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.

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif1

We isolated and characterized a novel light-regulated cDNA from the short-day plant Pharbitis nil that encodes a protein with a leucine (Leu) zipper motif, designated PNZIP (Pharbitis nil Leu zipper). The PNZIP cDNA is not similar to any other gene with a known function in the database, but it shares high sequence homology with an Arabidopsis expressed sequence tag and to two other sequences of unknown function from the cyanobacterium Synechocystis spp. and the red alga Porphyra purpurea, which together define a new family of evolutionarily conserved Leu zipper proteins. PNZIP is a single-copy gene that is expressed specifically in leaf photosynthetically active mesophyll cells but not in other nonphotosynthetic tissues such as the epidermis, trichomes, and vascular tissues. When plants were exposed to continuous darkness, PNZIP exhibited a rhythmic pattern of mRNA accumulation with a circadian periodicity of approximately 24 h, suggesting that its expression is under the control of an endogenous clock. However, the expression of PNZIP was unusual in that darkness rather than light promoted its mRNA accumulation. Accumulation of PNZIP mRNA during the dark is also regulated by phytochrome, since a brief exposure to red light in the middle of the night reduced its mRNA levels. Moreover, a far-red-light treatment at the end of day also reduced PNZIP mRNA accumulation during the dark, and that effect could be inhibited by a subsequent exposure to red light, showing the photoreversible response attributable to control through the phytochrome system.

Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans

Caenorhabditis elegans is an ideal organism for the study of the molecular basis of fundamental biological processes such as germ-line development, especially because of availability of the whole genome sequence and applicability of the RNA interference (RNAi) technique. To identify genes involved in germ-line development, we produced subtracted cDNA pools either enriched for or deprived of the cDNAs from germ-line tissues. We then performed differential hybridization on the high-density cDNA grid, on which about 7,600 nonoverlapping expressed sequence tag (EST) clones were spotted, to identify a set of genes specifically expressed in the germ line. One hundred and sixty-eight clones were then tested with the RNAi technique. Of these, 15 clones showed sterility with a variety of defects in germ-line development. Seven of them led to the production of unfertilized eggs, because of defects in spermatogenesis (4 clones), or defects in the oocytes (3 clones). The other 8 clones led to failure of oogenesis. These failures were caused by germ-line proliferation defect (Glp phenotype), meiotic arrest, and defects in sperm–oocyte switch (Mog phenotype) among others. These results demonstrate the efficacy of the screening strategy using the EST library combined with the RNAi technique in C. elegans.

Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo.

Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli. Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence. These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves. Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.

Interaction between human tRNA synthetases involves repeated sequence elements.

Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex. Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system. Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix. This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library. The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl- and prolyl-) tRNA synthetase, which is also known to be a component of the complex. Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively. Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs. Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex. The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.

Primordial emergence of the recombination activating gene 1 (RAG1): sequence of the complete shark gene indicates homology to microbial integrases.

The rearrangement of antibody and T-cell receptor gene segments is indispensable to the vertebrate immune response. All extant jawed vertebrates can rearrange these gene segments. This ability is conferred by the recombination activating genes I and II (RAG I and RAG II). To elucidate their origin and function, the cDNA encoding RAG I from a member of the most ancient class of extant gnathostomes, the Carcharhine sharks, was characterized. Homology domains identified within shark RAG I prompted sequence comparison analyses that suggested similarity of the RAG I and II genes, respectively, to the integrase family genes and integration host factor genes of the bacterial site-specific recombination system. Thus, the apparent explosive evolution (or "big bang") of the ancestral immune system may have been initiated by a transfer of microbial site-specific recombinases.

Gene recognition via spliced sequence alignment.

Gene recognition is one of the most important problems in computational molecular biology. Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored. Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes. This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons. On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99%. The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing. Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic. The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.

Sequence and expression of the murine Hoxd-3 homeobox gene.

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.

Cloning of rat MEK kinase 1 cDNA reveals an endogenous membrane-associated 195-kDa protein with a large regulatory domain.

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signal-regulated kinase 2.

Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.

Unexpected beta2-microglobulin sequence diversity in individual rainbow trout.

For mammals beta2-microglobulin (beta2m), the light chain of major histocompatibility complex (MHC) class I molecules, is invariant (or highly conserved) and is encoded by a single gene unlinked to the MHC. We find that beta2m of a salmonid fish, the rainbow trout (Oncorhynchus mykiss), does not conform to the mammalian paradigm. Ten of 12 randomly selected beta2m cDNA clones from an individual fish have different nucleotide sequences. A complex restriction fragment length polymorphism pattern is observed with rainbow trout, suggesting multiple beta2m genes in the genome, in excess of the two genes expected from the ancestral salmonid tetraploidy. Additional duplication and diversification of the beta2m genes might have occurred subsequently. Variation in the beta2m cDNA sequences is mainly at sites that do not perturb the structure of the mature beta2m protein, showing that the observed diversity of the trout beta2m genes is not primarily a result of pathogen selection.

Induction of cellular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNA-transfected Epstein-Barr virus-immortalized autologous B cells.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor.

The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.

Sequence verification of human creatine kinase (43 kDa) isozymes by high-resolution tandem mass spectrometry.

Amino acid sequencing by recombinant DNA technology, although dramatically useful, is subject to base reading errors, is indirect, and is insensitive to posttranslational processing. Mass spectrometry techniques can provide molecular weight data from even relatively large proteins for such cDNA sequences and can serve as a check of an enzyme's purity and sequence integrity. Multiply-charged ions from electrospray ionization can be dissociated to yield structural information by tandem mass spectrometry, providing a second method for gaining additional confidence in primary sequence confirmation. Here, accurate (+/- 1 Da) molecular weight and molecular ion dissociation information for human muscle and brain creatine kinases has been obtained by electrospray ionization coupled with Fourier-transform mass spectrometry to help distinguish which of several published amino acid sequences for both enzymes are correct. The results herein are consistent with one published sequence for each isozyme, and the heterogeneity indicated by isoelectric focusing due to 1-Da deamidation changes. This approach appears generally useful for detailed sequence verification of recombinant proteins.

In vitro integration of human immunodeficiency virus type 1 cDNA into targets containing protein-induced bends.

Integration of human immunodeficiency virus type 1 cDNA into a target DNA can be strongly influenced by the conformation of the target. For example, integration in vitro is sometimes favored in target DNAs containing sequence-directed bends or DNA distortions caused by bound proteins. We have analyzed the effect of DNA bending by studying integration into two well-characterized protein-DNA complexes: Escherichia coli integration host factor (IHF) protein bound to a phage IHF site, and the DNA binding domain of human lymphoid enhancer factor (LEF) bound to a LEF site. Both of these proteins have previously been reported to bend DNA by approximately 140 degrees. Binding of IHF greatly increases the efficiency of in vitro integration at hotspots within the IHF site. We analyzed a series of mutants in which the IHF site was modified at the most prominent hotspot. We found that each variant still displayed enhanced integration upon IHF binding. Evidently the local sequence is not critical for formation of an IHF hotspot. LEF binding did not create preferred sites for integration. The different effects of IHF and LEF binding can be rationalized in terms of the different proposed conformations of the two protein-DNA complexes.