An increased proportion of transgenic plants in the progeny of rapeseed (Brassica napus L.) transformants.

Cotyledon and leaf explants of two spring rapeseed varieties were transformed with Agrobacterium tumefaciens harboring a genetic construct with the gfp marker gene. In order to reduce the proportion of hyperhydrated shoots, which appeared during regenerant formation, we optimized sucrose content in the regeneration media. Analysis of the progeny obtained from T0 regenerants showed that in a number of lines the distribution of the gfp marker did not follow Mendelian segregation of a monogenic trait in self-pollinated plants, while in the progeny of the other lines of transgenic plants, the gfp marker was completely absent, although its presence had been confirmed in all selected T0 plants. We also found that in individual transformants gfp is randomly inherited throughout the central peduncle; its presence in the genome of seedlings does not depend on the location of the pod. Thus, both transformed and non-transformed cells were involved in the formation of gametes in T0 plants. In addition, marker segregation was different in plants of the T1 line obtained by nodal cuttings of a primary transformant, depending on the location of the cuttings on the stem of the original plant, indicating that the nature of T1 generation plants was also chimeric. Furthermore, we showed that propagation of plants by cutting followed by propagation by seeds formed as a result of self-pollination led to an increase in the proportion of transgenic plants in subsequent generations. The results obtained during the course of this study show that the transformants were chimeric, i. e. their tissues contained both transgenic and non-transgenic cells, and this chimeric nature was passed on to subsequent generations. We found that, in addition to nutrient media composition, other factors such as plant genotype and explant type also contribute to the rising of chimeric plants during transformation. Based on these results, we developed a simplified method, which consists of several rounds of a combination of cutting, seed production by self-pollination, and subsequent culling of wild-type plants, which significantly enriched descendent populations of the original rapeseed transformants with plants transgenic for the gfp marker.

Expression of P-Type ATPases of Marine Green Microalga Dunaliella maritima under Hyperosmotic Salt Shock.

Partial sequences of P-type ATPases were cloned from the marine microalgae Dunaliella maritima, two putative H+-ATPases (DmHA1 and DmHA2) and two putative Ca2+-ATPases (DmCA1 and DmCA2). The probable functions of the cloned proteins were suggested on the basis of their primary structure similarity with the proteins whose functions have been already characterized. The transcriptional response of the cloned D. maritima ATPase genes to a sharp increase in the NaCl concentration in the culture medium (from 100 to 500 mM) was investigated by quantitative RT-PCR. Hyperosmotic salt shock led to a significant increase in the DmHA2 expression and to a slight increase in the DmCA2 expression, whereas the expression of the two other ATPases, DmHA1 and DmCA1, was decreased. These data indicate that the DmHA2 ATPase is involved in maintenance of ion homeostasis in D. maritima cells under hyperosmotic salt shock.

Molecular cloning and characterisation of SaCLCa1, a novel protein of the chloride channel (CLC) family from the halophyte Suaeda altissima (L.) Pall.

The SaCLCa1 gene, a putative orthologue of AtCLCa, the Arabidopsis thaliana gene encoding a NO3-/H+ antiporter, was cloned from the halophyte Suaeda altissima. It belonged to the CLC family, comprising anionic channels and anion/H+ antiporters. SaCLCa1 ion specificity was studied by heterologous expression of this gene in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encoded the only CLC family protein, the Cl- transporter Gef1p, in undisrupted strains of this organism. For comparison, the function of another recently identified S. altissima CLC family gene, SaCLCc1, was also characterised. Expression of SaCLCc1 in Δgef1 cells restored their ability to grow on selective media. This supported the chloride specificity of this transporter. By contrast, expression of SaCLCa1 did not complement the growth defect phenotype of Δgef1 cells. However, growth of the Δgef1 mutant on the selective media was partially restored when it was transformed with SaCLCa1(C562 T), encoding the modified protein SaCLCa1(P188S), in which proline responsible for NO3- selectivity in selective filter was replaced by serine providing chloride selectivity. Quantitative real-time polymerase chain reactions (qRT-PCR) showed that significant induction of SaCLCa1 occurred in the roots of S. altissima when plants were grown on nitrate-deficient medium, while SaCLCc1 activation was observed in S. altissima leaves of plants grown in increasing Cl- concentrations of nutrient solution. These results suggested that SaCLCa1 and SaCLCc1 function as anionic transporters with nitrate and chloride specificities, respectively.

Cloning and Functional Analysis of SaCLCc1, a Gene Belonging to the Chloride Channel Family (CLC), from the Halophyte Suaeda altissima (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe2+-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn2+. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl- ions.

Evidence for a novel pathway for the targeting of a Saccharomyces cerevisiae peroxisomal protein belonging to the isomerase/hydratase family.

We, and others, have identified a novel Saccharomyces cerevisiae peroxisomal protein that belongs to the isomerase/hydratase family. The protein, named Dci1p, shares 50% identity with Eci1p, a delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids. Both of these proteins are localized to peroxisomes, and both contain motifs at their amino- and carboxyl termini that resemble peroxisome targeting signals (PTS) 1 and 2. However, we demonstrate that the putative type 1 signaling motif is not required for the peroxisomal localization of either of these proteins. Furthermore, the correct targeting of Eci1p and Dci1p occurs in the absence of the receptors for the type 1 or type 2 peroxisome targeting pathway. Together, these data suggest a novel mechanism for the intracellular targeting of these peroxisomal proteins.

Global regulatory functions of Oaf1p and Pip2p (Oaf2p), transcription factors that regulate genes encoding peroxisomal proteins in Saccharomyces cerevisiae.

