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The thymic lesion of the nude mouse causes a profound block in T cell development. The failure of most T cells to mature in nude mice is likely to reflect a requirement for signals elaborated in the normal thymus. Interleukin 7 (IL-7), a lymphokine that is normally expressed in the thymus and has been implicated in T cell maturation, might be central to this process. To test this possibility, we introduced a transgene directing lymphoid expression of IL-7 into nude mice and found that it substantially alleviates the block in T cell maturation caused by the thymic defect. IL-7 transgenic nude mice have increased numbers of peripheral cells expressing the T cell marker Thy-1, the T cell antigen receptor complex, and the co-receptors CD4 and CD8. The IL-7 transgene also restores T cell-specific proliferation and activation responses to the peripheral cells of transgene-rescued nude mice. Such findings point toward a fundamental role for IL-7 in the thymic maturation of T cells.

We recently identified a novel 8-kDa transmembrane protein, Mat-8, that is expressed in a subset of murine breast tumors. We have now cloned a cDNA encoding the human version of Mat-8 and show that it is expressed both in primary human breast tumors and in human breast tumor cell lines. The extracellular and transmembrane domains of Mat-8 are homologous to those of phospholemman (PLM), the major plasmalemmal substrate for cAMP-dependent protein kinase and protein kinase C in several different tissues. PLM, which induces chloride currents when expressed in Xenopus oocytes, contains consensus phosphorylation sites for both cAMP-dependent protein kinase A and protein kinase C in its cytoplasmic domain. In contrast, the cytoplasmic domain of Mat-8 contains no such consensus phosphorylation sites and is, in fact, unrelated to the cytoplasmic domain of PLM. RNA blot analysis reveals that Mat-8 and PLM exhibit distinct tissue-specific patterns of expression. We show that expression of Mat-8 in Xenopus oocytes induces hyperpolarization-activated chloride currents similar to those induced by PLM expression. These findings suggest that Mat-8 and PLM, the products of distinct genes, are related proteins that serve as Cl channels or Cl channel regulators but have different roles in cell and organ physiology.

To study oncogenesis in the erythroid lineage, we have generated transgenic mice carrying the human c-MYC proto-oncogene under the control of mouse GATA-1 regulatory sequences. Six transgenic lines expressed the transgene and displayed a clear oncogenic phenotype. Of these, five developed an early onset, rapidly progressive erythroleukemia that resulted in death of the founder animals 30-50 d after birth. Transgenic progeny of the sixth founder, while also expressing the transgene, remained asymptomatic for more than 8 mo, whereupon members of this line began to develop late onset erythroleukemia. The primary leukemic cells were transplantable into nude mice and syngeneic hosts. Cell lines were established from five of the six leukemic animals and these lines, designated erythroleukemia/c-MYC (EMY), displayed proerythroblast morphology and expressed markers characteristic of the erythroid lineage, including the erythropoietin receptor and beta-globin. Moreover, they also manifested a limited potential to differentiate in response to erythropoietin. Studies in the surviving transgenic line indicated that, contrary to our expectations, the transgene was not expressed in the mast cell lineage. That, coupled with the exclusive occurrence of erythroleukemia in all the transgenic lines, suggests that the GATA-1 promoter construct we have used includes regulatory sequences necessary for in vivo erythroid expression only. Additional sequences would appear to be required for expression in mast cells. Further, our results show that c-MYC can efficiently transform erythroid precursors if expressed at a vulnerable stage of their development.

The IgE-triggered release of mast cell mediators in response to antigen is thought to be the primary event in immediate hypersensitivity reactions such as systemic anaphylaxis. Although mast cells and basophils can be activated in vitro by non-IgE stimuli, it is not known whether these triggers lead to physiological changes in vivo. To investigate this possibility, we generated mice with a homozygous null mutation of the C epsilon gene. Such mice make no IgE, but produce other immunoglobulin isotypes normally. We report that despite the IgE deficiency, sensitized mutant mice become anaphylactic on antigen challenge and display tachycardia and pulmonary function changes similar to those seen in wild-type animals. These responses are accompanied by vascular leak, sharply elevated plasma histamine and rapid death. IgE-independent anaphylaxis does not depend on complement activation, but, as indicated in studies using genetically immunodeficient RAG-2- and SCID mice, does require a functional immune system. Such results clearly demonstrate that non-IgE pathways for hypersensitivity reactions exist in mice.

B-less mice express a human immunoglobulin (Ig) lambda transgene that induces a severe deficiency of both immature pre-B and mature B lymphocytes. To understand this perturbation in B lymphopoiesis, we have devised a sensitive limiting dilution polymerase chain reaction assay that quantitates specific Ig rearrangements and thus quantitates B lineage cells at various stages of differentiation within unfractionated bone marrow. We find that there are significantly reduced frequencies of both VH-to-DJH and VK-to-JK rearrangements in the transgenic strain, whereas the frequency of D-to-JH rearrangements approximates that of wild type. Since Ig gene rearrangements occur in a stepwise fashion in which D-to-JH joining precedes that of VH-to-DJH and VK-to-JK, these results indicate that the major block of B lymphocyte development in the B-less strain occurs after D-to-JH rearrangement. Interestingly, sequence analysis of residual VHDJH junctions from transgenic pre-B lymphocytes reveals that an abnormally high proportion of these are out of frame and therefore nonproductive. Taken together, these data suggest that early expression of the transgenic lambda protein specifically prevents the development of a normal-sized population of precursor B lymphocytes coexpressing functional IgH. The transgene-induced immunodeficiency appears to arise by a precocious maturation process in which precursors bypass a developmental stage associated with cellular expansion.

The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c-mpl gene, a member of the cytokine receptor superfamily. We have isolated cDNA and genomic clones encoding murine c-mpl and localized the c-mpl gene to mouse chromosome 4. Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl. The chimera consists of the extracellular domain of the human interleukin-4 receptor and the cytoplasmic domain of mpl. A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin-4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal. In addition, we show that 25-40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full-length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells. Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells.

