNA.We show that inhibition of miR-375,which is expressed in the pancreatic islet and pituitary gland of the embryo[21],results in dispersed islet cells in later stages of embryonic development,whereas no effects were observed in the pituitary gland.The morpholino-mediated miRNA knockdown strategy presented here,is an extremely fast and well-controlled method to study miRNA function in development.Academic Editor:James C.Carrington,Oregon State University,United States of AmericaReceived October13,2006;Accepted May22,2007;Published July24,2007Copyright:Ó2007Kloosterman et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Abbreviations:dpf,days postfertilization;GFP,green fluorescent protein;hpf, hours postfertilization;LNA,locked nucleic acid;miRNA,microRNA;MO, morpholino oligonucleotide;RT-PCR,reverse transcriptase PCR*To whom correspondence should be addressed.E-mail:rene@niob.knaw.nlP L o S BIOLOGYResultsMorpholinos Targeting the Mature miRNA Deplete the Embryo of Specific miRNAsSince it is difﬁcult to obtain a genetic mutant for a miRNA in zebraﬁsh,we looked for alternative strategies to deplete the embryo of speciﬁc miRNAs.Antisense molecules such as 29-O-methyl and locked nucleic acid(LNA)oligonucleotides have been used to inhibit miRNAs in cell lines[16,18,22], Drosophila embryos[23],and adult mice[17].We tried to use these molecules to inhibit the function of endogenous miRNAs in the zebraﬁsh embryo.Although they can be used to suppress the effects of miRNA overexpression[24], injection of higher concentrations required to obtain good knockdown of endogenous miRNAs resulted in toxic effects, when injecting1nl solution at a concentration of approx-imately10l M and50l M for LNA and29-O-methyl oligonucleotides,respectively(unpublished data).Therefore, we switched to morpholinos because these are widely used to inhibit mRNA translation and splicing in zebraﬁsh embryos [19],and have also been shown to target miRNAs in the embryo[2,20,24].We injected1nl of600l M morpholino solution with a morpholino complementary to the mature miR-206in one-or two-cell–stage embryos.Subsequently, embryos were harvested at24,48,72,and96hours postfertilization(hpf),and subjected to in situ hybridization and Northern blotting(Figure1A and1B).This analysis showed that the mature miRNA signal is suppressed up to4d after injection of the morpholino.The knockdown effect was speciﬁc for this miRNA;parallel in situ analysis of the same embryos with a probe for miR-124did not show any effects on expression of this miRNA(Figure1B).Thus,miRNA detection can be speciﬁcally and efﬁciently suppressed during embryonic and early larval stages of zebraﬁsh development using morpholinos antisense to the mature miRNA.The zebraﬁsh embryo can be used to monitor the effect of miRNAs on greenﬂuorescent protein(GFP)reporters fused to miRNA target sites[24].To determine the effect of a morpholino in this assay system,we constructed a GFP reporter for miR-30c and tested it in the presence and absence of a mature miR-30c duplex.Injected miR-30c silences this GFP reporter,which is in line with previous reports using similar strategies in the embryo(Figure1C) [2,20,24].Co-injection of the miR-30c duplex and a morpho-lino targeting mature miR-30c rescues the reporter signal, whereas injection of a control morpholino did not reverse the silencing by miR-30c.These data indicate that a morpholino can block the activity of a mature miRNA duplex in a functional assay.There are three possible explanations for the observed reduction in the detection signal for a miRNA that is targeted by a morpholino.First,the hybridization of a morpholino could disturb isolation of the miRNA.Second,the morpho-lino could destabilize the miRNA.Third,the morpholino could inhibit the maturation of the miRNA.To examine the effect of a morpholino on the isolation of a mature miRNA,we incubated a mature miR-206duplex and a control duplex(miR-205)with a morpholino against miR-206 in vitro.After isolation,samples were analyzed by Northern blotting for the presence of miR-206and miR-205.We could still detect miR-206,indicating that there is no effect of the morpholino on the RNA isolation procedure(Figure1D). However,when morpholino and miRNA duplex were incubated together in vitro and loaded on a denaturing gel without isolation,we observed a decrease in the signal for miR-206,indicating that the morpholino can bind to the miRNA in vitro and still does so in the denaturing gel. Next,we wanted to know whether a morpholino could affect the stability of a mature miRNA in vivo.Therefore,we injected a mature miR-206and a control duplex(miR-205) together with a morpholino against miR-206in the embryo. After incubation for8h,RNA was isolated and subjected to Northern blot analysis to probe for injected miR-206and injected miR-205.In contrast to the data obtained for endogenous miR-206,there was no decrease observed in the amount of injected miR-206in the morpholino-injected embryos(Figure1D)(endogenous miR-206is not yet ex-pressed at this stage).Since these data show that there is no effect of a morpholino on miRNA isolation or stability,we conclude that morpholinos deplete the embryo of miRNAs by inhibit-ing miRNA maturation.If this is the case,then we expect morpholinos targeting other regions of the miRNA precursor to act as well as the morpholinos designed against the mature miRNA,and this is indeed what weﬁnd(see next section). Morpholinos Targeting the miRNA Precursor Interfere with Primary miRNA ProcessingInjection of antisense oligos in embryos might result in off-target effects.Thus,phenotypic data retrieved from antisense knockdown experiments should be treated with caution.In Drosophila,29-O-methyl oligo–mediated knockdown of embry-onically expressed miRNAs caused defects that clearly differed from the phenotype of the corresponding knockout ﬂy[9,23].In sea urchin experiments,off-target effects of morpholino knockdowns are well documented,though low incubation temperatures favor off-target interactions[25]. Toﬁlter out off-target effects,we sought a control strategy that would allow us to compare effects of morpholinos withAuthor SummaryThe striking tissue-specific expression patterns of microRNAs (miRNAs)suggest that they play a role in tissue development. These small RNA molecules(;22bases in length)are processed from long primary transcripts(pri-miRNA)and regulate gene expression at the posttranscriptional level.There are hundreds of different miRNAs,many of which are strongly conserved.Vertebrate embryonic development is most easily studied in zebrafish,but genetically disrupting miRNA genes to see which miRNA does what is technically challenging.In this study,we interfere with miRNA function during the first few days of zebrafish embryonic develop-ment by introducing specific antisense morpholino oligonucleotides (morpholinos have been used previously to interfere with the synthesis of the much larger mRNAs).We show that morpholinos targeting the miRNA precursor can block processing of the pri-miRNA or directly inhibit the activity of the mature miRNA.We also used morpholinos to study the developmental effects of miRNA knockdown.Although we did not observe gross phenotypic defects for many miRNAs,we found that zebrafish miR-375is essential for formation of the insulin-secreting pancreatic islet.Loss of miR-375 results in dispersed islet cells by36hours postfertilization, representing one of the first vertebrate miRNA loss-of-function phenotypes.independent sequences targeted to the same miRNA.Because our data on morpholinos targeting the mature miRNA suggested that miRNA biogenesis might be affected,we designed morpholinos targeting the Drosha and Dicer cleavage sites of the precursor miRNA (Figure 2A).We decided to test this strategy on miR-205,since it is expressed relatively early,and there are only two,but identical,copies in the ﬁsh genome.Four different morpholinos were designed to inhibit miR-205biogenesis:two targeting the Drosha cleavage site complementary to either the 59or 39arm of the stem,and two morpholinos similarly targeting the Dicer cleavage site (Figure S1).These morpholinos were injected under similar conditions as described for miR-206and compared to the morpholino targeting mature miR-205.Interestingly,all ﬁve morpholinos induced complete or near-complete loss of miR-205(Figure 2B).Many miRNAs are highly expressed during later stages of embryonic development [21].Therefore,we tested how long the effect of the morpholinos would last.Although for this series of morpholinos the knockdown is best at 24hpf,the effect is still signiﬁcant up to 72hpf (Figure 2C).Next,we tested a similar series of morpholinos against the miR-30c precursor and analyzed miR-30c expression by Northern blotting (Figure S2).However,we only observed knockdown for the morpholino targeting mature miR-30c,but not for the other four morpholinos targeting the miR-30c precursor.This could be because miR-30c resides in a family of closely related species,with more sequence variability in the regions outside of the mature miRNA.The precursors ofthe family members might not all be targeted by these morpholinos (Figure S2).Thus,not all miRNAs are equally prone to knockdown by morpholinos that target the miRNA precursor.To investigate the effect of morpholinos on exogenously introduced pri-miR-205,we injected mRNA derived from a GFP construct with pri-miR-205in the 39UTR.Again,we could not detect mature miR-205derived from this construct after targeting by morpholinos (Figure 2D).Interestingly,the miR-205precursor also could not be detected in the embryos co-injected with morpholinos,whereas pre-miR-205could be detected in the absence of morpholinos (Figure 2D).Because pri-miR-205was cloned in the 39UTR of GFP,we monitored GFP ﬂuorescence after injection of this construct.In the presence of a morpholino,GFP ﬂuorescence increased (Figure 2E),suggesting accumulation of the primary miRNA.Therefore,we performed reverse transcriptase PCR (RT-PCR)on 8-h-old embryos injected with GFP-pri-miR-205and a control mRNA (luciferase)(Figure 2F).In the presence of a morpholino,the GFP-pri-miR-205mRNA level is higher compared to control embryos that were not injected with morpholinos.This experiment conﬁrms the GFP data and shows that morpholinos targeting the miRNA precursor inhibit Drosha cleavage.Next,we tested whether processing of the pre-miRNA might also be inhibited by morpholinos.Therefore,we injected a miR-205precursor in the one-cell–stage embryo.Northern analysis showed that the precursor was processed into mature miRNA in the embryo (Figure 2G).However,co-Figure 1.Morpholinos Targeting the Mature miRNA Deplete the Zebrafish Embryo of Specific miRNAs(A)Northern blot for miR-206in wild-type and MO miR-206–injected embryos at 24,48,and 72hpf.5S RNA serves as a loading control.(B)In situ analysis of miR-206and miR-124expression in different stage embryos after injection of MO miR-206.(C)Effect of a morpholino targeting miR-30c on a silencing assay with miR-30c and a responsive GFP sensor construct.(D)In vivo and in vitro effects of a morpholino on the stability and RNA extraction of a synthetic miR-206duplex.miR-205serves as a loading control.doi:10.1371/journal.pbio.0050203.g001Figure2.Morpholinos Targeting the Precursor miRNA Interfere with miRNA Maturation(A)Design of morpholinos targeting the precursor miRNA.(B)Northern blot analysis of miR-205in30-h-old embryos injected with different morpholinos against pri-miR-205.5S RNA serves as a loading control.