突脐孢属Brnl基因核苷酸序列比较及系统发育研究

对所有供试突脐孢菌株的Brnl基因(1,3,8-三羟基萘还原酶基因)扩增均获得PCR产物。序列比较表明：在种内各菌株间没有核苷酸序列长度变化,存在核苷酸序列简单代换;在种间核苷酸序列长度有变化,核苷酸的缺失或插入发生在内含子区;所有菌株编码区核苷酸序列长度相同;在种内水平氨基酸序列没有差别,显示出高度的保守性。利用最大简约法(Maximum Parsimony)和邻近结合法(Neighbor-joining)构建系统发育树,两个系统发育树的拓扑结构相似,不同种在不同的分支上。Brnl基因适合突脐孢属种级水平的分子系统学研究。     

突脐孢属Brn1基因核苷酸序列比较及系统发育研究

对所有供试突脐孢菌株的Brn1基因 (1, 3, 8 -三羟基萘还原酶基因) 扩增均获得PCR产物。序列比较表明:在种内各菌株间没有核苷酸序列长度变化,存在核苷酸序列简单代换;在种间核苷酸序列长度有变化,核苷酸的缺失或插入发生在内含子区;所有菌株编码区核苷酸序列长度相同;在种内水平氨基酸序列没有差别,显示出高度的保守性。利用最大简约法(Maximum Parsimony)和邻近结合法(Neighbor-joining)构建系统发育树,两个系统发育树的拓扑结构相似,不同种在不同的分支上。Brn1基因适合突脐孢属种级水平的分子系统学研究。     

红角辉蝽和紫翅果蝽线粒体完整基因组测序及蝽亚科系统发育分析(半翅目: 蝽科)(英文)

【目的】本研究对红角辉蝽Carbula crassiventris和紫翅果蝽Carpocoris purpureipennis完整线粒体基因组测序,以探究蝽亚科(Pentatominae)线粒体基因组特征并重建其系统发育关系。【方法】使用Illumina MiSeq测序平台测定红角辉蝽和紫翅果蝽线粒体基因组全序列,并进行组装和注释。基于这2个种和其他30个蝽亚科分类单元线粒体基因组的13个蛋白质编码基因的第1和2位密码子以及2个rRNA基因的核苷酸序列,利用贝叶斯和最大似然法重建蝽亚科系统发育树。【结果】红角辉蝽和紫翅果蝽的线粒体基因组全长分别为15 824 和16 575 bp, 包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个控制区。蝽亚科内线粒体基因组基因排列顺序保守且没有发现基因重排。此外,蝽亚科内的碱基组成、密码子使用和RNA结构均较为保守; 控制区重复序列拥有不同的长度、类型和拷贝数。基于贝叶斯法和最大似然法重建的系统发育树显示二星蝽族(Eysarcorini)、果蝽族(Carpocorini)、稻绿蝽族(Nezarini)和Antestiini构成一个稳定分枝。【结论】系统发育分析支持辉蝽属Carbula应属于二星蝽族,而果蝽属Carpocoris、斑须蝽属Dolycoris和珠蝽属Rubiconia同属于果蝽族。     

中国库蚊属四十个种(亚种)的分子系统发育关系研究(双翅目:蚊科)