Mutations at the mouse limb deformity (ld) locus result in defects of growth and patterning of the limb and kidney during embryonic development. The gene responsible for this phenotype is large and complex, with the capacity to generate a number of alternatively spliced messenger RNA transcripts encoding nuclear protein isoforms called "formins." We have made polyclonal antibodies to specific formin peptides and have confirmed the authenticity of the antibodies' reactivity, using cell lines derived from mice with molecularly defined mutations at the ld locus. In addition, we have used these antibodies to detect and characterize polypeptides encoded by both wild-type and mutant ld alleles. In so doing, we show that a formin isoform (i) is modified by posttranslational phosphorylation at serine and threonine residues and (ii) when present in a crude nuclear extract, is retained by DNA-cellulose.

Recent advances in molecular biology have enabled incorporation of proto-oncogenes into the mouse germline. In this study we use a transgenic mouse line that overexpresses the fibroblastic growth factor (FGF) family member, int-2, under the control of mouse mammary tumor virus (MMTV) regulatory elements. One of the tissues targeted by MMTV is the mouse prostate. Expression of the MMTV-int-2 transgene in male transgenic mouse carriers results in a dramatic enlargement of the prostate gland which on histologic examination closely resembles the epithelial/glandular BPH observed in human and canine models. Pre- and postpubertal transgenic (NR) and wild-type (WT) FVB/N male mice were evaluated for the effects of hormonal manipulation by orchiectomy and orchiectomy followed by androgen replacement. Orchiectomy results in a significant decrease in size of the prostate in both NR and WT mice (p < 0.05), regardless of sexual maturity. Exogenous hormonal replacement with testosterone or dihydrotestosterone following orchiectomy results in significant regrowth of the prostate in both NR and WT mice. Flutamide, a potent nonsteroidal anti-androgen, resulted in a 55% reduction in size of the NR prostate (p < 0.002) and a similar 44% reduction in size of the WT prostate. Similarly, treatment of both NR and WT mice with leuprolide, a GnRH agonist, resulted in a significant decrease in prostate size (p < 0.05). Treatment of both NR and WT mice with finasteride (MK-906), a 5-alpha reductase inhibitor, failed to produce any significant regression in prostatic tissue. Based upon these data, we conclude that this transgenic mouse model, expressing int-2, produces an epithelial BPH histologically similar to other animal models. This transgenic model is hormonally sensitive and appears to represent a unique model for the investigation of BPH and growth factor induced epithelial cell hyperplasia.

To investigate the role of interleukin 7 (IL-7) in the development of the lymphoid system, we have generated two lines of transgenic mice carrying an IL-7 cDNA fused to an immunoglobulin heavy chain promoter and enhancer. This transgene is expressed in the bone marrow, lymph nodes, spleen, thymus, and skin provoking a perturbation of T cell development characterized by a marked reduction of CD4+ CD8+ (double-positive) thymocytes. Quite unexpectedly, however, both lines also develop a progressive cutaneous disorder involving a dermal lymphoid infiltrate that results in progressive alopecia, hyperkeratosis, and exfoliation. Although the infiltrate is primarily composed of T lineage cells, its development is not impeded in the athymic nu/nu background. Furthermore, the phenotype can be transmitted horizontally by transplanting lymphoid tissues or skin to syngeneic wild-type mice. Thus, the phenotype is conveyed by skin-homing, mobile cells (presumably the infiltrating lymphocytes) in a cell-autonomous fashion. In addition to the skin phenotype, this transgene also provokes the development of a lymphoproliferative disorder that induces B and T cell lymphomas within the first 4 mo of life. These findings suggest potential physiologic actions of IL-7 in T cell development and in cutaneous immunity. They also demonstrate that IL-7 can act as an oncogene in the living organism.

IP-10 is a member of the -C-X-C-chemokine superfamily of proinflammatory cytokines whose secretion is induced by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). To date no function has been described for IP-10. We have genetically engineered tumor cells to secrete high levels of murine IP-10 and demonstrate that while IP-10 has no effect on the growth of these tumor cells in culture, it elicits a powerful host-mediated antitumor effect in vivo. The IP-10 antitumor response is T lymphocyte dependent, non-cell autonomous, and appears to be mediated by the recruitment of an inflammatory infiltrate composed of lymphocytes, neutrophils, and monocytes. These results document an important biologic property of IP-10 and raise the possibility that some of the T cell-directed effects of IFN-gamma and LPS may be mediated by this chemokine.

Marshall Nirenberg and Heinrich Matthaei initiated their biochemical approach to elucidating the genetic code in 1959, 6 years following the discovery of the double helix structure of DNA (1). At the time, geneticists were using T4 bacteriophage mutagenesis/reversion of plaque morphology toward the same goal by inferring that three mutations were needed to specify an amino acid (2). The genetic code challenge was to link the nucleic acid sequence composed of four bases to translation of 20 amino acids. Nirenberg presented his breakthrough announcement twice at the 1961 International Congress of Biochemistry (ICB) in Moscow: the first presentation to a small audience and the second, at the urging and support of Francis Crick, to the entire congress.

The heparin-binding growth factors include a family of seven structurally related proteins that can potentially interact with four known high affinity receptors. We have cloned the murine homologues of fibroblast growth factor receptors 1 and 3 (mFR1 and mFR3). To define the ligand specificity of these receptors, we have characterized their binding properties with respect to acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) and their biologic activity with respect to aFGF, bFGF, FGF-4/K-FGF, and FGF-5. Unlike mFR1, which binds both aFGF and bFGF, mFR3 preferentially binds aFGF. mFR3-mediated mitogenicity also favors aFGF and FGF-4 with a 10-12-fold lower response to bFGF and no response to FGF-5. Both receptor binding and growth factor-mediated mitogenicity are dependent on heparin. Heparin-binding growth factor activity can thus be regulated by proteoglycans and by the type of FGF receptor expressed on the target cell.

