Six actin gene subfamilies map to five chromosomes of Petunia hybrida

The Mitchell variety of Petunia hybrida possesses a superfamily of actin genes which contains between 100 and 200 members that can be divided into at least six highly divergent subfamilies. The segregation of restriction fragment length polymorphisms among 96 plants from two backcrosses between the Violet 23 and Red 51 Petunia varieties and the Violet 23 x Red 51 hybrid was examined using gene-specific probes from six Petunia actin gene subfamilies. These data were compared with the genotypes of each plant at 11 marker loci which are distributed among the seven chromosomes of Petunia and which determine flower, pollen, and isozyme phenotypes. From these analyses, members of these six actin gene subfamilies were mapped to five locations on five Petunia chromosomes: the PAc9, PAc1, PAc4, and PAc2 subfamilies are on chromosomes I, II, III, and VII respectively; the PAc3 and PAc7 subfamilies are tightly linked on chromosome IV. All members of the PAc4 subfamily cosegregated as a cluster of genes. These data are discussed regarding gene amplification in plants.     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

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     

Molecular characterization of the rbcS multi-gene family of Petunia (Mitchell)

Summary The small subunit (RbcS) of ribulose bisphosphate carboxylase (RuBPCase) is encoded by eight genes in Petunia (Mitchell). These genes can be divided into three subfamilies (51, 117 and 71) based upon hybridization to three petunia rbcS cDNA clones. The nucleotide sequence of six of the eight petunia rbcS genes is presented here and the structure of the genes is discussed with respect to their genomic linkage and their expression levels in petunia leaf tissue. The rbcS genes belonging to the same subfamily encode an identical mature RbcS polypeptide, however the different subfamilies encode distinguishable polypeptides. All the genes, except one, contian two introns within the mature subunit coding region; one gene contains one extra intron within the coding region. There are large regions of nucleotide sequence homology within the introns of genes within a subfamily, but significantly less homology between the introns of genes of different subfamilies. A complex pattern of homology within the multiple genes of the 51 subfamily is observed. There are regions within these genes which share high levels of sequence homology; this homology does not extend throughout the whole gene and the regions of homology do not always occur in adjacent genes. Two 3 rbcS gene fragments which we isolated from the petunia genome show high levels of homology to two of the intact rbcS genes.     

Structure,expression, chromosomal location and product of the gene encoding ADH1 inPetunia

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated fromPetunia hybrida cv. V30 by screening aPetunia genomic library with a maizeAdh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of twoAdh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay inPetunia protoplasts. We have designated this genePetunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction ofAdh1 mRNA.     

Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression

In order to scan the 5 flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5 end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from –67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from –142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (–59 and –52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.     

Evolution of thesrc-related protein tyrosine kinases

A phylogenetic analysis ofsrc-related protein tyrosine kinases (PTKs) showed that one group of these genes is quite ancient in the animals, its divergence predating the divergence of the diploblast and triploblast phyla. Three other major groupings of genes were found to predate the divergence of protostome and deuterostome phyla. Most knownsrc-related PTKs of mammals were found to belong to five well-differentiated families: srcA, srcB, abl, csk, and tec. One srcA gene (fyn) has an alternatively spliced seventh exon which shows a different pattern of relationship from the remainder of the gene; this suggests that this exon may have been derived by a recombinational event with another gene, perhaps one related tofgr. The recently published claim that mammalian members of this family expressed in the nervous system evolve more slowly at nonsynonymous nucleotide sites than do those expressed in the immune system was not supported by an analysis of 13 pairs of human and mouse orthologues. Rather, T-cell-specificsrc-related PTKs were found to have higher rates of nonsynonymous substitution than were those having broader expression. This effect was particularly marked in the peptide binding site of the SH2 domain. While the SH2 binding site was highly conserved among paralogous mammalian members of the srcA and srcB subfamilies, no such effect was seen in the comparison of paralogous members of the csk and tec subfamilies. This suggests that, while the peptide binding function of SH2 is conserved within both srcA and srcB subfamilies, paralogous members of the csk and tec subfamilies have diverged functionally with respect to peptide recognition by SH2.     

Insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins

Summary Invertebrate actins resemble vertebrate cytoplasmic actins, and the distinction between muscle and cytoplasmic actins in invertebrates is not well established as for vertebrate actins. However, Bombyx and Drosophila have actin genes specifically expressed in muscles. To investigate if the distinction between muscle and cytoplasmic actins evidenced by gene expression analysis is related to the sequence of corresponding genes, we compare the sequences of actin genes of these two insect species and of other Metazoa. We find that insect muscle actins form a family of related proteins characterized by about 10 muscle-specific amino acids. Insect muscle actins have clearly diverged from cytoplasmic actins and form a monophyletic group emerging from a cluster of closely related proteins including insect and vertebrate cytoplasmic actins and actins of mollusc, cestode, and nematode. We propose that muscle-specific actin genes have appeared independently at least twice during the evolution of animals: insect muscle actin genes have emerged from an ancestral cytoplasmic actin gene within the arthropod phylum, whereas vertebrate muscle actin genes evolved within the chordate lineage as previously described.Offprint requests to.: N. Mounier     

Diversity of secondary endosymbiont-derived actin-coding genes in cryptomonads and their evolutionary implications

In the secondary endosymbiotic organisms of cryptomonads, the symbiont actin genes have been found together with the host one. To examine whether they are commonly conserved and where they are encoded, host and symbiont actin genes from Pyrenomonas helgolandii were isolated, and their specific and homologous regions were digoxigenin (DIG) labeled separately. Using these probes, Southern hybridization was performed on 13 species of cryptomonads. They were divided into three groups: (1) both host and symbiont actin gene signals were detected, (2) only the host actin gene signal was detected, and (3) host and unknown actin signals were detected. The phylogenetic analysis of these actin gene sequences indicated that the evolutionary rates of the symbiont actin genes were accelerated more than those of the hosts. The unknown actin signals were recognized as the highly diverged symbiont actin genes. One of the diverged symbiont actin sequences from Guillardia theta is presumed to be as a pseudogene or to its precursor. Southern hybridizations based on the samples divided by pulsed-field gel electrophoresis showed that all actin genes were encoded by the host nuclei. These results possibly represent the evolutionary fate of the symbiont actin gene in cryptomonads, which was firstly transferred from the symbiont nucleus or nucleomorph, to the host nucleus and became a pseudogene and then finally disappeared there.     

The chalcone synthase multigene family of Petunia hybrida (V30): differential,light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.     

Independent gene evolution in the potato actin gene family demonstrated by phylogenetic procedures for resolving gene conversions and the phylogeny of angiosperm actin genes

Summary Nine different actin DNA sequences were isolated from the common potato,Solanum tuberosum, and the nucleotide sequence of five actin loci and of two allelic variants are presented. Unlike the wide variation in intron position among animal actin genes, the potato actin genes have three introns situated in the same positions as reported for all other angiosperm actin genes. Using a novel combination of analytical procedures (G-test and compatibility analysis), we could not find evidence of frequent large or small nonreciprocal exchanges of genetic material between the sequenced loci, although there were a few candidates. Resolution of such gene conversion events and the quantification of independence of gene evolution in multigene families is critical to the inference of phylogenetic relationships. Comparison with actin genes in other angiosperm species suggests that the actin multigene family can be divided into a number of subfamilies, evolved by descent rather than gene conversion, which are of possible functional origin, with one major subfamily diversification occurring before the divergence of monocots and dicots. The silent rate of nucleotide substitution was estimated to be similar to that suggested for a number of other plant nuclear genes, whereas the replacement rate was extremely slow, suggestive of selective constraints.     