【目的】对在中国有分布的库蚊属Culex种(亚种)的COⅠ序列和ITS2序列进行测序,构建和讨论这些种(亚种)的分子系统发育关系。【方法】测定了库蚊属20个种(亚种)的COⅠ和ITS2序列,并从NCBI数据库中下载了库蚊属另外20个种(亚种)的COⅠ和3个种(亚种)的ITS2序列。对库蚊属40个种(亚种)的COⅠ序列和其中23个种(亚种)的ITS2序列进行碱基构成、种间遗传距离和饱和度分析,并对COⅠ+ITS2序列进行ILD(incongruence length difference)检验。分别使用2种分子数据集(COⅠ和ITS2)的核苷酸序列,用最大似然法(ML)、贝叶斯法(BI)、邻接法(NJ)和最大简约法(MP)推断这些种的系统发育关系。通过Kishino-Hasegawa(KH)和ShimodairaHasegawa(SH)检验评估这4种系统树间的差异,确定最为合理的系统发育树。【结果】本研究新测序获得20种(亚种)的COⅠ和ITS2序列的长度范围分别为625~685 bp和300~559 bp。COⅠ和ITS2序列在库蚊属成对蚊种间的遗传距离分别是0.002~0.198和0.006~1.807。库蚊属23个种(亚种)的COⅠ+ITS2序列的ILD检验结果显示,数据集具有不相容性,因此COⅠ+ITS2序列不适用于这些种(亚种)的系统发育研究。经KH和SH检验显示,4种系统发育树中,基于COⅠ序列构建的BI树最为合理,而基于ITS2序列构建的MP树最为合理。基于COⅠ核苷酸序列所构建的BI树显示,除幼小库蚊Cx.infantulus和短须库蚊Cx.brevipalpis外,各亚属间成员聚类结果与传统的形态学归类结果吻合;路蚊亚属Lutiza和包蚊亚属Barraudius都归入库蚊亚属Culex内;梅蚊亚属Maillotia和新库蚊亚属Nexoculex聚为一支;库状蚊亚属Culiciomyia、真黑蚊亚属Eumelanomyia和簇角蚊亚属Lophoceramyia均显示为单系。基于ITS2序列所构建的MP树显示,各亚属间和种(亚种)间关系混乱。【结论】重建的分子系统发育树表明库蚊亚属不是一个单系群。在重建库蚊属系统发育关系时,相较于ITS2和COⅠ+ITS2,COⅠ是更为理想的分子标记。本研究构建的分子系统发育关系为中国库蚊属中各亚属和各种(亚种)之间的亲缘关系的研究奠定了基础。     

甘薯NBS类抗病基因类似物的分离与序列分析

利用已克隆植物抗病基因NBS（Nucleotide binding site）序列中的保守模体（motif）“P-loop”和“GLPL”合成简并引物,以甘薯（Ipomoea batatas）栽培品种青农2号基因组DNA为模板进行PCR扩增,通过T/A克隆、测序和序列分析,共得到15条具有连续ORF的抗病基因类似物（Resistance gene analogues,RGAs）序列,它们之间核苷酸序列间的相似性系数在41.2%-99.4%之间,而相应推测的氨基酸序列间的相似性系数在20.6%-100%之间,同时对分离的RGAs的核苷酸和氨基酸序列进行系统发育树分析,表明甘薯RGAs可分为TIR（Drosophila Toll or human interleukin receptor-like）和nonTIR两类.对甘薯RGAs和5个已克隆植物NBS的氨基酸序列进行结构分析表明,它们包括“P-loop”、“Kinase-2”、“Kinase-3a”、“GLPL”4个抗病基因所共有的保守模体.这些表明甘薯与其它物种的NBS类RGAs可能具有同样的起源和进化机制.     

中国熊野藻属植物系统发育分析——基于叶绿体UPA序列

以中国产的6种熊野藻属(Kumanoa)和串珠藻属(Batrachospermum)植物为材料,测定其UPA序列,并在GenBank中下载相关基因序列,通过贝叶斯法、最大似然法和邻接法构建系统发育树,分析其系统发育。结果显示,UPA序列核苷酸变异位点占序列长度的43.1%,其中简约信息位点占序列长度的31.1%,种间差异度为0~1.81%,属间差异度大于4.19%,可以作为分子标记用于系统发育分析。中国产熊野藻属的两个种和来自世界范围内的多数该属种类聚为一大支,支持该属的建立。基于UPA序列的地理起源分析显示,两属的祖先分布区节点主要在北美洲和大洋洲,然后向其它地区扩散,熊野藻属向外扩散后,在南美洲分布较为集中。     

两种缘毛类纤毛虫胞质Hsp70基因序列的克隆及其系统发育分析

克隆得到2种缘毛类纤毛虫——钟形钟虫(Vorticella campanula)和螅状独缩虫(Carchesium polypinum)的胞质Hsp70基因部分序列,长度均为438bp,编码146个氨基酸。以细菌为外类群,利用最大似然法和邻接法构建包括其他5种纤毛虫在内的共26个物种的Hsp70基因氨基酸序列系统发育树,其拓扑结构显示：V．campanula和C．polypinum聚在一起,并与另2种寡膜纲的嗜热四膜虫(Tetrahymena thermophila)及草履虫(Paramecium tetraurelia)聚为姊妹枝,提示了缘毛类纤毛虫为单系,且隶属于寡膜纲的系统发育地位。     

我国禽脑脊髓炎病毒分离株全基因组的测定

测定了我国禽脑脊髓炎病毒(avian encephalomyelitis virus,AEV)分离株L2Z株的全基因组核苷酸序列.该病毒株的3′和5′非编码区核苷酸序列用3′和5′RACE(cDNA末端快速扩增)法获得.基因组全长为7 059个核苷酸残基,包括494个核苷酸残基的5′非编码区、6 402个核苷酸残基的开放阅读框和136个核苷酸残基的3′非编码区及poly(A)尾巴.与已发表的AEV疫苗株1 143的基因组序列比较发现,它们之间核苷酸和氨基酸的同源性分别为98%和97.6%.结构蛋白(VP1～VP4)中,主要宿主保护性免疫原蛋白VP1氨基酸之间差异较小.与小RNA病毒科其它病毒属相比,在非结构蛋白3D中,预测的8个RNA依赖性RNA聚合酶主要结构域中的4个高度保守.从而进一步确认了AEV的分子特性.     

两种毛蚜线粒体基因组比较分析

【目的】线粒体基因组分析已被应用于昆虫系统发育研究。本研究以蚜科Aphididae重要类群毛蚜亚科物种为代表,测定并比较分析了该类蚜虫的线粒体基因组特征,探讨了基于线粒体基因组信息的蚜虫系统发育关系重建。【方法】以毛蚜亚科三角枫多态毛蚜Periphyllus acerihabitans Zhang和针茅小毛蚜Chaetosiphella stipae Hille Ris Lambers,1947为研究对象,利用长短PCR相结合的方法测定线粒体基因组的序列,分析了基因组的基本特征;基于在线t RNAscan-SE Search Server搜索方法预测了t RNA的二级结构;基于12个物种(本研究获得的2个物种和10个Gen Bank上下载的物种数据)的蛋白编码基因(PCGs)序列,利用最大似然法和贝叶斯法重建了蚜科的系统发育关系。【结果】两种毛蚜均获得了约94%的线粒体基因组数据,P.acerihabitans获得了14 908 bp,控制区为1 205 bp;C.stipae获得了13 893 bp,控制区为609 bp。两种毛蚜同时获得33个基因,包含接近完整的13个蛋白编码基因(PCGs)(nad5不完整),18个tRNA,2个rRNA基因;ka/ks值表明,C.stipae的进化速率更快。从基因组组成、基因排列顺序、核苷酸组成分析、密码子使用情况、t RNA二级结构等特征来分析,两种蚜虫线粒体基因组基本特征相似。系统发育重建结果表明毛蚜亚科、蚜亚科的单系性得到了支持,毛蚜亚科位于蚜科的基部位置。【结论】两种毛蚜线粒体基因组的基本特征相似,符合蚜虫线粒体基因组的一般特征,两种线粒体基因组的长度差异主要来自控制区长度的不同;系统发育重建支持毛蚜亚科与蚜亚科的单系性,毛蚜亚科位于蚜科较为基部的位置。研究结果为蚜虫类系统发育重建提供了参考。     