Interleukin 4 (IL-4) is a potent, pleiotropic lymphokine that affects a variety of cells, especially those of hematopoietic origin. Although murine and human IL-4 are homologous proteins, they display a species specificity in which murine IL-4 acts only upon mouse cells, and human IL-4 only upon human cells. We have used a mutagenesis strategy to define both the structural determinants of this specificity and a receptor binding domain of murine IL-4. To do this, we developed convenient solid-phase binding assays for mouse and for human IL-4, each utilizing receptor-immunoglobulin fusion proteins and alkaline phosphatase-tagged ligands. These were employed to assess the receptor binding activities of wild type and mutant forms of IL-4. In a separate biological assay, we measured the ability of each version of IL-4 to induce proliferation of a cultured mouse T-cell line. By replacing regions of mouse IL-4 with homologous segments of human IL-4, we found that the amino-terminal 16 residues and the carboxyl-terminal 20 residues of murine IL-4 are required for species-specific receptor binding as well as for T-cell proliferation. A major portion of the amino acid sequence between these regions can be substituted between mouse and human without loss of receptor binding or biological activity. Further, alanine-scanning mutagenesis revealed specific residues in the amino- and carboxyl-terminal regions (Glu-12, Ile-14, Leu-104, Asp-106, Phe-107, and Leu-111) that bear side chains critical for function. An analysis of the carboxyl-terminal region of murine IL-4 and its comparison with carboxyl-terminal regions of other related cytokines suggest an evolutionary conservation of structural and functional features.

We have created several transgenic mouse strains that bear the human lambda light chain gene driven by its own promoter and a mouse immunoglobulin heavy chain enhancer. The transgene is expressed in many tissues, with particularly high levels of expression in the bone marrow, thymus, spleen, and lymph nodes. One of these transgenic lines, B-less, displays a dramatic phenotype characterized by an acute susceptibility to bacterial and viral infections. Analysis of this strain shows it to be profoundly deficient in both immature (pre-B) and mature B cells, as well as in circulating immunoglobulin. The pre-B and B cell defects are cell autonomous, as judged by cell culture and bone marrow graft chimeras. Despite this B cell deficiency, the T cell lineage appears grossly normal as assessed by flow cytometric analysis and by its response to mitogen stimulation. Since an independently derived transgenic strain bearing the same human lambda construct displays a partial B-less phenotype, it is likely that the B lineage deficiency is due to a dominant effect of transgene expression rather than to the insertional perturbation of an endogenous mouse gene. It is interesting that the deficiency phenotype is fully expressed in the FVB/N genetic background, but is suppressed in F1 hybrids formed between the FVB/N and C57BL/6 inbred strains. Evidently, there are one or more dominant genetic suppressors of B-less in the C57BL/6 genome.

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Proper limb development requires concerted communication between cells within the developing limb bud. Several molecules have been identified which contribute to the formation of a circuitry loop consisting in large part of secreted proteins. The intracellular actin nucleator, Formin 1 (Fmn1), has previously been implicated in limb development, but questions remain after the identification of a Gremlin transcriptional enhancer within the 3' end of the Fmn 1 locus. To resolve this issue, a knockout mouse devoid of Fmn1 protein was created and characterized. The mice exhibit a reduction of digit number to four, a deformed posterior metatarsal, phalangeal soft tissue fusion as well as the absence of a fibula to 100% penetrance in the FVB genetic background. Importantly, this mutant allele does not genetically disrupt the characterized Gremlin enhancer, and indeed Gremlin RNA expression is upregulated at the 35 somite stage of development. Our data reveal increased Bone Morphogenetic Protein (Bmp) activity in mice which carry a disruption in Fmn1, as evidenced by upregulation of Msx1 and a decrease in Fgf4 within the apical ectodermal ridge. Additionally, these studies show enhanced activity downstream of the Bmp receptor in cells where Fmn1 is perturbed, suggesting a role for Fmn1 in repression of Bmp signaling.

Damage initiates a pleiotropic cellular response aimed at cellular survival when appropriate. To identify genes required for damage survival, we used a cell-based RNAi screen against the Drosophila genome and the alkylating agent methyl methanesulphonate (MMS). Similar studies performed in other model organisms report that damage response may involve pleiotropic cellular processes other than the central DNA repair components, yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and contextual importance has been lacking. Further, by comparing data from different model organisms, identification of conserved and presumably core survival components should be forthcoming. We identified 307 genes, representing 13 signaling, metabolic, or enzymatic pathways, affecting cellular survival of MMS–induced damage. As expected, the majority of these pathways are involved in DNA repair; however, several pathways with more diverse biological functions were also identified, including the TOR pathway, transcription, translation, proteasome, glutathione synthesis, ATP synthesis, and Notch signaling, and these were equally important in damage survival. Comparison with genomic screen data from Saccharomyces cerevisiae revealed no overlap enrichment of individual genes between the species, but a conservation of the pathways. To demonstrate the functional conservation of pathways, five were tested in Drosophila and mouse cells, with each pathway responding to alkylation damage in both species. Using the protein interactome, a significant level of connectivity was observed between Drosophila MMS survival proteins, suggesting a higher order relationship. This connectivity was dramatically improved by incorporating the components of the 13 identified pathways within the network. Grouping proteins into “pathway nodes” qualitatively improved the interactome organization, revealing a highly organized “MMS survival network.” We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. A biologically intuitive, highly interconnected MMS survival network was revealed after we incorporated pathway data in our interactome analysis., Author Summary Cellular damage is known to elicit a pleiotropic response, but the relative importance of the constituent components in cell survival is poorly understood. To provide an unbiased identification of the proteins utilized in damage survival, we performed an RNAi survival screen in fly cells with methyl methanesulfonate (MMS). The genes identified are involved in 13 biologically diverse pathways. Comparison with analogous yeast data demonstrated a lack of conservation of the individual MMS survival genes but a conservation of pathways. We went on to demonstrate the MMS responsiveness for five representative pathways in both fly and mouse cells. We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. Incorporation of pathway data in interactome analysis also improved connectivity and, more importantly, revealed a biologically intuitive, highly inter-connected “MMS survival network.” This pathway conservation and inter-connectivity implies extensive interaction between pathways; for diseases such as cancer, such crosstalk may dictate disparate cellular responses not necessarily expected and confound treatments that are not tailored to the individual molecular context.

