Ectopic expression of the<Emphasis Type="Italic"> Suppressor of Underreplication</Emphasis> gene inhibits endocycles but not the mitotic cell cycle in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.Communicated by G. P. Georgiev     

Members of the<Emphasis Type="Italic"> S</Emphasis>-receptor kinase multigene family in<Emphasis Type="Italic"> Senecio squalidus</Emphasis> L. (Asteraceae), a species with sporophytic self-incompatibility

While the molecular basis of sporophytic self-incompatibility (SSI) has been investigated extensively in the Brassicaceae, almost nothing is known about the molecular regulation of SSI in other families, such as the Asteraceae. In species of Brassica and in Arabidopsis lyrata, a stigma-specific serine-threonine receptor kinase (SRK) and its cognate ligand, a pollen coating-borne cysteine-rich protein (SCR/SP11), determine the female and male sides of the SSI response, respectively. Here we have used RT-PCR with degenerate primers to conserved regions of SRK to amplify three SRK-like gene fragments expressed in stigmas of Senecio squalidus (Asteraceae). The Senecio S-receptor-like kinase (SSRLK) sequences share ~43% amino acid sequence identity with Brassica SRK₃ but higher amino acid sequence identity (~50%) with two Solanum bulbocastanum receptor-like kinase genes of unknown function. Despite expression in stigmas, all three SSRLKs were expressed at varying levels in floral and vegetative tissues. No allelic polymorphism was detected for the three SSRLKs in two S homozygous lines of S. squalidus or three other lines of S. squalidus carrying different S alleles. A full-length cDNA clone was obtained for SSRLK1 and its predicted amino acid sequence revealed significant structural differences to Brassica SRKs, most notably a major N-terminal truncation of 169 amino acids and the presence of just 8 conserved cysteine residues within the putative receptor domain instead of 12. Together, the sequence characteristics and expression characteristics of SSRLKs suggest that they are unlikely to be directly involved in the SSI response of S. squalidus. These findings are discussed in terms of the evolution of the SRK multigene family and the molecular basis of SSI in S. squalidus and the Asteraceae.     

Characterization of candidate class A,B and E floral homeotic genes from the perianthless basal angiosperm <Emphasis Type="Italic">Chloranthus spicatus</Emphasis> (Chloranthaceae)

The classic ABC model explains the activities of each class of floral homeotic genes in specifying the identity of floral organs. Thus, changes in these genes may underlay the origin of floral diversity during evolution. In this study, three MADS-box genes were isolated from the perianthless basal angiosperm Chloranthus spicatus. Sequence and phylogenetic analyses revealed that they are AP1-like, AP3-like and SEP3-like genes, and hence these genes were termed CsAP1, CsAP3 and CsSEP3, respectively. Due to these assignments, they represent candidate class A, class B and class E genes, respectively. Expression patterns suggest that the CsAP1, CsAP3 and CsSEP3 genes function during flower development of C. spicatus. CsAP1 is expressed broadly in the flower, which may reflect the ancestral function of SQUA-like genes in the specification of inflorescence and floral meristems rather than in patterning of the flower. CsAP3 is exclusively expressed in male floral organs, providing the evidence that AP3-like genes have ancestral function in differentiation between male and female reproductive organs. CsSEP3 expression is not detectable in spike meristems, but its mRNA accumulates throughout the flower, supporting the view that SEP-like genes have conserved expression pattern and function throughout angiosperm. Studies of synonymous vs nonsynonymous nucleotide substitutions indicate that these genes have not evolved under changes in evolutionary forces. All the data above suggest that the genes may have maintained at least some ancestral functions despite the lack of perianth in the flowers of C. spicatus. Nucleotide sequences data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY316311, AY397762 and AY379963.     

Protease inhibitors clitocypin and macrocypin are differentially expressed within basidiomycete fruiting bodies

Clitocypin and macrocypin are cysteine protease inhibitors of the mycocypin family which is unique to basidiomycetes. We have established that Clitocybe nebularis and Macrolepiota procera each contain genes for both macrocypin and clitocypin. Both are expressed in M. procera but only clitocypin in C. nebularis. Further analysis of mycocypin expression at the mRNA and protein levels in mature fruiting bodies of M. procera revealed that clitocypin is expressed evenly throughout the fruiting body, while the level of expression of macrocypins varies, and, at the protein level, is much higher in the veil fragments and the ring. The expression patterns of various mycocypins were determined in Coprinopsis cinerea, using promoters linked to a reporter gene. The expression profile of the clitocypin promoter was similar to that of the constitutive promoter gpdII from Agaricus bisporus, while that of the macrocypin 4 promoter was limited to the outer edges of the fruiting body throughout development. In addition, the activity of the macrocypin 3 promoter was different, indicating different regulation of expression for different macrocypin genes. The complex, tissue specific expression patterns for mycocypin genes suggest different biological roles for the products, either in regulation of endogenous proteases or in defense against pathogens or predators.     

