Showing papers by "Natasha V. Raikhel published in 1994"

Genes Galore: A Summary of Methods for Accessing Results from Large-Scale Partial Sequencing of Anonymous Arabidopsis cDNA Clones

Abstract: High-throughput automated partial sequencing of anonymous cDNA clones provides a method to survey the repertoire of expressed genes from an organism. Comparison of the coding capacity of these expressed sequence tags (ESTs) with the sequences in the public data bases results in assignment of putative function to a significant proportion of the ESTs. Thus, the more than 13,400 plant ESTs that are currently available provide a new resource that will facilitate progress in many areas of plant biology. These opportunities are illustrated by a description of the results obtained from analysis of 1500 Arabidopsis ESTs from a cDNA library prepared from equal portions of poly(A+) mRNA from etiolated seedlings, roots, leaves, and flowering inflorescences. More than 900 different sequences were represented, 32% of which showed significant nucleotide or deduced amino acid sequence similarity to previously characterized genes or proteins from a wide range of organisms. At least 165 of the clones had significant deduced amino acid sequence homology to proteins or gene products that have not been previously characterized from higher plants. A summary of methods for accessing the information and materials generated by the Arabidopsis cDNA sequencing projects is provided.

A Small GTP-Binding Protein from Arabidopsis thaliana Functionally Complements the Yeast YPT6 Null Mutant

Abstract: A clone designated A.t.RAB6 encoding a small GTP-binding protein was isolated from a cDNA library of Arabidopsis thaliana leaf tissue. The predicted amino acid sequence was highly homologous to the mammalian and yeast counterparts, H.Rab6 and Ryh1/Ypt6, respectively. Lesser homology was found between the predicted Arabidopsis protein sequence and two small GTP-binding proteins isolated from plant species (44% homology to Zea mays Ypt1 and 43% homology to Nicotiana tabacum Rab5). Conserved stretches in the deduced amino acid sequence of A.t.Rab6 include four regions involved in GTP-binding, an effector region, and C-terminal cysteine residues required for prenylation and subsequent membrane attachment. Northern blot analysis demonstrated that A.t.Rab6 mRNA was expressed in root, leaf, stem, and flower tissues from A. thaliana with the highest levels present in roots. Escherichia coli produced histidine-tagged A.t.Rab6 protein-bound GTP, whereas a mutation in one of the guanine nucleotide-binding sites (asparagine122 to isoleucine) rendered it incapable of binding GTP. Functionally, the A.t.RAB6 gene was able to complement the temperature-sensitive phenotype of the YPT6 null mutant in yeast. The isolation of this gene will aid in the dissection of the machinery involved in soluble protein sorting at the trans-Golgi network of plants.

The basic domain in the bZIP regulatory protein Opaque2 serves two independent functions: DNA binding and nuclear localization

Abstract: For organisms to grow and develop, transcriptional regulatory proteins must localize to the nucleus. This movement is mediated by nuclear localization sequences (NLSs) which consist of short stretches of basic amino acids that are part of the structure of mature nuclear proteins. Two NLSs have been previously identified in the maize regulatory protein Opaque2 (O2), a member of the basic-domain, leucine-zipper (bZIP) class of proteins. In this report, it has been determined that both of these NLSs are necessary for import of O2, and the focus has been on a functional analysis of the more efficient, bipartite NLS that is present in the basic or DNA-binding domain. A mutation which contains altered amino acids on both parts of this NLS severely reduced nuclear targeting, and led to the definition of two classes of bipartite NLSs. Since the bipartite NLS is located in the highly conserved. DNA-binding domain, the dual role of this domain was examined using the maize mutant o2-676, in which a conservative mutation eliminates the DNA-binding function. The o2-676 protein localized to the nucleus in maize and the bZIP domain from the mutant protein was sufficient to redirect a reporter protein to the nucleus in transgenic plant cells. Thus, it was possible to show that the nuclear targeting function of this domain is independent of DNA binding. Sequence conservation of the basic domain of other bZIP proteins suggests that the bifunctionality of this domain may be conserved in all members of this class; a consensus sequence for a bipartite NLS in bZIP proteins is suggested.

Identification of an enhancer/silencer sequence directing the aleurone-specific expression of a barley chitinase gene.

