Inheritance of a mutation at the Rb-1 locus, which has been mapped to band q14 of human chromosome 13, results in predisposition to retinoblastoma. Cloned DNA segments homologous to arbitrary loci of human chromosome 13 and which reveal polymorphic restriction endonuclease recognition sequences, have been used to look for somatic genetic events that might occur during tumorigenesis. A comparison of constitutional and tumour genotypes from several cases indicates that tumorigenesis may result from the development of homozygosity for the mutant allele at the Rb-1 locus. The homozygosity in these cases results from mitotic nondisjunction, resulting in loss of the homologous wild-type chromosome, or from a mitotic recombination event.

A system for fractionating populations of Jiving cells by velocity sedimentation in the earth's gravitational field i s described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes pIace primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be signBcantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 -/hour) it is 1.5 X 1 0 ' cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r2/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

Mice deficient for the RB gene (RB-/-), prior to death at embryonic day 14.5, show increased cell death in all tissues that normally express RBI: the nervous system, liver, lens, and skeletal muscle precursor cells. We have generated transgenic mice (RBlox) that express low levels of pRb, driven by an RB1 minigene. RBIox/RB -/-mutant fetuses die at birth with specific skeletal muscle defects, including increased cell death prior to myoblast fusion, shorter myotubes with fewer myofibrils, reduced muscle fibers, accumulation of elongated nuclei that actively synthesized DNA within the myotubes, and reduction in expression of the late muscle-specific genes MCK and MRF4. Thus, insufficient pRb results in failure of myogenesis in vivo, manifest in two ways. First, the massive apoptosis of myoblasts implicates a role of pRb in cell survival. Second, surviving myotubes failed to develop normally and accumulated large polyploid nuclei, implicating pRb in permanent withdrawal from the cell cycle. These results demonstrate a role for pRb during terminal differentiation of skeletal muscles in vivo and place pRb at a nodal point that controls cell proliferation, differentiation, and death.[Key Words: Retinoblastoma gene; differentiation; myogenesis; cell cycle control; apoptosis] Received July 25, 1996; revised version accepted October 16, 1996.Terminal differentiation is a dynamic process coupled to cell cycle arrest that requires continuous active control (Blau 1993). The retinoblastoma gene (RB1) product (pRB) has been implicated in cell cycle exit and terminal differentiation. For example, SV40 large T antigen, which binds and inactivates pRb, can stimulate differentiated myotubes in culture to reenter the cell cycle (Gu et al. 1993). RB1 is a tumor suppressor gene, absence of which predisposes individuals to retinoblastoma in infancy and, to a lesser extent, osteosarcoma in the second decade of life, at times when these tissues normally undergo terminal differentiation (for review, see Zacksenhaus et al. 1993a). Inactivation of RB1 also contributes to the malignant progression of a wide spectrum of tumors including breast, prostate, lung, and bladder. Moreover, tumors with apparently normal RB1 frequently contain mutations in the pathway that regulates pRb function, 4These authors contributed equally to this work. 5Corresponding authors. resulting in inactivation of pRb (for review, see Weinberg 1995).pRb is a member of a family of proteins including p107 (Ewen et al. 1991) and p130 (Harmon et al. 1993;Li et al. 1993) that interact with transcription factors and viral oncoproteins through shared conserved domains. The RB family of proteins exerts a negative effect on cell proliferation by modulating the activity of certain transcription factors (Defeo-Jones et al.

No abstract

No abstract

Mice homozygous for the scid mutation on chromosome 16 have a severe combined immune deficiency as a result of their inability to correctly rearrange their immunoglobulin and T-cell receptor genes. In scid mice, when precursors for B and T lymphocytes reach the stage of development requiring expression of these surface receptors, a defective recombinase system aberrantly cuts and rejoins the receptor gene segments greatly reducing the efficiency of producing functional receptors. As a result, most scid mice have no detectable B or T lymphocytes. We have demonstrated that the scid defect is not specific to lymphocyte development. Myeloid cells and fibroblasts from scid mice show a marked increase in sensitivity to ionizing radiation, indicating that the scid mutation leads to an inability to repair DNA damage induced by ionizing radiation as well as interfering with rearrangement of the immunoglobulin and T-cell receptor genes.