(C)Time series of miR-205expression after injection of mature,no lap loop,and drosha star morpholinos against pri-miR-205.(D)Northern blot analysis of miR-205derived from embryos injected with a GFP-pri-miR-205transcript and four different morpholinos targeting pri-miR-205.Co-injected miR-206serves as an injection and loading control.Embryos were collected8h after injection.(E)GFP expression in24-h embryos injected with morpholinos and a GFP-pri-miR-205construct as used in(C).Pri-miR-205is positioned just upstream of the polyA signal in the39UTR of the GFP mRNA.Red fluorescent protein(RFP)serves as an injection control.(F)RT-PCR analysis of injected GFP-pri-miR-205mRNA with(þ)and without(À)co-injected morpholinos.Luciferase serves a an injection control. Embryos were collected8h after injection.(G)Northern analysis of the effect of morpholinos on an injected miR-205precursor.Embryos were collected8h after injection.WT,wild type.doi:10.1371/journal.pbio.0050203.g002injection of the overlap loop and non-overlapping loop morpholinos blocked processing completely.There was only a little effect of morpholinos targeting the Drosha cleavage site,probably because they only partially overlap the precursor.A similar analysis was performed for miR-375,which is expressed in the pancreatic islet and pituitary gland[21],and has two copies in the zebraﬁsh genome,which differ in the regions outside the mature miRNA.Overlap loop and loop morpholinos were designed for both miR-375–1and miR-375–2,and a morpholino against the miRNA star sequence could be used to target both copies of miR-375simultaneously(Figure3A).The efﬁcacy of all morpholinos was assessed by determining their effect on injected pri-miR-375–1or pri-miR-375–2transcripts(Figure 3B).As expected,each morpholino targeted the transcript to which it was directed.However,the star miR-375morpholino did not knock down miR-375completely.In addition, morpholino oligonucleotide(MO)miR-375did not interfere with processing of miR-375from pri-miR-375–1,possibly because this primary transcript forms a more stable hairpin. In all cases,the lack of a signal for mature miR-375coincided with the absence of pre-miR-375,which could be detected in the absence of a complementary morpholino.Next,all morpholinos were injected separately and in combination,and embryos were subjected to Northern blotting to determine endogenous miR-375expression at24 and48hpf(Figure3C).In contrast to the results obtained by in situ hybridization(see last section),the morpholino to mature miR-375only slightly decreased the expression of miR-375.However,MO miR-375could inhibit the activity of a mature miR-375duplex in a GFP-miR-375-target reporter assay(Figure3E).The morpholinos targeting only one copy of miR-375reduced miR-375expression,with the strongest effect for the morpholinos targeting pri-miR-375–1.How-ever,simultaneous injection of morpholinos targeting pri-miR-375–1and pri-miR-375–2completely knocked down mature miR-375,indicating that both transcripts are ex-pressed.To further determine the contribution of each transcript to mature miR-375accumulation,we performed in situ hybridization for pri-miR-375–1and pri-miR-375–2(Figure 3D).Both transcripts could not be detected in wild-type embryos.However,pri-miR-375–1was detected in the pancreatic islet and the pituitary gland in embryos injected with the miR-375–1loop morpholino and the morpholino to miR-375star.Similarly,pri-miR-375–2was only detected in embryos injected with the miR-375–2loop morpholino,the morpholino to miR-375star and mature miR-375.Thus,both transcripts are expressed in the pituitary gland and the pancreatic islet,similar to miR-1in the developing mouse heart[15].Together,this indicates that these morpholinos inhibit primary miRNA processing and result in primary miRNA accumulation,as we described for miR-205.In conclusion,our data demonstrate that morpholinos targeting the miRNA precursor can interfere with primary miRNA processing at either the Drosha or Dicer cleavage step and that morpholinos targeting the mature miRNA can inhibit their activity in a functional assay.Taken together, our data show that different morpholinos targeting the same miRNA may serve as positive controls for miRNA knockdown phenotypes in the embryo.Knockdown of Many miRNAs Does Not Result in Any Observed Developmental DefectsTo identify functions for individual miRNAs in zebraﬁsh embryonic development,we knocked down a series of11 conserved vertebrate miRNAs(Table S1)and analyzed their expression after morpholino knockdown.Injected embryos were monitored phenotypically by microscopic observation until four days postfertilization(dpf).Knockdown of most miRNAs resulted in loss of in situ staining for the respective miRNA.However,we could not observe gross morphological malformations after knockdown of these miRNAs(Figure4A). Therefore,we analyzed embryos injected with morpholinos against miR-182,miR-183,or miR-140in more detail,because we could easily stain the tissues that express these miRNAs (Figure4B).Embryos injected with morpholinos against miR-182or miR-183,which are expressed in the lateral line neuromasts and hair cells of the inner ear,were treated with DASPEI,which stains hair cells.Embryos injected with a morpholino against miR-140,which is expressed in cartilage, were subjected to Alcian Blue staining,a cartilage marker. However,staining of these speciﬁc cell types that express the miRNA did not uncover any defects upon knockdown(Figure 4B).In conclusion,knockdown of many miRNAs does not appear to signiﬁcantly affect zebraﬁsh embryonic develop-ment,at least not to the extent that can be visualized by the methods used in these examples.Knockdown of miR-375Affects Pancreatic Islet MorphologyMiR-375is known to be expressed in the pancreatic islet and the pituitary gland,and wasﬁrst isolated from pancreatic beta cells[21,26].This miRNA is conserved in vertebrates and may regulate insulin secretion by inhibiting myotrophin[26]. We injected a morpholino against mature miR-375into the one-cell–stage embryo.This morpholino effectively knocked down miR-375in theﬁrst4d of development(Figure5A),and it could also block the activity of an injected miR-375duplex, as monitored by its effect on a GFP reporter silenced by miR-375(Figure3E).During theﬁrst5dpf,there was no clear developmental defect except for a general delay in development.At around7 dpf,approximately80%of the injected embryos died.Next, we analyzed the development of both the pituitary gland and the pancreatic islet,by in situ hybridization with pit1and insulin markers.This analysis revealed no change in the formation of the pituitary gland(Figure5B).However, analysis of insulin expression showed a striking malformation of the islet cells in3-d-old morphant embryos(Figure5B). Wild-type embryos have a single islet at the right side of the midline,whereas the miR-375knockdown embryos have dispersed insulin-positive cells.The effect is sequence speciﬁc,because a morpholino complementary to the mature miR-375morpholino inhibited the pancreatic islet pheno-type(Figure5E).The pancreatic islet consists of four cell types,a,b,d,and PP,expressing glucagon,insulin,somatostatin,and pancre-atic polypeptide,respectively.Insulin is theﬁrst hormone expressed,and somatostatin co-localizes partially with in-sulin,whereas glucagon-expressing cells are distinct[27].A more detailed analysis using somatostatin and glucagon asmarker genes revealed a similar pattern of scattered islet cells in the miR-375morphant (Figure 5C).In zebraﬁsh,insulin is ﬁrst expressed at the 12-somite stage in a few scattered cells located at the midline,dorsal to the yolk [28].Insulin-positive cells migrate posteriorly and converge medially to form an islet by 24hpf.To look at the development of the pancreatic islet in time,we collected MO miR-375and noninjected control embryos at different stages,Figure 3.Specific Morpholinos Deplete the Embryo of miR-375(A)Sequence alignment of the two miR-375genes from zebrafish and design of morpholinos targeting the dre-miR-375–1and dre-miR-375–2precursors.(B)Northern blot analysis of the effect of morpholinos on the expression of miR-375derived from injected pri-miRNA mRNAs for miR-375–1and miR-375–2.MO-375–1overlap loop and loop morpholinos target exclusively the pri-miR-375–1construct,and MO-375–2overlap loop and loop morpholinos target exclusively the pri-miR-375–2construct.Co-injected miR-206serves as a loading and injection control.Embryos were collected 8h after injection.(C)Northern blot analysis of the effect of morpholinos on endogenous miR-375expression at 24hpf and 48hpf.MiR-206serves as loading control.(D)In situ hybridization for pri-miR-375–1and pri-miR-375–2on wild-type (WT)and morpholino-injected embryos.Arrowheads indicate the pituitary gland and the pancreatic islet.(E)Analysis of GFP expression in 24-h embryos injected with a miR-375GFP sensor construct,a synthetic miR-375duplex and MO miR-375.Red fluorescent protein (RFP)serves as an injection control.NIC,noninjected control.doi:10.1371/journal.pbio.0050203.g003Figure 4.Knockdown of Many miRNAs Does Not Affect Zebrafish Embryonic Development(A)Phenotypes and in situ analysis of 3-and 4-d-old embryos after injection of morpholinos against 11different mature miRNAs.(B)Daspei staining of 72-h-old embryos injected with MO miR-182and MO miR-183,and wild-type control (upper panel).Alcian Blue staining of 72-h-old embryos injected with MO miR-140and noninjected control (lower panel).doi:10.1371/journal.pbio.0050203.g004and investigated the expression of insulin (Figure 5D).At the 16-somite stage,insulin-positive cells are scattered at the midline in both noninjected and MO miR-375–injected embryos,and a presumptive islet is formed by 24hpf.Subsequently,when the insulin-positive islet is moving to the right side of the embryo in later stages,the islet breaks apart and insulin-positive cells become scattered in morphantembryos (Figure 5D).Also,in later stages,the phenotype persists,although miR-375is re-expressed at approximately 5dpf in morpholino-injected embryos (Figure 5A).Next,we analyzed the effect of all miR-375control morpholinos described in the previous section,by staining for insulin (Figure 6A).Both the dispersion phenotype and the knockdown were striking for embryos injected withMOFigure 5.Knockdown of miR-375Results in Aberrant Migration of Pancreatic Islet Cells(A)In situ analysis of miR-375knockdown in MO miR-375–injected embryos and noninjected controls at 24,48,72,and 120hpf.Arrowheads indicate the pituitary gland and the pancreatic islet.(B)In situ analysis of the pancreatic islet (insulin staining)and the pituitary gland (pit1staining)in miR-375morphants and noninjected controls.Arrowheads indicate the pituitary gland and the pancreatic islet.(C)In situ analysis of pancreatic islet development in wild-type and morphant embryos using insulin,somatostatin,and glucagon as markers.(D)Time series of insulin expression in wild-type and morphant embryos injected with MO miR-375.(E)Insulin expression in 72-hpf embryos injected with MO miR-375and a complementary morpholino.doi:10.1371/journal.pbio.0050203.g005Figure6.Specific Effects of miR-375Knockdown on the Development of the Endocrine Pancreas(A)In situ analysis of miR-375and insulin expression in72-hpf embryos injected with morpholinos against the miR-375precursor and negative control morpholinos for let-7and miR-124.(B)Expression of islet1,foxa2,and ptf1a in wild-type and miR-375knockdown embryos.Arrows indicate the pancreatic islet.WT,wild typedoi:10.1371/journal.pbio.0050203.g006。

