Analysis of transcriptional activity of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> ornithine-delta-aminotransferase gene promoter

Cloning of the Arabidopsis thaliana genomic DNA fragment presumably corresponding to the promoter region of the ornithine-delta-aminotransferase (OAT) gene is reported. The reporter-gene construct, containing the Escherichia coli beta-glucouronidase gene under control of the OAT gene promoter was generated. The Nicotian tabacum SR1 transformants carrying this construct were obtained. It was demonstrated that under normal conditions, expression of the reporter gene was associated with the meristems and the zones of intensive shoot growth. Possible role of the OAT gene in nitrogen metabolism and shoot development is discussed.     

Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings

Summary The objective of this study was to separate and determine effects on the field performance of transgenic potatoes that originate from the tissue culture process of transformation and from the genes inserted. The constructs introduced contained the reporter gene for betaglucuronidase (GUS) under the control of the patatin promoter (four different constructs) and the neomycin phosphotransferase gene under the control of the nopaline synthase promoter. Both genes might be expected to have a neutral effect on plant phenotype. The field performance of transgenic plants (70 independent transformants) was compared with non-transgenic plants regenerated from tuber discs by adventitious shoot formation and from shoot cultures established from tuber nodal cuttings. Plants from all three treatments were grown in a field trial from previously field-grown tubers, and plant performance was measured in terms of plant height at flowering, weight of tubers, number of tubers, weight of large tubers and number of large tubers. There was evidence of somaclonal variation among the transgenic plants; mean values for all characters were significantly lower and variances generally higher than from plants derived from nodal shoot cultures. A similar change in means and variances was observed for the non-transgenic tuber-disc regenerants when compared with shoot culture plants. Plant height, tuber weight and tuber number were, however, significantly lower in transgenic plants than in tuber-disc regenerants, suggesting an effect on plant performance either of the tissue culture process used for transformation or of the genes inserted. There were significant differences between constructs for all five plant characters. The construct with the smallest segment of patatin promoter and the lowest level of tuber specificity for GUS expression had the lowest values for all five characters. It is proposed that the nature of GUS expression is influencing plant performance. There was no indication that the NPTII gene, used widely in plant transformation, has any substantial effect on plant performance in the field.     

番茄果实特异性启动子的克隆与遗传转化研究

为了实现外源基因在番茄果实中的高效和特异表达,克隆了番茄果实特异基因多聚半乳糖醛酸酶基因( Polygalacturonase,PG)的启动子.以中蔬四号番茄为材料,建立并优化了以子叶为外植体的番茄高效再生和遗传转化体系;以GUS为报告基因,构建PG:GUS植物表达载体,转化番茄.结果表明,在1.0 mg/L ZT的MS分化培养中,番茄子叶的发芽率最高,芽的诱导率高达91％,且发生畸态芽和褐化的外植体最少;通过抗生素浓度对农杆菌的抑制效果试验发现,当头孢霉素的浓度为200 mg/L时,抑制农杆菌的效果最好;成功克隆了番茄PG启动子,将PG启动子驱动的GUS基因转入番茄,对转基因后代果实的GUS染色表明,PG启动子驱动的外源基因在果实中特异表达.     

Functional expression of the rat organic anion transporter 1 (rOAT1) in Saccharomyces cerevisiae

Organic anion transporter 1 (OAT1) is localized in the basolateral membrane of the proximal tubule in the kidney and plays an essential role in eliminating a wide range of organic anions, preventing their toxic effects on the body. Structural and functional studies of the transporter would be greatly assisted by inexpensive and rapid expression in the yeast Saccharomyces cerevisiae. The gene encoding rat OAT1 (rOAT1) contains many yeast non-preferred codons at the N-terminus and so was modified by fusion of the favored codon sequence of a hemagglutinin (HA) epitope preceding the start codon. The modified gene was cloned into several yeast expression plasmids, both integrative and multicopy, with either ADH1 promoter or GAL1 promoter in order to find a suitable expression system. Compared with the wild type gene, a substantial increase in rOAT1 expression was achieved by modification in the translational initiation region, suggesting that the codon chosen at the N-terminus influenced its expression. The highest inducible expression of rOAT1 was obtained under GAL1 promoter in 2 mu plasmid. A large fraction of rOAT1 was glycosylated in yeast, unaffected by growth temperature. The recombinant yeast expressing rOAT1 showed an increase in the uptake of p-aminohippurate (PAH) and this showed a positive correlation with rOAT1 expression level. Location of rOAT1 predominantly in the yeast plasma membrane confirmed correct processing. The importance of glycosylation for rOAT1 targeting was also shown. To our knowledge, this is the first successful functional expression of rOAT1 in the yeast S. cerevisiae.     