Abstract: Chitinases are expressed in various plant tissues where they are thought to play a role in defense against chitin-containing pathogens. Transient gene expression assays have been used in tissues of barley to delineate promoter sequences involved in the regulation of an aleurone-specific chitinase gene (Chi26), and of a vegetatively expressed chitinase gene (Chi33). The assays measured the activities of transcriptional fusions between chitinase 5' upstream sequences and GUS reporter genes after DNA delivery by particle bombardment. Analysis of Chi26 5' and 3' promoter deletions indicated that sequences between -200 and -140 confer developmental and aleurone-specific expression. Deletions/replacements covering this part of the promoter indicated that sequences between -179 and -147 (E-region) direct expression in aleurone cells. The ability of the 33bp E-region of the Chi26 promoter to activate transcription specifically in aleurone was confirmed by constructing and testing two types of chimeric promoters. The first type, which contained two copies of the E-region fused to the CaMV 35S TATA box, conferred aleurone-specific expression of a GUS reporter gene. The second type, which contained a single copy of the E-region inserted into a deleted, inactive Chi33 promoter derivative, was also capable of directing transcription in aleurone but not in leaves. The pattern of expression of this and other Chi26/Chi33 chimeric promoters suggest that the E-region contains cis-acting sequences which activate transcription in aleurone and silence transcription in leaves. DNA sequence motifs implicated in the regulation of Chi26 and Chi33 are described.

A carboxy-terminal plant vacuolar targeting signal is not recognized by yeast

Abstract: Three different classes of signals for plant vacuolar targeting have been defined. Previous work has demonstrated that the carboxyl-terminal propeptide (CTPP) of barley lectin (BL) is a vacuolar targeting signal in tobacco plants. When a mutant BL protein lacking the CTPP is expressed in tobacco, the protein is secreted. In an effort to determine the universality of this signal, the CTPP was tested for its ability to target proteins to the vacuole of Saccharomyces cerevisiae. Genes encoding fusion proteins between the yeast secreted protein invertase and BL domains were synthesized and transformed into an invertase deletion mutant of yeast. Invertase assays on intact and detergent-solubilized cells demonstrated that invertase+CTPP was secreted, while nearly 90% of the invertase::BL+CTPP (fusion protein between invertase and BL containing the CTPP) and invertase::BL-CTPP proteins (fusion between invertase and BL lacking the CTPP) were retained intracellularly. These fusions were secreted in a mutant of yeast that normally secretes proteins targeted to the vacuole. With this and previous work, proteins representing all three classes of plant vacuolar targeting signals have now been tested in yeast, and in all cases, the experiments indicate that the plant proteins are directed to the yeast vacuole using signals other than those recognized by plants.

Targeting of proteins to the vacuole

Abstract: The plant vacuole is a dynamic multifunctional organelle that is essential for the regulation and maintenance of plant cell growth and development [1, 2]. A wide array of functions or characteristics associated with the vacuole are performed by proteins. A majority of these enzymes and proteins are delivered to the vacuole by way of the secretory pathway [3–6]. Electron microscopy (EM) has been useful in defining vacuolar morphology in various cell types and EM immunocytochemical localization has made a significant contribution in the identification of proteins associated with this organelle [7]. However, EM by itself has its own limitations and therefore the development of biochemical and molecular approaches was necessary for the analysis of protein sorting to the vacuole. Over the last three to four years, these newly developed approaches have been instrumental in the identification and characterization of the vacuolar targeting signals in plants [8–19]. The main focus of current research is the isolation and characterization of the components involved in the sorting and delivery of proteins to the plant vacuole. In this chapter, we will describe some of the basic techniques used to study protein targeting to the vacuole in tobacco plants.

Specific Nuclear Localization Sequence Binding to Plant Nuclei

Abstract: The import of proteins into the nucleus is mediated by nuclear localization sequences (NLSs), which are short polypeptide regions having a high content of basic amino acids (for reviews, see Roberts, 1989; Garcia-Bustos et al., 1991; Raikhel, 1992). NLSs are found at many locations within proteins but are not removed following translocation as are signals for chloroplastic (Keegstra, 1989), mitochondrial (Hartl and Neupert, 1990) and vacuolar (Chrispeels and Raikhel, 1992) import. The best studied NLSs are those that resemble the import signal of the simian virus 40 (SV40) large T-antigen (PKKKRKV; Kalderon et al., 1984; Lanford and Butel, 1984). Two additional classes of NLSs have also been identified. These are exemplified by the yeast mating type α-2 (Mat α-2) NLS (Hall et al., 1984), which possesses hydrophobic and basic amino acids (KIPIK), and bipartite NLSs which are composed of two short regions of basic amino acids separated by approximately ten residues (for review, see Dingwall and Laskey, 1991).