改进的CRISPR基因组编辑使用小型高活性和特异性的工程RNA引导核酸酶说明书

Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases Moritz J. Schmidt1, Ashish Gupta1, Christien Bednarski1, Stefanie Gehrig-Giannini1, Florian Richter1, Christian Pitzler1, Michael Gamalinda1, Christina Galonska1, Ryo Takeuchi2, Kui Wang2, Caroline Reiss2, Kerstin Dehne1, Michael J Lukason3, Akiko Noma2, Cindy Park-Windhol2, Mariacarmela Allocca2, Albena Kantardzhieva2, Shailendra Sane2, Karolina Kosakowska2, Brian Cafferty2, Jan Tebbe1, Sarah J Spencer3, Scott Munzer2, Christopher J. Cheng2, Abraham Scaria2, Andrew M. Scharenberg2, André Cohnen1* and Wayne M. Coco1* Supplementary Figure 1. Selection of four small, novel Staphylococcus Cas9s.(a) Relatedness and genomic context of four uncharacterized Staphylococcus Cas9 genes: S. hyicus (Shy), S. lugdunensis (Slu), S. microti (Smi) and S. pasteuri (Spa). Each Cas9 locus includes several direct repeats (DRs) and a tracr sequence upstream of the nuclease. Loci not drawn to scale. (b) Sequences and structure of RNA components. The associated elements shared 98.8 % (tracrs) and 97.2 % (DRs) sequence identity with the corresponding SauCas9 sequences1. Top left: Complementarity between Slu genome locus DRs and tracr sequence. Bottom left: Alignment of the four DR sequences. Blue box: non-identities. Right: sgRNA design for SluCas9 with GAAA tetraloop (Loop 1) fusing DR and tracr2. Secondary structure predictions indicated similar folding for the four tracrRNAs. (c) Alignment of known and putative PAM interacting motifs in the selected Staphylococcus Cas9s suggests PAM-interacting residues. Amino acid numbering based on SauCas9. Strictly conserved amino acids are inblue, chemically similar amino acids are green. Red boxes correspond to residues responsible for PAM recognition in SauCas93. (d) PAM sequences as web logos. PAMs of the Cas9s were identified by in vitro-cleavage assays on a heptanucleotide (N7) DNA library 3’ of VEGFA_T24 and confirmed by bacterial survival assays carrying the same libraries. In contrast to SauCas9, 3 of the 4 chosen Cas9s recognized the short, non-degenerate 5’-NNGG-3’ PAM. PAM numbering begins with the first position after the last 3’ guide nucleotide. Source data are pr ovided in the source d ata file. (e) Alignment to SauCas9 nuclease active site residues. Corresponding amino acids for each orthologue are highlighted with red boxes.Supplementary Figure 2. Genome editing with four Cas9s in HEK293T cells assayed by amplicon sequencing. Shy, Smi and Slu Cas9 activity on endogenous loci in HEK293T cells using SluCas9 tracrRNA. Spa, Smi and Slu were tested for 2 targets with 5’-NNGG-3’ PAM (guide_87 and guide_102 targeting the HBB_R01_T2 and VEGFA_T22 loci, respectively) and Shy with 5’-NNARMM-3’ PAM (guide_1-4 targeting the HBB_R01_T1, VEGFA_T1 and FANCF_T1 and FANCF_T2 loci). Cas9s were delivered as RNPs via nucleofection and editing was analyzed via amplicon sequencing (AmpSeq). For negative controls, the respective nucleases were nucleofected in absence of sgRNA. Editing values were normalized against background, n = 2 independent biological replicates, source data are provided in the source data file.Supplementary Figure 3. Screening assays for functional sRGN variants. (a) Screening approach used to identify improved sRGNs by protein engineering. The succession of screening assays is shown on the left with the corresponding number of sRGN variants that progressed on the right. 25 sRGN variants were identified as top hits in the final BFP disruption screen in HEK293T cells5. (b) Schematic for “live/dead” (L/D) bacterial survival assay. Cells were generated harbouring an arabinose-inducible toxic ccdB gene reporter plasmid, a second plasmid harbouring a transcription cassette for the corresponding sgRNA, and a third plasmid encoding an IPTG-inducible, Trc promot er-controlled nuclease gene. Active Cas9/sgRNA complexes successfully cleave the toxic reporter, which is inactivated by cleavage at the VEGFA-T2 target site (guide_113), and cells survive under selection conditions. Open circles are cartoons of plasmids, green circle depicts ccdB gene product, purple circle represents nuclease. (c) L/D assay validation using SpyCas9 and SpyCas9 mutants. WT = SpyCas9; D10A and H840A = nickases; and dSpyCas9 = catalytically inactive SpyCas9. Upon botharabinose and IPTG induction, SpyCas9-or D10A-expressing cells survive while those that express H840A or dSpyCas9 do not, WTSpyCas9 n = 2 independent biological replicates, all other data n =3 independent biological replicates, data are presented as mean ± SD. Source data are provided in the source data file. (d) Fluorescence polarization assay (FP Assay) schematic. Biotinylated and ATTO647N-labelled oligonucleotide duplexes were immobilized on streptavidin coated plates. RNP complexes were formed, and cleavage of the dsDNA was monitored by following decreasing anisotropy and increasing fluorescence intensity, arb.unit = arbitrary units.Supplementary Figure 4. Rational exchange of PAM-interacting domains. (a) Shuffling fragment architecture of screening hits sRGN1, sRGN2, sRGN3 and sRGN4. Dark blue = SluCas9 segments, light blue = ShyCas9 segments, green = SmiCas9 segments, pink = SpaCas9 segments. (b) Rationally swapping PI-domains (PID) and WEDGE/PI-domains from the NNGG-recognizing SluCas9 to ShyCas9 alters PAM motif recognition and creates functionally active proteins. Chimera 1 and 2 substitute SluCas9 WEDGE/PI domain amino acids 739-1053 and 910-1053, respectively, into the ShyCas9 gene. Bacterial live/dead growth analysis on the VEGFA_T2 target sequence (guide_113) with a NNGG-PAM, indicates that both chimeric constructs gained the ability to effectively employ an NNGG PAM motif, while the unaltered ShyCas9, as expected, cannot. Data presented is the mean of n =2 independent biological replicates, source data are provided in the source data file. PLL = phosphate lock loop, CTD = C-terminal domain, WED = WEDGE domain, TOPO = topoisomerase domain, Ara = arabinose.Supplementary Figure 5. Catalytic turnover and indel patterns. (a) Plasmid containing the on-target sequence, 5'-TCGTAAAGTGGTGCGTTCTC-3', was mixed with the indicated nuclease at a DNA:RNP molar ratio of 2:1. At the indicated times, reactions were quenched and analyzed, data presented are n =1 biological replicate. Stoichiometric product formation ratios greater than 1 indicate that single nuclease molecules cleave d multiple substrate molecules. (b) Insertion and deletion (indel) pattern for SpyCas9, SluCas9 and sRGN3.1 on eight target sites within the VEGFA locus (for guide sequences, see Supplementary Table 1). Amplicon sequencing was performed and indel identity was calculated by CRISPResso. Data are presented as mean of n =2-3 independent biological replicates, except Spy (guide_37) n = 1 independent biological replicate, source data are provided in the source data file. Unaltered reads (indel = 0 bp, black squares) were excluded from the analysis.Supplementary Figure 6. Genome editing in a murine hepatoma cell line. Genome editing performance of SpyCas9, SluCas9, sRGN3.1, sRGN3.3, sRGN1, sRGN2, and sRGN4 variants in murine cells was assessed by transfection of Hepa1-6 cells with the respective albumin locus-targeting mRNAs (see Supplementary Table 2) and sgRNAs (Alb-T1 target, guide 112, see Supplementary Table 1 for sequence), n = 3 independent biological replicates for sRGNs and SluCas9, data are presented as mean ± SD, and n = 2 for SpyCas9). Source data are provided in the source data file.Supplementary Figure 7. Extended activity and specificity assessments. (a) Activity comparison plots of sRGN3.1 vs. SpyCas9 and SluCas9 on 96 targets. 48 therapeutically relevant targets and 48 rationally designed targets (engineered to explore GC-content between 20% and 80 %, see Supplementary Table 1 for sequences) were probed with SpyCas9, SluCas9 and sRGN3.1 in the FP cleavage assay. Each dot depicts the mean initial slope for each reaction, arb.unit = arbitrary units. Line visualizes x = y; data are presented in mean and are n = 2 independent biological replicates. (b) Left: Nuclease specificity assessment by cell-free FP assay at all possible single nucleotide target::gRNA mismatches. Heatmap ranges from white (no off-target cleavage) to dark blue (extensive off-target cleavage), purple box = WT nucleotide. PAM (not depicted) is to the right of the displayed sequences. Middle: alternative depiction of these data with relative initial rate values (slope) for each mismatch position, colors indicate the respective mutation at each position. Overall specificity for SpyCas9 and SluCas9 was about equal, while overall specificity of sRGN3.1 was 15% higher than SpyCas9. Right: on-target cleavage in these experiments relative to SpyCas9, data are presented as mean with n =2 independent biological replicates. (c) On-target editing observed with the nuclease concentrations selected by titration as input for the GUIDE-Seq experiment, with or without double-stranded oligodeoxynucleotide (dsODN), required for capturing of off-targets in GUIDE-Seq experiments. 60 pmol SpyCas9 and 30 pmol each of SluCas9 and sRGN3.1 were used for targeting HBB-R01, except for sRGN3.1 with 22nt guide, 8pmol were used. For targeting VEGFA_T2, 60 pmol SpyCas9, 8 and 18 pmol sRGN3.1 (for 20nt and 22nt guide, respectively) and 6 and 10 pmol SluCas9 (for 20nt and 22nt guide, respectively), n = 1.(d) Quantification of off-targets retrieved for each nuclease by GUIDE-Seq by number of mismatches (left) or overall number of off-targets (right). Results for both targets (HBB_R01 and VEGFA_T2) were combined for these plots. Source data for all subfigures are provided in the source data file.Supplementary Figure 8. LNP-mediated in vivo editing of sRGNs. (a)UPLC analysis of sRGN3.1 and SpyCas9 LNPs. Left: Representative chromatogram. sRGN3.1 mRNA LNPs had a size of 74 nm, polydispersity index (PDI) of 0.11, and RNA entrapment of 96%; SpyCas9 mRNA LNPs had a size of 74 nm, PDI of 0.13, and RNA entrapment of 91%. Lipid standards (right) were analyzed to identify peaks. sRGN3.1 LNPs showed a slight reduction in amino lipid(AL) and an increase in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1(Δ9-Cis)PE (DOPE) phospho-lipid (Phos) content compared to SpyCas9 LNPs, suggesting altered lipid-RNA associations compared to SpyCas9 mRNA, arb.unit = arbitrary units, CH = cholesterol, PEG =poly-ethylene-glycol, black line = background. (b) CryoTEM morphology analysis of sRGN3.3 and SpyCas9 LNPs. sRGN LNPs displayed improved circularity and multilamellar structure. One representative image of preparations shown in 8 c. (c) SpyCas9-LNP and sRGN3.3-LNP functional stability at 4°C. Liver editing in mice was assessed on indicated days upon intravenous administration. Left: dose of 2 mg/kg, n = 4 independent biological replicates, mean ± S.D. Right: Normalization of the data to day 1 showed 3.6-fold activity reduction for SpyCas9-LNPs and 1.3-fold activity reduction for sRGN3.3-LNPs. (d) Functional in vivo evaluation via TIDE analysis of uridine depletion for sRGN3.1 and sRGN3.3 mRNA constructs with different uridine-substituted base modifications. sRGN3.3 mRNA with (N1)-methylpseudouridine (m1Ψ) or sRGN3.1 with pseudouridine (Ψ) showed no significant difference with uridine depletion; whereas sRGN3.1 mRNA with m1Ψ, 5-methoxyuridine (5moU), and no modification showed significantly increased editing with uridine depletion, (dose of 1 mg/kg, n = 4 independent biological replicates, mean ± SD). For all other in vivo studies m1Ψ modification and the non-uridine depleted constructs were used. Significance was determined using the Mann Whitney test, (*) = p < 0.05, ns = not significant. (e) In vivo evaluation of sgRNA modification approaches in mice via TIDE analysis. Tested were internal chemical modifications (Internal mod 1 and 2) and increasing protospacer length from 20 to 23 nt (Supplementary Table 1). Liver editing showed a dose response at 0.5, 0.75, and 1.5 mg/kg of total LNP-encapsulated RNA. Both modification strategies showed improved potency compared to standard modified sgRNAs. Protospacer length of 23 nt (standard modifications) showed highest potency. N = 4 independent biological replicates, mean ± SD. Source data are provided in the source data file.Supplementary Figure 9. Editing at the intron 40 of the USH2A gene locus. (a) The 7595-2144A > G Usher disease mutation in intron 40 of USH2A (IVS40) results in an additional exon being incorporated in the USH2A mRNA. The T429 IVS50 guide (guide_110) targets the mutant IVS40 allele, while WT-IVS40 targets the WT (Supplementary Table 1, guide_111). GUIDE-seq revealed no sRGN off-target sites above background for T428-IVS40-guide and confirmed via AmpSeq (source data file, Supplementary Fig. 9). A plasmid carrying the sRGN3.1 gene and T428-IVS40-guide was nucleofect ed into the homozygous IVS40 293FT (293FT-IVS40) cell line together with dsODN. Break sites in which dsODN was inserted were identified by NGS. Only genomic sequences quite distant (> 7 mismatches and multiple alignment gaps) from the on-target site for T428-IVS40-guide were captured by dsODN at relevant read counts, suggesting that these sites were not cleaved as off-targets by sRGN3.1 complexed with T428-IVS40-guide. Background was 0.26 % for SpyCas9 and 0.07 % for sRGN3.1. Three replicates for sRGN3.1are shown, identical scales for each replicate. (b) Comparison of editing with mutant IVS40 allel e targeting guide and its surrogate guide. 293FT-IVS40 cell line and its WT parent cell line were transfected with a plasmid carrying either SpyCas9 or sRGN3.1 and sgRNA that matched either WT (guide_111) or the IVS40 SNP allele(guide_110). WT-IVS40-guide differs from T428-IVS40-guide by a single nucleotide and completely matches the wild type USH2A intronic sequence of NHP and human. Insertions and deletions (indels) were quantified using cells harvested 7 days after transfection via TIDE analysis, n = 2 biologically independent experiments, 2 technical replicas each, each datapoint is shown. Source data are provided in the source data file.Supplementary Figure 10. Gating strategy for FACS analysis of BFP-disruption. HEK293T cells, harboring a BFP cassette in the AAVS1 locus were transfected with nuclease expression plasmid (with T2A-GFP) and guide expression plasmid (guide 5-9 and 11-16 for sRGNs and guide_19-23and 25-30 for SpyCas9). Cells were FACS-analyzed 7 days post transfection. Shown is the gating strategy for the control (nuclease only) and a nuclease plus guide treated sample, as used for evaluation of % BFP disruption as presented in Figure 2. A signal from minimum of 10,000 cells was assayed using the V450 filter set in the BD FACS canto II and software diva 8.0.1 and FlowJo 10.7.2.Supplementary References1. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature520,186–191 (2015).2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptivebacterial immunity. Science337, 816–821 (2012).3. Nishimasu, H. et al. Crystal Structure of Staphylococcus aureus Cas9. Cell162, 1113–1126 (2015).4. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases inhuman cells. Nature Biotechnology31, 822–826 (2013).5. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAMspecificities. Nature523, 481–485 (2015).。

