A modified polyoma virus genome has been constructed which can encode the middle-T protein, but not the large-T or small-T proteins. This was achieved, starting with the full length viral DNA inserted into a plasmid vector, by replacing a small genomic restriction fragment spanning the middle-T intervening sequence with the equivalent fragment from a cloned partial cDNA copy of the middle-T protein mRNA. Transfection of the modified viral DNA into cultured rat cells efficiently induced the formation of transformed cell foci which gave rise to cell lines that grew as tumours after injection into Fisher rats. The only viral early-region antigen synthesized by the cell lines was the middle-T protein. Expression of the middle-t protein is therefore sufficient to establish and maintain a transformed state. The viral mRNA produced by two of the transformed cell lines was structurally indistinguishable from the normal middle-T mRNA found in productively infected cells, suggesting that RNA splicing is not an essential step in the biogenesis of this messenger.

No abstract

Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human e-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human (-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase (1-globin mRNA levels 8-to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed.The human p-like globin gene locus consists of six linked genes in the 5' to 3' order e-yGyA_4p__B spanning =50

The receptor subunit gp130 transduces multiple cell type–specific activities of the leukemia inhibitory factor (LIF)/interleukin (IL)-6 family of cytokines through the signal transducer and activator of transcription (STAT) and src homology 2 domain–bearing protein tyrosine phosphatase (SHP)-2/ras/Erk pathways. To define STAT-dependent physiological responses, we generated mice with a COOH-terminal gp130ΔSTAT “knock-in” mutation which deleted all STAT-binding sites. gp130ΔSTAT mice phenocopyed mice deficient for IL-6 (impaired humoral and mucosal immune and hepatic acute phase responses) and LIF (failure of blastocyst implantation). However, unlike mice with null mutations in any of the components in the gp130 signaling pathway, gp130ΔSTAT mice also displayed gastrointestinal ulceration and a severe joint disease with features of chronic synovitis, cartilaginous metaplasia, and degradation of the articular cartilage. Mitogenic hyperresponsiveness of synovial cells to the LIF/IL-6 family of cyto-kines was caused by sustained gp130-mediated SHP-2/ras/Erk activation due to impaired STAT-mediated induction of suppressor of cytokine signaling (SOCS) proteins which normally limits gp130 signaling. Therefore, the joint pathology in gp130ΔSTAT mice is likely to arise from the disturbance of the otherwise balanced activation of the SHP-2/ras/Erk and STAT signaling cascades emanating from gp130.

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Aberrant c-myc expression patterns occur in human Burkitt's lymphoma cells, which consistently exhibit c-myc chromosomal translocations, mutations within and flanking the translocated allele, a loss of the block to transcription elongation in exon 1, and a promoter shift to use of the upstream P1 promoter. To define the mechanism responsible for the loss of transcription elongation blockage and resulting c-myc deregulation in Burkitt's lymphoma, we analyzed transcription patterns after transfer of normal and Burkitt's lymphoma c-myc alleles into murine cells and Xenopus oocyte germinal vesicles. We have determined that although the mutations within and surrounding several Burkitt's lymphoma c-myc alleles are not sufficient, in themselves, to abrogate the transcription elongation block, transcription initiation from the P2 promoter may be necessary to obtain the block to transcription elongation. To test directly the role of c-myc promoters in programming transcription elongation blockage, we analyzed transcription patterns from in vitro mutagenized c-myc genes containing deletions of either the P1 or P2 promoter. These data confirm that Pl-initiated c-myc transcripts do not terminate at discrete sites near the 3' end of exon 1, whereas P2-initiated transcripts either terminate or read through the transcription block signals. Therefore, overexpression and/or constitutive expression from the c-myc P1 promoter may contribute to increased readthrough transcription in Burkitt's lymphoma cells and, hence, to aberrant expression patterns or levels of c-myc steady-state transcripts. In addition, the ability of normal cells to modulate c-myc P2-initiated transcription to either read through or to block elongation provides a fine control mechanism over c-myc steady-state RNA levels.

The presence of TGF alpha mRNA has been reported previously to occur in primary colon cancers. We report the expression of the normal 4.5 kb TGF alpha transcript in the mucosa of the normal human gastro-intestinal tract from oesophagus through to colon. The highest levels of human TGF alpha mRNA occurred in the duodenum but significant levels were present in all of the mucosa. Similarly, in the rat gastro-intestinal tract, TGF alpha transcripts were detected in the lower gastro-intestinal tract mucosa. The relative abundance of the TGF alpha mRNA appeared to decrease in distal regions of the gastro-intestinal tract. The level of the TGF alpha mRNA was similar in both the normal and the neoplastic colon tissue. Similarly, in 2 patients with carcinomas, the TGF alpha mRNA was expressed at similar levels in the tumour and in adjacent mucosa. Although TGF alpha mRNA is associated with transformed cells from the gastro-intestinal tract, the presence of this mRNA at equivalent concentrations in normal mucosa suggests that over-production of TGF alpha is not an essential feature of carcinomas in the gastro-intestinal tract.

Interaction of thrombopoietin (TPO) with its receptor, c‐Mpl, triggers cell growth and differentiation responses controlling primitive haemopoietic cell production and megakaryocytopoiesis. To examine the important receptor domains and signal transduction pathways involved in these cellular responses, c‐Mpl cytoplasmic domain truncation and tyrosine substitution mutants were generated. In the myelomonocytic leukaemia cell lines WEHI3B‐D+ and M1, ectopic expression of the wild‐type c‐Mpl receptor induced TPO‐dependent cellular differentiation characterized by increased cell migration through agar and acquisition of the morphology and molecular markers of macrophages. Consistent with the concept that proliferative and differentiation signals emanate from distinct receptor domains, the C‐terminal 33 amino acids of c‐Mpl were dispensable for a proliferative response in Ba/F3 cells but proved critical for WEHI3B‐D+ and M1 differentiation. Finer mapping revealed that substitution of Tyr599 by phenylalanine within this c‐Mpl domain was sufficient to abolish the normal differentiation response. Moreover, in contrast to the normal c‐Mpl receptor, this same mplY599F mutant was also incapable of stimulating TPO‐dependent Shc phosphorylation, the association of Shc with Grb2 or c‐Mpl and of inducing c‐fos expression. Thus activation of components of the Ras signalling cascade, initiated by interaction of Shc with c‐Mpl Tyr599, may play a decisive role in specific differentiation signals emanating from the c‐Mpl receptor.