Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida

Chalcone synthase-encoding genes (chs) in Petunia hybrida comprise a multigene family. Some of the chs genes have been grouped into a subfamily, based upon their strong cross-hybridization and tight genomic linkage. From genomic libraries eight 'complete' chs genes, two chs gene 5'-fragments and two chs gene 3'-fragments have been isolated. The nucleotide sequence of six complete chs genes is presented and discussed in relation to their evolutionary origin and expression in different tissues. Each member of the family consists of two exons separated by an intron of variable size and sequence, which is located at a conserved position. The chs gene fragments represent single exons. Homology between non-linked chs genes is approx. 80% at the DNA level and restricted to protein-coding sequences. Homology between subfamily members (which are tightly linked) is higher (90-99%) and extends into untranslated regions of the gene, strengthening the view that they arose by recent gene duplications. The chsD gene contains a mutated translation stop codon, suggesting that this is an inactive (pseudo)gene. None of the other members of the gene family exhibits characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must have been a recent event. Homology at the protein level between some (expressed) chs genes is surprisingly low. The possibility that these genes encode proteins with slightly different enzymatic activities is discussed.     

Cloning and characterization of a novel chalcone synthase gene from Phalaenopsis hybrida orchid flowers

Chalcone synthase (CHS, EC 2.3.1.74) is the key enzyme involved in flavonoid and anthocyanin biosynthesis. A complete DNA sequence of chalcone synthase gene designated Pchs1 was isolated by means of usual and then inverse polymerase chain reactions from genomic DNA of an orchid, Phalaenopsis hybrida, cv. Formosa rose. Nucleotide sequence analysis based on alignment with published Phalaenopsis chs cDNA revealed that Pchs1 contained an intact open reading frame of 1173-bp with one 109-bp intron at the conserved site. The deduced polypeptide (PCHS1) from Pchs1 comprised 390 amino acids with a predicted mol wt of 42.5 kD. PCHS1 showed 61–65% identities with CHS from other plants and retained most of the conserved residues. Some putative cis-regulatory elements were present at the 5′ and 3′ flanking regions of Pchs1. Southern blot analysis predicted at least four chs-like genes, thus indicating the presence of a small multigene chs family in P. hybrida. Relative quantitative RT-PCR showed that Pchs1 is expressed in petals at early flower development as well as in lip tissue when the flower has just opened. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 250–258. The text was submitted by the authors in English.     

Cloning and characterization of chalcone synthase from the moss, Physcomitrella patens

Since the early evolution of land plants from primitive green algae, flavonoids have played an important role as UV protective pigments in plants. Flavonoids occur in liverworts and mosses, and the first committed step in the flavonoid biosynthesis is catalyzed by chalcone synthase (CHS). Although higher plant CHSs have been extensively studied, little information is available on the enzymes from bryophytes. Here we report the cloning and characterization of CHS from the moss, Physcomitrella patens. Taking advantage of the available P. patens EST sequences, a CHS (PpCHS) was cloned from the gametophores of P. patens, and heterologously expressed in Escherichia coli. PpCHS exhibited similar kinetic properties and substrate preference profile to those of higher plant CHS. p-Coumaroyl-CoA was the most preferred substrate, suggesting that PpCHS is a naringenin chalcone producing CHS. Consistent with the evolutionary position of the moss, phylogenetic analysis placed PpCHS at the base of the plant CHS clade, next to the microorganism CHS-like gene products. Therefore, PpCHS likely represents a modern day version of one of the oldest CHSs that appeared on earth. Further, sequence analysis of the P. patens EST and genome databases revealed the presence of a CHS multigene family in the moss as well as the 3'-end heterogeneity of a CHS gene. Of the 19 putative CHS genes, 10 genes are expressed and have corresponding ESTs in the databases. A possibility of the functional divergence of the multiple CHS genes in the moss is discussed.     

