Pseidomonas exotoxin (PE), a single-chain polypeptide toxin of 613 amino acids, consists of three fuinctional domains: an amino-terminal receptor-binding domain, a middle translocation domain, and a carboxyl-terminal ADPribosylation domain. Deletion of as few as 2 or as many as 11 amino acids from the carboxyl terminus of PE does not affect ADP-ribosylation activity but produces noncytotoxic. molecules. Deletions and substitutions between positions 602 and 611 of PE show that the -last 5 amino acids of PE are very important for its cytotoxic action. The carboxyl-terminal sequence of PE is Arg-Glu-Asp-Leu-Lys. Mutational have been shown to be important for ADPribosylation activity (6-8). Recently, mutational analysis of domain II has shown that certain portions of this domain are absolutely required for the cytotoxicity of PE as well as a chimeric toxin made up of transforming growth factor a (TGFa) and a portion of PE (9, 10). In particular, the arginines at positions 276 and 279 are required for the cytotoxicity of PE (9).While constructing various chimeric toxins in which growth factors were fused to a form of PE (PE40) that was devoid of domain I, we observed that the recombinant fusion proteins made by attaching TGFa, interleukin 2 (IL2), or interleukin 4 (IL4) at the carboxyl end of PE40 had poor cytotoxic activity. PE40-TGFa was about 40-fold less active than TGFa-PE40 (11, 12), and PE40-IL2 and PE40-IL4 were inactive in killing target cells (unpublished results). Furthermore, immunoconjugates made from PE40 were less cytotoxic than anticipated, probably due to conjugation of the antibody with one of the three lysine residues present near the carboxyl end of domain III (13). These observations led us to examine the possibility that the carboxyl terminus of the activity domain of PE, domain III, may have an additional role in the cytotoxic action of PE. Here we present evidence that the carboxyl-terminal 5 amino acids of PE (ArgGlu-Asp-Leu-Lys, REDLK) have important information for the cytotoxicity of PE that is unrelated to ADP-ribosylation. Similar sequences are also found at the carboxyl end of Escherichia coli heat-labile toxin (14) and Cholera toxin A chain (15). The sequence REDLK may be a recognition sequence required for entry of the ADP-ribosylation domain of PE into the cytosol. MATERIALS AND METHODSMaterials. Sources of most reagents have been described (11,16). The polymerase chain reaction (PCR) was performed using a GeneAmp kit (Perkin-Elmer/Cetus).Mutants and Plasmid Constructions. Mutants were created by oligonucleotide-directed mutagenesis using plasmid pVC45f+T as described (5, 9) or using PCR as described below. pVC45f+T carries a PE gene under control of a T7 promoter and also contains a T7 transcriptional terminator and an fl phage origin (9,16 308The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
While common in viral infections and neoplasia, spontaneous cell-cell fusion, or syncytialization, is quite restricted in healthy tissues. Such fusion is essential to human placental development, where interactions between trophoblast-specific human endogenous retroviral (HERV) envelope proteins, called syncytins, and their widely-distributed cell surface receptors are centrally involved. We have identified the first host cell-encoded protein that inhibits cell fusion in mammals. Like the syncytins, this protein, called suppressyn, is HERV-derived, placenta-specific and well-conserved over simian evolution. In vitro, suppressyn binds to the syn1 receptor and inhibits syn1-, but not syn2-mediated trophoblast syncytialization. Suppressyn knock-down promotes cell-cell fusion in trophoblast cells and cell-associated and secreted suppressyn binds to the syn1 receptor, ASCT2. Identification of the first host cell-encoded inhibitor of mammalian cell fusion may encourage improved understanding of cell fusion mechanisms, of placental morphogenesis and of diseases resulting from abnormal cell fusion.
The epidermal growth factor (EGF) receptor is the functional target of the mitogen EGF and the cellular homolog of the avian erythroblastosis virus erbB oncogene product. Regulation of expression of the proto-oncogene encoding the EGF receptor can be elucidated by studying the structure and function of the gene promoter outside the confines of the cell. Previously, we reported the isolation of the human EGF receptor gene promoter. The promoter is highly GC rich, contains no TATA or CAAT box, and has multiple transcription start sites. An Si nuclease-sensitive site has now been found 80 to 110 base pairs (bp) upstream from the maijor in vivo transcription initiation site. Two sets of direct repeat sequences were found in this area; both conform to the motif TCCTCCTCC. When deletion mutations were made in this region of the promoter by using either Bal 31 exonuclease or S1 nuclease, we found that in vivo activity dropped three-to fivefold, on the basis of transient-transfection analysis. Examination of nuclear protein binding to normal and mutated promoter DNAs by gel retardation analysis and DNase I footprinting revealed that two specific factors bind to the direct repeat region but cannot bind to the S1 nuclease-mutated promoter. One of the specific factors is the transcription factor Spl. The results suggest that these nuclear trans-acting factors interact with the S1 nuclease-sensitive region of the EGF receptor gene promoter and either directly or indirectly stimulate transcription.The epidermal growth factor (EGF) is a potent mitogen capable of stimulating protein and RNA synthesis, DNA replication, and, ultimately, cellular proliferation (5,19,30). It elicits these cellular responses by binding to the Nterminus of a 170-kilodalton cell surface glycoprotein, the EGF receptor. The receptor possesses a very active tyrosine kinase at its C-terminus and is capable of phosphorylating itself and other substrates upon EGF binding (4). By virtue of its extensive homology to the erbB oncogene product of the avian erythroblastosis virus, the EGF receptor is considered the cellular erbB proto-oncogene (7,33,47,53). This proto-oncogene has been found to be overexpressed and occasionally amplified in a variety of malignant cell types (6,27,31,37,38,54 Promoter regions of active genes are frequently hypersensitive to the endonucleases DNase I and S1 (10,29,50). This sensitivity is thought to be the result of changes in the structure of the active chromatin, possibly because of the differential dissociation of nucleosomes and local alteration in DNA superhelicity (29,50). Isolated eucaryotic promoter regions cloned into plasmid vectors maintain sensitivity to S1 nuclease, provided the plasmid is supercoiled (12,14,34,35,40,44,55 (#1) is designated by the bent arrow at position -258. Heavy arrows underscore the location of the four sequences making up two sets of direct repeats (designated A and B). The cross-hatched box marks the general region sensitive to Si nuclease. The underlined sequences are those deleted by S1 nuc...
We have found that the imprinted H19 gene can be expressed either biallelically or monoallelically in the developing human placentae. H19 biallelic expression is confined to the placenta until 10 weeks of gestation, after which it becomes exclusively maternal, and does not affect allele-specificity or levels of IGF2 expression. The promoter region of H19 is hypomethylated at all stages of placental development, while the 3' portion shows progressive methylation of the paternal allele with gestation. Our observations demonstrate that the establishment of functional H19 imprinting occurs during the early development of the placenta and provide an opportunity to understand the mechanism by which the H19 primary imprint is manifested in somatic cells.
We have examined the imprinting of the Wilms' tumour suppressor gene (WT1) in human tissues. We confirm that WT1 is biallelically expressed in the kidney, however, in five of nine preterm placentae WT1 was expressed largely or exclusively from the maternal allele. Monoallelic expression of WT1 was also found in two fetal brains. These data demonstrate that WT1 can undergo tissue specific imprinting. Furthermore, because monoallelic expression of WT1 was not found in all placentae examined, WT1 imprinting may be genetically polymorphic within the human population.
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