MicroRNA-33b downregulates the differentiation

MicroRNA-33b downregulates the differentiation and development of porcine preadipocytesMasaaki Taniguchi •Ikuyo Nakajima •Koichi Chikuni •Misaki Kojima •Takashi Awata •Satoshi MikawaReceived:11February 2013/Accepted:20December 2013/Published online:8January 2014ÓThe Author(s)2014.This article is published with open access at Abstract Sterol regulatory element binding transcription factor (SREBF)is a key transcription regulator for lipid homeostasis.MicroRNA-33b (miR-33b)is embedded in intron 16of porcine SREBF1and is conserved among most mammals.Here,we investigated the effect of miR-33b on adipocyte differentiation and development in porcine sub-cutaneous pre-adipocytes (PSPA).PSPA were transiently transfected with miR-33b,and adipose differentiation was then induced.Delayed adipose differentiation and decreased lipid accumulation were observed in miR-33b-transfected putational predictions suggested that miR-33b may target early B cell factor 1(EBF1),an adipocyte activator of lipogenesis regulators such as CCAAT-enhancer binding protein alpha (C/EBP a )and peroxisome proliferator-activated receptor gamma (PPAR c ).Both gene and protein expression of EBF1were downregulated in miR-33b-transfected PSPA,followed by considerable decreases in the expression of C/EBP a and PPAR c and their downstream lipogenic genes.However,miR-33b transfection did not markedly affect mRNA and protein expression of SREBF1.We also investigated dif-ferences in the expression of miR-33b and lipogenic genesin subcutaneous fat tissues between 5-month-old crossbred gilts derived from Landrace (lean-type)and Meishan (fatty-type)ndrace-derived crossbred gilts expressed more miR-33b and less lipogenic genes than did gilts derived from Meishan.Our results suggest that miR-33b affected the differentiation and development of PSPA by attenuating the lipogenic gene expression cascade through EBF1to C/EBP a and PPAR c .The differential expression of miR-33b observed in crossbred gilts may in part account for differences in lipogenic gene expression and the fat:lean ratio between pig breeds.Keywords Adipocyte ÁGene expression ÁLipogenesis ÁMicroRNA ÁPPAR ÁSREBFIntroductionPork,together with chicken and beef,is an important protein source for humans.In the livestock industry,including pork production,carcass fat quantity and quality are major determinants of the productivity and palatability of meat.Previous work has shown that the molecular pathway of fat metabolism is regulated in a species-speciﬁc and fat-depot-speciﬁc manner [1,2].Therefore,it is essential to understand the molecular mechanisms that underlie adipogenesis and lipogenesis in porcine fat tissues.Previous studies have investigated the transcriptional regulation of genes associated with adipogenesis [3,4].Among the transcription regulators,sterol regulatory ele-ment-binding transcription factor (SREBF)is known to regulate the transcriptional activation of genes involved in the uptake and synthesis of cholesterol,fatty acids,tri-glycerides,and phospholipids [5–7].In addition to tran-scription factors,microRNAs (miRNA),which are *22-ntElectronic supplementary material The online version of this article (doi:10.1007/s11033-013-2954-z )contains supplementary material,which is available to authorized users.M.Taniguchi ÁM.Kojima ÁT.Awata ÁS.Mikawa (&)Animal Genome Research Unit,Agrogenomics Research Center,National Institute of Agrobiological Sciences,2-1-2Kannondai,Tsukuba,Ibaraki 305-8602,Japan e-mail:mikawa@affrc.go.jpI.Nakajima ÁK.ChikuniAnimal Products Research Division,National Institute of Livestock and Grassland Science,2Ikenodai,Tsukuba,Ibaraki 305-0901,JapanMol Biol Rep (2014)41:1081–1090DOI 10.1007/s11033-013-2954-znon-coding RNAs generated from the sequential process-ing of long RNA transcripts[8],are considered to play key roles in the regulation of gene expression at the post-transcriptional level in the diverse regulatory pathways of many cellular processes.MirBase,a public database of miRNA,contains331miRNAs that have been identiﬁed in the porcine genome(Release19available since August 2012,)[9].Recent studies[10,11] reported that miRNA(miR-33a)embedded in SREBF2 gene inﬂuences cholesterol metabolism in murine hepato-cytes and human macrophages by repressing adenosine-triphosphate-binding cassette transporter A1(ABCA1).In addition,Da´valos et al.[12]reported that miR-33a/b is associated with the repression of fatty acid oxidation and insulin signaling in hepatocytes.Further,identiﬁcation and functional studies of miRNAs have been done in livestock species including pig and cattle[13,14].These studies suggest that fat cell development is achieved via a com-plicated mechanism that is potentially regulated by miR-NAs in a species-speciﬁc manner.Therefore,to clarify the porcine-speciﬁc pathways in adipocyte differentiation and development,we asked whether miR-33b inﬂuences the regulation of adipogenesis and lipogenesis in porcine preadipocytes.First,we deter-mined the full-length nucleotide sequence of the porcine SREBF1gene.Second,we transfected miR-33b into por-cine subcutaneous pre-adipocytes(PSPA)to examine whether the transcriptional regulation of genes relevant to adipocyte differentiation and development was affected by miR-33b.We also investigated the mRNA and protein expression levels of an miR-33b target gene that was identiﬁed in an miRNA target prediction search and,as such,assumed to be involved in adipocyte differentiation and st,we discuss the possible effect of miR-33b on differences in backfat(BF)thickness and blood triglyceride levels between representative lean and fat crossbred pigs.Materials and methodsAnimal samplesEuropean crossbred(E)dams,including Landrace,Large White,and Duroc,were mated with Landrace(L)or Meishan(M)boars.Meishan pigs are substantially fatter, particularly in subcutaneous adipose tissue,than are typical European pig breeds[15,16].Seven each of EL and EM gilts were fed under the same nutritional con-ditions until slaughter at5months old at the National Institute of Livestock and Grassland Science(NILGS). Body weights(mean±SD)of the EL and EM at slaughter were82.0±3.8and82.8±6.2,respectively.There was no signiﬁcant body weight difference between the breed types(P=0.782by Student t test).Tissue samples for total RNA extraction were collected from the liver,longissimus dorsi(LD)muscle,and subcuta-neous adipose tissues at the mid-dorsal area of these animals.BF thickness measurements at the shoulder, back,and lumber positions were averaged and are pre-sented as such.All animal care and use in this study was in accordance with the animal experimentation guideline of the NILGS and was approved by the NILGS Animal Care Committee.Cell culturePSPA were cultured in DMEM growth medium containing a low glucose concentration(1.0g/L)and10%fetal bovine serum(FBS)(Invitrogen,Carlsbad,CA,USA).The subconﬂuent cells were passed every3days.To produce mature adipocytes,we plated the PSPA at2.19104cells/cm2 and grew them for3days to reach conﬂuence.After the cells reached conﬂuence(denoted as0day),adipose con-version was carried out in high-glucose(4.5g/L)DMEM containing10%FBS,5l g/mL insulin(Sigma-Aldrich, Basel,Switzerland),0.25l M dexamethasone(Sigma-Aldrich),33l M biotin(Wako Pure Chemicals,Osaka, Japan),17l M pantothenate(Sigma-Aldrich),and5mM octanoate(Sigma-Aldrich).The medium was changed every other day.To investigate the effect of miR-33b on these cells,we subjected groups of cells to the following three experimental conditions:(1)growth medium alone;(2)differentiation with a negative control miRNA (miR-NC)transfection;and(3)differentiation with miR-33b transfection.All cells were cultured at37°C in a humidiﬁed incubator in5%CO2.TransfectionTo investigate the effect of miR-33b,we transfected a commercially available mature miRNA molecule,Pre-miR TM miRNA Precursor(hsa-miR-33b)(Ambion,Austin, TX,USA),into conﬂuent cells.The transfectant hsa-miR-33b is not a hairpin construct but a mature miRNA molecule,so that although it was designed for use with human cells,it can be applicable to pigs,cattle,or dogs,because the mature miRNA sequence of miR-33b is100%identical among these species.For the miR-NC in experimental group2,we used Pre-miR TM Negative Control#1,which is a Pre-miR TM molecule designed to produce no identiﬁable effects on known miRNA function(Ambion).These transfectants were transfected by using the DharmaFECT1 transfection reagent according to the manufacturer’s instructions(ThermoFisher Scientiﬁc,Waltham,MA, USA).Four replicate samples of cells were harvested at2,4,8,12,and16days after transfection and induction of adipocyte differentiation.Cloning of the porcine SREBF1geneA porcine bacterial artiﬁcial chromosome(BAC)library constructed from a boar of Large White/Landrace/Duroc composite was screened by means of a PCR-based method with primers derived from the porcine SREBF1mRNA sequence(GenBank ID:NM_214157.1).BAC clones were sequenced to determine the full-length porcine SREBF1 gene in accordance with the previously established method [17].Gene expression assayTotal RNA including small RNA was extracted from por-cine tissue samples and PSPA by using ISOGEN IITable1Nucleotide sequences of primers and probes used for real-time PCR Gene Nucleotide sequence of primers and probes(50–30)Accession numberSREBF1Forward CGGACGGCTCACAATGCAB686492 Reverse GCAAGACGGCGGATTTATTCProbe TCAACGACAAGATCATCGAGPPAR c1Forward CTCGGACACCGGAGCTGGAJ006756 Reverse CAACCATGGTCACCTCGCTAAProbe CGCCAGGCCACCACCGCAGATTPPAR c2Forward GGTGAAACTCTGGGAGATTCTCTTAAF059245 Reverse CAACCATGGTCACCTCTTGTGAProbe CGATGCCTTCGACACGCTGTCTGCAAC/EBP aForward AGGAGGACGAGTCGAAGCAXM_003127015 Reverse GGCGGAGGGTGTGAATGCProbe CTTTCCCTACCAGCCACCGCCGCEBF1Forward ATGTTTGTCCATAATAACTCCAAGCXM_003359834 Reverse CTTTGATACAGGGAGTAGCATGTTProbe ACCCCTCGGAAGGTACGCCCTCTTATCFASNForward GCTGGCCTACACGCAGAGNM_001099930 Reverse GGCCCTGGAGCGGTATCAProbe CGCCTCCAGCACCTTGCCTTGCaP2Forward CAGGAATTTGATGAAGTCACTGCNM_001002817 Reverse GTGGTTGTCTTTCCATCCCACProbe TGACAGGAAAGTCAAGAGCACCATAACCTTADIPOQForward CACCACTGGCAAATTCCACTGNM_214370 Reverse CCTTCACATCCTTCAAGTAGACCProbe CCTGGGCTGTACTACTTCTCCTTCCACGCD36Forward CCTACTGGCTGAGTTATTGTGACNM_001044622 Reverse CACAGCATAGATTGACCTGCAAProbe TGGTACAGATGCAGCCTCATTTCCACCTSCD1Forward ACGGATATCGCCCTTATGACAAGNM_213781 Reverse CGCTGGCAGAATAGTCATAGGGProbe TGGAAGCCCTCACCCACAGCTCCC(NIPPON GENE,Toyama,Japan).Total RNAs were reverse-transcribed to synthesize single-strand DNA by using ReverTra Ace(TOYOBO,Osaka,Japan).Real-time PCR was performed with the TaqMan system(Applied Biosystems,Foster City,CA,USA)to examine the relative gene expression of SREBF1,C/EBPa,EBF1,fatty acid synthase(FASN),peroxisome proliferator-activated protein gamma1and2(PPAR c1and PPAR c2),stearoyl-CoA desaturase1(SCD1),adipocyte-fatty acid binding protein (aP2),adiponectin(ADIPOQ),and fatty acid translocase (CD36)by using primers and gene-speciﬁc probes (Table1).The TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assays(Applied Biosystems) designed for hsa-miR-33b were used for relative quantiﬁ-cation of miR-33b.The TaqMan Endogenous Control Eukaryotic18S rRNA gene(Applied Biosystems)was used for the relative quantiﬁcation of all of the genes examined. The comparative threshold cycle method(DD Ct)was employed to calculate the relative quantiﬁcation of gene expression based on the formula,2(-DD Ct)whereDD Ct¼ðCt targert geneÀCt18S rRNAÞtestÀðCt targert geneÀCt18S rRNAÞcalibrator[18,19].The DD Ct values obtained from0day,non-trea-ted control PSPA of experimental group1and from LD muscle were used as calibrators for the relative quantiﬁ-cation of gene expression in the PSPA and5-month-old gilts,respectively.Western blot analysisNuclear and cytoplasmic proteins were prepared by using the CelLytic NuCLEAR Extraction Kit(Sigma-Aldrich), according to the manufacturer’s instructions.The protein content was determined by using a bicinchoninic acid protein assay(Pierce,Rockford,IL,USA).Fifteen micro-grams of nuclear or cytoplasmic protein were separated by electrophoresis through12.5%SDS-polyacrylamide gels (ATTO,Tokyo,Japan).After they were electro-transferred onto nitrocellulose membranes by using the iBlot gel transfer system(Invitrogen),the proteins underwent Pon-ceau S staining(Sigma-Aldrich)to verify equal loading of the lanes.The membranes were then blocked overnight at 4°C in phosphate buffered saline(PBS)containing5% skim milk and0.1%Tween20,followed by a1-h incu-bation at room temperature with primary polyclonal anti-bodies speciﬁc to EBF1(Abcam,Cambridge,UK),the transcriptionally active form of SREBF1in the nucleus (Abnova,Aachen,Germany),and b-actin(ACTB)(Ana-Spec,San Jose,CA,USA).Horseradish peroxidase-con-jugated anti-rabbit antibodies(GE Healthcare, Buckinghamshire,UK)served as secondary antibodies. Antigen–antibody complexes were visualized by use of the ECL detection system(GE Healthcare),and the chemilu-minescence signal was scanned with a LAS-3000imaging system and quantiﬁed with Multi Gauge ver2.0,with which the imaging system was equipped(Fujiﬁlm,Tokyo,Japan). Lipid accumulationAt each time point of the culture period,PSPA were washed with PBS,ﬁxed with10%formaldehyde,and stained withﬁltered oil-red O solution(0.3%oil-red O in 60%isopropanol).The triglycerides in the PSPA and blood from the crossbred gilts were extracted with chlo-roform–methanol(2:1,v/v)and quantiﬁed enzymatically by using the Triglyceride E Test(Wako).Data analysisMeasurements of gene expression,protein expression and triglyceride(TG)content were repeated in three indepen-dent experiments.Differences in gene expression,protein expression,and TG content among the cell culture treat-ments were analyzed with Tukey’s multiple comparison tests(P\0.05).Body weight,gene expression levels,BF thickness,and blood triglyceride concentration were com-pared between crossbred gilts by using the Student t test (P\0.05).Results and discussionCharacterization of the full-length