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The bacteriophage lambda (λ) recombination system Red has been used for engineering large DNA fragments cloned into P1 and bacterial artificial chromosomes (BAC or PAC) vectors. So far, this recombination system has been utilized by transferring the BAC or PAC clones into bacterial cells that harbor a defective λ prophage. Here we describe the generation of a mini-λ DNA that can provide the Red recombination...
The sphingolipid, ceramide, is a natural dietary constituent and a potent mediator of apoptosis. If left undegraded, it may induce apoptosis and cause disruption of cellular integrity. A potential mechanism to prevent ceramide-induced apoptosis in various organs may involve ceramidases that facilitate the degradation of ceramide. In this study, we first isolated and characterized the murine neutral...
The mitochondrial nad9 and nad6 genes were analyzed in four lupin species: Lupinus luteus, Lupinus angustifolius, Lupinus albus and Lupinus mutabilis. The nucleotide sequence of these genes confirmed their high conservation, however, higher number of nucleotide substitution was observed in the L. albus genes. Southern hybridizations confirmed the presence of single copy number of these genes in L...
DNA alkylation tolerance is a major concern in cancer chemotherapy. It has been suggested that mutations in DNA mismatch repair genes may result in alkylation tolerance. This alkylation tolerant phenotype is often manifested in cells lacking an O6-methylguanine DNA methyltransferase (MTase) activity. However, deletion of each mismatch repair gene in the MTase mutant of a model eukaryotic yeast does...
Mammalian Polycomb group (PcG) proteins are known to function during the maintenance of spatially restricted expression of Hox cluster genes and cellular proliferation. To understand the molecular basis of PcG functions, it is important to identify the components of mammalian PcG complexes. We isolated mouse YAF2 as a protein that interacts with Ring1B, a known constituent of mammalian PcG complexes...
In the process of programmed cell death (PCD), a key role has been attributed to endonucleases capable to cleave nuclear DNA at internucleosomal sites. In barley (Hordeum vulgare L.), two such nucleases (Bnuc1 and BEN1) were individually identified in unrelated tissues. In the present work, we demonstrate that their genes are also expressed in immature anthers at different stages of pollen development...
The multicopy human U2 small nuclear (sn)RNA genes are transcribed by RNA polymerase (pol) II and contain two major promoter elements upstream of the transcription start site: an essential proximal sequence element (PSE) at around −55 and a distal sequence element (DSE) at around −220. We have carried out an in vivo footprinting analysis on these genes, and the results suggest that most, if not all,...
Slitrk family proteins are characterized as integral membrane proteins that have two leucine-rich repeat (LRR) domains and a carboxy-terminal domain that is partially similar to trk neurotrophin receptor proteins. The LRR domains are similar to those of slit proteins. In a previous study, we showed that mouse Slitrk genes are expressed predominantly in neural tissue and have neurite-modulating activity...
Early growth response factor 1 (Egr-1) is a key transcriptional factor to mediate gene expression after vascular injury. To better understand the role of Egr-1 in vasculature, we globally profiled Egr-1 target genes in human endothelial cells using adenoviral gene transfer and Affymetrix oligonucleotide-based microarray technology. More than 300 genes regulated by ≥3-fold with Egr-1 overexpression...
Ciliates are a group of microbial eukaryotes defined by the presence of dimorphic nuclei—each cell contains both a transcriptionally active macronucleus and a germline micronucleus. During the development of the macronucleus, germline chromosomes are rearranged through extensive fragmentation, removal of internally excised sequences (IESs) and DNA amplification. We have characterized three IESs in...
Bacterial virus phi29 is the most efficient in vitro DNA packaging system, with which up to 90% of the added DNA can be packaged into purified recombinant procapsid in vitro. The findings that phi29 virions can be assembled with the exclusive use of cloned gene products have bred a thought that phi29 has a potential to be a gene delivery vector since it is a nonpathogenic virus. gp12 of bacterial...
Pre-α-Inhibitor is a plasma protease inhibitor and a heterodimeric molecule whose one polypeptide chain is encoded by the ITIH3 gene. In order to understand the expression of this protein that is regulated in health and disease, we have analyzed the 5′ flanking region of ITIH3, specifically focussing on its proximal promoter. A combination of methods including wild-type (wt) or mutant promoter linked...
Two genes encoding ATP-binding cassette (ABC) transporters were isolated from the crop plant Zea mays (maize). The clones, designated ZmMRP1 and ZmMRP2, were highly homologous to members of the multidrug resistance associated protein (MRP) subfamily. Genomic Southern analysis and characterisation of bacterial artificial chromosome (BAC) clones demonstrated that both genes are present in two copies...
The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV) and the neomycin phosphotransferase II (Neo) selection marker from the same transcript. Envelope expression...
Regulation of RNA polymerase II (pol II) transcription is a highly dynamic process requiring the coordinated interaction of an array of regulatory proteins. Central to this process is the TATA-binding protein (TBP), the key component of the multiprotein complex TFIID. Interaction of TBP with core promoters nucleates the assembly of the preinitiation complex and subsequent recruitment of pol II. Despite...
Interferon-α with its antiproliferative activity is widely used for the treatment of viral infections and tumor therapy such as melanoma. Naturally occurring resistance to recombinant interferon alpha-2a (IFN-α) and severe side effects limit the therapeutic efficacy. Understanding of the molecular mechanisms involved in unresponsiveness may therefore lead to the development of novel formulations that...
In vitro fusion of weakly metastatic Cloudman S91 melanoma cells with macrophages from DBA/2J mice (syngeneic with Cloudman S91 melanoma) produced hybrids with metastatic potentials ranging from low to high, with more than half showing enhanced metastasis over the parental melanoma [Clin. Exp. Metastasis 16 (1998) 299]. These hybrids, derived from the same parental fusion partners, represent a unique...
A novel human gene, named as hTLP19, was isolated and characterized as secretory protein by combining bioinformatics tools with experiments. The hTLP19 encodes 172 amino acid residues with signal peptide in its N-terminal and a thioredoxin (Trx) domain that is homologous with some genes in Mus musculus, Xenopus laevis, etc. Moreover, the result from insulin reduction assay indicated that the hTLP19...
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