Two transcription factors, Oaf1p and Pip2p (Oaf2p), are key components in the pathway by which several Saccharomyces cerevisiae genes encoding peroxisomal proteins are activated in the presence of a fatty acid such as oleate. By searching the S. cerevisiae genomic database for the consensus sequence that acts as a target for these transcription factors, we identified 40 genes that contain a putative Oaf1p-Pip2p binding site in their promoter region. Quantitative Northern analysis confirmed that the expression of 22 of the genes identified is induced by oleate and that either one or both of these transcription factors are required for the activation. In addition to known peroxisomal proteins, the regulated genes encode novel peroxisomal proteins, a mitochondrial protein, and proteins of unknown location and function. We demonstrate that Oaf1p regulates certain genes in the absence of Pip2p and that both of these transcription factors play a role in maintaining the glucose-repressed state of one gene. Furthermore, we provide evidence that the defined consensus binding site is not required for the regulation of certain oleate-responsive genes.

A complex containing two transcription factors regulates peroxisome proliferation and the coordinate induction of beta-oxidation enzymes in Saccharomyces cerevisiae.

Expression of the POX1 gene, which encodes peroxisomal acyl coenzyme A oxidase in the yeast Saccharomyces cerevisiae, is tightly regulated and can be induced by fatty acids such as oleate. Previously we have shown that this regulation is brought about by interactions between trans-acting factor(s) and an upstream activating sequence (UAS1) in the POX1 promoter. We recently identified and isolated a transcription factor, Oaf1p, that binds to the UAS1 of POX1 and mediates its induction. A screening strategy has been developed and used to identify eight S. cerevisiae mutants, from three complementation groups, that are defective in the oleate induction of POX1. Characterization of one such mutant led to the identification of Oaf2p, a protein that is 39% identical to Oaf1p. Oaf1p and Oaf2p form a protein complex that is required for the activation of POX1 and FOX3 and for proliferation of peroxisomes. We propose a model in which these two transcription factors heterodimerize and mediate this activation process. The mutants that we have isolated, and further identification of the corresponding defective genes, provide us with an opportunity to characterize the mechanisms involved in the coordinate regulation of peroxisomal beta-oxidation enzymes.

Regulation of peroxisomal fatty acyl-CoA oxidase in the yeast. Saccharomyces cerevisiae.

Peroxisomes are specialized organelles found in most eukaryote cells, where their major functions are in cellular respiration and fatty acid oxidation. Proliferation of this organelle, and induction of peroxisomal enzymes, is a phenomenon that occurs in diverse species, and is stimulated by a number of physiological and pharmacological stimuli. A large number of chemically diverse compounds, including hypolipidemic drugs and industrial plasticizers, have been shown to cause peroxisome proliferation and the induction of peroxisomal enzymes in rodents. Chronic exposure to these compounds produces hepatocellular carcinomas, however, the mechanism by which this tumorigenic event occurs is unknown. In the yeast Saccharomyces cerevisiae peroxisomes are induced when a fatty acid such as oleate is supplied as a carbon source in the growth medium. In addition, many peroxisomal enzymes are induced by growth on oleate; these include enzymes of the peroxisomal beta-oxidation cycle. This regulation occurs at the transcription level, and is controlled by specific trans-acting factors. The research in our laboratory has focused on the mechanisms involved in this regulation, and on the identification and characterization of the proteins involved. Our recent results, and current research directions are summarized.

Yeast mutants deficient in ER-associated degradation of the Z variant of alpha-1-protease inhibitor.

Saccharomyces cerevisiae mutants deficient in degradation of alpha-1-proteinase inhibitor Z (A1PiZ) have been isolated and genetically characterized. Wild-type yeast expressing A1PiZ synthesize an ER form of this protein that is rapidly degraded by an intracellular proteolytic process known as ER-associated protein degradation (ERAD). The mutant strains were identified after treatment with EMS using a colony blot immunoassay to detect colonies that accumulated high levels of A1PiZ. A total of 120,000 colonies were screened and 30 putative mutants were identified. The level of A1PiZ accumulation in these mutants, measured by ELISA, ranged from two to 11 times that of A1PiZ in the parent strain. Further studies demonstrated that the increased levels of A1PiZ in most of the mutant strains was not the result of defective secretion or elevated A1PiZ mRNA. Pulse chase experiments indicated that A1PiZ was stabilized in several strains, evidence that these mutants are defective in ER-associated protein degradation. Genetic analyses revealed that most of the mutations were recessive, approximately 30% of the mutants characterized conformed to simple Mendelian inheritance, and at least seven complementation groups were identified.

Purification, identification, and properties of a Saccharomyces cerevisiae oleate-activated upstream activating sequence-binding protein that is involved in the activation of POX1.

Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids. Peroxisomes are the sole site for the beta-oxidation of fatty acids in yeast. The first and rate-limiting enzyme of this cycle is fatty acyl-CoA oxidase. The gene encoding this enzyme in Saccharomyces cerevisiae (POX1) undergoes a complex regulation that is dependent on the growth environment. When this yeast is grown in medium containing oleic acid as the main carbon source, peroxisomes are induced and POX1 expression is activated. When cells are grown in the presence of glucose, the expression of POX1 mRNA is repressed, whereas growth on a carbon source such as glycerol or raffinose causes derepression. This rigorous regulation is brought about by the complex interactions between trans-acting factors and cis-elements in the POX1 promoter. Previously, we characterized regulatory elements in the promoter region of POX1 that are involved in the repression and activation of this gene (Wang, T., Luo, Y., and Small, G. M. (1994) J. Biol. Chem. 269, 24480-24485). In this study we have purified and identified an oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene. The protein has a predicted molecular mass of approximately 118 kDa.