Membrane-bound immunoglobulin (mIg) is the antigen receptor on B lymphocytes mediating early events in antigen presentation and signal transduction. Wild-type human mIgM constructs transfected into the murine B-cell lymphoma A20 are expressed as transmembrane proteins with antigen presentation and signaling functions comparable to the endogenous mIgG2A; the transfected wild-type mIgM is internalized rapidly after anti-Ig cross-linking. Transfected constructs lacking the normal three-amino acid cytoplasmic tail are expressed exclusively as phosphatidylinositol-linked proteins, lack both antigen presentation and signal transduction functions, and are internalized slowly following anti-Ig binding. The molecular mass of the cytoplasmic tail-deleted phosphatidylinositol-linked Ig molecule is consistent with cleavage of the transmembrane residues during processing. Cytoplasmic domains may therefore regulate the mode of expression of membrane proteins and thereby influence their functional capabilities.

The ligand (KL) for the c-kit receptor is a growth factor encoded at the mouse steel (Sl) locus. KL exists in both cell surface and soluble forms, though little is known of the regulation and functional significance of these forms. We show here that tissue-specific alternative splicing gives two types of KL mRNA. Both encode a transmembrane domain, but in transfected cells one produced the soluble form of KL at relatively high levels, whereas the other preferentially gave the cell surface form. Cell surface KL not only stimulated proliferation, but also mediated cell-cell adhesion. The SId allele, which impairs development of hematopoietic cells, melanocytes, and germ cells, has a deletion in the KL gene removing the transmembrane and intracellular domains. Expression of a corresponding cDNA gave a soluble protein that stimulated cellular proliferation but was not associated with the cell surface. These results provide evidence that cell surface KL has a critical role in the intact organism.

The RecQ family helicase BLM is critically involved in the maintenance of genomic stability, and BLM mutation causes the heritable disorder Bloom’s syndrome. Affected individuals suffer from a predisposition to a multitude of cancer types and an ill-defined immunodeficiency involving low serum Ab titers. To investigate its role in B cell biology, we inactivated murine Blm specifically in B lymphocytes in vivo. Numbers of developing B lymphoid cells in the bone marrow and mature B cells in the periphery were drastically reduced upon Blm inactivation. Of the major peripheral B cell subsets, B1a cells were most prominently affected. In the sera of Blm-deficient naive mice, concentrations of all Ig isotypes were low, particularly IgG3. Specific IgG Ab responses upon immunization were poor and mutant B cells exhibited a generally reduced Ab class switch capacity in vitro. We did not find evidence for a crucial role of Blm in the mechanism of class switch recombination. However, a modest shift toward microhomology-mediated switch junction formation was observed in Blm-deficient B cells. Finally, a cohort of p53-deficient, conditional Blm knockout mice revealed an increased propensity for B cell lymphoma development. Impaired cell cycle progression and survival as well as high rates of chromosomal structural abnormalities in mutant B cell blasts were identified as the basis for the observed effects. Collectively, our data highlight the importance of BLM-dependent genome surveillance for B cell immunity by ensuring proper development and function of the various B cell subsets while counteracting lymphomagenesis.

Experimental carcinogenesis has led to a concept that defines two discrete stages in the development of skin tumors: (i) initiation, which is accomplished by using a mutagen that presumably activates a protooncogene, and (ii) promotion, which is a reversible process brought about most commonly by repeated application of phorbol esters. We have created a transgenic mouse strain that carries the activated v-Ha-ras oncogene fused to the promoter of the mouse embryonic alpha-like, zeta-globin gene. Unexpectedly, these animals developed papillomas at areas of epidermal abrasion and, because abrasion can also serve as a tumor-promoting event in mutagen-treated mouse skin, we tested these mice for their ability to respond to phorbol ester application. Within 6 weeks virtually all treated carrier mice had developed multiple papillomas, some of which went on to develop squamous cell carcinomas and, more frequently, underlying sarcomas. We conclude that the oncogene "preinitiates" carrier mice, replacing the initiation/mutagenesis step and immediately sensitizing them to the action of tumor promoters. In addition, treatment of the mice with retinoic acid dramatically delays, reduces, and often completely inhibits the appearance of promoter-induced papillomas. This strain has use in screening tumor promoters and for assessing antitumor and antiproliferative agents.

The membrane-bound form of immunoglobulin serves as an antigen-specific receptor for B cells mediating signal transduction and antigen presentation. We have developed an assay that reconstitutes both these physiologic responses with respect to the antigen phosphorylcholine. By introducing specific mutations in the human Ig mu chain gene, we have shown that certain transmembrane residues and the short cytoplasmic domain are crucial for these two activities. Moreover, elimination of a single transmembrane hydroxyl group severely inhibits antigen presentation without affecting signal transduction, suggesting that these two functions are mediated by different protein interactions.

The c- kit proto-oncogene, the gene at the mouse W developmental locus, is one of a substantial group of genes that appear to encode cell surface receptors but for which the ligands are unknown. We have characterized the kit ligand by a generally applicable approach: the receptor extracellular domain was genetically fused to placental alkaline phosphatase, producing a soluble receptor affinity reagent with an enzyme tag that could be easilly and sensitively traced. This fusion protein, APtag-KIT, was used to demonstrate a specific binding interaction (K D = 3 × 10 −8 M) with a ligand on 3T3 fibroblast lines. In situ staining showed labeling over the whole surface of the 3T3 cells, but not extending to adjacent nonexpressing cells. These findings provide direct molecular evidence that the kit ligand can exist as a cell surface protein. Binding was not detected on 3T3 fibroblasts carrying the steel ( SI ) mutation, confirming the biological significance of the binding activity and demonstrating that mutations at the SI locus affect the expression or structure of the kit ligand.

