Characterization of the single-strand-specific BPV-1 origin binding protein, SPSF I, as the HeLa Pur alpha factor.

SPSF I and II are two cellular proteins which bind specifically to single-stranded DNA. SPSF I and II binding sites are found in the minimal origin of replication of BPV-1 DNA and near the P2 promoter of the cellular c-myc gene. DNA-binding properties of the two proteins to single-stranded oligonucleotides of different lengths and sequences were quantified by determination of DNA-binding constants. The binding constant of SPSF proteins to the lower strand of the BPV-1 origin was determined to be 1.5 x 10(-10) M-1. Peptide sequences derived from purified SPSF I and II revealed the identity of at least one of the SPSF proteins with the so-called HeLa Pur alpha factor. The HeLa Pur alpha factor was identified previously by virtue of its capacity to bind to purine-rich strands of the PUR element found in initiation zones of DNA replication [Bergemann, A.D., Ma,Z.-W. and Johnson, E.M. (1992) Mol. Cell. Biol. 12, 5673-5682]. Expression of the Pur cDNA confirmed the identity of the Pur alpha protein with the 42 kDa SPSF I protein. Analysis of several Pur alpha cDNA clones revealed the existence of an extended 3'-untranslated region in all Pur mRNAs.     

Impaired Functionality of Antiviral T Cells in G-CSF Mobilized Stem Cell Donors: Implications for the Selection of CTL Donor

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells.CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry.G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF.To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.     

A new form of crystalline rubisco and the conversion to its common dodecahedral form

In this paper, we present a new purification procedure that yields a new crystalline form of rubisco and has enabled us to completely remove this most abundant protein from tobacco leaf extract. The crystals formed within 48 h after refrigeration at 4 degrees C at pH 5.6. However, these crystals were not well-ordered crystals and lacked well-defined facets or edges. The remaining leaf extract (fraction 2 protein) was void of rubisco. Conversion of this new crystalline form of rubisco to its common dodecahedral form was achieved by dialysing the protein solution in Tris buffer at pH 8.0 or purified water. Since the molecular size of its large subunit of rubisco (55 kD) is similar to that of the papillomavirus capsid protein, L1 (57 kD), its complete removal from fraction 2-protein may facilitate the detection, purification, and recovery of the Li protein.     

E. coli chromosomal DNA in a transgene locus created by microprojectile bombardment in tobacco

Transgenic Research -     

Inhibition of the Raf-1 kinase by cyclic AMP agonists causes apoptosis of v-abl-transformed cells.

Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as chronic myeloid leukemias.     

A multi-generation analysis of the stability of transgenic virus resistance in doubled-haploid tobacco lines

Plants can be genetically engineered for virus resistance by transformation with a viral gene. We transformed tobacco with the tomato spotted wilt virus (TSWV) nucleocapsid gene from the Hawaiian L isolate in order to obtain TSWV resistant breeding lines. Doubled-haploid lines were produced from primary transgenic plants that were selected for resistance to the virus. Several of these lines showed very high levels of resistance and were symptomless after inoculation with the Hawaiian L isolate of TSWV. The accumulation of only low levels of full-length transgene RNA and protein observed in these lines is consistent with an RNA-mediated mechanism of resistance. The lines that were highly resistant to the Hawaiian L isolate of TSWV were also found to be highly resistant to several other isolates of TSWV, while lines that were only moderately resistant to the Hawaiian L isolate were often susceptible to the other isolates. The highly resistant lines were advanced over several generations by self-pollination. Although these lines were fully homozygous, several lines lost resistance in later generations, indicating that the resistance was unstable. Selection for resistance in these unstable lines did not prevent the occurrence of susceptible progeny in subsequent generations. Therefore, testing over several generations is required to determine the stability of resistance when breeding crops with transgenic virus resistance.     

Comparison of hepatic drug metabolizing enzyme activities in several agricultural species

1. Several pathways of drug metabolizing enzyme activity were measured in hepatic fractions of cattle, sheep, goats, chickens, turkeys, ducks, rabbits and rats. The pathways examined included the O-demethylation of p-nitrophenol, microsomal ester hydrolysis of procaine and glucuronidation of p-nitrophenol, and the cytosolic acetylation of sulfamethazine and sulfation of 2-naphthol. 2. For most enzymatic pathways measured, goats were more similar to sheep (wether) than to cattle (steers). The exception was UDP-glucuronyltransferase activity, which was significantly higher for the goat than for any other species studied. 3. Within the avian subset, the chicken and turkey were usually the most similar species. 4. The activities of arylsulfotransferase isozymes III and IV were particularly low for the duck compared to the chicken and turkey. 5. N-acetyltransferase activity was very high for rabbits and very low for sheep and goats.     

Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape,<Emphasis Type="Italic"> Brassica napus</Emphasis> L.

The green fluorescent protein (GFP) holds promise as a field-level transgene marker. One obstacle to the use of GFP is fluorescence variability observed within leaf canopies. In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape (Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased. GFP fluorescence intensity was directly correlated with the concentration of soluble protein. The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development. The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.Communicated by M.C. Jordan     

Scaffold attachment regions increase reporter gene expression in stably transformed plant cells.

The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays.