Lipofection strategy for the study of Xenopus retinal development

The analysis of gene function during retinal development can be addressed by perturbing gene expression either by inhibition or by overexpression in desired regions and at defined stages of development. An in vivo lipofection strategy has been applied for stage-specific and region-specific expression of genes in Xenopus retina. Due to colipofection efficiency, this strategy enables us to study functional interaction of genes by lipofecting multiple expression constructs. This lipofection technique also allows us to transfect morpholino oligonucleotides into retinoblasts to block gene function. We present here various aspects of this technique, including recent improvements and modifications.     

The expression of a hunchback ortholog in the polychaete annelid Platynereis dumerilii suggests an ancestral role in mesoderm development and neurogenesis

Orthologs of the Drosophila gap gene hunchback have been isolated so far only in protostomes. Phylogenetic analysis of recently available genomic data allowed us to confirm that hunchback genes are widely found in protostomes (both lophotrochozoans and ecdysozoans). In contrast, no unequivocal hunchback gene can be found in the genomes of deuterostomes and non-bilaterians. We cloned hunchback in the marine polychaete annelid Platynereis dumerilii and analysed its expression during development. In this species, hunchback displays an expression pattern indicative of a role in mesoderm formation and neurogenesis, and similar to the expression found for hunchback genes in arthropods. These data suggest altogether that these functions are ancestral to protostomes.Pierre Kerner and Fabiola Zelada González contributed equally to this work.     

Expression of Barstar as a selectable marker in yeast mitochondria

We describe a new and potentially universal selection system for mitochondrial transformation based on bacterial genes, and demonstrate its feasibility in Saccharomyces cerevisiae. We first found that cytoplasmically synthesized Barnase, an RNase, interferes with mitochondrial gene expression when targeted to the organelle, without causing lethality when expressed at appropriate levels. Next, we synthesized a gene that uses the yeast mitochondrial genetic code to direct the synthesis of the specific Barnase inhibitor Barstar, and demonstrated that expression of this gene, BARSTM, integrated in mtDNA protects respiratory function from imported barnase. Finally, we showed that screening for resistance to mitochondrially targeted barnase can be used to identify rare mitochondrial transformants that had incorporated BARSTM in their mitochondrial DNA. The possibility of employing this strategy in other organisms is discussed.Communicated by R. G. Herrmann     

The ten Hox genes of the millipede Glomeris marginata

We have isolated the ten Hox genes from the pill millipede Glomeris marginata (Myriapoda:Diplopoda). All ten genes are expressed in characteristic Hox-gene-like expression patterns. The register of Hox gene expression borders is conserved and the expression profiles show that the anterior-most limb-bearing segment in arthropods (antennal/cheliceral segment) does not express any Hox gene, while the next segment (intercalary/second-antennal/premandibular/pedipalpal segment) does express Hox genes. The Hox expression patterns in this millipede thus support the conclusion that all arthropods possess a deuterocerebral segment. We find that there is an apparent posterior shift of Hox gene expression domains dorsally relative to their ventral patterns, indicating that the decoupling of dorsal and ventral segmentation is not restricted to the level of segment polarity genes but apparently includes the Hox genes. Although the mechanism for the decoupling of dorsal and ventral segmentation remains unsolved, the decoupling must be at a level higher in the hierarchy than that of the segment polarity and Hox genes. The expression patterns of Ultrabithorax and abdominal-A suggest a correlation between the function of these genes and the delayed outgrowth of posterior trunk appendages. This delay may be caused by an assumed repressor function of Ultrabithorax, which might partially repress the activation of the Distal-less gene. The Glomeris fushi tarazu gene is expressed in a Hox-like domain and in the developing central nervous system, but not in segmental stripes such as has been reported in another myriapod species, the centipede Lithobius. In contrast to the Lithobius fushi tarazu gene, there is no indication for a role in segment formation for the millipede fushi tarazu gene, suggesting that fushi tarazu first acquired its segmentation function in the lineage of the insects.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Liposome-mediated transfer of YAC DNA to tobacco cells