应用非序列联配方法对哺乳动物系统发育关系的探讨

非序列联配的序列分析方法,将序列中特定寡聚核苷酸的kmer统计频率作为特征,在序列间按特征进行比较和分析。这种方法综合考虑了所有变异类型对序列整体特征的影响,因而在组学数据分析上有独特的优势。但是,这类方法在复杂多细胞生物基因组系统发育中的适用性仍然有待检验。在本文中,我们使用基于非序列联配方法的CVTree软件,以45种哺乳动物的蛋白质组数据建立了系统发育关系NJ树,并据此探讨了哺乳动物系统发育的若干问题。在广受关注的真兽下纲四个总目的关系问题上,CVTree支持形态学的普遍结论即上兽类(Epitheria)假说。这与基于序列联配方法支持的外非洲胎盘类(Exafro-placentalia )假说不同。在哺乳动物内部目的层次上,CVTree树的结论与分子和形态所普遍接受的系统发育关系基本一致。但是在目的内部,CVTree树会有较多的差异。研究结果初步显示非序列联配方法在使用复杂多细胞生物的组学数据进行系统发育关系分析中的可行性。对非序列联配方法自身的改进及其与传统基于取代的序列联配方法之间的比较仍有待深入研究。     

Vesicles Bearing Toxoplasma Apicoplast Membrane Proteins Persist Following Loss of the Relict Plastid or Golgi Body Disruption

Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.     

A genetic screen in rodent malaria parasites identifies five new apicoplast putative membrane transporters,one of which is essential in human malaria parasites

The malaria‐causing parasite, Plasmodium, contains a unique non‐photosynthetic plastid known as the apicoplast. The apicoplast is an essential organelle bound by four membranes. Although membrane transporters are attractive drug targets, only two transporters have been characterised in the malaria parasite apicoplast membranes. We selected 27 candidate apicoplast membrane proteins, 20 of which are annotated as putative membrane transporters, and performed a genetic screen in Plasmodium berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were apicoplast‐localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4), and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were apicoplast‐localised. Interestingly, three apicoplast‐localised candidates lack a canonical apicoplast targeting signal but do contain conserved N‐terminal tyrosines with likely roles in targeting. An inducible knockdown of an essential apicoplast putative membrane transporter, PfDMT2, was only viable when supplemented with isopentenyl diphosphate. Knockdown of PfDMT2 resulted in loss of the apicoplast, identifying PfDMT2 as a crucial apicoplast putative membrane transporter and a candidate for therapeutic intervention.     

Replication and partitioning of the apicoplast genome of Toxoplasma gondii is linked to the cell cycle and requires DNA polymerase and gyrase

Apicomplexans are the causative agents of numerous important infectious diseases including malaria and toxoplasmosis. Most of them harbour a chloroplast-like organelle called the apicoplast that is essential for the parasites’ metabolism and survival. While most apicoplast proteins are nuclear encoded, the organelle also maintains its own genome, a 35 kb circle. In this study we used Toxoplasma gondii to identify and characterise essential proteins involved in apicoplast genome replication and to understand how apicoplast genome segregation unfolds over time. We demonstrated that the DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the apicoplast. We show in knockdown experiments that apicoplast DNA Gyrase A and B, and Prex are required for apicoplast genome replication and growth of the parasite. Analysis of apicoplast genome replication by structured illumination microscopy in T. gondii tachyzoites showed that apicoplast nucleoid division and segregation initiate at the beginning of S phase and conclude during mitosis. Thus, the replication and division of the apicoplast nucleoid is highly coordinated with nuclear genome replication and mitosis. Our observations highlight essential components of apicoplast genome maintenance and shed light on the timing of this process in the context of the overall parasite cell cycle.     

Evidence for Golgi-independent transport from the early secretory pathway to the plastid in malaria parasites

The malaria parasite Plasmodium falciparum harbours a relict plastid (termed the apicoplast) that has evolved by secondary endosymbiosis. The apicoplast is surrounded by four membranes, the outermost of which is believed to be part of the endomembrane system. Nuclear-encoded apicoplast proteins have a two-part N-terminal extension that is necessary and sufficient for translocation across these four membranes. The first domain of this N-terminal extension resembles a classical signal peptide and mediates translocation into the secretory pathway, whereas the second domain is homologous to plant chloroplast transit peptides and is required for the remaining steps of apicoplast targeting. We explored the initial, secretory pathway component of this targeting process using green fluorescent reporter protein constructs with modified leaders. We exchanged the apicoplast signal peptide with signal peptides from other secretory proteins and observed correct targeting, demonstrating that apicoplast targeting is initiated at the general secretory pathway of P. falciparum. Furthermore, we demonstrate by immunofluorescent labelling that the apicoplast resides on a small extension of the endoplasmic reticulum (ER) that is separate from the cis-Golgi. To define the position of the apicoplast in the endomembrane pathway in relation to the Golgi we tracked apicoplast protein targeting in the presence of the secretory inhibitor Brefeldin A (BFA), which blocks traffic between the ER and Golgi. We observe apicoplast targeting in the presence of BFA despite clear perturbation of ER to Golgi traffic by the inhibitor, which suggests that the apicoplast resides upstream of the cis-Golgi in the parasite's endomembrane system. The addition of an ER retrieval signal (SDEL) - a sequence recognized by the cis-Golgi protein ERD2 - to the C-terminus of an apicoplast-targeted protein did not markedly affect apicoplast targeting, further demonstrating that the apicoplast is upstream of the Golgi. Apicoplast transit peptides are thus dominant over an ER retention signal. However, when the transit peptide is rendered non-functional (by two point mutations or by complete deletion) SDEL-specific ER retrieval takes over, and the fusion protein is localized to the ER. We speculate either that the apicoplast in P. falciparum resides within the ER directly in the path of the general secretory pathway, or that vesicular trafficking to the apicoplast directly exits the ER.     

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis

Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.     

Genetic Evidence that an Endosymbiont-derived Endoplasmic Reticulum-associated Protein Degradation (ERAD) System Functions in Import of Apicoplast Proteins

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1_Ap results in loss of apicoplast protein import and subsequent death of the parasite.     

Isoprenoid Precursor Biosynthesis Is the Essential Metabolic Role of the Apicoplast during Gametocytogenesis in Plasmodium falciparum

The malaria parasite harbors a relict plastid called the apicoplast and its discovery opened a new avenue for drug discovery and development due to its unusual, nonmammalian metabolism. The apicoplast is essential during the asexual intraerythrocytic and hepatic stages of the parasite, and there is strong evidence supporting its essential metabolic role during the mosquito stages of the parasite. Supply of the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is the essential metabolic function of the apicoplast during the asexual intraerythrocytic stages. However, the metabolic role of the apicoplast during gametocyte development, the malaria stages transmitted to the mosquito, remains unknown. In this study, we showed that production of IPP for isoprenoid biosynthesis is the essential metabolic function of the apicoplast during gametocytogenesis, by obtaining normal gametocytes lacking the apicoplast when supplemented with IPP. When IPP supplementation was removed early in gametocytogenesis, developmental defects were observed, supporting the essential role of isoprenoids for normal gametocytogenesis. Furthermore, mosquitoes infected with gametocytes lacking the apicoplast developed fewer and smaller oocysts that failed to produce sporozoites. This finding further supports the essential role of the apicoplast in establishing a successful infection in the mosquito vector. Our study supports isoprenoid biosynthesis as a valid drug target for development of malaria transmission-blocking inhibitors.     

The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.     

Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway

The plastid of Plasmodium falciparum (or 'apicoplast') is the evolutionary homolog of the plant chloroplast and represents a vestige of a photosynthetic past. Apicoplast indispensability indicates that it still provides essential functions to parasites. Similar to plant chloroplasts, the apicoplast is dependent on many nucleus-encoded genes to provide these functions. The apicoplast is surrounded by four membranes, two more than plant chloroplasts. Thus, protein targeting to the apicoplast must overcome additional membrane barriers. In P.falciparum we have analyzed apicoplast targeting using green fluorescent protein (GFP). We demonstrate that protein targeting is at least a two-step process mediated by bipartite N-terminal pre-sequences that consist of a signal peptide for entry into the secretory pathway and a plant-like transit peptide for subsequent import into the apicoplast. The P.falciparum transit peptide is exceptional compared with other known plastid transit peptides in not requiring serine or threonine residues. The pre-sequence components are removed stepwise during apicoplast targeting. Targeting GFP to the apicoplast has also provided the first opportunity to examine apicoplast morphology in live P. falciparum.