Sequence and expression analysis of a Xenopus laevis cDNA which encodes a homologue of mammalian 14-3-3 zeta protein

We report the cloning and characterisation of a cDNA that encodes a novel member of the Xenopus laevis 14-3-3 protein family. Sequence analysis reveals that the cDNA-encoded protein shares 84% identity with the rat, human or sheep 14-3-3ζ isoform, and between 66% and 77% identity with bovine, human or rat β, bovine γ, human τ, Drosophila 14-3-3 and a previously isolated Xenopus member. The corresponding mRNA is present in all adult tissues examined with the highest levels in the brain. Although the gene is expressed throughout embryogenesis, higher levels of mRNA accumulate after gastrulation. Whole-mount in situ hybridisation on tailbud stage embryo reveals strong expression of the gene in the head, optic vesicles, spinal cord and branchial arches with weaker expression in the somites. In addition, expression along the notochord is observed at stage 45 (tadpole). This spatial and temporal expression profile along with recent studies implicating the importance of 14-3-3 proteins in the regulation of signal transduction pathways argues for a key role of this isoform in embryonic development.     

Effect of DNA fragment size on transformation frequencies in tobacco (Nicotiana tabacum) and maize (Zea mays)

During eukaryotic cell transformation, the transforming DNA must enter the host cell, traverse the cytoplasm and enter the nucleus before becoming stably integrated into the genome. The limiting step for plant protoplast transformation may lie at the cell membrane, the nuclear membrane, or at the integration step. We show here that the size of the DNA fragment containing the selectable marker used to monitor transformation can directly affect the efficiency of stable transformation. In both tobacco and maize protoplasts, the smallest DNA fragments gave the highest stable transformation frequencies.     

Cloning of the am (glutamate dehydrogenase) gene of Neurospora crassa through the use of a synthetic DNA probe

In a previous study the alteration in the amino acid sequence of Neurospora crassa NADP-specific glutamate dehydrogenase (GDH) resulting from two mutually compensating frameshift mutations was used to deduce the first 17 nucleotides of the coding sequence of the am gene. In the work reported here, a synthetic 17-mer corresponding to the deduced sequence was shown to hybridize strongly to a 9-kb HindIII fragment from N. crassa wild-type DNA but not to any corresponding fragment from the DNA of a mutant strain known to be deleted for most or all of the gene. Wild-type HindIII fragments were fractionated for size and a fraction centering around 9 kb was cloned in vector λL47. Two clones carrying the strongly hybridizing fragment were identified. The hybridization to the 17-mer was localized within a 2.7-kb BamHI fragment and, within this, to a 700-bp BamHI-BglII subfragment. 5' end-labelled polyadenylated RNA isolated from wild-type mycelium hybridized to the 2.7-kb BamHI fragment and not appreciably to flanking fragments. The partial sequence analysis of the BamHI-BglII fragment has confirmed that the 17-mer probe matches the coding sequence at the 5' end of the gene and has also revealed an intervening sequence 67 bp in length, interrupting codon 15. Both the 9-kb HindIII fragment and the 2.7-kb BamHI fragment have been shown to be capable of transforming the deletion mutant to prototrophy and ability to produce GDH. Analysis of one transformant showed that the am gene was integrated, together with a part of the long arm of the lambda vector, at an unusual locus. This transformant, in which the am gene does not show its normal linkage to the linkage group 5 marker inl, was found to produce GDH to about 20% of the normal level.     

Similar tissue-specific expression of the Adh genes from different Drosophila species is mediated by distinct arrangements of cis-acting sequences

In Drosophila melanogaster transformants, the alcohol dehydrogenase (Adh) genes from D. affinidisjuncta and D. grimshawi show similar levels of expression except in the adult midgut where the D. affinidisjuncta gene is expressed about 10- to 20-fold more strongly. To study the arrangement of cis-acting sequences responsible for this regulatory difference, homologous restriction sites were used to create a series of chimeric genes that switched fragments from the 5 and 3 flanking regions of these two genes. Chimeric genes were introduced into the germ-line of D. melanogaster, and Adh gene expression was analyzed by measuring RNA levels. Various gene fragments in the promoter region and elsewhere influence expression in the adult midgut and in whole larvae and adults. Comparison of these results with earlier studies involving chimeras between the D. affinidisjuncta and D. hawaiiensis genes indicates that expression in the adult midgut is influenced by multiple regulatory sequences and that distinct arrangements of regulatory sequences can result in similar levels of expression both in the adult midgut and in the whole organism.     