This article describes a set of protocols—for retrofitting, transformation and purification—that together enable the delivery of full-sized YAC-DNA to plant cells. To be able to equip YACs of interest with plant selectable markers, we have constructed a retrofitting vector that carriesnptII anduidA. Furthermore, we established a transformation protocol for plant protoplasts that is sufficiently efficient to support transfer of high-molecular-weight DNA. In this protocol lipofection is combined with PEG-mediated direct gene transfer. Large amounts of purified DNA are necessary for lipofection. To obtain sufficient quantities of concentrated, purified YAC-DNA, we used an optimized two-step, gel-purification method. Transient expression of a YAC-bornuidA demonstrates that both retrofitting vector and transformation protocol are effective.     

蒺藜苜蓿LBD转录因子基因家族全基因组分析

LBD是植物中所特有的转录因子基因家族,在调控植物侧生组织发育、营养代谢以及响应逆境胁迫等方面具有重要作用。该研究利用生物信息学手段,从全基因组水平筛选和鉴定了蒺藜苜蓿LBD基因家族,并对基因结构、系统进化、进化压力、保守域、染色体定位以及基因表达模式等进行了分析。研究结果共鉴定出2类5亚类共计56个蒺藜苜蓿LBD家族基因,在8条染色体上均有分布,但分布不均匀。该家族成员外显子数目都不超过2个,结构简单,基因间在进化时存在负向选择作用。基因表达模式分析发现,该家族成员的表达具有一定的时空特异性,并受干旱和氮素调控。该研究结果对蒺藜苜蓿LBD基因功能研究及进化分析具有重要的意义。     

森林草莓SUN基因家族的鉴定及其对逆境的响应

SUN基因是调控植物生长发育的关键基因。本研究鉴定了二倍体森林草莓(Fragaria vesca)的SUN基因家族,并对各成员的理化性质、基因结构、系统进化以及基因表达进行了分析。结果表明,森林草莓有31个FvSUN基因,其编码蛋白可聚类为7个组,同一组内成员具有高度相似的基因结构与编码蛋白保守域;FvSUNs蛋白的亚细胞定位主要在细胞核中。共线性分析表明森林草莓FvSUNs基因家族主要通过染色体片段复制产生,拟南芥与森林草莓存在23对直系同源基因。利用森林草莓的转录组数据,对FvSUNs基因的组织表达特征进行分析,发现主要可归为3类：各组织均表达、组织中几乎不表达、组织特异性表达,并通过实时荧光定量PCR (quantitative real-time polymerase chain reaction, qRT-PCR)进一步验证结果。此外,还对森林草莓进行不同的逆境胁迫处理,qRT-PCR分析了31个FvSUNs基因的表达情况,发现大部分基因均在不同程度上受低温、高盐或干旱胁迫的诱导表达。这些研究结果为深入揭示草莓SUN基因的生物学功能及其分子机制奠定了基础。     

Comparison of Gene Expression Profiles of Fenneropenaeus chinensis Challenged with WSSV and Vibrio

Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio. Wang and Li contributed equally to this work.     

马尾松R2R3-MYB基因特征及进化和表达分析

MYB类转录因子在植物生长发育、代谢、应答生物胁迫和非生物胁迫的响应等生物过程发挥重要作用。为探究马尾松R2R3-MYB基因结构及功能,该研究以转录组数据为研究区域,从中筛选获得了17个马尾松R2R3-MYB基因,利用生物信息学对基因进行理化性质、系统进化树等分析,同时利用荧光定量PCR技术分析基因的组织特异性以及在花发育时期和非生物胁迫下的表达模式。结果表明：(1)17个PmMYBs亚细胞定位于细胞核,均无跨膜结构,且均含有Motif1、Motif2保守基序。系统发育进化树将马尾松PmMYBs划分为9个亚家族,且与火炬松、白云杉等裸子针叶植物关系较近。(2)17个基因均属于组成型表达,但在不同组织的表达量不同;所有基因均参与了花发育和非生物胁迫,不同基因在花发育不同时期的表达存在差异,有7个基因可能参与了雌雄性状转变;大部分基因响应非生物胁迫上调表达,但响应胁迫的时间存在差异;少数基因在胁迫中下调表达,尤其是PmMYB11基因在所有胁迫中均明显下调表达。该研究较系统地分析了马尾松R2R3-MYB基因的结构特征、系统进化及其在花发育时期和非生物胁迫下的表达模式,为深入探究马尾松R2R3...     

