Isolation of the Ornithine-δ-Aminotransferase cDNA and Effect of Salt Stress on Its Expression in Arabidopsis thaliana1

To evaluate the relative importance of ornithine (Orn) as a precursor in proline (Pro) synthesis, we isolated and sequenced a cDNA encoding the Orn-δ-aminotransferase (δ-OAT) from Arabidopsis thaliana. The deduced amino acid sequence showed high homology with bacterial, yeast, mammalian, and plant sequences, and the N-terminal residues exhibited several common features with a mitochondrial transit peptide. Our results show that under both salt stress and normal conditions, δ-OAT activity and mRNA in young plantlets are slightly higher than in older plants. This appears to be related to the necessity to dispose of an easy recycling product, glutamate. Analysis of the expression of the gene revealed a close association with salt stress and Pro production. In young plantlets, free Pro content, Δ1-pyrroline-5-carboxylate synthase mRNA, δ-OAT activity, and δ-OAT mRNA were all increased by salt-stress treatment. These results suggest that for A. thaliana, the Orn pathway, together with the glutamate pathway, plays an important role in Pro accumulation during osmotic stress. Conversely, in 4-week-old A. thaliana plants, although free Pro level also increased under salt-stress conditions, the δ-OAT activity appeared to be unchanged and δ-OAT mRNA was not detectable. Δ1-pyrroline-5-carboxylate synthase mRNA was still induced at a similar level. Therefore, for the adult plants the free Pro increase seemed to be due to the activity of the enzymes of the glutamate pathway.

Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site.

The tobacco N and Arabidopsis RPS2 genes, among several recently cloned disease-resistance genes, share highly conserved structure, a nucleotide-binding site (NBS). Using degenerate oligonucleotide primers for the NBS region of N and RPS2, we have amplified and cloned the NBS sequences from soybean. Each of these PCR-derived NBS clones detected low-or moderate-copy soybean DNA sequences and belongs to 1 of 11 different classes. Sequence analysis showed that all PCR clones encode three motifs (P-loop, kinase-2, and kinase-3a) of NBS nearly identical to those in N and RPS2. The intervening region between P-loop and kinase-3a of the 11 classes has high (26% average) amino acid sequence similarity to the N gene although not as high (19% average) to RPS2. These 11 classes represent a superfamily of NBS-containing soybean genes that are homologous to N and RPS2. Each class or subfamily was assessed for its positional association with known soybean disease-resistance genes through near-isogenic line assays, followed by linkage analysis in F2 populations using restriction fragment length polymorphisms. Five of the 11 subfamilies have thus far been mapped to the vicinity of known soybean genes for resistance to potyviruses (Rsv1 and Rpv), Phytophthora root rot (Rps1, Rps2, and Rps3), and powdery mildew (rmd). The conserved N- or RPS2-homologous NBS sequences and their positional associations with mapped soybean-resistance genes suggest that a number of the soybean disease-resistance genes may belong to this superfamily. The candidate subfamilies of NBS-containing genes identified by genetic mapping should greatly facilitate the molecular cloning of disease-resistance genes.

Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection.

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised patients and those with cystic fibrosis genetic disease. To identify new virulence genes of P. aeruginosa, a selection system was developed based on the in vivo expression technology (IVET) that was first reported in Salmonella system. An adenine-requiring auxotrophic mutant strain of P. aeruginosa was isolated and found avirulent on neutropenic mice. A DNA fragment that can complement the mutant strain, containing purEK operon that is required for de novo biosynthesis of purine, was sequenced and used in the IVET vector construction. By applying the IVET selection system to a neutropenic mouse infection model, genetic loci that are specifically induced in vivo were identified. Twenty-two such loci were partially sequenced and analyzed. One of them was a well-studied virulence factor, pyochelin receptor (FptA), that is involved in iron acquisition. Fifteen showed significant homology to reported sequences in GenBank, while the remaining six did not. One locus, designated np20, encodes an open reading frame that shares amino acid sequence homology to transcriptional regulators, especially to the ferric uptake regulator (Fur) proteins of other bacteria. An insertional np20 null mutant strain of P. aeruginosa did not show a growth defect on laboratory media; however, its virulence on neutropenic mice was significantly reduced compared with that of a wild-type parent strain, demonstrating the importance of the np20 locus in the bacterial virulence. The successful isolation of genetic loci that affect bacterial virulence demonstrates the utility of the IVET system in identification of new virulence genes of P. aeruginosa.

Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.

Production of infectious HIV-1 virions is dependent on the processing of envelope glycoprotein gp160 by a host cell protease. The protease in human CD4+ T lymphocytes has not been unequivocally identified, yet members of the family of mammalian subtilisin-like protein convertases (SPCs), which are soluble or membrane-bound proteases of the secretory pathway, best fulfill the criteria. These proteases are required for proprotein maturation and cleave at paired basic amino acid motifs in numerous cellular and viral glycoprotein precursors, both in vivo and in vitro. To identify the gp160 processing protease, we have used reverse transcription-PCR and Northern blot analyses to ascertain the spectrum of SPC proteases in human CD4+ T cells. We have cloned novel members of the SPC family, known as the human PC6 genes. Two isoforms of the hPC6 protease are expressed in human T cells, hPC6A and the larger hPC6B. The patterns of SPC gene expression in human T cells has been compared with the furin-defective LoVo cell line, both of which are competent in the production of infectious HIV virions. This comparison led to the conclusion that the hPC6 gene products are the most likely candidates for the host cell protease responsible for HIV-1 gp160 processing in human CD4+ T cells.

Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94.

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2Kb major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility com- plex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.

Isolation of 10 differentially expressed cDNAs in p53-induced apoptosis: activation of the vertebrate homologue of the drosophila seven in absentia gene.

We report the isolation of 10 differentially expressed cDNAs in the process of apoptosis induced by the p53 tamor suppressor. As a global analytical method, we performed a differential display of mRNA between mouse M1 myeloid leukemia cells and derived clone LTR6 cells, which contain a stably transfected temperature-sensitive mutant of p53. At 32 degrees C wild-type p53 function is activated in LTR6 cells, resulting in programmed cell death. Eight genes are activated (TSAP; tumor suppressor activated pathway), and two are inhibited (TSIP, tumor suppressor inhibited pathway) in their expression. None of the 10 sequences has hitherto been recognized as part of the p53 signaling pathway. Three TSAPs are homologous to known genes. TSAP1 corresponds to phospholipase C beta 4. TSAP2 has a conserved domain homologous to a multiple endocrine neoplasia I (ZFM1) candidate gene. TSAP3 is the mouse homologue of the Drosophila seven in absentia gene. These data provide novel molecules involved in the pathway of wild-type p53 activation. They establish a functional link between a homologue of a conserved developmental Drosophila gene and signal transduction in tumor suppression leading to programmed cell death.

Isolation of a Pax-6 homolog from the ribbonworm Lineus sanguineus.

The Pax-6 genes of vertebrates and Drosophila encode transcription factors with highly conserved paired- and homeodomains. They are expressed in the nervous system and the developing eyes. Loss-of-function mutations in mammals and flies lead to a reduction or absence of the eyes. By ectopic expression of Pax-6 in Drosophila ectopic eyes can be induced, indicating a determinative role in eye morphogenesis. We have isolated a Pax-6 homolog of the ribbonworm Lineus sanguineus. This gene shares extensive sequence identity and several conserved splice sites with the mammalian and Drosophila genes. During head regeneration the L. sanguineus Pax-6 homolog is expressed in the central nervous system, in the cerebral organ, and in the eye region. These findings support the hypothesis that Pax-6 was present in primitive metazoa before the evolutionary separation of vertebrates and arthropods and suggest a fundamental role in eye and central nervous system development.

Isolation of Drosophila cyclin D, a protein expressed in the morphogenetic furrow before entry into S phase.

During Drosophila development, nuclear and cell divisions are coordinated in response to developmental signals. In yeast and mammalian cells, signals that control cell division regulate the activity of cyclin-dependent kinases (Cdks) through proteins such as cyclins that interact with the Cdks. Here we describe two Drosophila cyclins identified from a set of Cdk-interacting proteins. One, cyclin J, is of a distinctive sequence type; its exclusive maternal expression pattern suggests that it may regulate oogenesis or the early nuclear divisions of embryogenesis. The other belongs to the D class of cyclins, previously identified in mammalian cells. We show that Drosophila cyclin D is expressed in early embryos and in imaginal disc cells in a pattern that anticipates cell divisions. Expression in the developing eye disc at the anterior edge of the morphogenetic furrow suggests that cyclin D acts early, prior to cyclin E, in inducing G1-arrested cells to enter S phase. Our results also suggest that, although cyclin D may be necessary, its expression alone is not sufficient to initiate the events leading to S phase.