Comparative studies of zebrafish Danio rerio lipoprotein lipase (lpl) and hepatic lipase (lipc) genes belonging to the lipase gene family: evolution and expression pattern

In this study, bioinformatics analysis, tissue distribution and developmental expression pattern of lipoprotein lipase (lpl) and hepatic lipase (lipc) in zebrafish Danio rerio are reported. In adult D. rerio, lpl was highly expressed in liver. This is remarkably different from the tissue expression pattern of LPL in mammals, which is not detected in the adult liver. The expression of lipc was liver specific, which is consistent with that in mammals. During embryogenesis, lpl mRNA was increased gradually in concentration from 0·5 hpf (hour post fertilization) to 6 dpf (days post fertilization), but lipc was not expressed at the early stage of the embryo until 3 dpf. In situ hybridization further displayed the expression pattern of lpl mainly restricted to the head region including cells surrounding the mouth opening, branchial arches, pectoral fin and lateral line neuromast, whereas lipc was mainly restricted to the liver and part of head regions including lens. This lays a foundation for further investigation of lpl or lipc function and evolution in fishes.     

Molecular structure and chromosomal localization of major repetitive DNA families in the chickpea (Cicer arietinum L.) genome

Three major repetitive DNA sequences were isolated from a genomic library of chickpea (Cicer arietinum L.) and characterized with respect to their genomic organization and chromosomal localization. All repetitive elements are genus-specific and mostly located in the AT-rich pericentric heterochromatin. Two families are organized as satellite DNAs with repeat lengths of 162–168 bp (CaSat1) and 100 bp (CaSat2). CaSat1 is mainly located adjacent to the 18S rDNA clusters on chromosomes A and B, whereas CaSat2 is a major component of the pericentric heterochromatin on all chromosomes. The high abundance of these sequences in closely related species of the genus Cicer as well as their variation in structure and copy number among the annual species provide useful tools for taxonomic studies. The retrotransposon-like sequences of the third family (CaRep) display a more complex organization and are represented by two independent sets of clones (CaRep1 and CaRep2) with homology to different regions of Ty3-gypsy-like retrotransposons. They are distributed over the pericentric heterochromatin block on all chromosomes with extensions into euchromatic regions. Conserved structures within different crossability groups of related Cicer species suggest independent amplification or transposition events during the evolution of the annual species of the genus.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

C-型咽侧体抑制素(AST-C)和促前胸腺激素释放激素(PTTH)基因在家蚕脑组织中的时空表达分析

【目的】咽侧体抑制素在昆虫体内具有重要的调控功能。本研究比较了家蚕Bombyx mori C-型咽侧体抑制素(allatostatin C,AST-C)与促前胸腺激素释放激素(prothoracicotropic hormone,PTTH)基因在不同发育阶段家蚕脑组织中转录表达的模式及其在脑组织的表达定位,以期为家蚕AST-C的功能研究提供重要的线索。【方法】利用脑组织芯片数据分析比较AST-C和PTTH基因在家蚕脑组织中的发育表达特征,RT-PCR验证其芯片数据,分析AST-C在不同发育阶段家蚕中枢神经系统中的表达模式,并用全组织包埋原位杂交技术对AST-C和PTTH在脑组织的表达进行定位。【结果】AST-C和PTTH在不同发育阶段家蚕脑组织中有相似的转录表达模式,且都在脑组织外侧一对神经分泌细胞中表达。【结论】AST-C可能与PTTH以相同的转录表达模式共同参与家蚕的变态发育调控。     

鸭梨PAL基因的反义遗传转化及表达分析

苯丙氨酸解氨酶(phenylalanin ammonia-lyase,PAL,EC4.3.1.5)是植物通过苯丙烷代谢途径合成木质素的关键酶和限速酶,其通过影响木质素的合成而与果实中石细胞的分化、发育及果实品质密切相关。为了降低鸭梨中苯丙氨酸解氨酶的含量,该研究利用反义PAL基因遗传转化鸭梨、降低鸭梨内源PAL基因的表达。结果表明:(1)采用RT-PCR技术,利用根据Gen Bank中西洋梨PAL基因序列设计特异性引物,扩增得到496 bp的鸭梨PAL基因片段。(2)将扩增片段反向插入载体p BI121的MCS区域,构建植物PAL基因反义表达载体p BI121-As PAL。接着采用电转化法将反义表达载体转入农杆菌EHA105中,并制备出农杆菌工程菌液。(3)利用农杆菌介导法对鸭梨组培苗叶片外植体进行遗传转化,得到23株转基因鸭梨苗。PCR检测证实PAL反义基因片段转入鸭梨中,实时定量PCR检测表明转基因鸭梨苗体内PAL基因表达量均有所降低,为非转基因苗的65%~75%。该研究结果表明利用反义RNA技术获得了抑制内源性PAL基因表达的转基因鸭梨植株,为改善鸭梨果实品质、改良品种奠定了基础。     