Gene expression pattern at desiccation in the anther of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Although gene expression profile of pollen has been described, there is limited information regarding a particular phase during anther/pollen development. This work characterizes gene expression pattern at desiccation in lily (Lilium longiflorum Thunb. cv Snow Queen) anthers. We have applied a suppression-subtractive hybridization (SSH) strategy, through which 90 clones were identified and sequenced. These clones resulted in the identification of 42 individual cDNAs among which 33 genes were specifically expressed at the desiccation phase of anthers of >150-mm buds. Fourteen cDNAs were chosen for further examination. Six genes were both dehydration- and abscisic acid (ABA)-inducible whereas the other eight genes were apparently dehydration-irrelevant. The group of dehydration- and ABA-induced genes was also induced by desiccation that developmentally occurs in the anther. The application of fluridone has a significant effect of inhibition on mRNA accumulation of these genes in maturing anthers during which desiccation occurs. Pollen germination analysis indicated that, of those dehydration-irrelevant genes, three were ABA-responsive and the other five were not. Thus, three separate signal pathways that function in the activation of late genes at desiccation during anther development are established. The first is the ABA-dependent pathway induced by environmental stress of dehydration. The other two pathways of signaling triggered by developmental cues, through which one is ABA-dependent and another is ABA-independent. The 14 gene proteins showed spatial and temporal expression patterns and may participate in membrane/cell wall synthesis, cytoskeletal organization, signaling, RNA binding, ubiquitin-mediated degradation and transportation during germination and tube growth. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">kitb</Emphasis>, a second zebrafish ortholog of mouse <Emphasis Type="Italic">Kit</Emphasis>

The large numbers of duplicated pairs of genes in zebrafish compared to their mammalian counterparts has lead to the notion that expression of zebrafish co-orthologous pairs in some cases can together describe the expression of their mammalian counterpart. Here, we explore this notion by identification and analysis of a second zebrafish ortholog of the mammalian Kit receptor tyrosine kinase (kitb). We show that in embryos, kitb is expressed in a non-overlapping pattern to that of kita, in the anterior ventral mesoderm, Rohon-beardRohon–Beard neurons, the otic vesicle, and trigeminal ganglia. The expression pattern of kita and kitb in zebrafish together approximates that of Kit in mouse, with the exception that neither zebrafish kit gene is expressed in primordial germ cells, a site of kit expression in the mouse embryo. In addition, zebrafish kita is expressed in a site of zebrafish primitive hematopoiesis but not required for blood development, and we fail to detect kitb expression in sites of zebrafish hematopoiesis. Thus, the expression and function of zebrafish kit genes cannot be described as a simple partition of the expression and function of mouse Kit. We discuss the possibility that these unaccounted for expression domains and functions are derived from more ancestral gene duplications and partitioning instead of the relatively recent teleost teleost-specific duplication. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Strict regulation of gene expression from a high-copy plasmid utilizing a dual vector system

High-copy plasmids are useful for producing large quantities of plasmid DNA, but are generally inadequate for tightly regulating gene expression. Attempts to suppress expression of genes on high-copy plasmids often results in residual or “leaky” production of protein. For stringent regulation of gene expression, it is often necessary to excise the gene of interest and subclone it into a low-copy plasmid. Here, we report a dual plasmid technique that enables tight regulation of gene expression driven by the lac promoter in a high-copy vector. A series of plasmids with varying copies of the lacI^q gene have been constructed to permit titration of the LacI protein. When a high-copy plasmid is transformed along with the appropriate lacI^q-containing plasmid, tight gene regulation is achieved, thus eliminating the need to subclone genes into low-copy plasmids. In addition, we show that this dual plasmid technique enables high-copy gene expression of a protein lethal to Escherichia coli, the ccdB protein. In principle, this technique can be applied to any high-copy plasmid containing the popular pUC replication of origin and provides an easier means of obtaining rigid control over gene expression.     

