Spatial and temporal expression patterns directed by theAgrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot

We have analysed the patterns of expression of a gene encoding -glucuronidase (GUS) fused to the promoter of theAgrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot,Daucus carota L. Gene expression was monitored by a histochemical assay of -glucuronidase activity. The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development. The patterns of expression however, differed between embryos in different stages of development. In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo. In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons. During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices. Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development. This difference in organ specificity of expression is likely due to a large deletion of the promoter.     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies

It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, -glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of -glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked glycosylation sites within the GUS gene was altered by site-directed mutagenesis. The second N-linked glycosylation site was not altered because sequence analysis of nucleotide sequences around the second putative glycosylation site revealed that the published sequence was incorrect, and that no such site existed.     

Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora

To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100. Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with beta-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic beta-D-glucuronides of phenolphthalein and/or p-nitrophenol. These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial beta-glucuronidases to potent mutagenic aglycone(s).     

Isolation and expression in Escherichia coli of a cDNA clone encoding human beta-glucuronidase

Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of beta-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73%. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropyl-thio-beta-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.     

Biochemical and genetic factors in the heat inactivation of murine β-glucuronidase

The enzymes coded for by two alleles at the glucuronidase structural locus (Gus) were compared in their response to pH, buffering anion, buffer molarity, ionic strength, and temperature. The heat-labile Gus^h gene product responded in a qualitatively similar but quantitatively reduced manner compared to the relatively heat-stable Gus ^b gene product. In all buffers tested, the enzyme was most heat stable at pH 5.0. Ranking of the various buffer anions tested, according to increasing heat stabilization, was water acetate phosphate < citrate. Varying the molarity of the buffers from 0.01 to 0.6 m at pH 5.0 revealed further differences among the buffers. Increasing ionic strength exerted a destabilizing force on the protein. The half-life of the enzyme decreased by as much as a hundredfold between 71 and 75 C. The Gus^h/Gus^h genotype also results in decreased activity levels in all tissues, reportedly because of decreased synthesis. The heat inactivation curves of Gus^b/Gus^h heterozygotes were incompatible with any theoretical curve based on the assumption that the Gus^b and Gus^h chromosomes in the heterozygote behave in a manner similar to that seen in the homozygotes.This research was supported by a Basil O'Connor Starter Research Grant from the National Foundation—March of Dimes (R. J. M.) and by a grant from The Jane Coffin Childs Memorial Fund for Medical Research (K. H.).Fellow of The Jane Coffin Childs Memorial Fund for Medical Research.     

Transfer and expression of <Emphasis Type="Italic">npt</Emphasis>II and <Emphasis Type="Italic">bar</Emphasis> genes in cucumber (<Emphasis Type="Italic">Cucumis satavus</Emphasis> L.)

Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l⁻¹ phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT (2–6 mg l⁻¹). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l⁻¹) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization.     

Stable Transformation of Embryogenic Tissues of <Emphasis Type="Italic">Pinus nigra</Emphasis> Arn. using a Biolistic Method

The stable transformation of embryogenic tissues of Pinus nigra Arn., cell line E104, has been achieved using a biolistic approach. The introduced DNA consisted of the uidA reporter gene under the control of the double CaMV 35S promoter and the nptII selection gene controlled by the single CaMV 35S promoter. Three days after bombardment, putative transformed tissues were selected for continued proliferation on a medium containing 20 mg geneticin l⁻¹. Resistant embryogenic tissue recovery required 10–12 weeks. The integration of the nptII and uidA genes was confirmed by both histochemical/fluorimetric GUS assays and PCR amplification of the inserts in the five geneticin resistant sub-lines of line E104. The activity of the uidA reporter gene in transgenic, embryogenic tissue lines was stable and could be detected after one year of culture. Somatic embryo maturation was, however, poor and no plantlet regeneration could be obtained.