Organization of soybean chalcone synthase gene clusters and characterization of a new member of the family

Chalcone synthase (CHS; EC 2.3.1.74), the first committed enzyme of the multibranched pathway of flavonoid/isoflavonoid biosynthesis is encoded by a multigene family in soybean, (Glycine max L. Merrill). Our results suggest that this gene family comprises at least seven members, some of which are clustered. We have identified four chs clusters in the allo-tetraploid G. max genome and chs5, a newly characterized member of the chs gene family is present in two of them. We describe the complete nucleotide sequence of chs5, the identification of its immediate neighbors and the organization of the four hitherto identified chs clusters in the Gm genome.     

Cloning and analysis of two genes for chalcone synthase from Petunia hybrida

Summary With the help of a cDNA probe for a chalcone synthase gene of Petroselinum a cDNA clone for a chalcone synthase gene of Petunia hybrida could be identified. The homologous cDNA allowed the cloning of two genomic EcoRI fragments from Petunia hybrida containing complete chalcone synthase genes. It could be demonstrated that the genes on the two fragments are different and are not allelic but members of a gene family. The two genes are found in a variety of different Petunia lines including in the two conditional mutants affected in chalcone synthase expression in floral buds, White Joy and Red Star. The structure of the two chs genes from Petunia is compared to the chs gene from Antirrhinum majus.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

化州柚查尔酮合成酶基因克隆与序列分析

利用CTAB-LiCl法提取高质量的化州柚总RNA,采用RT-PCR技术克隆查尔酮合成酶基因,获得广东道地药材化橘红资源化州柚的查尔酮合成酶基因。该基因编码区全长1176bp,编码391个氨基酸残基,与同样来源于柑橘属的查尔酮合成酶基因同源性高达98%。CTAB-LiCl法能提取高质量的化州柚总RNA,可以用于后续基因克隆和分析;克隆获得的查尔酮合成酶具有编码区,与同属植物相同基因具有高度序列同源性。     

Genome-wide identification and phylogenetic analysis of the chalcone synthase gene family in rice

Journal of Plant Research - The enzymes of the chalcone synthase family are also known as type III polyketide synthases (PKS), and produce a series of secondary metabolites in bacteria, fungi and...     

水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化,对纯化蛋白进行酶活检测,结果表明融合蛋白具有查尔酮合酶活性,可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。     

Cloning and characterization of the gene for phosphatidylcholine synthase

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the CDP-choline pathway or the methylation pathway. In prokaryotes only the methylation pathway was thought to occur. Recently, however, we could demonstrate (de Rudder, K. E. E., Sohlenkamp, C., and Geiger, O. (1999) J. Biol. Chem. 274, 20011-20016) that a second pathway for phosphatidylcholine biosynthesis exists in Sinorhizobium (Rhizobium) meliloti involving a novel enzymatic activity, phosphatidylcholine synthase, that condenses choline and CDP-diacylglyceride in one step to form PC and CMP. Using a colony autoradiography method we have isolated mutants of S. meliloti deficient in phosphatidylcholine synthase and which are no longer able to incorporate radiolabeled choline into PC. Complementation of such mutants with a sinorhizobial cosmid gene bank, subcloning of the complementing fragment, and sequencing of the subclone led to the identification of a gene coding for a presumptive CDP-alcohol phosphatidyltransferase. Amplification of this gene and its expression in Escherichia coli demonstrates that it codes for phosphatidylcholine synthase. Genomes of some pathogens (Pseudomonas aeruginosa and Borrelia burgdorferi) contain genes similar to the sinorhizobial gene (pcs) for phosphatidylcholine synthase. Although pcs-deficient S. meliloti knock-out mutants show wild type-like growth and lipid composition, they are unable to perform rapid PC biosynthesis that normally is achieved via the phosphatidylcholine synthase pathway in S. meliloti wild type.     

