Pseudorecombinant viruses (i.e., those containing a reassorted genome of closely related multipartite viruses) are often not as competitive as the parental viruses. The role of the 2b gene in hypervirulence and maintenance of a progressive infection was assessed in a pseudorecombinant virus formed between RNAs 1 plus 2 of Cucumber mosaic virus (CMV) and RNA 3 of Tomato aspermy virus (TAV). The presence of RNA 3 of TAV was found to affect the level of RNA accumulation but not the level of virulence. By contrast, the 2b genes of both TAV and a hypervirulent strain of CMV (WAII-CMV) were found to affect the virulence of the pseudorecombinant viruses but not the levels of viral RNA accumulation. The 2b gene rather than the overlapping open reading frame encoding the C-terminal 41 amino acids of 2a protein of the corresponding virus was found to be essential for promoting infection of the pseudorecombinant viruses in planta. However, the 2b gene was not essential for replication of pseudorecombinant viruses containing CMV RNAs 1 plus 2 and TAV RNA 3. These results indicate that the 2b protein is involved in promoting the cell-to-cell movement of the pseudorecombinant viruses. These data also suggest the existence of specific interaction between the TAV 2b protein and either RNA 3 or its encoded proteins, which may be critical for promoting or maintaining infection or both.
Risk-assessment studies of virus-resistant transgenic plants (VRTPs
The stem cell leukemia (SCL) gene encodes a basic helix-loop-helix transcription factor with a critical role in the development of both blood and endothelium. Loss-of-function studies have shown that SCL is essential for the formation of hematopoietic stem cells, for subsequent erythroid development and for yolk sac angiogenesis. SCL exhibits a highly conserved pattern of expression from mammals to teleost fish. Several murine SCL enhancers have been identified, each of which directs reporter gene expression in vivo to a subdomain of the normal SCL expression pattern. However, regulatory elements necessary for SCL expression in erythroid cells remain to be identified and the size of the chromosomal domain needed to support appropriate SCL transcription is unknown. Here we demonstrate that a 130-kilobase (kb) yeast artificial chromosome (YAC) containing the human SCL locus completely rescued the embryonic lethal phenotype of scl ؊/؊ mice. Rescued YAC ؉ scl ؊/؊ mice were born in appropriate Mendelian ratios, were healthy and fertile, and exhibited no detectable abnormality of yolk sac, fetal liver, or adult hematopoiesis. The human SCL protein can therefore substitute for its murine homologue. In addition, our results demonstrate that the human SCL YAC contains the chromosomal domain necessary to direct expression to the erythroid lineage and to all other tissues in which SCL performs a nonredundant essential function. IntroductionThe stem cell leukemia (SCL) gene (also known as TAL-1) encodes a basic helix-loop-helix (bHLH) transcription factor with an essential role in the development of both blood and endothelial cells. 1 Mice lacking a functional SCL protein failed to develop yolk sac hematopoiesis and blastocyst reconstitution experiments have demonstrated that SCL is required for both definitive and primitive hematopoiesis. 2,3 These data suggest that SCL plays a pivotal role in the formation or behavior of hematopoietic stem cells and are consistent with the observation that expression of antisense SCL suppressed the proliferation, cell cycle progression, and selfrenewal of a multipotent hematopoietic cell line. 4 SCL is likely to perform additional functions following lineage commitment because enforced SCL expression enhanced erythroid differentiation of hematopoietic cell lines 5,6 and increased erythroid and megakaryocytic differentiation of normal CD34 ϩ progenitors. 7,8 Several lines of evidence demonstrate that SCL is critical for normal endothelial development. scl knockout mice exhibit defective yolk sac angiogenesis thought to reflect an essential function for SCL during vessel formation. 9 SCL is also likely to play an earlier role during the formation of endothelial cells. In zebrafish and in murine embryonic stem (ES) cell systems, SCL is expressed in hemangioblasts, bipotent progenitors of blood and endothelium. 10,11 Moreover, SCL expression can partially rescue both blood and endothelial defects of the zebrafish cloche mutant 12 and ectopic expression of SCL during early development alters ...
The stem cell leukaemia gene (Scl) encodes a basic helix-loop-helix transcription factor with a pivotal role in both haematopoiesis and endothelial development. During mouse development, Scl is first expressed in extra-embryonic mesoderm, and is required for the generation of all haematopoietic lineages and normal yolk sac angiogenesis. Ectopic expression of Scl during zebrafish development specifies haemangioblast formation from early mesoderm. These results suggest that SCL is essential for establishing the transcriptional programme responsible for the formation of haematopoietic stem cells and have focused attention on the transcriptional regulation of Scl itself. Previous studies have identified a panel of Scl enhancers each of which directed expression to a subdomain of the normal Scl expression pattern. Among them, a 3′ enhancer directed expression during development to vascular endothelium and haematopoietic progenitors but not to Ter119+ erythroid cells. The expression in haematopoietic stem cells, however, remained undetermined. We demonstrate that this 3′ enhancer directs lacZ expression in transgenic mice to most foetal and adult long-term repopulating haematopoietic stem cells, and therefore functions as a stem cell enhancer. Consistent with these results, expression in Scl–/– embryos of exogenous Scl driven by the stem cell enhancer rescued the formation of early haematopoietic progenitors and also resulted in normal yolk sac angiogenesis. By contrast, erythropoiesis remained markedly deficient in rescued embryos. This observation is consistent with the inactivity of the stem cell enhancer in erythroid cells and reveals an essential role for SCL during erythroid differentiation in vivo.
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