). Although the mechanism of action of pllOR"I remains unknown, several lines of evidence suggest that it plays a role in the regulation of transcription. We now show that overexpression of pllOl causes repression of the adenovirus early promoter EIIaE and the promoters of two cellular genes, c-myc and RBI, both of which contain E2F-binding motifs. Mutation of the E2 element in the c-myc promoter abolishes pllOWl repression.We also demonstrate that a p110"R1 mutant, which is refractory to cell cycle phosphorylation but intact in Ela/large T antigen-binding properties, represses EHaE with 50-to 80-fold greater efficiency than wild-type pllOR11. These data provide evidence that hypophosphorylated pllOIWl actively represses expression of genes with promoters containing the E2F-binding motif (E2 element).Progression through the eukaryotic cell cycle appears to be regulated at a number of restriction points. For example, following mitogenic stimulation of quiescent fibroblasts, progression through G, towards the S phase requires a rapid induction of the proto-oncogene c-myc. Inhibition of c-myc expression with antisense c-myc oligonucleotides prevents cells from entering the S phase (20,28). In addition to positive factors such as c-myc, however, negative factors also play an important role in the regulation of the cell cycle. Furthermore, as with positive factors, progression to the transformed phenotype involves deregulation of these negative factors (52).The first negative regulator of the cell cycle to be identified was the product of the retinoblastoma susceptibility gene (RB1) (18), pllOl, a nuclear phosphoprotein with a relative molecular mass of 110 to 116 kDa (36; for a review, see reference 23). Negative regulation of the cell cycle by RBJ was implied from the model proposed by Knudson (33) and Comings (9), which predicted that retinoblastoma arose because of mutation of both alleles of RBI. This prediction was subsequently verified by characterization of mutations in retinoblastoma tumors (12)(13)(14)29). More recently, a number of observations have supported the model that pllO1l acts, in part, to control progression to the S phase. First, pllOl is modified in a cell cycle-dependent manner (5,7,11,43

It was recently shown that the E2F-pRB complex is a negative transcriptional regulator. However, it was not determined whether the whole complex or pRB alone is required for repression. Here we show that pRB and the related protein p107 are capable of direct transcriptional repression independent of E2F. When fused to the DNA binding domain of GAL4, pRB or p107 represses transcription of promoters with GAL4 binding sites. Thus, E2F acts as a tether for pRB or p107 but is not actively involved in repression of other enhancers. This function of pRB maps to the pocket and is abrogated by mutation of this domain. This result suggests an intriguing model in which the pocket has a dual function, first to bind E2F and second to repress transcription directly, possibly through interaction with other proteins. We also show that direct transcriptional repression by pRB is regulated by phosphorylation. Mutations which render pRB constitutively hypophosphorylated potentiate repression, while phosphorylation induced by cyclin A or E reduces repression ninefold.The childhood eye cancer retinoblastoma (RB) results from loss of function of the protein expressed by the RB1 locus (72). Consistent with its role as a tumor suppressor, the RB protein (pRB) is able to block the growth of some but not all cell types (72). A close relative of pRB, p107, is also capable of growth suppression (74), although its involvement in tumor growth has not been documented. Three members of the RB family have now been isolated: pRB, p107 (16), and pRB2/p130 (26,43,48). Homology is greatest in the so-called pocket region, which consists of A and B domains separated by a spacer (26,43,48). The pocket was originally identified as the minimal region of pRB required to bind the adenovirus E1A and simian virus 40 (SV40) large T oncoproteins (32) and has also been shown to be essential for the interaction of pRB with a variety of cellular proteins (10,11,15,21,33,37,54,56,57,66).The function of pRB is tightly regulated by phosphorylation. It is hypophosphorylated in the G 1 phase of the cell cycle but becomes progressively more phosphorylated upon entry into S phase (3,7,9). Phosphorylation appears to disable pRB in several functional assays. Thus, hypo-but not hyperphosphorylated pRB binds to the viral proteins E7 and large T (14, 47), various cellular proteins (11,20,28,36,60,66,69), and components of the cell which allow nuclear tethering of pRB (52,62). In addition, overexpression of pRB blocks the RB Ϫ cell line SAOS-2 in G 1 , where pRB is hypophosphorylated (18,63), and this inhibition of growth is overcome by cotransfection of cyclins which mediate the phosphorylation of pRB through cyclin-dependent kinases (cdks) (30). Finally, transcriptional activation by E2F or Elf-1 is more sensitive to repression by a mutant pRB molecule which is constitutively hypophosphorylated than to wild-type pRB (25, 66).The molecular mechanism behind the phenotypic effects of pRB presumably lies in its ability to modulate the expression of various genes. However, exac...