薇甘菊颈盲蝽细胞色素P450 PmCYP4基因克隆及表达分析

【目的】薇甘菊颈盲蝽是入侵植物薇甘菊的天敌昆虫。CYP4家族基因在专食性昆虫与宿主植物的相互作用中发挥着极其重要的作用,探明其在不同部位的表达情况,可为薇甘菊生物控制提供科学依据。【方法】采用RACE技术克隆薇甘菊颈盲蝽CYP基因,实时荧光定量PCR检测其在不同部位的表达情况。【结果】PmCYP4C1基因全长1713 bp,其中ORF长1500 bp,共编码500个氨基酸,理论分子质量为57.44 ku,无信号肽;与其他昆虫CYP4家族基因的同源性大于40%,与温带臭虫CYP的亲缘关系最近。该基因在雌、雄虫各部位均有表达,且都是足部的表达量明显地高于其他部位;雌、雄虫的表达差异在于雄虫翅膀中的表达量明显地高于触角和残体,但在雌虫中这3个部位的表达量无显著差异,且雄虫翅膀中的表达量显著地高于雌虫,是其2.37倍。【结论】薇甘菊颈盲蝽PmCYP4基因除参与代谢有毒物质外,其主要功能可能是编码与薇甘菊颈盲蝽运动相关的酶。     

Use of PCR and partial sequencing of the large-subunit rRNA gene to identify Alexandrium catenella (Dinophyceae) from the South of Chile

A fragment of the large-subunit ribosomal DNA gene (LSU rDNA) from Chilean Alexandrium catenella clones isolated from two different geographic regions (XI and XII) was amplified by PCR and the products cloned and sequenced. Based on the analysis of the PCR products it is possible to distinguish two strains of A. catenella, denominated strain type 1 (a single PCR product band) and strain type 2 (two PCR product bands). These two strains proliferate in both, the XI and XII regions. Only in the XI region, there is evidence that they bloom simultaneously. The LSU rDNA sequence analysis indicate that the Chilean A. catenella isolated clones are more related to the North American ribotype-Western subribotype.     

Cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus)

The Deleted in Azoospermia (DAZ) family of RNA binding proteins consists of highly conserved genes boule, daz and daz-like (dazl) essential for germ cell development. boule is known for its unisexual meiotic expression in invertebrates and mammals, but meiotic-specific female expression plus meiosis-preferential male expression in trout, and meiosis-preferential bisexual expression in medaka. dazl shows highly conserved bisexual expression throughout gametogenesis in diverse species. Here we report the cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus), an important aquaculture fish. Molecular cloning and sequence analysis led to the identification of tilapia boule and dazl cDNAs. The predicted partial Boule contains a conserved RRM motif and Dazl has the C-terminal sequence. On a phylogenetic tree, tilapia Boule and Dazl are in separate clades of Boule and Dazl homologs from other species, indicating their divergence during early vertebrate evolution. By RT-PCR analysis, boule and dazl showed bisexual gonad-specific expression. By in situ hybridization analysis, both boule and dazl RNAs were restricted to female and male germ cells of adult gonads but absent in gonadal soma. In the ovary, boule and dazl RNAs were abundant in oocytes. In the testis, boule and dazl RNAs were prominent in meiotic spermatocytes but barely detectable in meiotic products. These data show that boule and dazl are expressed bisexually in germ cells and provide useful markers to study gametogenesis in the adult tilapia.     

Developmental regulation of the Drosophila tropomyosin I (TmI) gene is controlled by a muscle activator enhancer region that contains multiple cis-elements and binding sites for multiple proteins

Developmental gene regulation in vertebrate somatic muscles involves the cooperative interaction of MEF2 (myocyte-specific enhancer-binding factor 2) and members of the b-HLH (basic helix-loop-helix) family of myogenic factors. Until recently, however, nothing was known about the factors that control the developmental regulation of muscle genes during embryogenesis in Drosophila. The Drosophila Tropomyosin I (TmI) gene contains a proximal and distal muscle enhancer within the first intron that regulates its expression in embryonic/larval and adult muscles. We have recently shown that the 355-bp proximal enhancer contains a binding site for the Drosophila homologue of vertebrate MEF2 and that MEF2 acts cooperatively with a basal level muscle activator region to direct high level muscle expression in transgenic flies. The 92-bp muscle activator region, however, does not contain any consensus E-box (CANNTG) binding site sequences for b-HLH myogenic factors, suggesting the MEF2 may interact with other factors to regulate muscle genes in Drosophila. In this study we have used mutation analysis and germ-line transformation to analyze the cis-acting elements within the muscle activator region that regulate its expression in transgenic flies. We have identified a 71-bp region that is sufficient for low basal level temporal- and muscle-specific expression in the embryo, larva, and adult. Substitution mutations within the muscle activator region have identified several cis-element regions spanning 60-bp that are required for either full or partial muscle activator function. An analysis of proteins that bind to this region by gel mobility shift assay and copper nuclease footprinting has allowed us to identify the sites in this region at which multiple proteins complex and interact. We propose that these cis-elements and the proteins that they bind regulate muscle activator function and together with MEF2 are capable of regulating high level muscle expression. Dev. Genet. 20:297–306, 1997. © 1997 Wiley-Liss, Inc.