甜荞查尔酮合酶基因Chs的克隆及序列分析

利用RT-PCR技术从甜荞中克隆得到查耳酮合酶(CHS)的cDNA开放阅读框(ORF)序列,命名为FeChs,NCBI登录号为GU172166.1.该序列长1 179 bp,编码392个氨基酸,与其它植物CHS基因的同源性为78%～92%,其推导的氨基酸序列含有CHS高度保守的活性位点及CHS的标签序列GFGPG.     

Cloning and characterization of chitin synthase gene fragments from Penicillium chrysogenum

Abstract We constructed a 3'-directed cDNA library of cleistothecia and Hülle cells of Aspergillus nidulans to examine gene expression patterns of the sexual structures and to have probes necessary to isolate sexual structure-specific genes. Sequencing of 360 randomly selected cDNA clones yielded 272 expressed sequence tags (ESTs), most of which probably represent frequently or less expressed genes in sexual structures of A. nidulans . Among the 272 ESTs, 33 ESTs (87 cDNA clones) appeared more than once and 2 ESTs appeared 6 times; 9 ESTs matched GenBank entries. When compared with sequences obtained from a mycelial 3'-directed cDNA library of A. nidulans , 28 out of 33 ESTs seem to be sexual structure-specific. Northern blot analyses of 20 ESTs showed that 17 are sexual structure-specific. The remaining three ESTs also hybridized with RNA isolated from vegetative mycelia. These results suggest that analyses of ESTs from different cell types or tissues can readily demonstrate gene expression patterns of specific cell types and identify cell type-specific cDNA probes.     

Molecular characterization of the chalcone isomerase gene family in Deschampsia antarctica

Deschampsia antarctica (Poaceae) is one of the two vascular plants known to have colonized the Antarctic region. Studies examining the biosynthesis of flavonoids, compounds which plants use, for example, for protection against overexposure to UV light or as antioxidants that scavenge free radicals and other oxidative species, in D. antarctica may provide clues to its success in that extreme environment. We characterized the family of genes encoding chalcone isomerase (CHI EC 5.5.1.6), an important enzyme involved in flavonoid biosynthesis, in D. antarctica. Sequence analysis of the three family members revealed differences in numbers of introns and lengths of coding regions among the three and suggest that DaCHI3 is likely a pseudogene (ψDaChi2). Salinity stress resulted in differential mRNA expression of the DaCHI genes with ψDaCHI2 exhibiting the earliest response (3-h post-treatment), induced by as much as sevenfold, while DaCHI1 and DaCHI2 mRNAs accumulated later (3d and 5d post-treatment, respectively) and, in the case of DaCHI2, with a response of nearly sixfold. We discuss how differences in the proposed gene structures, deduced protein characteristics, and mRNA expression patterns suggest that the members of this gene family may have unique functions in the phenylpropanoid pathway in D. antarctica.     

Transformation of antisense constructs of the chalcone synthase gene superfamily into Gerbera hybrida: differential effect on the expression of family members

Suppression of gene expression using antisense technology has been successful in various applications. In this paper we report differential inhibition of gene expression of the chalcone synthase (chs) gene superfamily members in transgenic Gerbera hybrida (Asteraceae) plants. We have transformed two different cDNAs of the chs gene family, gchs 1 [4] and gchs2, in antisense orientation under control of the CaMV 35S promoter into gerbera. Gchs1 codes for an enzyme with chalcone synthase activity while gchs2 is a more diverged member of the gene family having distinct structure and expression pattern. Furthermore, gchs2 is evidently not involved in anthocyanin synthesis and encodes an enzyme with novel catalytic properties. In both cases effective blocking of the resident sense gene expression was detected. In addition, the transformation affected differentially the expression of other members of the chs gene family. The degree of inhibition appeared to depend on the sequence homology between the antisense and the target genes. In the unevenly coloured inflorescences detected among anti-gchs1 transformants during their growth, relaxation of the antisense effect was here shown to start from the most distant member of the gene family, further demonstrating the influence of sequence homology in the stability of antisense inhibition.     

All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity

Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase K_m and V_max for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.     

Cloning and characterization of additional members of the G protein-coupled receptor family

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.