New vectors for manipulation and selection of functional yeast artificial chromosomes (YACs) containing human DNA inserts.

A set of fragmentation vectors is described which produce a deletion series of smaller yeast artificial chromosomes (YACs) from a larger parent YAC with the insertion of a eukaryotic selectable marker. In addition, new vectors were designed to permit integration of the genes encoding neomycin (neo) or hygromycin B (hyg) resistance into YACs containing inserts of human DNA. All these vectors are compatible with the yeast host strain AB1380, in which most human genomic YAC libraries are maintained. Linearized vector DNA is used to transform yeast cells in which homologous recombination between human DNA in the YAC and the Alu sequence in the fragmentation or integrating vector produces terminal deletions from the acentromeric (URA3) end of the YAC or insertion of the vector into the YAC, respectively. A set of directional deletions of a YAC is useful for genomic mapping, restriction analysis and functional measurements of large chromosomal regions. The neo and hyg eukaryotic markers permit the study of gene function after introduction of deleted YACs into mammalian cells. Transformation of YACs with the fragmentation vectors resulted in fragmentation in 21-46% of the clones examined; transformation with the integrating vector resulted in integration in 46% of the clones examined.

Protection against glutamate toxicity through inhibition of the p44/42 mitogen-activated protein kinase pathway in neuronally differentiated P19 cells.

Excessive levels of the neurotransmitter glutamate trigger excitotoxic processes in neurons that lead to cell death. N-Methyl-D-aspartate (NMDA) receptor over-activation is a key excitotoxic stimulus that leads to increases in intracellular calcium and activation of downstream signaling pathways, including the p44/42 mitogen-activated protein (MAP) kinase pathway. In the present study, we have demonstrated that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a potent and selective inhibitor of the p44/42 MAP kinase signaling pathway, prevents glutamate-induced death in neuronally differentiated P19 cells. In addition, we show that differentiated, but not undifferentiated, P19 cells expressed zeta1, epsilon1, and epsilon2 subunits of the NMDA receptor. Differentiated P19 cells exhibited specific NMDA receptor binding and intracellular calcium responses to glutamate that were blocked by the selective NMDA receptor antagonist [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not U0126. Glutamate treatment of differentiated P19 cells triggered a rapid and sustained induction in p42 MAP kinase phosphorylation that was blocked by U0126. Pretreatment of differentiated P19 cells with U0126, but not other classes of protein kinase inhibitors, protected against glutamate-induced cell death. Post-treatment with U0126, even as late as 6 hr after glutamate application, also protected against glutamate toxicity. These results suggest that the p44/42 MAP kinase pathway may be a critical downstream signaling pathway in glutamate receptor-activated toxicity.

Antimitotic drugs cause increased tumorigenicity of multidrug resistant cells.

Taxol and vinblastine are widely and effectively used in the treatment of cancer, but the initial response to chemotherapy is often hampered by the development of multidrug resistant (MDR) cells. The collateral effects of these two antimitotic drugs on MDA-435 human breast carcinoma cells were investigated. When used at concentrations below those required to depolymerize microtubules, both drugs increased cyclin dependent kinase activity and stimulated the MAPK signal transduction pathway. The activity of MAPK pathway elements and cyclin dependent kinases were found to be constitutively elevated in an MDR cell line that was selected with taxol and expresses high levels of P-glycoprotein. The MDR cells maintained these alterations and also overexpressed hyperphosphorylated RB. This was manifested in a higher growth rate for MDR cells in low serum and an increased ability to form colonies in soft agar. These observations suggest that despite high levels of P-glycoprotein in MDR cells, a sufficient amount of taxol remains intracellular to accelerate the cell cycle machinery and activate the MAPK pathway. These alterations accumulate in resistant cells and contribute to a more transformed phenotype. Thus, in addition to the development of MDR, exposure of tumor cells to antimitotic agents produces further cellular changes that may contribute to the failure of chemotherapy.

Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2.

We report the characterization of BMS-911543, a potent and selective small-molecule inhibitor of the Janus kinase (JAK) family member, JAK2. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in cell lines dependent upon JAK2 signaling, and had little activity in cell types dependent upon other pathways, such as JAK1 and JAK3. BMS-911543 also displayed anti-proliferative responses in colony growth assays using primary progenitor cells isolated from patients with JAK2(V617F)-positive myeloproliferative neoplasms (MPNs). Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2 signaling, with sustained pathway suppression being observed after a single oral dose. At low dose levels active in JAK2-dependent PD models, no effects were observed in an in vivo model of immunosuppression monitoring antigen-induced IgG and IgM production. Expression profiling of JAK2(V617F)-expressing cells treated with diverse JAK2 inhibitors revealed a shared set of transcriptional changes underlying pharmacological effects of JAK2 inhibition, including many STAT1-regulated genes and STAT1 itself. Collectively, our results highlight BMS-911543 as a functionally selective JAK2 inhibitor and support the therapeutic rationale for its further characterization in patients with MPN or in other disorders characterized by constitutively active JAK2 signaling.

Amplification of specific gene products from human serum.

