Machiko Sawada

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine-guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.

Multiple Vacuolar Sorting Determinants Exist in Soybean 11S Globulin

The sorting determinants of glycinin, a soybean (Glycine max) 11S globulin, which mediates protein targeting to the protein storage vacuole (PSV), were investigated in maturing soybean cotyledons by transient expression assays. A C-terminal stretch of 10 amino acids of A1aB1b, a glycinin group I subunit, was sufficient to direct green fluorescent protein (GFP) to the PSV. This peptide may correspond to a C-terminal vacuolar sorting determinant (ctVSD). Because functional inhibition of this putative ctVSD of A1aB1b did not block PSV sorting of A1aB1b, we used the three-dimensional structure of A1aB1b to identify candidates for a sequence-specific determinant (ssVSD). We found that the sequence downstream of disordered region 4 could direct GFP to the PSV and that Ile-297 is critical for sorting. However, functional inhibition of the ctVSD, combined with the Ile297Gly mutation, did not abolish the vacuolar sorting of A1aB1b, suggesting that A1aB1b has a third sorting determinant in addition to ctVSD and ssVSD. A glycinin group II subunit, A3B4, lacked a ctVSD but contained a VSD reminiscent of an ssVSD and an additional sorting determinant. We also demonstrate, by expression of dominant negative mutants of small GTPases and drug treatment experiments, that the trafficking of A1aB1b is COPII vesicle–dependent and wortmannin- and brefeldin A–sensitive.

Role of Protein Tyrosine Phosphorylation in Etoposide-Induced Apoptosis and NF-κB Activation

Abstract When a human myeloid cell line, U937, was incubated with etoposide (10 μg/mL), morphologically apoptotic cells first appeared at 3 hr and increased with time to 50% at 6 hr. Pretreatment of U937 cells for 30 min with a potent tyrosine kinase inhibitor, herbimycin A (10 μM), significantly suppressed the appearance of apoptotic morphological changes. Concomitantly, herbimycin A pretreatment prevented both high molecular weight and internucleosomal DNA fragmentation induced by etoposide. Two major bands at 30 and 66 kDa with enhanced tyrosine phosphorylation inhibited by herbimycin A were detectable after 30 min of incubation with etoposide. In addition, herbimycin A prevented etoposide-induced NF-κB activation. The expressions of Bcl-2 and Bax, on the other hand, were not affected by herbimycin A pretreatment. Herbimycin A was also found to inhibit 1-β- d -arabinofuranosylcytosine-induced apoptotic changes and NF-κB activation. These results suggest that activation of tyrosine kinase(s) may play an important role in apoptotic processes induced by a variety of anti-cancer drugs.

Evaluation of mutant frequencies at the hprt and the T-cell receptor loci in pediatric cancer patients before treatment

Mutant frequencies (Mfs) at the two genetic loci, the hypoxanthine phosphoribosyl transferase (hprt) gene and the T-cell receptor (TCR) gene were evaluated in pediatric cancer patients before starting chemotherapy or radiotherapy. The study population consisted of 27 patients with various solid tumors (mean age +/- SD; 5.5 +/- 5.1 years, range; 0.2-14.5 years), 5 patients with acute leukemia (10.3 +/- 6.1, 1.3-17.0 years), and 26 healthy controls (11.6 +/- 4.0, 4.4-22.2 years). Although the age distributions were different, the mean Mf values of the hprt and the TCR loci were comparable among these three groups. On an individual basis taking the age factor into consideration, the hprt-Mfs of 3 patients with solid tumors, i.e., two patients with Hodgkins disease and one patient with Askin tumor, were found to be well above the 95% confidence limit. There were no patients with a TCR-Mf exceeding the 95% confidence limit. These data suggest the possibility that some patients with solid tumors may be predisposed to mutational susceptibility before treatment. The assay of the hprt-Mf appears more sensitive than the TCR-Mf assay in distinguishing these patients.

