Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele.     

Identification of medaka magnetoreceptor and cryptochromes

Magnetoreception is a hallmark ability of animals for orientation and migration via sensing and utilizing geomagnetic fields. Magnetoreceptor (MagR) and cryptochromes (Cry) have recently been identified as the basis for magnetoreception in Drosophila. However, it has remained unknown whether MagR and Cry have conserved roles in diverse animals. Here we report the identification and expression of magr and cry genes in the fish medaka (Oryzias latipes). Cloning and sequencing identified a single magr gene, four cry genes and one cry-like gene in medaka. By sequence alignment, chromosomal synteny and gene structure analysis, medaka cry2 and magr were found to be the orthologs of human Cry2 and Magr, with cry1aa and cry1ab being coorthologs of human Cry1. Therefore, magr and cry2 have remained as single copy genes, whereas cry1 has undergone two rounds of gene duplication in medaka. Interestingly, magr and cry genes were detected in various stages throughout embryogenesis and displayed ubiquitous expression in adult organs rather than specific or preferential expression in neural organs such as brain and eye. Importantly, magr knockdown by morpholino did not produce visible abnormality in developing embryos, pointing to the possibility of producing viable magr knockouts in medaka as a vertebrate model for magnet biology.     

Effective control of yellow stem borer and rice leaf folder in transgenic rice indica varieties Basmati 370 and M 7 using the novel δ-endotoxin cry2A Bacillus thuringiensis gene

Transgenic rice indica varieties Basmati 370 and M 7 expressing the novel cry2A (Bt) insecticidal gene were generated by particle bombardment. Molecular and biochemical analyses in R0 and R1 populations confirmed stable integration and expression of this novel Bt transgene. We estimated that the gene product was expressed up to 5% of total leaf protein. Insect feeding bioassays demonstrated that the Cry2A protein was effective against the yellow stem borer and the rice leaf folder, two major rice pests in the Indian Subcontinent. This is the first report of the control of major rice pests using this specific Bt gene. The cry2A gene can now be used in combination with other insecticidal genes for pyramiding resistance against insect pests. This will delay, or perhaps in combination with integrated pest management practices, prevent evolution of insect populations resistant to single insecticidal genes.     

苏云金杆菌辅助蛋白P19对杀虫晶体蛋白Cry11Aa表达的影响

苏云金杆菌以色列亚种的p19基因、cry11Aa基因和p20基因位于同一操纵子上,据推测辅助蛋白P19可能与Cry11Aa蛋白的晶体化相关。本研究利用穿梭载体pHT3101构建了两个重组质粒pHcy1和pHcy3,两质粒均携带cry11Aa基因,但后者完全缺失了cry11Aa基因上游的p19基因。将重组质粒电激转化至苏云金杆菌无晶体突变株4Q7中进行蛋白表达,SDS-PAGE结果表明在4Q7(pHcy1)和4Q7(pHcy3)中均能检测到正常表达的Cry11Aa蛋白,但单位体积培养液的Cry11Aa蛋白在辅助蛋白P19存在时的表达量明显高于其单独表达的表达量;透射电镜观察显示两菌株中的Cry11Aa蛋白形成了大小相近、形状相似的双梯形晶体;另外,生物测定结果表明重组菌株4Q7(pHcy1)和4Q7(pHcy3)对三龄致倦库蚊的杀虫活性没有显著性差异。该现象说明辅助蛋白P19的缺失对Cry11Aa蛋白的晶体形成和杀蚊活性没有影响,但P19作为分子伴侣在一定程度上帮助提高了Cry11Aa蛋白的表达水平。     

The role of cryptochrome 2 in flowering in Arabidopsis

We have investigated the genetic interactions between cry2 and the various flowering pathways in relation to the regulation of flowering by photoperiod and vernalization. For this, we combined three alleles of CRY2, the wild-type CRY2-Landsberg erecta (Ler), a cry2 loss-of-function null allele, and the gain-of-function CRY2-Cape Verde Islands (Cvi), with mutants representing the various photoreceptors and flowering pathways. The analysis of CRY2 alleles combined with photoreceptor mutants showed that CRY2-Cvi could compensate the loss of phyA and cry1, also indicating that cry2 does not require functional phyA or cry1. The analysis of mutants of the photoperiod pathway showed epistasis of co and gi to the CRY2 alleles, indicating that cry2 needs the product of CO and GI genes to promote flowering. All double mutants of this pathway showed a photoperiod response very much reduced compared with Ler. In contrast, mutations in the autonomous pathway genes were additive to the CRY2 alleles, partially overcoming the effects of CRY2-Cvi and restoring day length responsiveness. The three CRY2 alleles were day length sensitive when combined with FRI-Sf2 and/or FLC-Sf2 genes, which could be reverted when the delay of flowering caused by FRI-Sf2 and FLC-Sf2 alleles was removed by vernalization. In addition, we looked at the expression of FLC and CRY2 genes and showed that CRY2 is negatively regulated by FLC. These results indicate an interaction between the photoperiod and the FLC-dependent pathways upstream to the common downstream targets of both pathways, SOC1 and FT.     

苏云金芽孢杆菌的cry2A芽孢期启动子和分子伴侣ORF1-ORF2对Cry11Aa蛋白表达的影响

[目的]分析苏云金芽孢杆菌的cry2A型芽孢期启动子对晶体蛋白Cry11Aa的协调作用和分子伴侣ORF1-ORF2对Cry11Aa表达的促进功能.[方法]3个包括cry11Aa编码区的重组质粒pHcy1、pHcy2和pHcy4被构建并电激转化到苏云金芽孢杆菌晶体缺陷株4Q7中,其中pHcy1质粒携带cry11Aa基因自身启动子和分子伴侣p19基因,pHcy2携带cry2A型芽孢期启动子和分子伴侣orf1-orf2基因,pHcy4质粒在pHcy1的上游插入了cry2A型芽孢期启动子和分子伴侣orf1-orf2基因.SDS-PAGE分析了Cry11Aa蛋白在各重组苏云金菌株中的表达情况,并通过生物测定确定了其对蚊虫的生物活性.[结果]SDS-PAGE结果表明,Cry11Aa蛋白在4Q7(pHcy1)和4QT(pHcy4)均获得了表达,在4Q7(pHcy2)中未检测到Cry11Aa蛋白,推测晶体蛋白Cry11A不能利用cry2A型启动子进行表达调控;Cry11Aa蛋白在等体积4Q7(pHcy4)培养液中的表达量是4Q7(pHcy1)菌株的1.25倍,暗示着分子伴侣ORF1-ORF2在某种程度上能提高Cry11Aa的蛋白表达量.4Q7(pHcy1)和4Q7(pHcy4)形成的Cry11Aa蛋白晶体的形状和大小相似,两者对致倦库蚊的生物活性没有明显差异,LC50s分别为59.33 ng/mL和66.21 ng/mL,.[结论]推测晶体蛋白Cry11A能否成功表达与其使用启动子的类型和两者的协调配合有关.分子伴侣ORF1-ORF2虽然在某种程度上能提高Cry11Aa的蛋白表达量,但对提高Cry11Aa蛋白的杀蚊毒力没有显著性帮助.     

An Improved PCR-Restriction Fragment Length Polymorphism (RFLP) Method for the Identification of cry1-Type Genes

The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.     

Cloning and expression of cryptochrome2 cDNA in the rat.

Cryptochromes (CRY) are blue-light photoreceptors that regulate the circadian rhythm in animals and plants. In mammals, two types of CRY are involved in the regulation of circadian rhythm, but rat cryptochromes have not yet been identified. Therefore, we isolated and characterized cry2 cDNA from the rat brain. The cloned rat cry2 cDNA consists of 2,131 nucleotides and has a single open-reading frame that encodes the rat CRY2 of 594 amino acids with start and stop codons. The deduced amino acid sequence of the rat CRY2 was 97% identical with that of mice and 93% with humans, but it showed a relatively low identity of 64% with that of zebrafish. It also exhibited a high homology (about 70%) with CRY1 of mice and humans. A Northern blot analysis showed that rat cry2 was expressed in all of the tissues examined. Rat cry2 was expressed at a relatively higher level in peripheral tissues than in the brain. In situ hybridization in the whole brain indicated that the strong signal of cry2 mRNA is mainly present in the suprachiasmatic nucleus (SCN) region, but very weak in other brain regions. Therefore, present results indicate that rat cry2 may function in circadian photoreception in the rat brain.     

Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content

Cryptochromes are blue light photoreceptors found in plants, bacteria, and animals. In Arabidopsis, cryptochrome 2 (cry2) is involved primarily in the control of flowering time and in photomorphogenesis under low-fluence light. No data on the function of cry2 are available in plants, apart from Arabidopsis (Arabidopsis thaliana). Expression of the tomato (Solanum lycopersicum) CRY2 gene was altered through a combination of transgenic overexpression and virus-induced gene silencing. Tomato CRY2 overexpressors show phenotypes similar to but distinct from their Arabidopsis counterparts (hypocotyl and internode shortening under both low- and high-fluence blue light), but also several novel ones, including a high-pigment phenotype, resulting in overproduction of anthocyanins and chlorophyll in leaves and of flavonoids and lycopene in fruits. The accumulation of lycopene in fruits is accompanied by the decreased expression of lycopene beta-cyclase genes. CRY2 overexpression causes an unexpected delay in flowering, observed under both short- and long-day conditions, and an increased outgrowth of axillary branches. Virus-induced gene silencing of CRY2 results in a reversion of leaf anthocyanin accumulation, of internode shortening, and of late flowering in CRY2-overexpressing plants, whereas in wild-type plants it causes a minor internode elongation.     

Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light-dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light-dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light-induced phosphorylation. These results demonstrate that blue light-dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.     

Expression of a recombinant Cry1Ac crystal protein fused with a green fluorescent protein in Bacillus thuringiensis subsp. kurstaki Cry-B

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis Cry(-)B strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the cry1Ac gene (pProAc-GFP). The B. thuringiensis Cry(-)B strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immunoblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single species, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis Cry(-)B strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuringiensis.     

拟南芥中光信号系统中关键基因CRY1、CRY2和COP1启动子的表达模式分析

通过构建表达光信号系统关键基因CRY1、CRY2和COP1启动子与GUS融合基因的拟南芥转基因植株,并对转基因植株进行GUS组织化学染色的结果表明,CRY1、CRY2和COP1的表达模式不受光条件的调控,并且在各器官有广泛的表达。分别分析CRY1基因启动子在cop1突变体以及COP1基因启动子在cry1突变体遗传背景中表达模式的结果表明,CRY1和COP1在转录水平上不存在明显的相互调控关系。