Agrobacterium-mediated transformation of tarwi (Lupinus mutabilis Sweet), a potential platform for the production of plant-made proteins

A robust, reproducible method of Agrobacterium-mediated transformation was developed for Lupinus mutabilis Sweet (tarwi), a large-seeded Andean legume. Initially, a regeneration and transformation protocol was developed using a plasmid which contained a bifunctional fusion gene conferring both β-glucuronidase (gus) and neomycin phosphotransferase activities, under the control of a constitutive 35S35SAMV promoter. The tissue explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. Using a series of tissue culture media for cocultivation, shoot initiation, shoot elongation, and rooting, kanamycin-resistant transgenic plants were recovered from approximately 1% of the explants. This transformation protocol was further used with a construct that contained the human adenosine deaminase (hADA) gene under the control of a legumin seed-specific promoter, also with a kanamycin resistance cassette for chemical selection. Changes made during the course of this study, which included adjustments to the antibiotic concentration during the shoot elongation and rooting phases plus the incorporation of techniques to improve ventilation in the tissue culture system, resulted in major improvements in shoot quality and, most significantly, rooting. The outcome was an increased frequency of transgenic plant recovery (7.4%), with a low (9.6%) rate of plants that escaped selection. The inheritance of the hADA gene was documented and showed the expected Mendelian segregation pattern. The produced hADA protein was a fully functional enzyme and localized only in the seed, as expected. Thus, this legume species is an excellent candidate for a nonfood plant host platform for the production of plant-made proteins.     

Inheritance of ornithine aminotransferase gene, mRNA, and enzyme defect in a family with gyrate atrophy of the choroid and retina.

We studied the human ornithine aminotransferase (OAT) gene, mRNA, and enzyme activity in fibroblasts from a family with gyrate atrophy (G.A.) of the choroid and retina, using a normal human OAT cDNA as a probe. The family consists of an affected patient, who is heterozygous for a partial deletion of the functional OAT gene and whose cells produce no mRNA, and of his father, mother, two sons, and a daughter. Southern blot analysis of the OAT gene showed the partial deletion in the patient and in his father and daughter and in one son. Northern blot analysis revealed no OAT mRNA in the patient and approximately 50% of the normal level of OAT mRNA in the father, mother, two sons, and daughter. Assay showed that the OAT activity in these individuals mirrored the OAT mRNA levels. The results indicate that an active allele of the OAT gene expresses 50% of the total normal OAT mRNA and activity and that both alleles of the gene are inactive in the patient in this pedigree, a situation resulting in a complete absence of the OAT mRNA, in accordance with the autosomal recessive mechanism of this disease; they also indicate a 50% decrease of OAT mRNA and enzyme activity in obligate heterozygous carriers carrying one defective allele and that these defects are stably inherited.     

The ornithine aminotransferase gene in gyrate,atrophy of the retina: Analysis of expression and gross structure of this gene in cultured fibroblasts

Summary Gyrate atrophy (GA), a degenerative disease of the human chorioretina, is associated with a deficiency of ornithine aminotransferase (OAT) activity, hyperornithinemia, and ornithinuria. We have characterized a cDNA clone for OAT (HLOAT) that was isolated from a cDNA library constructed from mRNA prepared from Hep G2, cells, a human hepatoma cell line. We have used HLOAT and a nearly full length OAT cDNA clone isolated from, a rat liver library (RLOAT) to examine in cultured fibroblasts from individuals with GA and control individuals, the expression of OAT mRNA and the gross structure of the OAT gene. Northern blot analyses of total cellular RNA indicated that 3 of 3 control cell lines and 5 of 6 GA cell lines are capable of expressing an OAT related mRNA of approximately 2100 bases, the size of OAT mRNA. To date, this is the only case of GA in which a complete lack of OAT mRNA has been observed. Southern blot analyses of DNA isolated from these cell lines indicated that the gross structure of the OAT gene is usually not detectably altered in individuals with GA. However, a unique pattern, of restriction fragments was observed upon digestion with Eco RI or Hind III of DNA from the GA cell line that does not express OAT mRNA. These unique Eco RI and Hind III fragments arise from the OAT structural gene and will serve as useful molecular markers that allow this particular defective OAT allele to be identified. When the cellular DNAs were digested with Hinf I and examined with a probe that corresponds to at least a portion of the active site of the enzyme, i. e., the pyridoxal phosphate binding site, identical patterns of fragments were detected in all samples. Therefore, it appears unlikely that the loss of OAT activity associated with these GA cases, 4 of which are pyridoxal phosphate responders, is the result of insertions or deletions in this region of the OAT gene. This study indicates that the lack of OAT enzyme activity associated with GA is the result of a variety of different molecular defects within the OAT gene. This project was initiated in the laboratory of H. C. P. and was supported by grants CA07175, CA22484, and 5 T32 CA09020 from the National Cancer Institute and Postdoctoral Fellowship PF-2414 from the American Cancer Society. The continuing work in the laboratory of J. D. S. was supported by grants CA36727 and HD24189 from the National, Institutes of Health, grants SIG-16, ACS-IN165A, and a Junior Faculty Research Award (JFRA-227) from the American Cancer Society, and by University of Nebraska Medical Center Seed Research Grant 88-10.     

A single-base change at a splice acceptor site in the ornithine aminotransferase gene causes abnormal RNA splicing in gyrate atrophy

Gyrate atrophy (GA) is an autosomal recessive eye disease involving a progressive loss of vision due to chorioretinal degeneration in which the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is defective. Two sisters with GA are described in this study in whom an A-to-G substitution at the 3 splice acceptor site of intron 4 in one allele of the OAT gene results in a truncated OAT mRNA devoid of exon 5 sequence. The mutation in the other allele was identified to be a missense mutation at codon 318 by denaturing gradient gel electrophoresis and direct sequencing of the polymerase chain reaction (PCR)-amplified DNA. Thus, these GA patients are compound heterozygotes with respect to mutations in the OAT gene that result in inactivation of OAT.