sequenceof the porcine SREBF1geneFirst,we determined the20,099-nt sequence of the full-length porcine SREBF1gene including the50-untranslated region(UTR),the19exons that encode the3,456-nt open reading frame,the18introns,and the30-UTR(GenBank ID:AB686492).Unlike the human SREBF1gene(Gen-Bank ID:NG_029029.1),the porcine SREBF1gene sequence that we determined,together with NCBI refer-ence sequence(RefSeq)information,revealed that there is only one SREBF1isoform,and no transcriptional variants have been identiﬁed in the pig genome(GenBank ID: NM_214157.1)or in cattle[20].Porcine SREBF1shares 86and83%homology to the human SREBF1c mRNA and amino acid sequences,respectively.In the full-length porcine SREBF1gene sequence which we determined in this study,intron16included a sequence that is highly conserved among mammals and contains a pre-miRNA sequence(GenBank ID:AB686493)(Supplementary Fig.1),while the corresponding region in recently pub-lished porcine genome sequence(NW_003540979)is notidentiﬁed.The pre-miRNA sequence expressed mature porcine miR-33b (ssc-miR-33b),which shared high homology with several other mammalian species except for mouse (Supplementary Fig.1).Effect of miR-33b on lipid accumulation in PSPA To investigate the effect of miR-33b on lipid homeo-stasis,we transfected miR-33b into PSPA,which repre-sent an ideal experimental model to study porcine adipocyte physiology [21].Non-transfection/non-differ-entiation-induced control cells continued to proliferate and showed no signs of morphological change or lipid accumulation throughout the time course,whereas dif-ferentiation induction clearly induced lipogenesis after 4days (Fig.1).Transfection of miR-33b at differentia-tion induction decreased lipid accumulation in PSPA after 8days compared with transfection of miR-NC (negative control microRNA),suggesting that miR-33b affected the differentiation and development of the PSPA (Fig.1).Consistent with the observation of lipid accu-mulation by oil-red O staining,the TG content of the miR-33b-transfected cells was signiﬁcantly (P \0.05)decreased by 70.3%(4days),71.4%(8days),67.6%(12days)and 77.4%(16days)compared with that of the miR-NC-transfected cells (Fig.2).These results clearly demonstrate that miR-33b attenuates the differ-entiation and development of PSPA.MiR-33b target gene predictionTo investigate the biological function of miR-33b in PSPA,we explored putative miR-33b target genes by using algo-rithms including PITA [22],TargetScan v6.0[23],and [24].These computational predictions sug-gested that miR-33b may target porcine early B-cell factor 1(EBF1)mRNA,because multiple possible miR-33b recog-nition sites were found in the 30-UTR of porcine EBF10 d (A)control2 d 4 d 8 d 12 d 16 d(B)miR-NC(C)miR-33bFig.1Morphological characterization of PSPA.PSPA were treated with:a growth medium;b differentiation medium with negative-control miRNA (miR-NC)transfection;c Differentiation mediumwith miR-33b transfection.Lipid accumulation was visualized with oil-red O staining.Scale bar 50l mTime, day2481216T r i g l y c e r i d e , m g /d L2004008001,0001,2001,400a ab a bb a bc a b ca b c Fig.2Differences in triglyceride content in PSPA.PSPA were treated as follows:growth medium (white ),differentiation medium with negative-control miRNA transfection (shaded ),and differenti-ation medium with miR-33b transfection (black ).Triglyceride content was measured at least three parisons of relative gene expression were performed by using Tukey’s multiple comparison tests.Different letters show a signiﬁcant difference (P \0.05)mRNA,one of which was 100%identical to that of numer-ous other mammalian species (Supplementary Fig.2a,b).In contrast,none of the master regulators of adipocyte dif-ferentiation and development were predicted to be miR-33b targets with these methods.These ﬁndings suggest that ssc-miR-33b has a conserved effect on the post-transcrip-tional regulation of EBF1expression in porcine adipocytes.The BioSystems Database describing ‘‘Transcriptional Regulation of White Adipocyte Differentiation’’in the NCBI [24]suggested that EBF1binds and activates the PPAR c promoter (/biosystem s?term=205243).In addition,Jimenez et al.[26]reported that EBF1promotes adipogenesis by inducing the expres-sion of the C/EBP a and PPAR c 1promoters,and repressing GATA-2,which is considered to negatively affect adipo-genesis in mice [4].Like SREBF1,C/EBP a and PPAR c are master regulators that are recognized as molecular markers of adipocyte differentiation and development [27–29].Fig.3Changes in adipogenic and lipogenic genes in PSPA.PSPA were treated as follows:growth medium (white ),differentiation medium with negative-control miRNA transfection (shaded ),and differentiation medium with miR-33b transfection (black ).Relativegene expression was measured by using the 2-DD Ct method at least three parisons of relative gene expression were performed by using Tukey’s multiple comparison tests.Different letters show a signiﬁcant difference (P \0.05)Therefore,we assumed that the observed decrease in lipogenesis in PSPA was caused by ssc-miR33b via the attenuation of adipogenic and lipogenic pathways that are regulated by C/EBP a and PPAR c ,which are ordinarily activated by EBF1.We examined the promoter regions of the porcine PPAR c 1,c 2,and C/EBP a genes by using TFSERACH ver1.3based on TRANSFAC [28]and found that the promoters of C/EBP a and PPAR c genes contained EBF1and C/EBP a binding sites,respectively (Supple-mentary Figs.3,4,5).These results suggest that porcine EBF1may be associated with the transcriptional regulation of C/EBP a and,indirectly,of PPAR c genes.Effect of miR-33b on SREBF1and EBF1expression Transfection of precursor miRNAs into PSPA successfully induced the transitional expression of miR-33b.The expression level of miR-33b in miRNA precursor-transfectedPSPA was signiﬁcantly higher (P \0.05)than those in the control and in miR-NC-transfected PSPA,although the endogenous miR-33b level was relatively low and did not ﬂuctuate over time (Fig.3).Because of the genomic localization of ssc-miR-33b,we examined whether miR-33b inﬂuences its host SREBF1gene.SREBF1gene expression did not appear to be ele-vated during the PSPA differentiation induction time course.The mRNA level of SREBF1upon miR-33b transfection tended to be lower throughout the time course,but no signiﬁcant differences were detected (Fig.3).In addition,the relative protein expression level of SREBF1after miR-33b transfection was not different from that after transfection of miR-NC (Fig.4).These results suggest miR-33b had relatively little effect on SREBF1expression.In contrast,the mRNA levels of EBF1in cells with miR-33b transfection were signiﬁcantly (P \0.05)decreased by 66.9%(4days),70.2%(8days),and 80.8%(12days)compared with those after miR-NC transfection (Fig.3).In addition to this mRNA abundance,the protein expression level of EBF1in miR-33b-transfected PSPA was signiﬁ-cantly (P \0.05)decreased by 80.8%(2days),61.2%(8days),and 84.8%(16days)compared with that after miR-NC transfection (Fig.4).These results suggest that miR-33b affects EBF1expression at both the transcrip-tional and post-transcriptional levels.Effect of miR-33b on C/EBP a ,PPAR c and their downstream adipo/lipogenic genesGiven our ﬁnding of decreased EBF1expression upon miR-33b transfection,we investigated the gene expression of the adipogenic and lipogenic master regulators C/EBP a and PPAR c and their downstream genes,including aP2,CD36,ADIPOQ ,SCD1,and FASN (Fig.3).The relative mRNA level of PPAR c 2gradually increased in the differentiation-induced PSPA.However,the PPAR c 2mRNA level after miR-33b transfection sig-niﬁcantly decreased compared with that after miR-NC transfection from the 2-day time point onward (P \0.05).In agreement with the PPAR c 2mRNA expression,the relative gene expression of PPAR c 1,another isoform of PPAR c broadly expressed in various tissue types,was also decreased in miR-33b-transfected PSPA compared with that in miR-NC-transfected PSPA (P \0.05).Similarly,the relative gene expression of C/EBP a in miR-33b-trans-fected PSPA was signiﬁcantly decreased compared with that in miR-NC-transfected PSPA from the 2-day time point onward (P \0.05).These results demonstrate that the changes in the expression patterns of PPAR c and C/EBP a were similar,although the levels of gene expres-sion were different.The results of the gene expression assays for PPAR c 2and C/EBP a suggest that thedecreaseFig.4Changes in SREBF1and EBF1protein expression levels in PSPA.PSPA were treated as described above.Relative gene expression was determined by Western blotting at least three times.The image shown is a representative parisons of relative protein expression were performed by using Tukey’s multiple comparison tests.1H:Positive control (nucleoprotein extracted from HeLa cells).2miR-NC:negative control miRNA.Different alphabets show a signiﬁcant difference (P \0.05)in expression of these genes was affected by EBF1deg-radation,as indicated in the BioSystems Database of the NCBI and by Jimenez et al.[26].Genes known to be regulated by PPAR c ,such as aP2,CD36,and ADIPOQ ,were highly expressed in adipose differentiation-induced PSPA,whereas their expression was signiﬁcantly decreased in miR-33b-transfected PSPA (Fig.3).In addition,the same ﬂuctuating expression pat-tern was observed for the PPAR c 2gene,suggesting that the decreased expression of the aP2,CD36,and ADIPOQ genes was the result of decreased PPAR c 2in miR-33b-transfected PSPA.In human adipocytes,transcription of aP2,CD36,and ADIPOQ is reported to be regulated by PPAR c 2,and these genes are associated with fat formation and metabolism [31–33].The mRNA level of SCD1was generally increased in line with the degree of lipogenesis in adipose differentia-tion-induced PSPA,but the mRNA level after miR-33b transfection was at least 30%less than that after miR-NC transfection from the 4-day time point onward (P \0.05)(Fig.3).The observed decrease in SCD1mRNA expres-sion levels was similar to that of the PPAR c 2mRNA level,because SCD1gene transcription can be regulated bySREBF1or PPAR c 2[5,34].The mRNA level of FASN after miR-33b transfection was 41%less than that after miR-NC transfection at 4days (P \0.05).In contrast to the SCD1mRNA level,changes in the FASN mRNA level were small,suggesting that FASN transcription may be modestly regulated by PPAR c 2,as demonstrated in pre-vious studies [35,36].Taken together,these results suggest that expression of adipogenic and lipogenic genes is well regulated by the master regulators PPAR c 2and C/EBP a in PSPA and that the observed decrease of lipid accumulation in PSPA can be explained by the decrease in expression of these key genes.Furthermore,the delay in adipogenesis and decrease in lipogenesis observed in PSPA after miR-33b transfection was characterized by the degradation of PPAR c 2and C/EBP a but not that of SREBF1,possibly due to decreased EBF1transcriptional regulation.Possible effect of miR-33b on fat formation differences between pig breedsTo investigate the effect of miR-33b on fat formation in fattening pigs and on the generation of different fat traitsinFig.5Comparison of miR-33b,lipogenic genes,and fat formation indices betweenMeishan-and Landrace-derived crossbred gilts.Expression of miR-33b and lipogenic genes in muscle (LD muscle),liver and Sc fat (subcutaneous fat)tissues.Open squares and shaded circles indicate individual crossbred gilts derived from Landrace (EL)and Meishan (EM),respectively.Mean values are denoted with horizontal barspigs that were crossbred with typical lean(Landrace)-and fatty(Meishan)-type breeds,we analyzed differences in BF thickness and blood TG levels and also in miR-33b, SREBF1,EBF1,PPAR c,and C/EBP a gene expression in the subcutaneous tissues of EL and EM crossbred gilts.We found that EM gilts showed signiﬁcantly higher blood TG levels(P\0.01)and BF thickness(P\0.001) than did EL gilts,in agreement with a previousﬁnding [37].In addition to this difference in physiological char-acteristics between these pig breeds,gene expression assays indicated that miR-33b expression in EL tended to be higher than that in EM(P=0.08)(Fig.5).In contrast, EL gilts showed signiﬁcantly lower expression of lipogenic genes,including SREBF1(P\0.01),EBF1(P\0.01), PPAR c2(P\0.01),and C/EBP a(P\0.05)than did EM gilts.These results suggest that the difference in miR-33b expression between these pig breeds is associated with differences in the transcriptional regulation of lipogenic genes,leading to observed differences in fat-related traits such as BF thickness and blood TG levels.It is important to note that only seven gilts were used to obtain these data; therefore,given the small sample size,it is difﬁcult to draw conclusions regarding correlations between miR-33b and lipogenic gene regulation and fat-related traits.In addition, genetic polymorphisms in SREBF1and miR-33b may have certain effect on their expressions,although so far there is no evidence for a difference in the SREBF1gene sequences between Landrace and Meishan pigs.As described in a review by Dodson et al.[2],meat animals have recently begun to be utilized as good experimental models for lipid metabolic research.In addition,the lack of miR-33b in Srebf1gene in traditional model animals such as mouse and rat(Supplementary Fig.1)[11,12]may mean that the pig is a better model for biomedical research on lipid metabolism.Future studies of miR-33b will include evaluating cor-relations between miR-33b,lipogenic genes,and pork carcass characteristics related to fat tissues by using a larger number of pigs.Acknowledgments This work was supported by KAKENHI (22780256).Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use,dis-tribution,and reproduction in any medium,provided the original author(s)and the source are credited.References1.Hishikawa D,Hong YH,Roh SG,Miyahara H,Nishimura Y,Tomimatsu 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21世纪是生命科学的世纪20世纪后叶分子生物学的突破性...