We determined the DNA sequence of two large regions of chromosome 22: 33.7 kb containing the C lambda complex; and 5.2 kb 5' of the functionally rearranged lambda gene from the human myeloma, U266. Analysis of these sequences reveals the complete structure of the human C lambda complex and a previously undescribed seventh C lambda region that may encode the Ke+Oz- lambda protein. The seven constant regions are organized in a tandem array, and each is preceded by a single J lambda region. lambda 1, lambda 2, lambda 3, and lambda 7 are apparently active genes, while lambda 4, lambda 5, and lambda 6 are pseudogenes. There are no other J lambda or C lambda regions within a 60-kb region surrounding the C lambda complex; however, there are at least four other lambda-like genes and lambda pseudogenes in the human genome. The lambda genes appear to have evolved via a series of gene duplication events resulting from unequal crossing over or gene conversion between the highly conserved C lambda regions on mispaired chromosomes. The lack of Alu sequences in this large segment of DNA suggests that the C lambda complex resulted from a recent amplification of a smaller Alu-free segment of DNA. Illegitimate recombination between repeated sequences containing lambda 2 and lambda 3 may be responsible for variable amplification of the lambda genes. We also found a 1,377-bp open reading frame (ORF) located on the opposite strand in the region containing lambda 7. While this ORF is flanked by potential RNA splicing signals, we have no evidence that it is part of a functional gene. We also discovered a V lambda pseudogene, called psi V lambda 1, 3 kb upstream of the U266 lambda gene. Using primer extension analysis to map the transcription start in the human lambda gene, we have identified its initiation point 41 bp upstream of the initiation codon. Analysis of the lambda promoter reveals that it contains a TATAA box at position -29 relative to the transcription initiation site and an octamer sequence at -67. Computer analysis of 40 kb of DNA sequences surrounding the human lambda locus has revealed no sequences resembling the kappa or IgH transcriptional enhancers, nor have in vitro analyses for function revealed enhancer activity. A comparison of these results with those obtained in separate studies with transgenic mice point to a complex, developmentally linked mechanism of transcriptional activation.

Background Regulation of the cytoskeleton is a central feature of cell migration. The formin family of proteins controls the rate of actin nucleation at its barbed end. Thus, formins are predicted to contribute to several important cell processes such as locomotion, membrane ruffling, vesicle endocytosis, and stress fiber formation and disassociation. Methodology/Principal Findings In this study we investigated the functional role of Formin1-isoform4 (Fmn1-IV) by using genetically null primary cells that displayed augmented protrusive behaviour during wound healing and delayed cell spreading. Cells deficient of Fmn1-IV also showed reduced efficiency of focal adhesion formation. Additionally, we generated an enhanced green fluorescence protein (EGFP)-fused Fmn1-IV knock-in mouse to monitor the endogenous subcellular localization of Fmn1-IV. Its localization was found within the cytoplasm and along microtubules, yet it was largely excluded from adherens junctions. Conclusions/Significance It was determined that Fmn1-IV, as an actin nucleator, contributes to protrusion of the cell's leading edge and focal adhesion formation, thus contributing to cell motility.

Oncogenesis is a progressive process often involving collaboration between various oncogenes and tumor suppressors. To identify those genes that collaborate with oncogenic ras, we took advantage of the Tg.AC transgenic mouse, a line that harbors the v-Ha-ras transgene and spontaneously develops an array of malignant tumors. By crossing Tg.AC mice on an inbred FVB background to other inbred strains, F1 mice were created that could be analysed using genome wide, single nucleotide polymorphism (SNP) screens. Loss of heterozygosity (LOH) in tumors and tumor cell lines marked a somatic event, possibly the inactivation of tumor suppressor gene(s). LOH could also represent DNA damage, a sign of genomic instability in the pretransformed cell. Nonetheless, the screens showed no evidence of such generalized genomic instability. Instead, they revealed a single region of LOH on chromosome 4 that occurred via somatic recombination/gene conversion, generating a region of isoparental disomy. This LOH provided a clue that linked v-Ha-ras to the inactivation of the Ink4a locus in 25 of 32 tumor cell lines. This collaboration is seen regardless of tumor type or genetic background. In contrast, tumors that develop in bitransgenic mice bearing both the v-Ha-ras gene and a heterozygous mutant p53 allele tend to retain the Ink4a locus and instead lose the p53 wild-type allele. This suggests that different strategies can be selected to collaborate with v-Ha-ras in tumorigenesis.

Background The mechanism involved in the maintenance and differentiation of embryonic stem (ES) cells is incompletely understood. Results To address this issue, we have developed a retroviral gene trap vector that can target genes expressed in undifferentiated ES cells. This gene trap vector harbors both GFP and Neo reporter genes. G-418 drug resistance was used to select ES clones in which the vector was integrated into transcriptionally active loci. This was then followed by GFP FACS profiling to identify ES clones with reduced GFP fluorescence and, hence, reduced transcriptional activity when ES cells differentiate. Reduced expression of the GFP reporter in six of three hundred ES clones in our pilot screening was confirmed to be down-regulated by Northern blot analysis during ES cell differentiation. These six ES clones represent four different genes. Among the six integration sites, one was at Zfp-57 whose gene product is known to be enriched in undifferentiated ES cells. Three were located in an intron of a novel isoform of CSL/RBP-Jkappa which encodes the key transcription factor of the LIN-12/Notch pathway. Another was inside a gene that may encode noncoding RNA transcripts. The last integration event occurred at a locus that may harbor a novel gene. Conclusion Taken together, we demonstrate the use of a novel retroviral gene trap vector in identifying genes preferentially expressed in undifferentiated ES cells.

Regulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, we have conducted a genome-wide RNAi screen in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related CG16975/dSfmbt. These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila , domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth.

Bloom's syndrome (BS) is a genetic disorder characterized cellularly by increases in sister chromatid exchanges (SCEs) and numbers of micronuclei. BS is caused by mutation in the BLM DNA helicase gene and involves a greatly enhanced risk of developing the range of malignancies seen in the general population. With a mouse model for the disease, we set out to determine the relationship between genomic instability and neoplasia. We used a novel two-step analysis to investigate a panel of eight cell lines developed from mammary tumors that appeared in Blm conditional knockout mice. First, the panel of cell lines was examined for instability. High numbers of SCEs were uniformly seen in members of the panel, and several lines produced chromosomal instability (CIN) manifested by high numbers of chromosomal structural aberrations (CAs) and chromosome missegregation events. Second, to see if Blm mutation was responsible for the CIN, time-dependent analysis was conducted on a tumor line harboring a functional floxed Blm allele. The floxed allele was deleted in vitro, and mutant as well as control subclones were cultured for 100 passages. By passage 100, six of nine mutant subclones had acquired high CIN. Nine mutant subclones produced 50-fold more CAs than did nine control subclones. Finally, chromosome loss preceded the appearance of CIN, suggesting that this loss provides a potential mechanism for the induction of instability in mutant subclones. Such aneuploidy or CIN is a universal feature of neoplasia but has an uncertain function in oncogenesis. Our results show that Blm gene mutation produces this instability, strengthening a role for CIN in the development of human cancer.

