Showing papers in "Molecular Genetics and Genomics in 1996"

Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.).

Abstract: Microsatellite markers containing simple sequence repeats (SSR) are a valuable tool for genetic analysis. Our objective is to augment the existing RFLP map of rice with simple sequence length polymorphisms (SSLP). In this study, we describe 20 new microsatellite markers that have been assigned to positions along the rice chromosomes, characterized for their allelic diversity in cultivated and wild rice, and tested for amplification in distantly related species. Our results indicate that the genomic distribution of microsatellites in rice appears to be random, with no obvious bias for, or clustering in particular regions, that mapping results are identical in intersubspecific and interspecific populations, and that amplification in wild relatives of Oryza sativa is reliable in species most closely related to cultivated rice but becomes less successful as the genetic distance increases. Sequence analysis of SSLP alleles in three related indica varieties demonstrated the clustering of complex arrays of SSR motifs in a single 300-bp region with independent variation in each. Two microsatellite markers amplified multiple loci that were mapped onto independent rice chromosomes, suggesting the presence of duplicated regions within the rice genome. The availability of increasing numbers of mapped SSLP markers can be expected to increase the power and resolution of genome analysis in rice.

Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides

Abstract: We report the isolation of cDNA clones containing the full 6.3-kb coding sequence of the para-type sodium channel gene of the housefly, Musca domestica. This gene has been implicated as the site of knockdown resistance (kdr), an important resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. The cDNAs predict a polypeptide of 2108 amino acids with close sequence homology (92% identity) to the Drosophila para sodium channel, and around 50% homology to vertebrate sodium channels, Only one major splice form of the housefly sodium channel was detected, in contrast to the Drosophila para transcript which has been reported to undergo extensive alternative splicing. Comparative sequence analysis of housefly strains carrying kdr or the more potent super-kdr factor revealed two amino acid mutations that correlate with these resistance phenotypes. Both mutations are located in domain II of the sodium channel. A leucine to phenylalanine replacement in the hydro-phobic IIS6 transmembrane segment was found in two independent kdr strains and six super-kdr strains of diverse geographic origin, while an additional methionine to threonine replacement within the intracellular IIS4-S5 loop was found only in the super-kdr strains. Neither mutation was present in five pyrethroid-sensitive strains. The mutations suggest a binding site for pyrethroids at the intracellular mouth of the channel pore in a region known to be important for channel inactivation.

Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics.

Abstract: Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the speciesS. officinarum andS. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n+n transmission of the parental chromosomes instead of the peculiar 2n+n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n=107–115) about 10% were identified as originating fromS. spontaneum and about 10% were identified as recombinant chromosomes between the two speciesS. officinarum andS. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.

Stable nuclear transformation of the diatom Phaeodactylum tricornutum.

Abstract: A nuclear transformation system has been developed for the diatomPhaeodactylum tricornutum using microparticle bombardment to introduce thesh ble gene fromStreptoalloteichus hindustanus into cells. Thesh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from theP. tricornutum fcp genes were used to drive expression ofsh ble. Between 10–100 transformants were recovered/108 cells. Transformants were able to grow on at least 500 µg/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations thesh ble gene was present in 1–3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1–2.0 µg/mg total protein). Thecat reporter gene fused to afcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed inP. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.

A general system for generating unlabelled gene replacements in bacterial chromosomes

Abstract: A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

A new family of DNA binding proteins includes putative transcriptional regulators of the Antirrhinum majus floral meristem identity gene SQUAMOSA.

Abstract: Several sites of nuclear protein interaction within the promoter region of theAntirrhinum majus floral meristem identity geneSQUAMOSA were detected using an electrophoretic mobility shift assay. One of these sites displayed a particularly clear interaction with nuclear protein extracted from inflorescences but not with nuclear protein extracted from young, non-flowering plants. This site could thus represent a binding motif for a transcriptional activator. A South-western screen of an inflorescence cDNA expression library resulted in the isolation of several cDNAs representing two different genes namedSBP1 andSBP2 (forSQUAMOSA-pROMOTERBINDINGPROTEIN gene 1 and 2). Both genes encode highly similar protein domains which were found to be necessary and sufficient for binding DNA in a sequence-specific manner. This DNA-binding domain showed no similarity to known proteins in the databases. However, it is characteristic for a small family of gene products inA. majus and other plant species. Expression ofSBP1 and2 is developmentally regulated and their transcriptional activation precedes that ofSQUAMOSA. The data presented support the idea that members of the newly identifiedSBP gene family function as transcription factors involved in the control of early flower development.

The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form.

Abstract: Thecre1 genes of the filamentous fungiTrichoderma reesei andT. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor genecreA ofAspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C2H2 type. Thecre1 promoters contain several sequence elements that are identical to the previously identified binding sites forA. nidulans CREA. Steady-state mRNA levels forcre1 of theT. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest thatcre1 expression may be autoregulated. TheT. reesei strain Rut-C30, a hyperproducer of cellulolytic enzymes, was found to express a truncated form of thecre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-lengthcre1 gene into this strain caused glucose repression ofcbh1 expression, demonstrating thatcre1 regulates cellulase expression.

Ebb and flow of the chloroplast inverted repeat

Abstract: The endpoints of the large inverted repeat (IR) of chloroplast DNA in flowering plants differ by small amounts between species. To quantify the extent of this movement and define a possible mechanism for IR expansion, DNA sequences across the IR-large single-copy (IR-LSC) junctions were compared among 13 Nicotiana species and other dicots. In most Nicotiana species the IR terminates just upstream of, or somewhere within, the 5' portion of the rps19 gene. The truncated copy of this gene, rps19', varies in length even between closely related species but is of constant size within a single species. In Nicotiana, six different rps19' structures were found. A phylogenetic tree of Nicotiana species based on restriction site data shows that the IR has both expanded and contracted during the evolution of this genus. Gene conversion is proposed to account for these small and apparently random IR expansions. A large IR expansion of over 12 kb has occurred in Nicotiana acuminata. The new IR-LSC junction in this species lies within intron 1 of the clpP gene. This rearrangement occurred via a double-strand DNA break and recombination between poly (A) tracts in clpP intron 1 and upstream of rps19. Nicotiana acuminata chloroplast DNA contains a "molecular fossil' of the IR-LSC junction that existed prior to this dramatic rearrangement.

Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis.

Abstract: Pathogenic development ofUstilago maydis, the causative agent of corn smut disease, is a multistep process. Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously. Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur. In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) fromAequorea victoria as a vital marker inU. maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo.

Reciprocal regulation of Delta(1)-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants

Abstract: Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745 bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the delta 1-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.

Self-incompatibility (S) alleles of the Rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily.

Abstract: Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.

Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica)

Abstract: Using reverse transcription polymerase chain reactions (RT-PCR), the DNA sequence for the main membrane-spanning region (IS3 through IVS6) of the gene encoding the α-subunit of the para sodium channel of the German cockroach, Blattella germanica, has been determined. The overall structure of the open reading frame region of this B. germanica gene is very similar to that of the para gene of Drosophila melanogaster, and that of the partially sequenced para gene of Musca domestica. On the other hand, it is distinctly different from that of the DSC gene (Drosophila sodium channel). As a result of a side-by-side comparison of the para gene sequences of the susceptible CSMA strain and the kdr resistant VT strain of B. germanica, on mutation (TTG to TTC) at the approximate center of the IIS6 membrane-spanning segment was found to result in an amino acid change from L to F. While the functional meaning of this mutation for the operation of the para sodium channel remains to be studied, this region is very highly conserved among all sodium channels identified so far, and is one of the most hydrophobic areas of the entire α-subunit. For comparison, we have studied the same region of the para sodium channel of both kdr and susceptible SBO strain of the housefly, Musca domestica. We found the homologous type of mutation, CTT to TTT, resulting in the same amino acid alteration (L to F) at this site. However, in the case of houseflies both kdr and susceptible strains contained both L and F versions of the protein. The ratio of TTT to CTT was significantly higher in the kdr strain of M. domestica than in the three susceptible strains examined.

The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas.

Abstract: A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5′ and 3′ untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg+ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (PmR) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the PmR transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a PmR transformant to wild-type strains. Direct selection of PmR transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.

Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress.

Abstract: We have isolated several mutants ofSaccharomyces cerevisiae that are sensitive to oxidative stress in a screen for elevated sensitivity to hydrogen peroxide. Two of the sixteen complementation groups obtained correspond to structural genes encoding enzymes of the pentose phosphate pathway. Allelism of thepos10 mutation (POS forperoxidesensitivity) to thezwf1/met1 mutants in the structural gene for glucose 6-phosphate dehydrogenase was reported previously. The second mutation,pos18, was complemented by transformation with a yeast genomic library. The open reading frame of the isolated gene encodes 238 amino acids. No detectable ribulose 5-phosphate epimerase activity was found in thepos18 mutant, suggesting that the corresponding structural gene is affected in this mutant. For that reason the gene was renamedRPE1 (forribulose 5-phosphateepimerase).RPE1 was localized to chromosome X. The predicted protein has a molecular mass of 25 966 Daltons, a codon adaptation index (CAI) of 0.32, and an isoelectric point of 5.82. Database searches revealed 32 to 37% identity with ribulose 5-phosphate epimerases ofEscherichia coli, Rhodospirillum rubrum, Alcaligenes eutrophus andSolanum tuberosum. We have characterizedRPE1 by testing enzyme activities inrpe1 deletion mutants and in strains that overexpressRPE1, and compared the hydrogen peroxide sensitivity ofrpe1 mutants to that of other mutants in the pentose phosphate pathway. Interestingly, all mutants tested (glucose 6-phosphate dehydrogenase, gluconate 6-phosphate dehydrogenase, ribulose 5-phosphate epimerase, transketolase, transaldolase) are sensitive to hydrogen peroxide.