第一章绪论一简答题1. 21世纪是生命科学的世纪。

20世纪后叶分子生物学的突破性成就，使生命科学在自然科学中的位置起了革命性的变化。

试阐述分子生物学研究领域的三大基本原则，三大支撑学科和研究的三大主要领域？答案：（1）研究领域的三大基本原则：构成生物大分子的单体是相同的；生物遗传信息表达的中心法则相同；生物大分子单体的排列（核苷酸，氨基酸）导致了生物的特异性。

（2）三大支撑学科：细胞学，遗传学和生物化学。

（3）研究的三大主要领域：主要研究生物大分子结构与功能的相互关系，其中包括DNA和蛋白质之间的相互作用；激素和受体之间的相互作用；酶和底物之间的相互作用。

2. 分子生物学的概念是什么？答案：有人把它定义得很广：从分子的形式来研究生物现象的学科。

但是这个定义使分子生物学难以和生物化学区分开来。

另一个定义要严格一些，因此更加有用：从分子水平来研究基因结构和功能。

从分子角度来解释基因的结构和活性是本书的主要内容。

3 二十一世纪生物学的新热点及领域是什么？答案：结构生物学是当前分子生物学中的一个重要前沿学科，它是在分子层次上从结构角度特别是从三维结构的角度来研究和阐明当前生物学中各个前沿领域的重要学科问题，是一个包括生物学、物理学、化学和计算数学等多学科交叉的，以结构（特别是三维结构）测定为手段，以结构与功能关系研究为内容，以阐明生物学功能机制为目的的前沿学科。

这门学科的核心内容是蛋白质及其复合物、组装体和由此形成的细胞各类组分的三维结构、运动和相互作用，以及它们与正常生物学功能和异常病理现象的关系。

分子发育生物学也是当前分子生物学中的一个重要前沿学科。

人类基因组计划，被称为“21世纪生命科学的敲门砖”。

“人类基因组计划”以及“后基因组计划”的全面展开将进入从分子水平阐明生命活动本质的辉煌时代。

目前正迅速发展的生物信息学，被称为“21世纪生命科学迅速发展的推动力”。

尤应指出，建立在生物信息基础上的生物工程制药产业，在21世纪将逐步成为最为重要的新兴产业；从单基因病和多基因病研究现状可以看出，这两种疾病的诊断和治疗在21世纪将取得不同程度的重大进展；遗传信息的进化将成为分子生物学的中心内容”的观点认为，随着人类基因组和许多模式生物基因组序列的测定，通过比较研究，人类将在基因组上读到生物进化的历史，使人类对生物进化的认识从表面深入到本质；研究发育生物学的时机已经成熟。

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2000;60:5012-5016. Published online September 1, 2000.Cancer ResGeorge I. Salti, Taline Manougian, Miguel Farolan, et al.in Intermediate-thickness Cutaneous Malignant MelanomaMicropthalmia Transcription Factor: A New Prognostic MarkerUpdated Version/content/60/18/5012Access the most recent version of this article at:Cited Articles/content/60/18/5012.full.html#ref-list-1This article cites 14 articles, 8 of which you can access for free at:Citing Articles/content/60/18/5012.full.html#related-urls This article has been cited by 18 HighWire-hosted articles. Access the articles at:E-mail alerts related to this article or journal.Sign up to receive free email-alerts Subscriptions Reprints and .pubs@ Department at To order reprints of this article or to subscribe to the journal, contact the AACR Publications Permissions.permissions@ Department at To request permission to re-use all or part of this article, contact the AACR Publications[CANCER RESEARCH60,5012–5016,September15,2000]Advances in BriefMicropthalmia Transcription Factor:A New Prognostic Marker inIntermediate-thickness Cutaneous Malignant Melanoma1George I.Salti,2Taline Manougian,Miguel Farolan,Anne Shilkaitis,Dibyen Majumdar,and Tapas K.Das Gupta Departments of Surgical Oncology[G.I.S.,T.M.,A.S.,T.K.D.G.]and Pathology[M.F.],College of Medicine,and Department of Mathematics,Statistics,and Computer Sciences[D.M.],University of Illinois at Chicago,Chicago,Illinois60612AbstractMicropthalmia transcription factor(Mitf)is involved in melanocyte development and differentiation.The current study was undertaken to determine whether there is a relationship between Mitf expression and survival in patients with intermediate-thickness(1.0–4.0mm)melanoma. Original paraffin blocks or slides of the primary tumor were accessible in 63such patients.Mitf expression was evaluated by immunocytochemistry and analyzed visually.Slides were graded as follows according to the percentage of cells whose nuclei stained positive for Mitf:(a)0,0%;(b)؉1,1–25%;(c)؉2,26–50%;(d)؉3,51–75%;and(e)؉4,>75%.Median follow-up was50months.Mean thickness was2.2؎0.7mm.Mean overall survival was171.90؎13.12months.Mean disease-free survival was 168.53؎13.96months.Fifty-two melanomas(82.5%)stained positive for Mitf.By univariate analysis,mean overall survival and disease-free sur-vival in patients whose melanomas did not express Mitf were 80.89؎17.98months(median,51months)and71.36؎19.87months (median,40months),respectively.This compares with187.90؎13.41 months(median,not reached)and186.78؎13.84months(median,not reached),respectively,for patients whose melanomas expressed Mitf (P؍0.0086and P؍0.0054).These findings persisted in multivariate analysis.In addition,patients with>50%Mitf expression had signifi-cantly fewer nodal metastases after node dissection than patients with <50%Mitf expression(P؍0.04).Our data suggest that Mitf may be a new molecular prognostic marker in patients with intermediate-thickness melanoma.IntroductionThe incidence of cutaneous malignant melanoma continues to rise. It is estimated that in the year2000,47,700new cases will be diagnosed,with7,700deaths(1).The prognosis of primary cutaneous melanoma is usually estimated by several histopathological criteria, such as tumor thickness,ulceration,levels of invasion,and mitoses/ mm2(2).However,limitations in the accuracy of these prognostic markers have led investigators to search for additional clues in the microenvironment in primary cutaneous melanomas.Notable cellular and molecular markers include expression of various types of inte-grins(3,4).Although these markers have helped define several subsets of patients with different natural histories,the search contin-ues for additional prognostic markers.A number of genetic and cell biological models indicate that pigment cell development and function depend on both cell-extrin-sic and-intrinsic factors.Although how these factors are function-ally integrated is poorly understood,it is generally agreed that the basic helix-loop-helix-zipper Mitf3relates to the downstream targets responsible for cell proliferation,fate determinism,and melanin biosynthesis(5,6).Mutations in Mitf are found from fishes to man.In fact,Mitf is critical not only for neural crest-derived melanocytes of the integument and inner ear(7)but also for the cells of the retinal pigment layer,which develop from the neural epithelium of the optic cup.Mouse mutations at the micropthalmia locus(mi)are characterized by small nonpigmented eyes,lack of integumentary melanocytes,and deficiencies in the inner ears.Also,these mutations are associated with a deficiency in mast cells and osteoclasts,resulting in a condition similar to osteopetrosis in humans.In humans,Mitf mutation is characterized by hearing deficiencies,white forelock,and ocular anomalies(Waardenburg syndrome IIA;Ref.8).It is believed that Mitf can induce pigment cell-specific transactivation of tyrosinase and tyrosinase-related protein1(9–11).In addition,␣-MSH has been shown to up-regulate the pigment gene expression via a cyclic AMP-mediated signal transduction pathway that stimulates the expression of Mitf(12).Moreover,it was reported that the c-Kit signaling pathway in melanocytes targets transcription factor for micropthalmia simultaneously for short-lived activation and net degradation(13). Recently,King et al.(14)reported that Mitf is a sensitive and specific melanocyte marker for melanoma diagnosis.Because Mitf appears to be involved in melanocyte development and differentiation,we hypothesized that its expression correlates with survival in patients with intermediate-thickness(1–4mm)melanoma. Our findings suggest that Mitf expression in the primary tumor correlates with patient survival.Materials and MethodsPatients and Tumor Specimens.We analyzed the primary lesions of63 patients with intermediate-thickness(1.0–4.0mm)malignant melanomas treated in the Department of Surgical Oncology at the University of Illinois at Chicago.The slides and blocks were obtained from our melanoma tissue and sera bank.All patients have been managed by the Department of Surgical Oncology at the University of Illinois at Chicago.The clinical and follow-up data of these patients are maintained in the melanoma research registry.Immunohistochemistry.Four-␮m-thick sections were used for immuno-cytochemical studies.Standard avidin-biotin-peroxidase immunohistochemis-try was performed with the mouse monoclonal IgG1micropthalmia antibody Ab-2(Neomarkers,Union City,CA).Antigen retrieval was accomplished with 1m M EDTA buffer.We used1%dried skim milk to block nonspecific binding before incubation with the primary antibody for2h(1:25dilution).Amin-oethylcarbazine was used as a chromagen.Evaluation of Staining.The stained slides were graded as follows accord-ing to the percentage of cells whose nuclei stained positive for Mitf:(a)0,0%;(b)ϩ1,1–25%;(c)ϩ2,26–50%;(d)ϩ3,51–75%;and(e)ϩ4,Ͼ75%. Cytoplasmic staining was excluded from the analysis.Representative immuno-histochemical staining of two different primary melanomas is shown in Fig.1.Received6/1/00;accepted8/3/00.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section1734solely to indicate this fact.1Supported by NIH Training Grant T32-CA09432and a grant from the Cancer Federation,Inc.2To whom requests for reprints should be addressed,at Department of SurgicalOncology(M/C820),College of Medicine,University of Illinois at Chicago,840South Wood Street,Chicago,IL60612.Phone:(312)996-3288;Fax:(312)996-9365;E-mail: geosalti@.3The abbreviations used are:Mitf,micropthalmia transcription factor;OS,overall survival;DFS,disease-free survival.Statistical Analysis.Survival curves were generated by the Kaplan-Meier product limit estimate.Statistical comparisons were performed using the log-rank test.Outcomes evaluated were DFS and OS.We evaluated the effect of Mitf staining (graded from 0to ϩ4)on both DFS and OS.In addition,we arbitrarily evaluated the effect of three different cutoff levels of Mitf staining on survival:(a )0(negative)versus ϩ1to ϩ4(positive);(b )0to ϩ1(Յ25%)versus ϩ2to ϩ4(Ͼ25%);and (c )0to ϩ2(Յ50%)versus ϩ3to ϩ4(Ͼ50%).Pairwise association between Mitf staining and known prognostic variables such as thickness,histology,location of primary tumor,number of mitoses/mm 2,ulceration,and nodal status was also evaluated.Differences in distribu-tion of variables were assessed by the Mann-Whitney and Kruskal-Wallis tests.Multivariate analysis was performed by using the Cox proportional hazards model.P Ͻ0.05was considered statistically significant.Death from melanoma was considered the only event for OS.DFS events included regional recur-rences and distant metastases.ResultsThere were 36males and 27females.The mean age at diagnosis was 53.25Ϯ15.49years (age range,17–81years).The primary cutaneous melanomas were located on the trunk in 23patients (36.5%),the lower extremities in 23patients (36.5%),the upper extremities in 10patients (15.9%),and the head and neck in 7patients (11.1%).Thirty-one (49.2%)melanomas were superficial spreading melanomas,18(28.5%)were nodular melanomas,9(14.3%)were acral lentiginous melanomas,and 5(7.9%)were lentigo maligna melanomas.Mean thickness was 2.2Ϯ0.7mm (range,1–4mm).Twenty-one melanomas were ulcerated (33.3%),and 42(66.7%)were nonulcerated.The mean number of mitoses/mm 2per high-power field was 4.3Ϯ2.62(range,1–12).The majority of melanomas had Clark’s level IV invasion (52of 63melanomas,82.5%).Of 63patients,11(17.5%)did not stain forMitf,17(27%)had ϩ1staining,13(20.6%)had ϩ2staining,13(20.6%)had ϩ3staining,and 9(14.3%)had ϩ-plete node dissection was performed in 56of 63(88.8%)patients.Twenty-six (46.4%)of these patients were node positive,and 37(58%)were node negative.The median follow-up was 50months.The mean DFS (Fig.2A )was 168.53Ϯ13.96months (5-year DFS,74.1%).Mean OS (Fig.2B )was 171.9Ϯ13.12months (5-year OS,74.5%).Nodal status significantly affected survival in our cohort of patients.Mean DFS and OS in those patients who had nodal disease were 105.37months (5-year DFS,58.4%)and 109.90months (5-year OS,58.6%).In contrast,patients without nodal disease had a DFS of 192.55months (5-year DFS,82%;P ϭ0.04)and an OS of 193.54months (5-year OS,82.2%;P ϭ0.05).In this small series of 63patients,only thickness significantly influenced survival when evaluated by univariate analysis.The sur-vival advantage was most marked when patients had primary mela-noma thicker than 2.6mm.In those instances,the DFS and OS were 66.48months (5-year DFS,60%)and 67.1months (5-year OS,58%),respectively,whereas the DFS and OS were 180.52months (5-year DFS,76.5%;P ϭ0.067)and 183.24months (5-year OS,78%;P ϭ0.039%),respectively,in patients with primary lesions Ͻ2.6mm thick.Curiously,when stratified according to the presence or absence of ulceration in the primary tumor,ulceration was significant only in patients whose melanomas were Ͼ2mm thick.In patients with ulceration,the DFS and OS were 119.41months (5-year DFS,58.9%)and 123.20months (5-year OS,60%),respectively,com-pared with 160months (5-year DFS,80.7%;P ϭ0.086)andFig.1.Representative immunostaining of dif-ferent levels of Mitf expression in two primary cutaneous malignantmelanomas.Fig.2.Kaplan-Meier survival curves of the entire popu-lation.A ,DFS.B ,OS.165.14months (5-year OS,82.7%;P ϭ0.05)in patients with nonulcerated primaries.Expression of Mitf as determined immunohistochemically in the primary lesion appears to significantly influence both DFS and OS(Table 1;Fig.3).Moreover,by applying semiquantitative methods,it appears that the amount of Mitf expression could be directly correlated to both DFS and OS.Mean DFS and OS in the 11patients whose melanomas did not stain positive for Mitf were 71.36months (median,40months;5-year DFS,37.6%)and 80.89months (median,51months;5-year OS,42.1%),respectively.These figures are significantly lower than those for patients who showed evidence of Mitf expression,for whom the mean DFS and OS were 186.78months (5-year DFS,83.1%;P ϭ0.0086)and 187.9months (5-year OS,84%;P ϭ0.0054),respectively.Mean DFS and OS with Ͼ25%of the melanoma cells staining positive for Mitf expression were 137.84months (5-year DFS,92.2%;P ϭ0.023)and 147.32months (5-year OS,92%;P ϭ0.039)compared with cells in which Ͻ25%of the cells stained positive for Mitf expression,in which the DFS and OS were 127.14months (5-year DFS,55.8%)and 136.68months (5-year OS,58%),re-spectively.This observation was most marked in DFS where Ͼ50%of the cells expressed Mitf.DFS was 212.57months(5-yearFig.3.Kaplan-Meier survival curves that compare patients with different levels of Mitf expression.DFS and OS were significantly better for those patients whose melanomas stained positive for Mitf [A (DFS),P ϭ0.0086;B (OS),P ϭ0.0054)]and for those patients whose melanomas expressed Ͼ25%Mitf [C (DFS),P ϭ0.023;D (OS),P ϭ0.039].DFS was significantly better for those patients whose melano-mas expressed Ͼ50%Mitf (E ,P ϭ0.035).OS tended to be better for those patients whose melanomas ex-pressed Ͼ50%Mitf (F ,P ϭ0.054).Table 1Influence of Mitf expression on DFS and OSMitf expression N DFS aOS a01171.36(P ϭ0.048)80.89(P ϭ0.077)ϩ117162.32172.73ϩ213119.62120.05ϩ313bbϩ49105.8106.8Absent 1171.36(P ϭ0.0086)80.89(P ϭ0.0054)Present 52186.78187.9Յ25%28127.14(P ϭ0.023)136.68(P ϭ0.039)Ͼ25%35137.84147.32Յ50%41127.27(P ϭ0.035)133.37(P ϭ0.054)Ͼ50%22212.57212.64a Mean values in months.bAll observations were censored;mean values were not calculated.DFS,92.3%)in these patients as compared with patients in whom Ͻ50%of the cells expressed Mitf,where the mean DFS was 127.27months(5-year DFS,64.8%).The P between these two survival figures was0.035.In this series,location of the primary tumor,age,sex,histological subtype,and level of invasion did not play any role in either DFS or OS.When multivariate analysis was performed on known primary prognostic variables,patients whose melanomas wereϾ2.6mm thick had worse DFS(Pϭ0.005)and OS(Pϭ0.032).When the nodal status was included as a covariate,both thickness and nodal status significantly influenced survival:(a)DFS,Pϭ0.078and Pϭ0.05,respectively;and(b)OS,Pϭ0.005and Pϭ0.048, respectively.When Mitf expression with a cutoff value of25%was included with the other primary tumoral prognostic variables, it significantly affected DFS(Pϭ0.014)and OS(Pϭ0.018). When all covariates were analyzed together(Table2),Mitf expression emerged as the most significant variable influencing DFS(Pϭ0.008).By stepwise Cox regression,the final model consisted of Mitf expression(Pϭ0.015)and thickness (Pϭ0.035)influencing DFS,and regional nodal status (Pϭ0.048)influencing OS.A significant relationship was noted between quantitative Mitf expression and the presence or absence of metastases in the regional nodes.WhenՆ50%of melanoma cells expressed Mitf,the incidence of nodal metastases was significantly lower.Of the26patients with nodal metastases,6(23%)had primary lesions withϾ50%Mitf expression,whereas20(77%)of the primary melanomas hadϽ50% of the cells expressing Mitf(Pϭ0.04).DiscussionWe have evaluated the biological significance of Mitf expression in primary cutaneous melanoma.It appears that Mitf expression is a molecular marker that can be used clinically along with such estab-lished markers as thickness of the primary lesion,ulceration,and incidence of regional node metastases.Our data suggest that Mitf expression is a marker for improved survival.This assumption is substantiated by the fact that6of11patients whose primary tumors did not stain positive for Mitf died of melanoma,and the mean DFS in these patients was40months.Furthermore,using a semiquantita-tive immunocytochemical analysis,it appears that the higher the number of melanoma cells showing Mitf expression in a given pri-mary tumor,the better the prognosis.For example,when25%of the melanoma cells expressed Mitf,both DFS and OS increased signifi-cantly compared with the primary lesions in which no Mitf was expressed.If the cutoff point for positivity of Mitf expression is assumed to be50%of the cells expressing Mitf,then the DFS of this group of patients increased significantly,and OS tended to increase significantly.Clinically,nodal status is probably the most important prognostic marker in cutaneous melanoma,and in the primary lesion,thickness and ulceration are considered the most significant markers for sur-vival.In this study,we confirmed that all of these three aspects are clinically reliable markers.However,in this small series,thickness and ulceration became statistically significant when the lesions were Ն2mm.Mitf expression,in contrast,remains significant in all in-stances when compared by univariate analysis.In multivariate anal-ysis,using the stepwise Cox regression model,Mitf expression and nodal status emerged as the most important variables influencing DFS and OS.This prognostic correlation between nodal status and Mitf ex-pression suggests that Mitf expression or lack thereof in primary lesions is an indicator of regional node metastases.This certainly was the case in our study.Patients with Mitf expression ofϾ50% had a significantly lower incidence of nodal disease than those with a Mitf expression ofՅ50%.The biological role of Mitf in melanocyte differentiation continues to show new facets.Our findings suggest that increased Mitf expression in patients with intermediate-thickness melanomas may lead to more differentiated tumors.This,in turn,leads to decreased relapse and nodal metas-tases and improved survival.In the study reported by King et al.(14),all melanomas evaluated stained positive for Mitf,but not for other melanocytic markers evaluated.In our study,82.5%of melanomas stained positive for Mitf.It should be noted that in addition to tissue blocks,we examined archival specimens only for which slides from outside institutions were sent to us for evaluation.The difference in tissue handling may explain,in part,the difference in staining.In addition,we have used a commercially available antibody for immunostaining.However,it is germane to emphasize that Mitf expression is a sign of differentiation; thus,dedifferentiated tumors will either not express or will barely express Mitf,and therefore there will be some lesions in which no Mitf expression will be detected.Thus,lack of expression should not be attributed merely to random occurrence.Our data also suggest that Mitf expression may not be as highly conserved in melanomas as suggested previously.In summary,the data presented above suggest for the first time that Mitf expression,as determined by immunocytochemistry,can be used as a new prognostic molecular marker in melanoma.Additional pro-spective studies are warranted to determine whether or not these findings will be validated.AcknowledgmentsWe thank Genoveva Murillo for technical help and Kevin Grandfield for editing the manuscript.References1.Greenlee,R.T.,Murray,T.,Bolden,S.,and Wingo,P.A.Cancer statistics,2000.CACancer J.Clin.,50:7–33,2000.2.Ronan,S.G.,Han,M.C.,and Das Gupta,T.K.Histologic prognostic indicators incutaneous malignant melanoma.Semin.Oncol.,15:558–565,1988.3.Hieken,T.J.,Farolan,M.,Ronan,S.G.,Shilkaitis,A.,Wild,L.,and Das Gupta,T.K.␤3Integrin expression in melanoma predicts subsequent metastasis.J.Surg.Res.,63: 169–173,1996.4.Hieken,T.J.,Ronan,S.G.,Farolan,M.,Shilkaitis,A.L.,Kim,D.K.,and Das Gupta,T.K.␤1Integrin expression in malignant melanoma predicts occult lymph node metastases.Surgery(St.Louis),118:669–675,1995.5.Hodgkinson,C.A.,Moore,K.J.,Nakayama,A.,Steingrimsson,E.,Copeland,N.G.,Jenkins,N.A.,and Arnheiter,H.Mutations at the mouse micropthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein.Cell,74:395–404,1993.6.Hughes,M.J.,Lingrel,J.B.,Karkowsky,J.M.,and Anderson,K.P.A helix-loop-helix transcription factor-like gene is located at the mi locus.J.Biol.Chem.,268: 20687–20690,1993.7.Opdecamp,K.,Nakayama,A.,Nguyen,M.T.,Hodgkinson,C.A.,Pavan,W.J.,andArnheiter,H.Melanocyte development in vivo and in neural crest cell cultures:crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor.Develop-ment(Camb.),124:2377–2386,1997.Table2Multivariate Cox regression analysis of all prognostic variables Variable DFS OS Thickness0.016a0.008 Ulceration0.3820.586 Histology0.3590.366 Location of primary tumor0.5620.335 Mitoses/mm20.0350.033 Nodal status0.0120.016 Mitf expression25%0.0080.011 a Significance shown in bold.8.Hughes,A.E.,Newton,V.E.,Liu,X.Z.,and Read,A.O.A gene for WaardenburgSyndrome type2maps close to the human homologue of the micropthalmia gene at chromosome3p12–p14.1.Nat.Genet.,7:509–512,1994.9.Yasumoto,K.,Yokoyama,K.,Takahashi,K.,Tomita,Y.,and Shibahara,S.Functionalanalysis of micropthalmia-associated transcription factor in pigment cell-specific tran-scription of the human tyrosinase family genes.J.Biol.Chem.,272:503–509,1997.10.Fang,D.,and Setaluri,V.Role of micropthalmia transcription factor in regulation ofmelanocyte differentiation marker mun.,256: 657–663,1999.11.Bentley,N.J.,Eisen,T.,and Goding,C.R.Melanocyte-specific expression of thehuman tyrosinase promoter:activation by the micropthalmia gene product and the role of the initiator.Mol.Cell.Biol.,14:7996–8006,1994.12.Price,E.R.,Horstmann,M.A.,Wells,A.G.,Weilbaecher,K.N.,Takemoto,C.M.,Landis,M.W.,and Fisher,D.G.␣-Melanocyte-stimulating hormone signaling regulates expression of micropthalmia,a gene deficient in Waardenburg Syndrome.J.Biol.Chem.,273:33042–33047,1998.13.Wu,M.,Hemesath,T.J.,Takemoto,C.M.,Horstmann,M.A.,Wells,A.G.,Price,E.R.,Fisher,D.Z.,and Fisher,D.E.C-Kit triggers dual phosphorylations,whichcouple activation and degradation of the essential melanocyte factor Mi.Genes Dev., 14:301–302,2000.14.King,R.,Weilbaecher,K.N.,McGill,G.,Cooley,E.,Mihim,M.,and Fisher,D.E.Micropthalmia transcription factor:a sensitive and specific melanocyte marker for melanoma diagnosis.Am.J.Pathol.,155:731–738,1999.。