A NOVEL MISSENSE MUTATION IN THE DKC1 GENE IN A JAPANESE FAMILY WITH X-LINKED DYSKERATOSIS CONGENITA

The authors report 2 male patients with dyskeratosis congenita (DC) in a Japanese kindred. Sequencing of the complementary DNA of the dyskerin gene (DKC1) revealed a T-to-C transition at nucleotide 1285 in exon 12 that resulted in a novel missense mutation L398P. Despite harboring the same mutation in the DKC1 gene, one patient had significantly milder hematological symptoms than the other, indicating that there may be other factors that determine the severity of DC.

Biomarkers in long survivors of pediatric acute lymphoblastic leukemia patients: late effects of cancer chemotherapy.

In order to elucidate the late effects of cancer chemotherapy, mutant frequencies (Mfs) at the hypoxanthine phosphoribosyl transferase (hprt) locus were evaluated in pediatric patients with early pre-B acute lymphoblastic leukemia (ALL). Hprt-Mfs were measured at least 2 years after completion of chemotherapy. Ten out of 15 patients were found to have hprt-Mfs exceeding the 99% confidence limits as calculated from observations of healthy controls. Although there was some intraindividual variation, serial measurements of hprt-Mfs with intervals of more than 6 months revealed that hprt-Mfs were fairly stable. Patients with high Mfs tended to have sibling clones as detected by clonality analysis using the T-cell receptor (TCR) rearrangement pattern, but clonality did not have a major effect on the Mfs. On the other hand, Mfs at the TCR locus and sister chromatid exchange frequency were within the normal range in all patients. These data suggest that chemotherapy can cause persistent genotoxicity in vivo in a subset of pediatric ALL patients and that the hprt-Mf is a useful method for measuring such an effect.

Methotrexate inhibits superoxide production and chemotaxis in neutrophils activated by granulocyte colony-stimulating factor

Treatment of circulating human neutrophils with recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) for 30 min augmented superoxide generation and chemotaxis induced by N‐formylmethionyl‐leucyl‐phenylalanine (fMLP) in a dose dependent manner. When neutrophils were treated with 1 μM of methotrexate (MTX) for 60 min after incubation with rhG‐CSF (10 ng/ml), the effects of rhG‐CSF on superoxide generation and chemotaxis were inhibited by approximately 49 and 29%, respectively. Although inhibitory effects of MTX were also seen in neutrophils not pretreated with rhG‐CSF, the degree of inhibition was much less. The addition of either hypoxanthine or guanosine at a concentration of 100 μM to the culture medium significantly attenuated the effects of MTX. However, in neutrophils obtained from a patient with Lesch‐Nyhan syndrome, which lacked hypoxanthine‐guanine phosphoribosyl transferase activity, neither hypoxanthine nor guanosine had any rescue effect. These results suggest that MTX inhibits superoxide generation and chemotaxis in rhG‐CSF‐activated neutrophils, at least in part, by disturbing purine nucleotide biosynthesis.

Measurement of Mutation Frequency at the HPRT Locus in Peripheral Lymphocytes

Validity of measurement of somatic cell mutation frequency (Mf) at the hprt locus for evaluating cancer risk of the given individual was determined in pediatric patients. Peripheral lymphocytes (PL) from patients with various diseases, including acute lymphoblastic leukemia (ALL) and Hodgkins disease (HD), DNA repair deficient syndromes or short stature receiving growth hormone (GH), were isolated through Ficoll-Hypaque sedimentation with informed consent. Mf at the hprt locus of PL was determined by limiting dilution assay using 6-thioguanine (6-TG). Results were as follows. (1) ALL patients after chemotherapy had higher Mf than that of age-matched controls. (2) Patients with HD tended to have higher Mf after chemotherapy. (3) Among DNA-repair deficient syndromes, diseases which are susceptible to cancer (Xeroderma pigmentosum, Ataxia telangiectasia) have high Mf, but those without any cancer disposition (Cockayne syndrome, Rothmund-Thomson syndrome) have normal Mf. (4) GH-receiving patients have normal Mf, regardless of total doses of GH. Measurement of Mf at HPRT locus may be useful for evaluating cancer risk of pediatric patients.