A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

The retinoblastoma protein (pRB) plays a key role in the control of normal development and proliferation through the regulation of the E2F transcription factors. We generated a mutant mouse model to assess the in vivo role of the predominant E2F family member, E2F4. Remarkably, loss of E2F4 had no detectable effect on either cell cycle arrest or proliferation. However, E2F4 was essential for normal development. E2f4-/- mice died of an increased susceptibility to opportunistic infections that appeared to result from craniofacial defects. They also displayed a variety of erythroid abnormalities that arose from a cell autonomous defect in late stage maturation. This suggests that E2F4 makes a major contribution to the control of erythrocyte development by the pRB tumor suppressor.

SEK1 (MKK4͞JNKK) is a mitogen-activated protein kinase activator that has been shown to participate in vitro in two stress-activated cascades terminating with the SAPK and p38 kinases. To define the role of SEK1 in vivo, we studied stress-induced signaling in SEK1؊͞؊ embryonic stem and fibroblast cells and evaluated the phenotype of SEK1 ؊͞؊ mouse embryos during development. Studies of SEK1؊͞؊ embryonic stem cells demonstrated defects in stimulated SAPK phosphorylation but not in the phosphorylation of p38 kinase. In contrast, SEK1؊͞؊ fibroblasts exhibited defects in both SAPK and p38 phosphorylation, demonstrating that crosstalk exists between the stress-activated cascades. Tumor necrosis factor ␣ and interleukin 1 stimulation of both stress-activated cascades are severely affected in the SEK1 ؊͞؊ fibroblast cells. SEK1 deficiency leads to embryonic lethality after embryonic day 12.5 and is associated with abnormal liver development. This phenotype is similar to c-jun null mouse embryos and suggests that SEK1 is required for phosphorylation and activation of c-jun during the organo-genesis of the liver.Three vertebrate mitogen-activated protein kinase (MAPK) cascades have been shown to regulate signal transduction necessary for the development and function of many tissues (1-3). The stress-activated kinase cascade involving MEKK, SEK1, and SAPK leads to the phosphorylation of c-jun and is activated by stress stimuli such as interleukin 1 (IL-1), tumor necrosis factor ␣ (TNF␣), heat shock, UV irradiation, and protein synthesis inhibition. The p38 MAPK cascade is activated by similar stimuli. The upstream kinases MKK3 and MKK6 function to activate p38(4-7). The MAPK SEK1 (also called MKK4 or JNKK) is capable of phosphorylating and activating SAPK in vitro. Furthermore, kinase inactive mutants of SEK1 specifically prevent SAPK activation by stress stimuli (4). SEK1 also has been shown to phosphorylate and activate p38 MAPK in vitro (4), suggesting that SEK1 may participate in the regulation of two independent MAPK cascades.Two recent reports have evaluated the function of SEK1 in murine embryonic stem (ES) cells (8, 9). These targeted gene disruption experiments demonstrated a significant defect in SAPK activity in response to stress signals such as anisomycin and heat shock. The activation of SAPK in response to UV irradiation was affected less dramatically in these SEK1-deficient ES cells, suggesting the existence of another kinase that activates SAPK. A candidate could be the recently cloned SEK1 family member MKK7 (10), which has been shown to specifically activate SAPK but not p38. Although these reports show that the activation of SAPK is affected in SEK1 Ϫ͞Ϫ ES cells, there are no defects seen in p38 kinase activity, implying that SEK1 only regulates the SAPK cascade in vivo and that the activation of p38 by SEK1 is an in vitro phenomenon.These reports do not address the role of SEK1 in TNF␣ and IL-1 signaling. ES cells do not express receptors for TNF␣ and IL-1. To evaluate this, we generated SEK1 ...

During a screen to identify c-Jun activators, we isolated a cysteine protease, SuPr-1, that induced c-Jun-dependent transcription independently of c-Jun phosphorylation. SuPr-1 is a member of a new family of proteases that hydrolyze the ubiquitin-like modifier, SUMO-1. SuPr-1 hydrolyzed SUMO-1-modified forms of the promyelocytic leukemia gene product, PML, and altered the subcellular distribution of PML in nuclear PODs (PML oncogenic domains). SuPr-1 also altered the distribution of other nuclear POD-associated proteins, such as CBP and Daxx, that act as transcriptional regulators. SuPr-1 action on transcription was enhanced by PML, and SuPr-1 failed to activate transcription in PML-deficient fibroblasts. Our studies establish an important role for SUMO proteases in transcription.

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Cyclin D1 is an important regulator of the transition from G1 into S phase of the cell cycle. The level to which cyclin D1 accumulates is tightly regulated. One mechanism contributing to the control of cyclin D1 levels is the regulation of its ubiquitination. SK-UT-1B cells are de®cient in the degradation of D-type cyclins. We show here that p27, a substrate of the SCF Skp2 ubiquitin ligase complex, is coordinately stabilized in SK-UT-1B cells. Further, we show that expression of Skp2 in SK-UT-1B cells rescues the cyclin D1 and p27 degradation defect observed in this cell line. These results therefore indicate that the SCF Skp2 ubiquitin ligase complex aects the ubiquitination of cyclin D1. In addition, we show that SK-UT-1B cells express a novel splice variant of Skp2 that localizes to the cytoplasm and that cyclin D1 ubiquitination takes place in the nucleus. We propose that the translocation of Skp2 into the nucleus is required for the ubiquitination of cyclin D1 and that the absence of the SCF Skp2 complex in the nucleus of SK-UT-1B cells is the mechanism underlying the ubiquitination defect observed in this cell line. Finally, our data indicates that dierential splicing of F-box proteins may represent an additional level of regulation of the F-box mediated ubiquitination pathway. Oncogene (2001) 20, 3641 ± 3650.

An important goal of tissue engineering is to achieve reconstitution of specific functionally active cell types by transplantation of differentiated cell populations derived from normal or genetically altered embryonic stem cells in vitro. We find that mast cells derived in vitro from wild-type or genetically manipulated embryonic stem cells can survive and orchestrate immunologically specific IgE-dependent reactions after transplantation into mast cell-deficient Kit W ͞Kit W-v mice. These findings define a unique approach for analyzing the effects of mutations of any genes that are expressed in mast cells, including embryonic lethal mutations, in vitro or in vivo.

SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate control of cellular death and proliferation. In lymphocytes, the SAPK pathway has been implicated in signaling from antigen, costimulatory, and death receptors; SEK1, which directly activates SAPK, is required for early embryonic development and has also been reported to be essential for normal lymphocyte development. In contrast to the latter findings, we have used RAG-2-deficient blastocyst complementation to show that SEK1-deficient embryonic stem cells support unimpaired T and B lymphocyte development. Moreover, mature SEK1-deficient lymphocytes are capable of SAPK activation. Surprisingly, however, aging SEK1-deficient chimeric mice frequently develop lymphadenopathy and polyclonal B and T cell expansions. Thus, SEK1 is not required for lymphocyte development, but is required for maintaining peripheral lymphoid homeostasis.