Surface melting and the surface phase diagram.
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Journal Article↗
Biomedical subjects
G An
Publications and source records attributed to G An.
Identification of upstream regulatory elements involved in the developmental expression of the Arabidopsis thaliana cab1 gene.
We studied cis regulatory elements controlling the light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene (cab1) by stably transforming tobacco plants using a tumor-inducing (Ti) plasmid vector system. The results from the 5' and internal deletion analyses indicate that there are at least three cis-acting elements that are involved in the light-dependent developmental expression of cab1 gene. Two such elements are located at the immediate upstream regulatory region and the other element is located at the further upstream region. The 1120-base-pair (bp) DNA fragment containing the immediate and far upstream region can confer light-inducible organ specificity on the truncated nos promoter. However, deletion of the 39-bp DNA fragment at the immediate upstream regulatory region from this hybrid promoter resulted in a nonfunctional promoter, revealing that the 39-bp region is important for the cab promoter specificity. Further analyses of this region suggest that a potential Z-DNA-forming sequence (ATACGTGT) is involved in light-dependent developmental expression of the cab1 gene. Two additional Z-DNA-forming sequences (ACACATAT) that are inverted repeats of this sequence are also found in the upstream region where the additional regulatory elements are expected.
Base Sequence↗
Organ-specific and developmental regulation of the nopaline synthase promoter in transgenic tobacco plants.
Control regions of the nopaline synthase (nos) gene have been widely used to express foreign genes in plants since the promoter is active in a wide variety of plant tissues. We report here the characteristics of the nos promoter activity in transgenic tobacco (Nicotiana tabacum) plants at various developmental stages. The promoter was highly active in the lower parts of a plant and gradually decreased in the upper parts. This vertical gradient was maintained throughout plant growth until the flowering stage when the overall promoter strength decreased significantly in the vegetative organs. However, in various flower organs, the nos promoter activities increased dramatically. Higher activity was observed in calyx, corolla, and stamens although the maximum promoter activity in each organ was found at different stages of flower development. The promoter activity in pistils was low and gradually increased in the ovaries after anthesis. In developing fruits, the nos promoter activity was strongly induced during the mid-stage of embryogenesis. These results indicate that the expression of the nos promoter is developmentally regulated and organ specific in transgenic tobacco plants.
Journal Article↗
Identification of an essential upstream element in the nopaline synthase promoter by stable and transient assays.
We studied the fine structure of the nopaline synthase (nos) promoter, which is active constitutively in a wide range of plant tissues, by both transient and stable transformation expression analyses. 3' and 5' deletion fragments were linked to form a set of internal deletion and duplication mutants that scanned the nos promoter. These mutated promoters were linked to the gene for the marker chloramphenicol acetyltransferase (CATase) as a means to readily assay promoter strength. The stable transformation analysis revealed the functional importance of an extended CCAAT box region (-97 to -63). Deletion of an upstream region (-112 to -101) containing an octameric repeated element resulted in a reduction in promoter strength by a factor of 30. A further deletion (-119 to -101) disrupted a potential Z-DNA-forming element as well, totally eliminating promoter function. Thus, a 19-base deletion across a repeated octamer and a potential Z-DNA-forming element identifies an essential upstream activator in the nos promoter. Duplication of the upstream element tripled promoter activity. Electroporation-mediated transient analysis was unable to distinguish downstream promoter elements. However, the upstream element behaved similarly in both assays in that deletion of the entire upstream element resulted in no promoter activity and that duplication of the element significantly enhanced the promoter strength.
Journal Article↗
Wound-inducible expression of a potato inhibitor II-chloramphenicol acetyltransferase gene fusion in transgenic tobacco plants.
A potato inhibitor II gene (IIK) was isolated from a library of potato genes in lambda bacteriophage. An 8-kilobase-pair (kbp) insert was identified using a tomato inhibitor II cDNA as a hybridization probe, and a 2.6-kbp fragment containing the gene was subcloned into the plasmid pUC13 and characterized. The nucleotide sequence of the isolated gene exhibited 87% identity with the wound-inducible tomato inhibitor II cDNA sequence. The amino acid sequence of inhibitor IIK, deduced from the potato gene, exhibited 84% identity with the tomato inhibitor II protein. A 1000-bp restriction fragment from the 5' flanking region of the gene was fused to the open reading frame of the chloramphenicol acetyltransferase (CAT) gene. This fusion was terminated in two ways: (i) with a terminator sequence from the potato inhibitor II gene and (ii) with a terminator from the 6b gene of Ti plasmid pTiA6. These chimeric genes were transferred into tobacco cells via a binary Ti vector system, and transgenic plants were regenerated. The CAT gene was expressed in leaves of transformed plants in response to wounding when fused with the inhibitor IIK promoter and terminator regions. The chimeric gene containing the 6b terminator did not express CAT in response to wounding. The wound-inducible expression of CAT activity was systemic and was induced in tissues distal to the wounded tissues. The time course of wound induction of CAT activity in transgenic tobacco leaves is similar to that found for wound-inducible inhibitor I and II mRNAs in tomato leaves. These results demonstrate that sequences necessary and sufficient for wound inducibility are present within approximately 1000 bp of the control regions of the inhibitor IIK genes and that wound-inducible components of tobacco leaf cells can regulate these sequences.