SMB基因与拟南芥细胞脱分化

应用Affymetrix基因芯片技术研究脱分化过程的全基因组表达情况,分析拟南芥叶柄细胞在脱分化过程中的差异表达基因。结果显示:(1)脱分化叶柄有4 222个基因表现出2倍或以上的表达差异,其中1 684个基因表达上调,2 538个基因表达下调。(2)半定量RT-PCR对部分差异表达基因进行验证,进一步筛选出参与拟南芥脱分化候选基因SMB(SOMBRERO)。(3)实时PCR结果表明,野生型叶柄诱导脱分化时,SMB基因表达量随着诱导时间延长而逐渐增加。(4)SMB基因功能缺失突变体smb-3经CIM诱导很难形成愈伤组织;在激素诱导不同时间段,smb-3叶柄没有明显脱分化现象,SMB基因缺失造成拟南芥叶柄脱分化障碍。研究表明,SMB基因参与拟南芥叶柄细胞脱分化过程。     

Stable transformation of insect cells to coexpress a rapidly selectable marker gene and an inhibitor of apoptosis

Summary We have constructed several plasmid expression vectors to express foreign genes in stably transformed insect cells. Unlike baculovirus-based expression vectors by which genes of interest are expressed transiently before lysis of the virus-infected cells, genes can be expressed continuously over many passages in a stable cell line. Furthermore, the function of a gene or genes expressed in a stable cell line from an insect-specific promoter that is constitutively expressed can be studied in the absence of virus infection and viral gene expression. In this study, we have expressed a novel, selectable marker gene, puromycin acetyltransferase, under the control of the Drosophila melanogaster hsp70 promoter or under the control of the AcMNPV ie-1 promoter which is active in Spodoptera frugiperda cells in the absence of virus infection. In addition, we have constructed expression vectors which coexpress two genes from separate promoters, the pac gene which confers resistance to puromycin and a baculovirus gene which inhibits apoptosis, derived from Orygia pseudotsugata nuclear polyhedrosis virus. Both genes were expressed in stable populations of S. frugiperda cells in the absence of continuous drug selection.     

拟南芥叶形态建成基因ABL1的图位克隆及鉴定

前期研究表明ABL1可能在植物叶发育过程中扮演重要的角色,其突变表现为叶片生长迟缓、成熟叶片叶缘缺刻明显等生长缺陷特征。该研究利用图位克隆及其精细定位技术,将ABL1基因锁定在2个SSLP标记T23K8和T8F5之间,该区间包含44个基因。通过生物信息学成功找到ABL1突变基因为拟南芥FAS1,该基因编码染色质组装因子CAF1的一个亚基,在植物顶端分生组织生长调控中扮演重要角色。RT-PCR结果显示,该基因表达受阻,功能互补实验证实abl1突变体的确是FAS1基因的一个新等位突变。研究结果暗示,ABL1/FAS1在植物叶形态建成中也起着重要作用。     

Genetic pathways controlling carpel development inArabidopsis thaliana

Carpel development inArabidopsis is known to be controlled by the organ identity geneAGAMOUS. However, even in the absence of AGAMOUS function, many carpel properties can arise suggesting that other genes are also involved. Two new carpel genes,CRABS CLAW andSPATULA, have been recognised by their specific disruptions to carpel development in mutant plants. These disruptions suggest thatCRABS CLAW normally plays a role in promoting the growth of specific regions of the carpel wall, whereasSPATULA apparently has a primary function in promoting development of the transmitting tract. When the function of these genes is also compromised along with that ofAGAMOUS in multiply mutant plants, carpelloid properties vanish. ThusAGAMOUS, CRABS CLAW andSPATULA act together in specifying carpel development, although none can do this alone. BecauseSPATULA mutants are epistatic to mutants of another carpel development gene,ETTIN, the latter may normally act by suppressing the action ofSPATULA in specific regions of the developing gynoecium. There is indirect evidence thatETTIN, and another morphogenetic gene,PINOID, act through regulating auxin-induced growth in specific regions of the developing flower, but it is not yet known how this could result in the suppression of SPATULA function. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”