Chalcone synthase-like gene in the liverwort, <Emphasis Type="Italic">Marchantia paleacea</Emphasis> var. <Emphasis Type="Italic">diptera</Emphasis>

A chalcone synthase (CHS)-like gene, MpCHSLK1, was isolated from liverwort, Marchantia paleacea var. diptera. Phylogenetic analysis revealed that MpCHSLK1 is closely related to stilbene synthase of the whisk fern, Psilotum nudum. Southern blot analysis using an MpCHSLK1 probe revealed that the gene belongs to a small gene family. Northern blot analysis indicated that CHS-like genes were expressed in either the mother plants or photoautotrophic cells. In photoautotrophic cells, the CHS-like genes were expressed light-dependently, and this expression was completely inhibited by the photosynthetic electron transport inhibitor, DCMU.Abbreviations CHS Chalcone synthase - DCMU 3-(3,4-Dichlorophenyl)-1-1-dimethylurea - POR Protochlorophyllide oxidoreductase - STS Stilbene synthase     

The Drosophila gene zfh2 is required to establish proximal-distal domains in the wing disc

Three main events characterize the development of the proximal-distal axis of the Drosophila wing disc: first, generation of nested circular domains defined by different combinations of gene expression; second, activation of wingless (wg) gene expression in a ring of cells; and third, an increase of cell number in each domain in response to Wg. The mechanisms by which these domains of gene expression are established and maintained are unknown. We have analyzed the role of the gene zinc finger homeodomain 2 (zfh2). We report that in discs lacking zfh2 the limits of the expression domains of the genes tsh, nub, rn, dve and nab coincide, and expression of wg in the wing hinge, is lost. We show that zfh2 expression is delimited distally by Vg, Nub and Dpp signalling, and proximally by Tsh and Dpp. Distal repression of zfh2 permits activation of nab in the wing blade and wg in the wing hinge. We suggest that the proximal-most wing fate, the hinge, is specified first and that later repression of zfh2 permits specification of the distal-most fate, the wing blade. We propose that proximal-distal axis development is achieved by a combination of two strategies: on one hand a process involving proximal to distal specification, with the wing hinge specified first followed later by the distal wing blade; on the other hand, early specification of the proximal-distal domains by different combinations of gene expression. The results we present here indicate that Zfh2 plays a critical role in both processes.     

The transmembrane protein,Tincar, is involved in the development of the compound eye in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We previously cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. Here, we have studied the expression pattern and functions of tinc during developmental processes. tinc mRNA is expressed in the central and peripheral nervous systems, and midgut during embryogenesis. In the third-instar larval eye disc, tinc mRNA is strongly expressed in all the differentiating ommatidial cells within and in the vicinity of the morphogenetic furrow. Loss-of-function analysis using the RNA-interference method revealed severe defects of eye morphogenesis during the late developmental stages. Our results suggested that tinc may have an indispensable role in the normal differentiation of ommatidial cells.Edited by C. Desplan     

<Emphasis Type="Italic">OsMADS22</Emphasis>, an <Emphasis Type="Italic">STMADS11</Emphasis>-like MADS-box gene of rice,is expressed in non-vegetative tissues and its ectopic expression induces spikelet meristem indeterminacy

We report the cDNA sequence and gene expression patterns of OsMADS22, a novel member of the STMADS11-like family of MADS-box genes, from rice. In contrast to previously reported STMADS11-like genes, whose expression is detected in vegetative tissues, OsMADS22 is mainly expressed during embryogenesis and flower development. In situ hybridization analysis revealed that OsMADS22 expression is localized in the L1 layer of embryos and in developing stamen primordia. Ectopic expression of OsMADS22 in transgenic rice plants resulted in aberrant floral morphogenesis, characterized by a disorganized palea, an elongated glume, and a two-floret spikelet. The results are discussed in terms of rice spikelet development and a novel non-vegetative role for a STMADS11-like gene.     