Journal Article↗
Transformation of Tobacco, Tomato, Potato, and Arabidopsis thaliana Using a Binary Ti Vector System.
Using a binary tumor-inducing (Ti) plasmid vector system, several plant species were transformed with a kanamycin resistance marker (neomycin phosphotransferase gene). Four Nicotiana species, seven tomato cultivars, two potato cultivars, and Arabidopsis thaliana were transformed by the binary vector transformation method. In this method, various plant organ pieces were co-cultivated with Agrobacterium tumefaciens cells carrying the binary vector, pGA472, and a helper Ti plasmid. We have also demonstrated that a wild type Ti plasmid can be used as a helper to obtain a transformed plant.
Journal Article↗
Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells.
I have developed promoter expression binary vectors based on the tumor-inducing plasmid of Agrobacterium tumefaciens to facilitate elucidation of plant gene regulation. Promoter activity can be determined by inserting DNA fragments into the multiple cloning sites of the vectors forming transcriptional and/or translational fusions between the cat structural gene and an inserted promoter region. The activity of the nopaline synthase (nos) promoter was demonstrated with the vector. However, three animal promoters tested with this system showed no measurable activity in plant cells. Examination of 40 independently derived transformed tissues revealed a 200-fold difference in the nos promoter activity. Furthermore, there is no apparent correlation between the neomycin phosphotransferase and chloramphenicol acetyltransferase activities, although both genes are closely linked and under control of identical nos promoters. These results indicate that vast differences in promoter activity of transferred genes can occur within the same cell, as well as in independently derived cell lines.
Journal Article↗
[Ultrastructural changes of the human gastric cancer cell line MGc 80-3 caused by HpD-laser in vitro].
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Adenocarcinoma, Mucinous↗
New cloning vehicles for transformation of higher plants.
We have constructed a set of small vectors based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which allow the transfer of exogenous DNA into plant chromosomes. These vectors contain: (i) a chimeric gene containing the transcriptional control signals from the nopaline synthase gene and the coding sequence for neomycin phosphotransferase; (ii) the ColE1 replicon; (iii) the cos site of bacteriophage lambda; (iv) the border sequences from the ends of the T-DNA region of the Ti plasmid; and (v) a wide host range replicon. Due to the small size of these cosmid vectors, DNA fragments up to 35 kbp can be inserted by an in vitro packaging method in Escherichia coli. The ability of these vectors to be stably replicated in both E. coli and A. tumefaciens allows their subsequent transfer to and maintenance in Agrobacterium without intermediate genetic manipulations. We demonstrate that DNA cloned into these vectors in A. tumefaciens can efficiently transform plants when in trans with a wild-type Ti plasmid which donates the functions necessary for DNA transfer and integration. We also show that only the right border of the T-DNA is necessary for DNA transformation.
Journal Article↗
A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium.
The construction and use of a Tn3-lac transposon, Tn3-HoHo1, is described. Tn3-HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta-galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3-HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta-galactosidase activity. Tn3-HoHo1 carries a non-functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3-HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3-HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3-HoHo1 generated lac fusions.
Amino Acid Sequence↗
Amphotericin B enhances efficiency of DNA-mediated gene transfer in mammalian cells.
Using plasmids containing the genes for thymidine kinase (tk) and neomycin resistance (neo), we have shown that DNA-mediated genotypic transformation of L and Chinese hamster ovary (CHO) cells is increased several-fold by the presence of the sterol-binding polyene antibiotic, amphotericin. Transformation into the same host cells, using genomic DNA, was also enhanced by amphotericin. Phenotypic expression of beta-galactosidase activity of a plasmid containing the gene for the enzyme was also markedly elevated when the antibiotic was added at transfection. Other sterol-binding polyene antibiotics also showed activity in these DNA-mediated gene transfer assays.
Amphotericin B↗
High efficiency transformation of cultured tobacco cells.
Tobacco calli were transformed at levels up to 50% by cocultivation of tobacco cultured cells with Agrobacterium tumefaciens harboring the binary transfer-DNA vector, pGA472, containing a kanamycin resistance marker. Transformation frequency was dependent on the physiological state of the tobacco cells, the nature of Agrobacterium strain and, less so, on the expression of the vir genes of the tumor-inducing plasmid. Maximum transformation frequency was obtained with exponentially growing plant cells, suggesting that rapid growth of plant cells is an essental factor for efficient transformation of higher plants.