Isolation of an oxygen-sensitive FNR protein of Escherichia coli: interaction at activator and repressor sites of FNR-controlled genes.

The Escherichia coli fnr gene product, FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth. FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state. To investigate this process, a fnr mutant that encodes an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis. In vivo, the mutant behaved like a wild-type strain under anaerobic conditions but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth. The altered fur gene was overexpressed in E. coli and the resultant FNR protein was purified to near homogeneity by using anaerobic chromatography procedures. An in vitro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein. The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mol of Fe per FNR monomer. In vitro DNase I protection studies were performed to establish the locations of the FNR-binding sites at the narG, narK, dmsA, and hemA promoters that are regulated by either activation or repression of their transcription. The sizes of the DNA footprints are consistent with the binding of two monomers of FNR that protect the symmetrical FNR-recognition sequence TTGAT-nnnnATCAA. Exposure of the FNR protein or protein-DNA complex to air for even short periods of time (approximately 5 min) led to the complete loss of DNA protection at a consensus FNR recognition site. A model whereby the FNR protein exists in the cell as a monomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed.

Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica.

Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 microM ZnCl2 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.

Isolation of HLA-DR1.(staphylococcal enterotoxin A)2 trimers in solution.

Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules. Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution. Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1.SEA2 trimers. The molar ratio of components was determined by dual isotope labeling. The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high-affinity MHC class II beta-chain binding was incapable of forming any complexes. Thus SEA binding to its MHC receptor is a two-step process involving initial beta-chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain.

Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: construction of alternate hosts with defined karyotypic alterations.

An intrinsic feature of yeast artificial chromosomes (YACs) is that the cloned DNA is generally in the same size range (i.e., approximately 200-2000 kb) as the endogenous yeast chromosomes. As a result, the isolation of YAC DNA, which typically involves separation by pulsed-field gel electrophoresis, is frequently confounded by the presence of a comigrating or closely migrating endogenous yeast chromosome(s). We have developed a strategy that reliably allows the isolation of any YAC free of endogenous yeast chromosomes. Using recombination-mediated chromosome fragmentation, a set of Saccharomyces cerevisiae host strains was systematically constructed. Each strain contains defined alterations in its electrophoretic karyotype, which provide a large-size interval devoid of endogenous chromosomes (i.e., a karyotypic "window"). All of the constructed strains contain the kar1-delta 15 mutation, thereby allowing the efficient transfer of a YAC from its original host into an appropriately selected window strain using the kar1-transfer procedure. This approach provides a robust and efficient means to obtain relatively pure YAC DNA regardless of YAC size.

Isolation of cDNA clones encoding RICH: a protein induced during goldfish optic nerve regeneration with homology to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases.

Using data derived from peptide sequencing of p68/70, a protein doublet induced during optic nerve regeneration in goldfish, we have isolated cDNAs that encode RICH (regeneration-induced CNPase homolog) from a goldfish regenerating retina cDNA library. The predicted RICH protein comprises 411 amino acids, possesses a pI of 4.48, and shows significant homology to the mammalian myelin marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase; EC 3.1.4.37). The mRNA encoding RICH was demonstrated, by both Northern blot analysis and RNase protection assays, to be induced as much as 8-fold in regenerating goldfish retinas at 20 days after nerve crush. Analysis of total RNA samples from various tissues showed a broad distribution of RICH mRNA, with the highest levels observed in gravid ovary. The data obtained strongly suggest that RICH is identical or very similar to p68/70. The molecular cloning of RICH provides the means for a more detailed analysis of its function in nerve regeneration. Additionally, the homology of RICH and CNPase suggests that further investigation may provide additional insight into the role of these proteins in the nervous system.

Isolation of a primate embryonic stem cell line.

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81) that are characteristic of human embryonal carcinoma cells. R278.5 cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers but differentiate or die in the absence of fibroblasts, despite the presence of recombinant human leukemia inhibitory factor. R278.5 cells allowed to differentiate in vitro secrete bioactive chorionic gonadotropin into the medium, express chorionic gonadotropin alpha- and beta-subunit mRNAs, and express alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. When injected into severe combined immunodeficient mice, R278.5 cells consistently differentiate into derivatives of all three embryonic germ layers. These results define R278.5 cells as an embryonic stem cell line, to our knowledge, the first to be derived from any primate species.