Molecular evolutionary analysis of the widespread<Emphasis Type="Italic"> piggyBac</Emphasis> transposon family and related "domesticated" sequences

piggyBac is a short inverted-repeat-type DNA transposable element originally isolated from the genome of the moth Trichoplusia ni. It is currently the gene vector of choice for the transformation of various insect species. A few sequences with similarity to piggyBac have previously been identified from organisms such as humans ( Looper), the pufferfish Takifugu rubripes (Pigibaku), Xenopus (Tx), Daphnia (Pokey), and the Oriental fruit fly Bactrocera dorsalis. We have now identified 50 piggyBac-like sequences from publicly available genome sequences and expressed sequence tags (ESTs). This survey allows the first comparative examination of the distinctive piggyBac transposase, suggesting that it might contain a highly divergent DDD domain, comparable to the widespread DDE domain found in many DNA transposases and retroviral integrases which consists of two absolutely conserved aspartic acids separated by about 70 amino acids with a highly conserved glutamic acid about 35 amino acids further away. Many piggyBac-like sequences were found in the genomes of a phylogenetically diverse range of organisms including fungi, plants, insects, crustaceans, urochordates, amphibians, fishes and mammals. Also, several instances of "domestication" of the piggyBac transposase sequence by the host genome for cellular functions were identified. Novel members of the piggyBac family may be useful in genetic engineering of many organisms.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Comparative genomics of yeast species: new insights into their biology

The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of DNA. However, gene function is much more conserved along evolution. Compared to S. cerevisiae and S. pombe, C. albicans has a larger number of specific genes, i.e., genes not found in other organisms, a fact that can account for the biological characteristics of this pathogenic dimorphic yeast which is able to colonize a large variety of environments.     

A conserved trans-regulatory landscape for scute expression on the notum of cyclorraphous Diptera

Bristles on the notum of many cyclorraphous flies are arranged into species-specific stereotyped patterns. The positions of bristles correlate with differences in the spatial expression of the scute (sc) gene in those species examined so far. However, a major upstream activator of scute, Pannier (Pnr), is expressed in a conserved domain over the entire medial notum. Here we examine the expression patterns in Calliphora vicina of stripe (sr), u-shaped (ush), caupolican (caup) and wingless (wg), genes known to modify the activity of Pnr or to act downstream of Pnr in Drosophila. We find that, with minor differences, their expression patterns are conserved. This suggests that the function of a trans-regulatory network of genes is relatively unchanged in derived Diptera and that many differences are likely to be due to changes in cis-regulatory sequences of scute.     

Identification and characterization of two rice autophagy associated genes, OsAtg8 and OsAtg4

Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has begun to be elucidated at the cellular level. A genome-wide search revealed significant conservation among autophagy genes in yeast and Arabidopsis. Up till now, however, there is no report about rice autophagy associated genes. Here we cloned OsAtg8 and OsAtg4 from Oryza sativa and detected their expression patterns in various tissues. Immunoblotting analysis showed that carboxyl terminus of OsAtg8 can be cleaved in yeast cell. Mutation analysis revealed that the conserved Gly117 residue of OsAtg8 was essential for its characteristic C-terminal cleavage as similar to that found in mammalian and yeast Atg8. We further proved that OsAtg8 interacted with OsAtg4, and this interaction was not affected by the conserved Gly117 mutation. Our results demonstrate that Atg8 conjugation pathway is conserved in rice and may play important roles in rice autophagy.     

毛白杨蔗糖合酶基因PtSS2克隆与表达分析

为了解毛白杨蔗糖合酶(sucrose synthase,SS)基因功能和表达模式,以毛白杨茎段来源的cDNA为模板,根据毛果杨PtrSS2CDS序列信息设计特异引物,采用RT-PCR技术分离克隆了PtSS2基因。测序分析表明,PtSS2基因序列全长为2 412bp,编码803个氨基酸,蛋白大小为92.14kD,理论等电点6.00,属于酸性蛋白;氨基酸序列同源性分析表明,PtSS2与毛果杨PtrSS2和PtrSS1一致性分别高达100%和98.63%;结构域分析表明,PtSS2含有2个高度保守的功能域,即蔗糖合酶(5~552)和糖基化转移酶(554~744)功能域。qRT-PCR检测表明,PtSS2在毛白杨根、茎、叶、营养芽、雄雌花芽等各个组织中均有表达,但在营养芽和花芽中表达量较高,根部相对较低,总体呈组成型表达模式;在干旱胁迫下,PtSS2表达水平提升。研究结果推测,蔗糖合酶基因PtSS2在毛白杨各组织器官发育过程及干旱胁迫响应过程中具有重要功能。     

Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam