One obvious phenotype of tumor cells is the lack of terminal differentiation. We previously classified rhabdomyosarcoma cell lines as having either a recessive or a dominant nondifferentiating phenotype. To study the genetic basis of the dominant nondifferentiating phenotype, we utilized microcell fusion to transfer chromosomes from rhabdomyosarcoma cells into C2C12 myoblasts. Transfer of a derivative chromosome 14 inhibits differentiation. The derivative chromosome 14 contains a DNA amplification. MDM2 is amplified and overexpressed in these nondifferentiating hybrids and in the parental rhabdomyosarcoma. Forced expression of MDM2 inhibits MyoD-dependent transcription. Expression of antisense MDM2 restores MyoD-dependent transcriptional activity. We conclude that amplification and overexpression of MDM2 inhibit MyoD function, resulting in a dominant nondifferentiating phenotype.Rhabdomyosarcomas are one of the most common solid tumors of childhood. Sarcomas have traditionally been classified as rhabdomyosarcomas on the basis of morphology and the expression of muscle structural genes, such as that for the myosin heavy chain (MHC) or desmin. Expression of MyoD has been shown to be the most sensitive marker for classifying sarcomas as rhabdomyosarcomas (4, 38). Rhabdomyosarcomas are grouped by histologic and cytogenetic criteria as either embryonal or alveolar rhabdomyosarcomas: a balanced translocation between chromosomes 2 and 13, t(2;13)(q35;q14), is associated with alveolar rhabdomyosarcomas (1). The PAX3 gene has been shown to be fused to a member of the forkhead gene family in the t(2;13) translocation (1, 39). Loss of heterozygosity on the short arm of chromosome 11 encompassing 11p15.5 is associated not only with embryonal rhabdomyosarcomas (38) but also with a number of other solid tumors (26), suggesting the location of a tumor suppressor gene(s) for multiple tumor types in this region.Recently, we have begun an analysis of five rhabdomyosarcoma cell lines (RD, Rh18, Rh28, Rh30, and RhJT) for expression and function of the MyoD family (42). We showed that even though MyoD is expressed in rhabdomyosarcoma cells, it is nonfunctional in inducing differentiation. Heterokaryon formation between 10T1/2 cells and RD, Rh28, Rh30, and RhJT cells results in differentiation of the heterokaryons into muscle and restoration of transcriptional activation by MyoD, indicating that these tumor lines display a recessive nondifferentiating phenotype. In contrast, heterokaryon formation with the rhabdomyosarcoma Rh18 and 10T1/2 cells did not result in myogenesis, suggesting that Rh18 cells display a dominant nondifferentiating phenotype.In this paper, we show that transfer of a derivative chromosome 14 from Rh18 cells into the differentiation-competent myoblast cell line C2C12 inhibits muscle differentiation and the ability of MyoD to function as a transcription factor. The derivative chromosome 14 contains a region of amplified DNA originating from chromosome 12 and contains a number of genes often amplified in sarcomas, ...

Muscle cell differentiation is controlled by a complex set of interactions between tissue restricted transcription factors, ubiquitously expressed transcription factors, and cell cycle regulatory proteins. We previously found that amplification of MDM2 in rhabdomyosarcoma cells interferes with MyoD activity and consequently inhibits overt muscle cell differentiation (1). Recently, we found that MDM2 interacts with Sp1 and inhibits Sp1-dependent transcription and that pRb can restore Sp1 activity by displacing MDM2 from Sp1 (2). In this report, we show that forced expression of Sp1 can restore MyoD activity and restore overt muscle cell differentiation in cells with amplified MDM2. Furthermore, we show that pRb can also restore MyoD activity and muscle cell differentiation in cells with amplified MDM2. Surprisingly, we found that the MyoD-interacting domain of pRb is dispensable for this activity. We show that the C-terminal, MDM2-interacting domain of pRb is both necessary and sufficient to restore muscle cell differentiation in cells with amplified MDM2. We also show that the C-terminal MDM2-interacting domain of pRb can promote premature differentiation of proliferating myoblast cells. Our data support a model in which the pRb-MDM2 interaction modulates Sp1 activity during normal muscle cell differentiation.We previously found that amplification of MDM2 1 in rhabdomyosarcoma cells inhibits MyoD function and inhibits muscle cell differentiation (1). The oncogenic properties of MDM2 are thought to result from interactions with several cell cycle regulatory proteins. MDM2 interacts directly with the tumor suppressor protein p53 (3) and blocks p53-mediated transactivation (4 -9). In addition, MDM2 has been shown to target p53 for rapid degradation (10, 11). MDM2 also interacts with a second tumor suppressor protein, the retinoblastoma-associated protein, pRb. This MDM2-pRb interaction results in inhibition of pRb growth regulatory function (12, 13). Furthermore, MDM2 interacts with the activation domains of the S-phasepromoting transcription factors E2F1 and DP1, resulting in stimulation of E2F1/DP1 transcriptional activity (14). Taken together, these observations suggest that MDM2 not only relieves the proliferative block mediated by either p53 or pRb but also promotes the G 1 -to-S-phase transition by stimulating E2F1/DP1 activity. The results presented here show that MDM2 can also modulate cellular differentiation through pRb and Sp1.Differentiating muscle cells fuse to form multinucleated myotubes, thereby withdrawing permanently from the cell cycle. This process is controlled by the MyoD family (MyoD, Myf-5, myogenin, and MRF4/Myf-6) of muscle-specific transcription factors (15). The MyoD family of basic helix-loop-helix transcription factors acts at multiple points in the myogenic lineage to establish muscle cell identity and control terminal differentiation. MyoD is found in a multiprotein complex that contains tissue-restricted (SRF or MEF2C) and ubiquitously expressed (E12/E47 and Sp1) transcription facto...

A genetic linkage map for loblolly pine (Pinus taeda L.) was constructed using segregation data from a three-generation outbred pedigree consisting of four grandparents, two parents, and 95 F2 progeny. The map was based predominantly on restriction fragment length polymorphism (RFLP) loci detected by cDNA probes. Sixty-five cDNA and three genomic DNA probes revealed 90 RFLP loci. Six polymorphic isozyme loci were also scored. One-fourth (24%) of the cDNA probes detected more than 1 segregating locus, an indication that multigene families are common in pines. As many as six alleles were observed at a single segregating locus among grandparents and it was not unusual for the progeny to segregate for three or four alleles per locus. Multipoint linkage analysis placed 73 RFLP and 2 isozyme loci into 20 linkage groups; the remaining 17 RFLP and 4 isozyme loci were unlinked. The mapped RFLP probes provide a new set of codominant markers for genetic analyses in loblolly pine.

Oventodted70-1090-year-old stands ofpondemsspineanmedium-tolow-qu were thinned in 1980 to 40.55. and 70 perant of n o d basal srea and c a n p a d to an unthinned cmuol. Mortality, diameter, and height in thesenorthem W o m i a stands wore m c a s u d annually from 1980 to 1987. After 8 years. mortality, primarily fmm mountain pine beetle (D~ndroclonrrr pondcrmae) and annosus rmt direare (Hcrcrobaridion mnosun), was reduced 100.95, and 86 percent relative to incming amounts of reserve basal area. Thinned stands averaged five times more cubic-foot volume gmwth than unthinned stands. More gmwth andlesr monality cwld result fmm mating similar stands elsewhere.

We report the identification of quantitative trait loci (QTL) influencing wood specific gravity (WSG) in an outbred pedigree of loblolly pine (Pinus taeda L.). QTL mapping in an outcrossing species is complicated by the presence of multiple alleles (> 2) at QTL and marker loci. Multiple alleles at QTL allow the examination of interaction among alleles at QTL (deviation from additive gene action). Restriction fragment length polymorphism (RFLP) marker genotypes and wood specific gravity phenotypes were determined for 177 progeny. Two RFLP linkage maps were constructed, representing maternal and paternal parent gamete segregations as inferred from diploid progeny RFLP genotypes. RFLP loci segregating for multiple alleles were vital for aligning the two maps. Each RFLP locus was assayed for cosegregation with WSG QTL using analysis of variance (ANOVA). Five regions of the genome contained one or more RFLP loci showing differences in mean WSG at or below the P = 0.05 level for progeny as grouped by RFLP genotype. One region contained a marker locus (S6a) whose QTL-associated effects were highly significant (P > 0.0002). Marker S6a segregated for multiple alleles, a prerequisite for determining the number of alleles segregating at the linked QTL and analyzing the interactions among QTL alleles. The QTL associated with marker S6a appeared to be segregating for multiple alleles which interacted with each other and with environments. No evidence for digenic epistasis was found among the five QTL.