The Ty1-copia group retrotransposons in Vicia species: copy number, sequence heterogeneity and chromosomal localisation.

Abstract: We present an in-depth study of theTy1-copia group of retrotransposons within the plant genusVicia, which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploidVicia species chosen to represent large (V. faba, 1C=13.3 pg), medium (V. melanops, 1C=11.5 pg) and small (V. sativa, 1C=2.3 pg) genomes within the genus. The copy numbers of the retrotransposons are all high but vary greatly, withV. faba containing approximately 106 copies,V. melanops about 1000 copies andV. sativa 5000 copies. The degree of sequence heterogeneity ofTy1-copia group elements correlates with their copy number within each genome, but neither heterogeneity nor copy number are related to the genome size of the host. In situ hybridization to metaphase chromosomes shows that the retrotransposons inV. faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotransposon evolution.

An Hsp70 antisense gene affects the expression of HSP70/HSC70, the regulation of HSF, and the acquisition of thermotolerance in transgenic Arabidopsis thaliana.

Abstract: The genes and proteins of the HSP70 family, are involved in important processes in cells and organelles at normal temperature and after heat stress. Constitutive Hsc70 and heat-inducible Hsp70 genes are known in all organisms including plants. The goal of our present investigation was to generate an Hsp70 mutation in Arabidopsis thaliana. In a transgenic approach a heat-inducible antisense Hsp70 gene was constructed, plants were transformed and screened for lack of heat-inducible HSP70 mRNA; two such lines were further investigated. In these plants the Hsp70 gene was not induced by heat shock, and the level of HSC70 RNA was also greatly reduced. This negative antisense effect was specific for genes of the HSP70 family and the induction of mRNAs encoding the small HSP18 class of heat shock protein (HSP) was not affected. The level of HSP70/HSC70 proteins was significantly reduced in transgenic plants, but HSP18 was induced to the same level in different transgenic lines and in untransformed plants. The acquisition of thermotolerance was negatively affected in artisense plants, the survival temperature being 2°C below the survival temperature of the wild type and other transgenic lines. Another major effect concerning the regulation of the endogenous heat shock transcription factor HSF was detected by testing the ability to form heterotrimers between authentic HSF and recombinant HSF-GUS (β-glucuronidase) proteins. The shut-off time, required to turn off HSF activity during recovery from heat stress, was significantly prolonged in antisense plants compared with wild-type and other transgenic lines. Our results imply a dual role of HSP70 in plants, a protective role in thermotolerance and a regulatory effect on HSF activity and hence the autoregulation of the heat shock response.

Selectable marker recycling in the chloroplast

Abstract: The bacterial geneaadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of theaadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking theaadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which theaadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas theaadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse theaadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.

Characterization and genetic mapping of simple repeat sequences in the tomato genome

Abstract: Tomato genomic libraries were screened for the presence of simple sequence repeats (SSRs) with seventeen synthetic oligonucleotide probes, consisting of 2- to 5-basepair motifs repeated in tandem. GAn and GTn sequences were found to occur most frequently in the tomato genome (every 1.2 Mb), followed by ATTn and GCCn (every 1.4 Mb and 1.5 Mb, respectively). In contrast, only ATn and GAn microsatellites (n > 7) were found to be frequent in the GenBank database, suggesting that other motifs may be preferentially located away from genes. Polymorphism of microsatellites was measured by PCR amplification of individual loci or by Southern hybridization, using a set of ten tomato cultivars. Surprisingly, only two of the nine microsatellite clones surveyed (five GTn, three GAn and one ATTn), showed length variation among these accessions. Polymorphism was also very limited betweenLycopersicon esculentum andL. pennelli, two distant species. Southern analysis using the seventeen oligonucleotide probes identified GATAn and GAAAn as useful motifs for the detection of multiple polymorphic fragments among tomato cultivars. To determine the structure of microsatellite loci, a GAn probe was used for hybridization at low stringency on a small insert genomic library, and randomly selected clones were analyzed. GAn based motifs of increasing complexity were found, indicating that simple dinucleotide sequences may have evolved into larger tandem repeats such as minisatellites as a result of basepair substitution, replication slippage, and possibly unequal crossing-over. Finally, we genetically mapped loci corresponding to two amplified microsatellites, as well as nine large hypervariable fragments detected by Southern hybridization with a GATA8 probe. All loci are located around putative tomato centromeres. This may contribute to understanding of the structure of centromeric regions in tomato.

Regulation of genes encoding subunits of the trehalose synthase complex in Saccharomyces cerevisiae: novel variations of STRE-mediated transcription control?

Abstract: Saccharomyces cerevisiae cells show under suboptimal growth conditions a complex response that leads to the acquisition of tolerance to different types of environmental stress This response is characterised by enhanced expression of a number of genes which contain so-called stress-responsive elements (STREs) in their promoters In addition, the cells accumulate under suboptimal conditions the putative stress protectant trehalose In this work, we have examined the expression of four genes encoding subunits of the trehalose synthase complex, GGS1/TPS1, TPS2, TPS3 and TSL1 We show that expression of these genes is coregulated under stress conditions Like for many other genes containing STREs, expression of the trehalose synthase genes is also induced by heat and osmotic stress and by nutrient starvation, and negatively regulated by the Ras-cAMP pathway However, during fermentative growth only TSL1 shows an expression pattern like that of the STRE-controlled genes CTT1 and SSA3, while expression of the three other trehalose synthase genes is only transiently down-regulated This difference in expression might be related to the known requirement of trehalose biosynthesis for the control of yeast glycolysis and hence for fermentative growth We conclude that the mere presence in the promoter of (an) active STRE(s) does not necessarily imply complete coregulation of expression Additional mechanisms appear to fine tune the activity of STREs in order to adapt the expression of the downstream genes to specific requirements

Structure and expression of three genes encoding ACC oxidase homologs from melon (Cucumis melo L.).

Abstract: The enzyme ACC oxidase catalyses the last step of ethylene biosynthesis in plants, converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. We have previously described the isolation and characterization of a cDNA clone (pMEL1) encoding an ACC oxidase homolog from melon (Cucumis melo L.). Here we report the isolation and characterization of three genomic clones, corresponding to three putative members of the ACC oxidase gene family in melon. All are transcriptionally active. The sequences of these genes have been determined. One genomic clone (CM-ACO1), corresponding to the cDNA previously isolated, presents a coding region interrupted by three introns. Its transcription initiation site has been defined with RNA from ripe fruit and ethylene-treated leaves. The other two genes (CM-ACO2, CM-ACO3) have only two introns, at positions identical to their counterparts inCM-ACO1. The degree of DNA homology in the coding regions ofCM-ACO2 andCM-ACO3 relative toCM-ACO1 is 59% and 75%, respectively.CM-ACO2 andCM-ACO3 are 59% homologous in their coding regions. These three genes have close homology toPH-ACO3, a member of the ACC oxidase multigene family of petunia. The predicted amino acid sequences of CM-ACO1 and CM-ACO3 are 77% to 81% identical to those encoded by the tomato and petunia genes, while the deduced amino acid sequence ofCM-ACO2 shows only 42% to 45% homology. RT-PCR analysis using gene-specific primers shows that the three genes are differentially expressed during development, ethylene treatment and wounding.CM-ACO1 is induced in ripe fruit and in response to wounding and to ethylene treatment in leaves.CM-ACO2 is detectable at low level in etiolated hypocotyls.CM-ACO3 is expressed in flowers and is not induced by any of the stimuli tested.

MAGGY, a retrotransposon in the genome of the rice blast fungus Magnaporthe grisea.

Abstract: Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.

Cell cycle-dependent expression of the mouse Rad51 gene in proliferating cells.

Abstract: The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouse Rad51 mRNA was examined in synchronized mouse m5S cells. The Rad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression of Rad51 mRNA. By immunohistochemical analyses, in mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouse Rad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes.

Identification of the pheromone response element in ustilago maydis

Abstract: Thea mating type locus ofUstilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. Binding of pheromone to its cognate receptor induces mating competence in haploid cells and stimulates filamentous growth of the dikaryon. We have analyzed transcription of genes located at thea locus and demonstrate that all genes are induced by pheromone. Transcriptional stimulation is mediated by a 9 bp DNA element (ACAAAGGGA) that occurs in multiple copies in both alleles of thea locus. By fusing multimers containing this 9 bp sequence to the pheromone gene promoter and to a heterologous promoter we demonstrate that this sequence acts as a pheromone response element. In addition, we show that expression of theb genes, which regulate pathogenic development of the dikaryon, is also stimulated by pheromone. Pheromone-inducible genes can be divided into three classes depending on whether their expression is reduced, maintained, or increased after cell fusion. These differences may suggest some regulatory cross-talk between thea andb loci.