Functional expression of single chain glycoprotein Ib alpha on the surface of COS cells and BHK cells.

The GpIb-IX complex constitutes the major receptor for von Willebrand factor (vWF) on the surface of blood platelets. The vWF-binding site has been mapped to one of the three constituent chains, GpIb alpha. Surface expression of GpIb alpha depends on the correct intracellular assembly with the GpIb beta and GpIX chains. We have now grafted a portion of the extracellular domain of GpIb alpha containing the vWF binding site onto the transmembrane/intracellular domain of the single chain surface molecule ICAM-1. Transient transfection of this chimeric protein in COS cells resulted in surface expression as assessed by immunostaining of live cells. Similar results were obtained after stable transfection into BHK cells. Purified vWF bound to the surface of transfected cells in the presence of ristocetin and botrocetin with a Kd of 52 ng/ml, comparable to the Kd for fixed platelets (65.5 ng/ml). This study indicates that functional expression of the vWF-binding domain of GpIb alpha on the surface of mammalian cells can be obtained in the absence of GpIb beta and GpIX. Furthermore, this model system simplifies existing methods for the assessment of the functional consequences of mutations in GpIb alpha as found in pseudo-von Willebrand disease and Bernard-Soulier syndrome.

Replicase-mediated resistance to alfalfa mosaic virus.

Tobacco plants transformed with the P1 and P2 replicase genes of alfalfa mosaic virus (AIMV) have been shown to produce functional replicase proteins, permitting their infection with AIMV inocula lacking the genome segments encoding P1 and P2, respectively. To see whether expression of a mutant P2 protein would interfere with the assembly of a functional replicase complex, tobacco plants were transformed with modified P2 genes. When plants were transformed with a P2 gene encoding an N-terminally truncated protein which mimicked the tobacco mosaic virus 54K protein, no resistance was observed with 10 independent lines of transformants. Similarly, when the GDD motif in the full-length P2 protein was changed into VDD, no resistance was observed in 14 transgenic lines. However, when the GDD motif was changed into GGD (5 lines), GVD (15 lines), or DDD (13 lines), 20 to 30% of the transgenic lines showed a high level of resistance to AIMV infection. This resistance was effective to inoculum concentrations of 10 to 25 micrograms/ml of virus and 100 micrograms/ml of viral RNA, causing severe necrosis of control plants. For all transgenic lines, the expression of the transgenes was analyzed at the RNA level. With the GGD, GVD, and DDD mutants, resistance was generally observed in plants with a relatively high expression level. This indicates that the resistance is due to the mutant replicase rather than to an RNA-mediated cosuppression phenomenon.

Biological activity of transcripts synthesized in vitro from full-length and mutated DNA copies of tobacco rattle virus RNA 2.

Full-length cDNA clones of RNA 2 of tobacco rattle virus (TRV) strain PLB have been cloned into the transcription vector pPM1. Products of in vitro transcription by Escherichia coli RNA polymerase, either capped or uncapped, were as infectious as native RNA 2 when coinoculated with RNA 1 of TRV strain TCM. At least 70% of the internal sequence of the cDNA could be deleted without reduction of the replication efficiency of the transcripts. Sequences of 340 nucleotides at the 5' end and 405 nucleotides at the 3' end of PLB RNA 2 were found to be sufficient for replication. The encapsidation of deletion mutants of PLB RNA 2 was investigated after addition of native PLB RNA 1 and RNA 2. Accumulation of these mutants was distinguished from that of wild-type RNA 2 by insertion of nonviral sequences in the deleted parts. Three mutant forms of RNA 2 with extensive deletions in the coat protein (CP) gene were replicated but failed to encapsidate, while mutants with nonviral sequences inserted downstream from the CP gene showed a large reduction in replication efficiency.

Genome structure of tobacco rattle virus strain PLB: further evidence on the occurrence of RNA recombination among tobraviruses.

RNA-2 (2196 nucleotides) of tobacco rattle virus (TRV) strain PLB was found to consist of a 5'-terminal sequence of 1376 nucleotides identical to the 5'-sequence of RNA-2 of TRV strain PSG and a 3'-terminal sequence of 820 nucleotides that is identical to the 3'-sequence of RNA-1 of strain PLB. Thus, in strains PLB and PSG the same coat protein gene is fused to different RNA-1 derived 3'-termini. By combining RNA-1 of TRV strain TCM with RNA-2 of strain PLB, a viable pseudo-recombinant was formed with genome segments that have nonidentical 3'-sequences. After 25 passages in tobacco each RNA retained its strain-specific 3'-sequences. This indicates that the perfect 3'-homology that occurs between the two genome segments of all natural TRV isolates analyzed so far is not a prerequisite for a stable genotype.

Relative performance of monohaploid potato clones and their diploid parents at plant level and after protoplast isolation and subsequent fusion.

Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.