Expression of Ornithine Decarboxylase Is Transiently Increased by Pollination, 2,4-Dichlorophenoxyacetic Acid, and Gibberellic Acid in Tomato Ovaries1

A cDNA encoding for a functional ornithine decarboxylase has been isolated from a cDNA library of carpels of tomato (Lycopersicon esculentum Mill.). Ornithine decarboxylase in tomato is represented by a single-copy gene that we show to be up-regulated during early fruit growth induced by 2,4-dichlorophenoxyacetic acid and gibberellic acid.

wrinkled1: A Novel, Low-Seed-Oil Mutant of Arabidopsis with a Deficiency in the Seed-Specific Regulation of Carbohydrate Metabolism1

During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling.

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.

The Role of Iron-Deficiency Stress Responses in Stimulating Heavy-Metal Transport in Plants1

Plant accumulation of Fe and other metals can be enhanced under Fe deficiency. We investigated the influence of Fe status on heavy-metal and divalent-cation uptake in roots of pea (Pisum sativum L. cv Sparkle) seedlings using Cd2+ uptake as a model system. Radiotracer techniques were used to quantify unidirectional 109Cd influx into roots of Fe-deficient and Fe-sufficient pea seedlings. The concentration-dependent kinetics for 109Cd influx were graphically complex and nonsaturating but could be resolved into a linear component and a saturable component exhibiting Michaelis-Menten kinetics. We demonstrated that the linear component was apoplastically bound Cd2+ remaining in the root cell wall after desorption, whereas the saturable component was transporter-mediated Cd2+ influx across the root-cell plasma membrane. The Cd2+ transport system in roots of both Fe-deficient and Fe-sufficient seedlings exhibited similar Michaelis constant values, 1.5 and 0.6 μm, respectively, for saturable Cd2+ influx, whereas the maximum initial velocity for Cd2+ uptake in Fe-deficient seedlings was nearly 7-fold higher than that in Fe-grown seedlings. Investigations into the mechanistic basis for this response demonstrated that Fe-deficiency-induced stimulation of the plasma membrane H+-ATPase did not play a role in the enhanced Cd2+ uptake. Expression studies with the Fe2+ transporter cloned from Arabidopsis, IRT1, indicated that Fe deficiency induced the expression of this transporter, which might facilitate the transport of heavy-metal divalent cations such as Cd2+ and Zn2+, in addition to Fe2+.

Formate Dehydrogenase, an Enzyme of Anaerobic Metabolism, Is Induced by Iron Deficiency in Barley Roots1

To identify the proteins induced by Fe deficiency, we have compared the proteins of Fe-sufficient and Fe-deficient barley (Hordeum vulgare L.) roots by two-dimensional polyacrylamide gel electrophoresis. Peptide sequence analysis of induced proteins revealed that formate dehydrogenase (FDH), adenine phosphoribosyltransferase, and the Ids3 gene product (for Fe deficiency-specific) increased in Fe-deficient roots. FDH enzyme activity was detected in Fe-deficient roots but not in Fe-sufficient roots. A cDNA encoding FDH (Fdh) was cloned and sequenced. Fdh expression was induced by Fe deficiency. Fdh was also expressed under anaerobic stress and its expression was more rapid than that induced by Fe deficiency. Thus, the expression of Fdh observed in Fe-deficient barley roots appeared to be a secondary effect caused by oxygen deficiency in Fe-deficient plants.

Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA1

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [14C]Arg, [14C]Orn, and [14C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [14C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.

The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).

APC mutations in colorectal tumors with mismatch repair deficiency.

We have investigated the influence of genetic instability [replication error (RER) phenotype] on APC (adenomatous polyposis coli), a gene thought to initiate colorectal tumorigenesis. The prevalence of APC mutations was similar in RER and non-RER tumors, indicating that both tumor types share this step in neoplastic transformation. However, in a total of 101 sequenced mutations, we noted a substantial excess of APC frameshift mutations in the RER cases (70% in RER tumors versus 47% in non-RER tumors, P < 0.04). These frameshifts were characteristic of mutations arising in cells deficient in DNA mismatch repair, with a predilection for mononucleotide repeats in the RER tumors (P < 0.0002), particularly (A)n tracts (P < 0.00007). These findings suggest that the genetic instability that is reflected by the RER phenotype precedes, and is responsible for, APC mutation in RER large bowel tumors and have important implications for understanding the very earliest stages of neoplasia in patients with tumors deficient in mismatch repair.

Growth retardation and cysteine deficiency in gamma-glutamyl transpeptidase-deficient mice.

gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme that catalyzes the first step in the cleavage of glutathione (GSH) and plays an essential role in the metabolism of GSH and GSH conjugates of carcinogens, toxins, and eicosanoids. To learn more about the role of GGT in metabolism in vivo, we used embryonic stem cell technology to generate GGT-deficient (GGTm1/GGTm1) mice. GGT-deficient mice appear normal at birth but grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts, and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels in the GGTm1/GGTm1 mice are elevated 6-fold and 2500-fold, respectively, compared with wild-type mice. Tissue GSH levels are markedly reduced in eye, liver, and pancreas. Plasma cyst(e)ine levels in GGTm1/GGTm1 mice are reduced to approximately 20% of wild-type mice. Oral administration of N-acetylcysteine to GGTm1/GGTm1 mice results in normal growth rates and partially restores the normal agouti coat color. These findings demonstrate the importance of GGT and the gamma-glutamyl cycle in cysteine and GSH homeostasis.

Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation.

Adaptive mutation sequences reproduced by mismatch repair deficiency.

Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous. The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E. coli occurs in the absence of mismatch repair. The postulate that mismatch repair is disabled transiently during adaptive mutation in E. coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth. Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution.