Gene amplification and analysis最新文献

The results summarized in this review show that the normal chicken myb gene codes for a protein of 77 kd which appears to play an important role in the control and/or differentiation of hematopoietic cells of myeloid and T lymphoid series. The activation of this gene has been observed in chicken and murine systems. In the avian system, this has been achieved by transduction of the myb oncogene into a retrovirus. Such a transduction resulted in the deletion of coding sequences from both the 5' and 3' ends of the gene. Initiation and terminator codons in helper viral sequences have been substituted for the analogous sequences in the proto-oncogene. Deletion of similar stretches of sequence in both the viruses suggested the possibility that these deletions may play an important role in the activation of this gene. The availability of the murine model system allowed us to examine this question further. In the ABPL tumor system, the activation of the myb locus occurred as a result of viral integration in a region immediately upstream to the v-myb related sequences. In NSF-60 cell line, the activation is due to the viral integration toward the 3' end of the gene. In both cases the viral integration results in the synthesis of aberrant mRNAs that have suffered deletions similar to those observed in the avian system. In all instances this results in the synthesis of truncated proteins which appear to mediate the transforming function. The availability of chicken and mouse c-myb cDNA clones makes it possible to test this hypothesis directly by construction of retroviruses containing various deletion mutations.

Site-directed mutagenesis techniques have been utilized to define important structural and functional domains within the RSV src gene product, pp60src. Deletion mutations within the amino terminal one-half of the src gene which impinge upon a region of the src protein delineated by amino acid residues 143 to 169 yielded transformation defective viruses. Src proteins encoded by such RSV mutants exhibited diminished tyrosine protein kinase activity in vitro and only slightly reduced levels of in vivo tyrosine protein kinase activity. We speculate that these structurally altered proteins are defective for target protein recognition. Point mutations and linker insertion mutations within the putative catalytic domain of pp60src served to block the transforming activity of mutant viruses. Mutant viruses encode src proteins that exhibited substantially reduced levels of tyrosine protein kinase activity both in vitro and in vivo.

The highly conserved, single copy c-myb gene has been independently transduced by two avian acute leukemia viruses, AMV and E26. This gene has also undergone insertional mutagenesis by non-acutely transforming murine leukemia viruses in a number of hematopoietic tumors. The common denominator of these retroviral activations of c-myb appears to be truncation of the normal coding region at either or both ends. The role of point mutations in myb-induced leukemogenesis is currently unknown. The products of the c-myb gene and its altered viral counterparts are nuclear proteins, a large fraction of which are associated with the nuclear matrix. In addition, the myb gene products have short half-lives and bind DNA in vitro. These features suggest that myb may act by regulating DNA replication or transcription. Consistent with this notion, the expression of c-myb is cell cycle dependent in several cell types. However, the abundant expression of c-myb in the thymus is not similarly regulated and may serve a different function. The expression of c-myb appears not to be limited to hematopoietic tissues as previously thought and the nature of the hematopoietic specificity of transformation by v-myb is not currently understood. Nevertheless, hematopoietic growth factors and their receptors appear to play an important role in such transformation. Two new experimental systems for studying myb have recently been described. First, the discovery of a myb-related gene in Drosophila should allow the application of powerful classical and molecular genetic approaches. The functional similarity of this distantly related gene to the much more closely related avian and mammalian myb genes is unknown. Second, recent studies of murine myb in normal and abnormal hematopoiesis offers several advantages relative to the avian system, such as in-bred animal strains, a wealth of specific cell-surface markers, and cloned hematopoietic growth factor and receptor genes. Isolation or construction of an acutely transforming murine myb retrovirus may thus be very useful. Several obvious goals for future research will be to define the function of myb proteins within the nucleus, to understand the regulation of myb expression during the cell cycle, to establish which molecular alterations are essential for converting c-myb into a transforming gene, and the determine the role of myb in human malignancies.

The collective evidence from several laboratories demonstrates that foreign genes from both prokaryotes and eukaryotes, can be expressed in B. subtilis if the heterologous gene is placed under the control of Bacillus promoters and ribosome binding sites. The nucleotide sequences of several Bacillus promoters are very similar to the consensus E. coli promoter sequence at both the -10 and -35 recognition sites. In spite of this apparent similarity, E. coli promoters appear to be poorly recognized in vitro by B. subtilis RNA polymerase35. Thus, a barrier to heterologous (E. coli) gene expression in B. subtilis may reside at the transcriptional level. In addition, there appears to be a barrier at the level of translation. Rabinowitz and co-workers have shown that ribosomes from gram-positive bacteria, including B. subtilis, are relatively inactive in the in vitro translation of mRNA gram-negative species. This appears to be due to an inability of ribosomes from gram-positive bacteria to use the relatively weak ribosome binding sites that commonly occur in RNA from gram-negative bacteria.