Journal Article↗
[Controlled hypotensive anesthesia in cleft palate surgery].
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Adolescent↗
The lethal effect of a plasmid resulting from transcriptional readthrough of rplJ from the rplKA operon in Escherichia coli.
A high-copy plasmid, pGA217, which carries a deletion (lacking the carboxy-terminal 20 amino acids) of the structural gene for ribosomal protein L10 (rplJ) is lethal to the cell in the absence of the gene (rplL) for r-proteins L7/L12, but only if the upstream operon for r-proteins L11 (rplK) and L1 (rplA) is present on the same plasmid. Measurements of beta-galactosidase activity of a hybrid protein expressed by a rplL-lacZ fusion indicated that the L10 fragment peptide which lacks the carboxy-terminal 20 amino acids is capable of exerting feedback regulation. Double transformation experiments with two compatible plasmids showed that the detrimental effect of the rplJ deletion on pGA217 can be reversed by the addition of a second plasmid which carries a functional gene for L7/L12. These two pieces of evidence suggest that the lethal effect of pGA217 is due to its property of feeding back on L7/L12 production from the chromosomal rplK gene. The upstream rplKA operon was inferred to have a cis-acting, stimulating effect on rplJ expression from the following evidence: (1) donor plasmids carrying the genes for L11 and/or L1 fail to exert a trans-acting effect, (2) deletion mutants which removed portions of rplK and/or rplA, but maintained the rplKA promoter, rplKp, still retained a severe growth-inhibiting effect. We suggest that these results can be explained by assuming that there is transcription from the rplKA promoter through rplJ and perhaps beyond.
Chromosome Deletion↗
Mutations in the rpIJ leader of Escherichia coli that abolish feedback regulation.
We have isolated mutants that fail to exhibit biosynthetic feedback regulation of a rpIJ-lacZ fusion. Analysis of these mutants and of others that were isolated earlier indicates that crucial sequences for both translational feedback regulation and efficient translation lie closely intermingled in the central region of the rpIJ mRNA leader 70-195 bases upstream from the translation start of rpIJ. We suggest that our point mutations define a region of the rpIJ leader mRNA to which L10 binds in effecting autogenous translational regulation.
Bacterial Proteins↗
Expression vehicles used in recombinant DNA technology.
We survey cloning vehicles whose function is to carry and express a gene in host cells including Escherichia coli, Saccharomyces cerevisiae and mammalian cells. In E. coli these include vehicles based on the lac operon, the trp operon, the rho leftward operon, and the recA gone; open reading frame cloning vehicles are also discussed, as are steps that can be taken to extrude a gene product from the cell and the use of plasmids with runaway replication. In S. cerevisiae we discuss vehicles based on the PGK gene, the ADH1 gene, the acid phosphatase gene and the GAL1-GAL10 gene cluster. In mammalian cells we discuss vehicles based on SV40 promoters, the metallothionein gene, retroviral LTR promoters, bovine papilloma virus and vaccinia virus.
Journal Article↗
Evidence for an internal promoter preceding tufA in the str operon of Escherichia coli.
We constructed plasmids carrying tufA from which the major promoter for the rpsL-rpsG-fus-tufA operon (also called the str operon) had been removed. These plasmids continued to express tufA, as judged by the ability to complement mocimycin resistance and by electrophoretic analysis of synthesized proteins. Tn5 transpositions into fus, the gene for elongation factor G, which lies immediately on the 5' side of tufA, failed to obstruct the expression of tufA. The subcloning of a 2,000-base-pair PstI-SmaI DNA fragment (containing the intercistronic region between tufA and fus, the distal portion of fus, and the proximal portion of tufA) next to promoterless tetracycline resistance genes (tet) yielded a plasmid that was capable of bestowing resistance to 12 microgram of tetracycline per ml. The removal of an EcoRI fragment that lies within fus destroyed the ability of the 2,000-base-pair PstI-SmaI fragment to promote the transcription of tet. These data indicate that, in addition to the operon's major promoter rpsLp, there is an internal promoter, tufAp, which can be used for the transcription of tufA, tufAp probably lies within fus, about 50 base pairs upstream from its 3' end and 120 base pairs from the start codon of tufA. The relative activities of tufB and of tufA-from-tufAp were estimated by a comparison of beta-galactosidase activities of almost identical EF-Tu-beta-galactosidase protein fusions; they were approximately equal.
Cloning, Molecular↗
Expression of bacterial beta-galactosidase in animal cells.
A recombinant plasmid containing the gene for bacterial beta-galactosidase, situated close to the simian virus 40 early promoter, has been constructed. Transfection of CHO, L, and COS-1 cells with this plasmid led to the expression and appearance of the enzyme. Using this system, we have developed a series of promoter cloning vehicles capable of accepting promoter signals for animal genes.
Animals↗