beta-Catenin, an oncogene, and P53, a tumor suppressor, are common targets of mutation in human cancers. It has been observed that P53 is often inactivated in tumors involving beta-catenin activation. In an attempt to model this situation in vivo, we crossed the previously characterized MMTV-DeltaN-beta-catenin mouse with the P53 knockout mouse. Female multiparous mice that carry the MMTV-DeltaN-beta-catenin transgene and that are heterozygous for P53 (Tg(DeltaN-betaCat)/+, P53+/-) display an increased tumor burden (2.05 vs 1.31 tumors/animal), with a generally more advanced pathology, and increased metastatic rate (39 vs 0%) relative to transgenic female mice that are wild type for P53 (Tg(DeltaN-betaCat)/+, P53+/+). These differences were not due to complete loss of P53 as only one of 21 tumors demonstrated loss of heterozygosity at the P53 locus. Furthermore, no mutations were present in tumors retaining a single wild-type allele. Tg(DeltaN-betaCat)/+, P53-/- male mice developed testicular teratomas and survived an average of 65 days, whereas non-Tg(DeltaN-betaCat), P53-/- males survived an average of 84 days. Sixty-two percent of Tg(DeltaN-betaCat), P53-/- mice developed testicular teratomas, whereas only 10% of the non-Tg(DeltaN-betaCat), P53-/- mice developed these tumors. These results indicate that the level of P53 and the tissue of origin are important factors in determining outcome of cancer caused by oncogene activation.

SummaryAlterations in cellular metabolism are among the most consistent hallmarks of cancer. Herein we have investigated the relationship between increased aerobic lactate production and mitochondrial physiology in tumor cells. To diminish the ability of malignant cells to metabolize pyruvate to lactate, lactate dehydrogenase A (LDH-A) levels were knocked down by means of LDH-A short hairpin RNAs. Reduction in LDH-A activity resulted in stimulation of mitochondrial respiration and decrease of mitochondrial membrane potential. It also compromised the ability of these tumor cells to proliferate under hypoxia. The tumorigenicity of the LDH-A-deficient cells was severely diminished, and this phenotype was reversed by complementation with the human ortholog LDH-A protein. These results demonstrate that LDH-A plays a key role in tumor maintenance.

The HER-2 oncoprotein is commonly overexpressed in a variety of human malignancies and has become an attractive antitumor target. A number of strategies to inhibit the HER-2 receptor tyrosine kinase are currently the focus of intensive preclinical and clinical research. In the present study, we have engineered a bifunctional peptide, BHAP, which consists of two modular domains: a HER-2-targeting/neutralizing domain and a mitochondriotoxic, proapoptotic domain. The chimeric peptide is biologically active and capable of selectively triggering apoptosis of HER-2-overexpressing cancer cells in culture, even those previously described as Herceptin resistant. Furthermore, BHAP slows down growth of HER-2-overexpressing human mammary xenografts established in SCID mice. This approach can be extended to the development of tailored targeted chimeric peptides against a number of overexpressed cellular receptors implicated in the development and progression of cancer.

Thalassemia is a disease caused by a variety of mutations affecting both the adult and embryonic alpha- and beta-globin loci. A mouse strain carrying an embryonic zeta-globin gene disrupted by the insertion of a PGK-Neo cassette displays an alpha-thalassemia-like syndrome. Embryonic survival of this zeta-null mouse is variable and strongly influenced by genetic background, the 129/SvEv mouse strain displaying a more severe phenotype than C57BL/6. We have identified two modifying loci on C57BL/6 chromosomes 2 and 5, which affect the penetrance of embryonic lethality in the 129/SvEv mouse. Through this work, we were able to observe an interesting effect on somatic recombination events in thalassemic embryos. We show that these events can occur on multiple chromosomes in very early embryonic cells, prior to their allocation to the germline. Our results demonstrate that somatic recombination events can be transmitted to subsequent generations.

We have mapped the mouse protein tyrosine phosphatase epsilon (PTP epsilon, gene symbol Ptpre) gene to the distal region of chromosome 7 by linkage analysis using two sets of multilocus genetic crosses. The human PTP epsilon gene (gene symbol PTPRE) was mapped to chromosome 10q26 by fluorescence in situ hybridization. We have previously documented the existence of two isoforms of PTP epsilon--a transmembranal, receptor-type isoform and a shorter, cytoplasmic one. Both isoforms have been suggested to arise from a single gene through the use of alternative promoters and 5' exons. The identification of a single PTP epsilon locus in both organisms is consistent with this suggestion.

The majority of patients who succumb to cancer die from metastatic disease progression rather than from the primary tumor. Elucidation of the mechanisms underlying tissue-specific metastasis is essential to the development of effective therapies. The mitogen-activated protein kinase kinase (MEK) pathway is frequently activated in human tumors and has been shown to regulate genes involved in proliferation, migration, and invasion. Studies with MEK-transformed EpH4 mouse mammary epithelial cells showed that these cells are highly tumorigenic but have a limited metastatic ability. Detachment of epithelial cells from the extracellular matrix causes disruption of the actin cytoskeleton and induces apoptosis. Several metastatic breast carcinoma cell lines have been shown to be resistant to cell death following actin disruption. This death-resistant phenotype can be modeled by overexpressing the antiapoptotic Bcl-2 protein in cells. This suggests that mechanisms that regulate survival of extravasated tumor cells may enhance metastatic efficiency. Therefore, we examined whether expression of Bcl-2 in MEK-transformed EpH4 mammary epithelial cells could provide a survival advantage and promote metastasis. Expression of Bcl-2 in parental EpH4 mammary epithelial cells or MEK-transformed cells was insufficient to induce increased migration, invasion, or tumor development. However, Bcl-2 expression markedly enhanced spontaneous lung metastasis from orthotopically implanted primary tumors. These results clearly show that mechanisms that regulate primary tumor development are distinct from those that promote metastasis and that assays designed to isolate genes involved in transformation may fail to identify genes that are critical regulators of metastasis.

Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1(-) fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G(2)/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

The function of DAXX, a highly conserved mammalian gene, has remained controversial; this is due, in part, to its identification in a variety of yeast two-hybrid screens. Targeted deletion in the mouse revealed that DAXX is essential for embryonic development. Furthermore, the increased levels of apoptosis observed in Daxx -knockout embryos and embryonic stem cell lines suggested that DAXX functions in an anti-apoptotic capacity. In contrast, overexpression studies showed that DAXX may promote apoptosis. Additional studies showed that, when overexpressed, DAXX could function as a transcriptional repressor. To clarify these matters, we have used RNAi to deplete endogenous DAXX and thereby assess DAXX function in cell lines previously tested in overexpression studies. Increased apoptosis was observed in DAXX-depleted cells, showing DAXX to be anti-apoptotic. The apoptosis induced by the absence of DAXX was rescued by Bcl-2 overexpression. In addition, transcriptional derepression was observed in RNAi-treated cells, indicating the ability of endogenous DAXX to repress gene expression and allowing for the identification of novel targets of DAXX repression, including nuclear factor κB (NF-κB)- and E2F1- regulated targets. Thus, depletion of DAXX by RNAi has verified the crucial role of endogenous DAXX as an anti-apoptotic regulator, and has allowed the identification of probable physiological targets of DAXX transcriptional repression.

Activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway is a frequent event in tumorigenesis, and analysis of human breast carcinomas demonstrates that 25-50% of these tumors express elevated levels of activated MAPK1/2. However, a direct role for MEK1 in regulating the invasive and metastatic potential of mammary epithelial cells remains to be established. To directly address the role of constitutive MEK1 signaling in transformation, we have selected the murine mammary epithelial cell line, EpH4, as a model system. EpH4 cells expressing constitutively activated MEK1 display invasive growth in 3-dimensional collagen gels and enhanced motility, and metastatic potential in modified Boyden chamber assays. Furthermore, analysis of markers of normal epithelial morphology by immunofluorescence revealed reorganization of the actin cytoskeleton, and mislocalization of beta-catenin and ZO-1 away from sites of cell-cell contact. However, in contrast to expectations, these changes occurred independently of an epithelial to mesenchymal transition, a change seen frequently in transformed epithelial cells. Moreover, transplantation of EpH4 cells expressing constitutively activated MEK1 into the cleared mammary fat pads of immune-competent hosts rapidly produced tumors that were highly invasive, well vascularized, and readily metastasized to distant organs. Gene expression profiling was performed to identify the downstream targets of MEK1 signaling. Constitutive MEK1 induced the expression of genes involved in proliferation and of matrix metalloproteinases, which regulate invasion and metastasis. These results demonstrate that constitutively activated MEK1 brings about robust tumorigenic changes in murine mammary epithelial cells, and mediates their invasiveness and metastasis in vivo without a requirement for epithelial to mesenchymal transition.

Tumorigenesis results from events that impinge on a variety of collaborating metabolic pathways. To assess their role in this process, we utilized a cell-based assay to perform a high-throughput, chemical library screen. In so doing, we identified F16, a small molecule that selectively inhibits proliferation of mammary epithelial, neu-overexpressing cells, as well as a variety of mouse mammary tumor and human breast cancer cell lines. F16 belongs to a group of structurally similar molecules with a delocalized positive charge. The compound is accumulated in mitochondria of responsive cells, driven by the membrane potential, and it compromises their functional integrity. Mitochondrial hyperpolarization is a shared feature of many tumor cell lines, explaining the broad action spectrum of this novel delocalized lipophilic cation.

Successful reproduction in mammals requires a competent egg, which is formed during meiosis through two assymetrical cell divisions. Here, we show that a recently identified formin homology (FH) gene, formin-2 (Fmn2), is a maternal-effect gene that is expressed in oocytes and is required for progression through metaphase of meiosis I. Fmn2(-/-) oocytes cannot correctly position the metaphase spindle during meiosis I and form the first polar body. We demonstrate that Fmn2 is required for microtubule-independent chromatin positioning during metaphase I. Fertilization of Fmn2(-/-) oocytes results in polyploid embryo formation, recurrent pregnancy loss and sub-fertility in Fmn2(-/-) females. Injection of Fmn2 mRNA into Fmn2-deficient oocytes rescues the metaphase I block. Given that errors in meiotic maturation result in severe birth defects and are the most common cause of chromosomal aneuploidy and pregnancy loss in humans, studies of Fmn2 may provide a better understanding of infertility and birth defects.

Tg.AC transgenic mice provide a sensitive assay for oncogenic agents and a convenient alternative to the two-stage initiation/promoter model of skin tumorigenesis. Although extensively used, this model has remained in part an enigma since mice that carry the Tg.AC transgene (consisting of v-Ha-Ras driven by an embryonic zeta-globin promoter) would not ordinarily be expected to develop skin and other adult tumors. Cloning and characterizing the inserted transgene has provided an insight into the Tg.AC phenotype. We find that the transgene is inserted into a Line-1 element in such a way as to create extended inverted repeats consisting of both transgene and Line-1 sequences. Such structures would be expected to contribute to the instability of the Tg.AC locus and we suggest that this instability is critical to the Tg.AC phenotype. Further, we strengthen this notion by introducing an inactivating mutation in the murine Wrn gene (a gene important in maintenance of genome stability) and showing that bigenic Tg.AC/Wrn(Deltahel/Deltahel) mice experience an eightfold increase in inactivating germline mutations at the Tg.AC locus. Similarly, Tg.AC/Wrn(Deltahel/Deltahel) mice that retain an intact and thus active Tg.AC locus experience a sharp increase in papillomas as compared to Tg.AC/Wrn(+/+) mice. This work demonstrates a genetic interaction between the instability of the multicopy transgene and the Werner Syndrome gene. From this, we conclude that genetic instability remains a key element in this tumor promoter model.

The Drosophila neuralized gene shows genetic interactions with Notch, Enhancer of split, and other neurogenic genes and is thought to be involved in cell fate specification in the central nervous system and the mesoderm. In addition, a human homologue of the Drosophila neuralized gene has been described as a potential tumor suppressor gene in malignant astrocytomas. We have isolated a murine homologue of the Drosophila and human Neuralized genes and, in an effort to understand its physiological function, derived mice with a targeted deletion of this gene. Surprisingly, mice homozygous for the introduced mutation do not show aberrant cell fate specifications in the central nervous system or in the developing mesoderm. This is in contrast to mice with targeted deletions in other vertebrate homologues of neurogenic genes such as Notch, Delta, and Cbf-1. Male Neuralized null mice, however, are sterile due to a defect in axoneme organization in the spermatozoa that leads to highly compromised tail movement and sperm immotility. In addition, female Neuralized null animals are defective in the final stages of mammary gland maturation during pregnancy.

Many tumor cells are impaired in adhesion-regulated apoptosis, which contributes to their metastatic potential. However, suppression of this apoptotic pathway in untransformed cells is not mediated only by adhesion to the extracellular matrix but also through the resulting ability to spread and adopt a distinct morphology. Since cell spreading is dependent on the integrity of the actin microfilament cytoskeleton, we sought to determine if actin depolymerization was sufficient to induce apoptosis, even in the presence of continuous attachment. For this study, we used a human mammary epithelial cell line (MCF10A), which is immortalized but remains adhesion dependent for survival. Treatment of MCF10A cells with latrunculin-A (LA), an inhibitor of actin polymerization, rapidly led to disruption of the actin cytoskeleton and caused cell rounding but preserved attachment. Initiation of apoptosis in LA-treated MCF10A cells was detected by mitochondrial localization of the Bax apoptotic protein, which was prevented by overexpression of Bcl-2. DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage in LA-treated MCF10A cells indicated progression to the execution phase of apoptosis. The MDA-MB-453 cell line, which was derived from a metastatic human mammary tumor, was resistant to PARP cleavage and loss of viability in response to actin depolymerization. Stable overexpression of Bcl-2 in the untransformed MCF10A cells was able to recapitulate the resistance to apoptosis found in the tumor cell line. We demonstrate that inhibition of actin polymerization is sufficient to stimulate apoptosis in attached MCF10A cells, and we present a novel role for Bcl-2 in cell death induced by direct disruption of the actin cytoskeleton.

The Wnt signal transduction pathway has been implicated in mammary tumorigenesis in the mouse. beta-catenin, a key downstream effector of this pathway interacts with and thus activates the Tcf/Lef family of transcription factors. Elevated levels of beta-catenin have been found in many human tumors, notably colon carcinomas. Recently, elevated levels of beta-catenin have been associated with poor prognosis in human adenocarcinoma of the breast. In order to assess the possible role of beta-catenin in mammary carcinoma, we have created transgenic mice bearing the MMTV-LTR driving an activated form of beta-catenin. These mice develop mammary gland hyperplasia and mammary adenocarcinoma, a phenotype very similar to that of transgenic mice expressing an MMTV-driven Wnt gene. Indeed, the histopathology of the mammary tumors in Wnt-mediated adenocarcinoma is identical to that observed in our beta-catenin-mediated disease model. Furthermore, putative beta-catenin transcriptional targets, cyclin D1 and c-myc, are elevated in beta-catenin-mediated mammary tumors and cell lines. These observations support the notion that the oncogenic Wnt pathway operates via beta-catenin and its targets in the context of mammary hyperplasia and carcinoma.

Transgenic mice expressing specific oncogenes usually develop tumors in a stochastic fashion suggesting that tumor progression is a multi-step process. To gain further understanding of the interactions between oncogenes and tumor suppressor genes during tumorigenesis, we have crossed a transgenic strain (TG.NK) carrying an activated c-neu oncogene driven by the MMTV enhancer/promoter with p53-deficient mice. c-neu transgenic mice have stochastic breast tumor formation and normal appearing salivary glands. However, c-neu mice heterozygous for a p53 deletion develop parotid gland tumors and loose their wild type p53 allele. c-neu mice with a homozygous p53 deletion have increased rates of parotid tumor onset suggesting that inactivation of p53 is required and sufficient for parotid gland transformation in the presence of activated c-neu. In contrast to the dramatic effect of p53 in parotid gland transformation, p53 loss has little effect on the rate or stochastic appearance of mammary tumors. In addition, p53 loss was accompanied by the down regulation of p21 in parotid gland tumors but not breast tumors. The parotid gland tumors were aneuploid and demonstrated increased levels of Cyclin D1 expression. These observations suggest that in c-neu transgenic mice, p53 alterations have differential tissue effects and may be influenced by the tissue specific expression of genes influencing p53 activity.

The Notch genes of C. elegans, Drosophila melanogaster and vertebrates encode receptors responsible for cell fate decisions during development. These Notch receptors and their ligands, Delta and Jagged, have been implicated in several human diseases. Truncated, constitutively active mutant forms of the Notch receptor appear to be involved in human T-cell leukemia, mammary carcinomas in mice, and a tumorous germline phenotype in C. elegans. Since activated Notch induces solitary tumors in transgenic mice, it is highly likely that collaborating genetic events are required for tumor formation. We have assessed four signal transduction pathways to determine which might play additional roles in malignant transformation in concert with activated Notch4. Our results suggest that transformation by Notch does not, as might have been expected, depend on the Src-like kinases Lck and Fyn, nor upon signals from protein kinase A and C (PKA, PKC). Rather, transformation by Notch requires active signals from the Erk/MAP kinase and PI-3 kinase pathways downstream of Ras. Oncogene (2000) 19, 4191 - 4198

We have generated transgenic mice that harbor a 140 kb genomic fragment of the human BRCA1 locus (TgN x BRCA1GEN). We find that the transgene directs appropriate expression of human BRCA1 transcripts in multiple mouse tissues, and that human BRCA1 protein is expressed and stabilized following exposure to DNA damage. Such mice are completely normal, with no overt signs of BRCA1 toxicity commonly observed when BRCA1 is expressed from heterologous promoters. Most importantly, however, the transgene rescues the otherwise lethal phenotype associated with the targeted hypomorphic allele (Brca1DeltaexllSA). Brca1-/-; TgN x BRCA1GEN bigenic animals develop normally and can be maintained as a distinct line. These results show that a 140 kb fragment of chromosome 17 contains all elements necessary for the correct expression, localization, and function of the BRCA1 protein. Further, the model provides evidence that function and regulation of the human BRCA1 gene can be studied and manipulated in a genetically tractable mammalian system. Oncogene (2000) 19, 4085 - 4090