The polymerase chain reaction (PCR) is an in vitro method for the primer-directed enzymatic amplification of specific DNA sequences. Ordinarily, sources with obvious DNA content such as cells, viruses, and plasmids serve as the origin of templates for the PCR. Here we report a simple and efficient method to obtain cellular DNA from serum suitable for use in PCR reactions or gene analysis. This procedure should facilitate the detection of disease and provide a basis for the examination of mutations in genes before the onset as well as during the progression of various diseases.

Human interferon-alpha A, -alpha 2, and -alpha 2(Arg) genes in genomic DNA.

The frequency in human genomic DNA of three human interferon (IFN) alpha genes which differ from each other by a single base substitution was examined in 11 normal individuals. The polymerase chain reaction (PCR) was used to amplify the coding sequence for the Hu-IFN-alpha 2, Hu-IFN-alpha A, and Hu-IFN-alpha 2(Arg) sequences from genomic DNA. The PCR products were then cloned and individual clones were sequenced. PCR products were also analyzed by restriction endonuclease analysis for the IFN-alpha 2 and IFN-alpha A genes by use of a HinfI site which is eliminated by the substitution of an A for a G in IFN-alpha A. The IFN-alpha A gene which was cloned from the myeloblastoid cell line KG-1 was not observed in any of the 201 clones sequenced from normal individuals or in the Namalwa cell line. It was detected in KG-1 cell genomic DNA where it represented 49% of the clones sequenced. Similarly the IFN-alpha 2(Arg) gene was not detected in normal individuals or in the KG-1 cell line but only in the lymphoblastoid cell line from which it was cloned. In Namalwa cells the IFN-alpha 2(Arg) sequence represented 35% of the clones sequenced while the IFN-alpha 2 sequence comprised 59%. Therefore, both the IFN-alpha A and IFN-alpha 2(Arg) sequences represent alleles of the IFN-alpha 2 gene.

Yeast artificial chromosome fragmentation vectors that utilize URA3 selection.

Two fragmentation vectors, pSE1 and pSE2, were developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA. Ura- yeast cells containing YACs were selected with 5-fluoro-orotic acid. Fragmented YACs were subsequently generated by transformation to a Ura+ phenotype. Over 80% of the transformants contained YACs of reduced molecular size. These fragmented YACs will prove to be useful in mapping the region of human chromosomes covered by the parental YAC. Fragmentation utilizing URA3 transformation provides a method for producing YAC deletion sets from YACs contained in AB1380 and other ura3- yeast stains. Linkage of a neomycin resistance gene to the URA3 gene facilitates functional analysis of these YACs in eukaryotic cells.

Sublocalization of the human interferon-gamma receptor accessory factor gene and characterization of accessory factor activity by yeast artificial chromosomal fragmentation.

A chromosomal fragmentation procedure was employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC, GART D142H8, known to map to human chromosome 21q and to encode the human interferon-gamma receptor (Hu-IFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene. When expressed in Chinese hamster ovary cells, these deleted YACs retain accessory factor activity, as judged by major histocompatibility complex class I antigen inducibility, until the deletions from the acentric end exceed 390 kilobases (kb). Therefore, the accessory factor (AF-1) gene can be localized to a 150-kb region at the left (centric) end of the parental 540-kb GART YAC. Cells containing functional YACs are also able to induce the ISGF3 gamma and gamma-activated factor (GAF) transcription factors, but were not protected against encephalomyocarditis virus (EMCV) upon treatment with Hu-IFN-gamma. Therefore, the Hu-IFN-gamma R and the AF-1 are sufficient for some, but not all, of the actions of Hu-IFN-gamma. We postulate that an additional accessory factor (AF-2) required for antiviral activity against EMCV is encoded on chromosome 21q.

EGF induces increased ligand binding affinity and dimerization of soluble epidermal growth factor (EGF) receptor extracellular domain.

The binding of epidermal growth factor (EGF) to its cell surface receptor (EGF-R) results in a number of intracellular responses including the activation of the receptor intracellular tyrosine kinase. Receptor oligomerization induced by ligand binding has been suggested to play an important role in signal transduction. However, the mechanisms involved in oligomerization and signal transduction are poorly understood. We have produced and purified several milligrams of recombinant extracellular domain of the EGF receptor (EGF-Rx) using the baculovirus/insect cell expression system. The baculovirus-generated EGF-Rx is glycosylated, has had its signal peptide correctly cleaved, and exhibits a dissociation constant for EGF similar to that for solubilized full-length receptor, of about 100 nM. The binding of EGF to EGF-Rx leads to the formation of receptor dimers and higher oligomerization states which are irreversibly captured using the covalent cross-linking agent disuccinimidyl suberate. Interestingly, purified receptor monomers and dimers, stabilized by the cross-linker in the presence of EGF, exhibit increased binding affinity toward EGF as compared with receptor monomers which have not been exposed to EGF. It appears that the high affinity state of receptor can be maintained by the covalent cross-linking agent. These results indicate that in addition to ligand binding, the extracellular domain of EGF receptor possesses the inherent ability to undergo ligand-induced dimerization and that the low affinity state is converted to a high affinity state by EGF.