Fluorescence in situ hybridization analysis of alien genes in Agrobacterium-mediated Cry1A(b)-transformed rice

The transgene in Agrobacterium-mediated Cry1A(b)-transgenic rice plants has been detected and its chromosomal location determined by fluorescence in situ hybridization (FISH). Eight of the nine transgenic lines tested showed hybridization signals. Signals were located on regions of the chromosome in which fraction length (FL) values varied from 26.2 (near the centromere) to 95.2 (distal regions). No signal was found on regions where the fraction length was less than 26.2, while six of the nine signals detected were located on regions with FL values of 75.3 or over. This demonstrates that Agrobacterium-mediated genes can integrate into multiple sites distributed in different parts of the chromosome, but that distal regions are the preferred sites and regions near the centromeres are colder for T-DNA integration. The donor DNA of the transformation was divided into two parts, labelled separately as probes for two-colour FISH. Results show that the transformed DNA sequences remained linked in the recipient genome. The relationship between integration position and transgene silencing, known as the 'position effect', is discussed.     

Biological Activity of Human Granulocyte-Macrophage Colony Stimulating Factor is Maintained in a Fusion with Seed Glutelin Peptide

Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants. Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression. Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5' end. With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active. Furthermore, native tobacco seed extracts inhibited the activity of E. coli-derived GM-CSF in this cytokine-dependent cell line. The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited. The feasibility of oral delivery of such stable seed-produced cytokines is discussed.     

Bt水稻中crylAb基因的遗传分析

用PCR、GUS染色和Western点杂交技术检测了Bt水稻杂交后代群体,在394株GUS阳性株中,有392株表达Bt蛋白,协同表达株率达99.49%。由此表明,在杂交后代中报告基因gus和目的基因crylAb紧密连锁遗传与表达。GUS组织染色和Southern杂交检测表明Bt水稻中的crylAb基因呈单位点显性基因遗传,且在有性世代中能稳定传递,还发现,在BC1、BC1F2和粳粳交F2群体中crylAb基因呈单位点显性基因遗传,而在籼粳交F2群体中偏离3：1分离。     

Conversion of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium absonum in culture and by immobilized cells

Abstract Volatile fatty acids (VFA) have interesting biological effects on eukaryotic cells, inducing alterations of cell shape, morphological differentiation, changes in the composition of the cytoplasmic membrane and growth inhibition. This paper describes the effects of VFA on granulocyte chemotaxis. The influences observed, usually inhibitory, were shown to depend on the concentration tested and the incubation atmosphere (anaerobic or 5% CO₂).
Because these metabolites are produced by several anaerobic bacteria both in vitro and at infected sites, one can speculate that they might act as potential 'leukotoxins' contributing to the pathogenicity of anaerobic bacteria.
We suggest that the well-known ability of anaerobes to inhibit their own phagocytosis and intracellular killing, as well as that of facultative anaerobes, might be at least partially due to the impairment of granulocyte functions induced by VFA.     

A gene showing sequence similarity to pectin esterase is specifically expressed in developing pollen of Brassica napus. Sequences in its 5′ flanking region are conserved in other pollen-specific promoters

Differential screening of a Brassica napus genomic library led to the isolation of the clone named Bp 19 containing a gene which is highly expressed during microspore development. The accumulation of Bp 19 mRNA starts in uninucleate microspores, increases during development reaching a peak in the late stages but declines considerably in mature pollen. The nucleotide sequence of the entire coding region and of extended portions of the 5 and 3 flanking regions was determined. Several homologous cDNA clones were also isolated and sequenced. The Bp 19 gene contains a single intron of 137 bp and gives origin to a mRNA of ca. 1.9 kb which codes for a polypeptide of 584 amino acids. Bp 19 protein has an estimated molecular weight of 63 kilodaltons and has a highly hydrophobic amino terminal region which shows features of a signal peptide. The carboxy half of the Bp 19 protein, starting at amino acid 269, has striking sequence similarity to the pectin esterases of tomato and of the plant pathogen Erwinia chrysanthemi. Four short domains are extremely well conserved in all the three proteins and therefore could represent catalytic sites responsible for enzyme activity. Comparison of the 5 flanking region of the Bp 19 gene with the sequence of other pollen-specific promoters revealed the presence of several conserved regions. These short promoter sequences could correspond to regulatory elements responsible for pollen-specific gene expression.     

Heat shock response during anther culture of broccoli (Brassica oleracea var italica)

Embryo formation from microspores of Brassica oleracea var Italica (Broccoli) and other Brassica species is greatly enhanced by an initial incubation at elevated temperatures (eg 35°C) followed by continued incubation of 25°C. In the present study we observed that a three hour high temperature treatment induced the formation of heat shock proteins in cultured anthers. These were identified in two dimensional gels by silver staining, and labelled heat shock proteins were synthesised in vitro from isolated anther RNA. The appearance of heat shock proteins in anthers followed a similar pattern and displayed similar characteristics to that from leaves. Comparison of the heat shock proteins induced in isolated cultured anthers of known highly embryogenic and less embryogenic plans did not reveal obvious qualitative differences.     

Characterization of oat vicilin-like polypeptides

The 7S and 3S globulin fractions were extracted and characterized from Avena sativa L. seeds which showed similar solubility characteristics and holoprotein size to those of the vicilin fraction in legumes. These holoproteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectrofocussing, and two-dimensional electrophoresis. Among the components of the oat reserve globulins, the 7S fraction was capable of binding to concanavalin A-Sepharose, thus indicating it to be a glycoprotein. This pattern of the glycosylation of the reserve proteins in oat resembles that observed in certain legume seeds such as Pisum sativum and Glycine max. The results support the notion that the protein components of cereal and legume globulins may be homologous.     

Role of endoplasmic reticulum in biosynthesis of oat globulin precursors

Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt-soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin α-β dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced α-β dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the α + β subunits, were labeled in protein bodies.

The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller α and β subunits.

     

Construction and rapid testing of synthetic and modified toxin gene sequences CryIA (b&c) by expression in maize endosperm culture

Summary The synthesis of two modified genes, Cry IA(b) and CryIA(c), each consisting of 1845 bp, is described in detail. The genes were synthesized using an improved PCR procedure based on recursive principles. The synthetic CryIA(c) gene was put under the control of a maize ubiquitin promoter. This construct was tested in a maize endosperm-derived suspension culture system. The use of maize endosperm culture as a quick and efficient system to test the activity of synthetic genes is described.     

Resistance to Tecia solanivora (Lepidoptera: Gelechiidae) in three transgenic Andean varieties of potato expressing Bacillus thuringiensis CrylAc protein

Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazúcar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 microg/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7-100% mortality, whereas the mortality levels on nontransgenic lines were 0-2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of CrylAc potatoes in the insect pest management strategies.