芥菜WOX基因家族的全基因组鉴定与分析

WUSCHEL相关-同源盒(WUSCHEL related-homeobox, WOX)基因家族是一类植物特有的转录因子基因家族,在植物的生长发育过程中发挥重要作用。本研究利用芥菜(Brassica juncea)基因组数据,通过HUMMER、Smart等软件进行检索筛选,共鉴定出51个WOX基因家族成员。利用Expasy在线软件对这些家族成员的蛋白质分子量、氨基酸序列长度、等电点等进行分析,并利用生物信息学软件对芥菜WOX基因家族进化关系、保守区域、基因结构等进行系统性分析,将芥菜WOX基因家族分为古老支、中间支和WUS支/现代支3个亚家族。结构分析表明,同一亚家族内的WOX转录因子家族成员的保守结构域的种类、组织形式以及基因结构具有高度的一致性,而不同亚家族之间呈现一定的多样性。51个WOX基因不均匀分布于芥菜18条染色体上,这些基因的启动子大多含有响应光、激素和非生物逆境胁迫相关的顺式作用元件。利用转录组数据和实时荧光定量PCR (real-time fluorescence quantitative PCR, qRT-PCR)分析发现,芥菜WOX基因的表达具有时空特异性,其中B...     

Complex patterns of actin molecular evolution in the red alga Stylonema alsidii (Stylonematophyceae,Rhodophyta)

The Stylonematophyceae is an early diverging red algal class that contains organisms in which sexual reproduction has not been reported. Previous studies have hypothesized a single copy of the actin gene in this class. Amplification of the actin gene in members of the Stylonematophyceae, including several isolates of Stylonema alsidii, reveals a more complex evolutionary history. The data support independent gene duplications in Goniotrichopsis reniformis and Stylonema alsidii. Three of the seven isolates of S. alsidii had three copies of actin. Analyses indicate that all copies are functional and not pseudogenes. Due to our lack of knowledge of the ploidy level in Stylonema and the asexual nature of these organisms, an ancient change in ploidy level, producing diploid or even triploid organisms, cannot be ruled out to explain two copies of actin within these organisms. Within Stylonema alsidii two of the actin copies (Groups 1 and 2) show similar levels of variation; p‐distances and the number of synonymous and non‐synonymous sites are roughly the same. Yet the changes are distributed differently. One group showed little shared variation among the samples, mutations therefore represent autoapomorphies, while variation in the other group is usually shared among samples (parsimony‐informative). Variation in the other group is usually shared among samples (parsimony‐informative). A third group of actin genes also observed in S. alsidii are highly divergent from the other two copies, yet they maintain all of the signatures of functional proteins. Our data reveal a complex evolutionary history of the actin gene in these species and highlight the lack of knowledge into the basic biology (ploidy level, life cycle characteristics) of this class.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

Increased expression of a plant actin gene during a biotrophic interaction between round-leaved mallow, Malva pusilla, and Colletotrichum gloeosporioides f. sp. malvae

Two actin genes, actA from the hemibiotrophic anthracnose fungus, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. malvae, and act1 from its host, Malva pusilla (Sm.) were cloned from a cDNA library developed from infected host tissue. The actin gene, actA, of C. gloeosporioides f. sp. malvae, which is similar to that of other euascomycetes, appears to be expressed constitutively. The actin gene of M. pusilla is most similar to one of the actin genes of Arabidopsis thaliana that is unique in being responsive to environmental stimuli such as wounding. Expression of actA was used to follow the growth of the fungus in the plant tissue. Low actA expression occurred until 72–96 h after inoculation and then increased rapidly, corresponding with the timing of the shift from slower biotrophic fungal growth to much more rapid necrotrophic growth. In contrast, expression of act1 approximately doubled during the biotrophic phase and then rapidly declined during the necrotrophic phase. Increased host actin expression could be due to host cytoskeleton rearrangement in response to biotrophic infection, and the subsequent decrease in host actin expression could be due to host cell disruption resulting from tissue maceration during necrosis. This is the first report of a host actin gene that can increase in expression during a compatible plant-pathogen interaction. Received: 15 March 1999 / Accepted: 1 May 1999     

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST 2 and RAT 1 genes of Saccharomyces cerevisiae

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.