SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
A human DOPA decarboxylase (DDC) cDNA probe of 747 base pairs has been used to map the DDC gene by in situ hybridization on mouse metaphase chromosomes. This result indicates that the gene is located on band 11A, near the erythroblastosis oncogene B (erb b) locus. This provides evidence for a synteny group on mouse chromosome 11 and human chromosome 7.
The cellular homologs of the ets gene from the avian erythroblastosis retrovirus E26 have been studied in chickens, humans, mice, and cats. In this report a further evolutionary step is taken by isolating and characterizing a Drosophila ets-related genomic clone. Sequence analysis of this clone has shown it to contain the 3' end of the v-ets gene, called ets-2, corresponding to the last two exons of chicken ets. The predicted amino acid sequence was found to have over 90% homology when compared to that of v-ets. This is the highest level of conservation observed for any previously characterized Drosophila oncogene homolog. Expression of the ets-2 gene occurs throughout development, but is highest during the embryonic and pupal stages. By in situ hybridization, the ets-2 chromosomal position was determined to be 58A/B which corresponds to no known phenotypic mutant. As this is a highly conserved gene, the Drosophila model system should prove useful for the determination of the ets gene function.
The locus SU(Lv)-ets-2 of the sea urchin Lytechinas variegatus related to the oncogene v-ets of avian erythroblastosis virus E26 has been molecularly cloned. The cloned DNA was found to contain a region with a high degree of homology to E26 v-ets. The sea urchin homology with v-ets starts at a consensus splice acceptor sequence and stops at the point where homology between v-ets and human c-ets ends. This region corresponds to the Hu-ets-2 homologous sequences defined by Watson et al. (1985, Proc. Natl. Acad. Sci, USA 82, 7294-7298). Ninety-one out of 97 (or 94%) predicted amino acids are identical between sea urchin c-ets and E26 v-ets over the region of homology. This degree of homology exceeds the maximum homology previously found between any oncogene and an invertebrate homolog. A somewhat weaker homology with the Hu-ets-2 sequences continues beyond, for 13 codons, ending at a common termination codon. Northern blot analysis of mature unfertilized eggs and early embryos from sea urchins of the species Strongylocentrotus purpuratus revealed a single 6.8-kb ets-related RNA that is expressed at a maximum level during the early stages of embryonic development. This RNA species is polyadenylated indicating that it is the message for the sea urchin ets-2 gene.
Mammalian erythroblasts and their leukemic counterparts contain characteristic disordered regions of plasma membrane identified as putative membrane protein collection sites. In order to determine whether erythroid cells which do not enucleate contain homologous membrane domains, immature avian erythroid precursor cells and avian erythroleukemic cells were examined using merocyanine 540 (MC540), a fluorescent dye whose binding is sensitive to the packing of membrane lipids. Results were found to contrast with previous studies of the murine equivalents of these cells. In birds, normal erythroid precursors, including basophilic erythroblasts from the bone marrow and spleen of anemic animals, contained no detectable (less than 0.1%) cells which were stained by the dye. But cells from chicks infected with avian erythroblastosis virus (AEV) did stain. Considering the pattern of staining observed on AEV-erythroblasts relative to other leukemic and normal phenotypes, however, we conclude that neither normal nor leukemic avian erythroid cells contain a functional equivalent to the membrane protein collection sites found on their mammalian counterparts.
Chicken erythroblasts transformed by a temperature-sensitive erythroblastosis virus (tsAEV) can terminally differentiate at the non-permissive temperature (42 degrees C). Morphological changes, as well as production of hemoglobin were shown to occur during maturation. The above system, as well as fractions of normal bone marrow cells were used to study the expression of cell surface antigens on erythroid cells. Four distinct reactivities were found with monoclonal antibodies (MAbs) and polyvalent sera: The MAb 4.5A5 was found to react with erythroblasts only. The MAb 4-M-12-26 reacted with tsAEV erythroblasts shifted to the non-permissive temperature (42 degrees C). The MAb 4.6C1 reacted weakly with erythroblasts and strongly with reticulocytes. The anti-Ery sera reacted with reticulocytes and erythrocytes. In addition the reactivity of MAb 4-M-12-26 was found only on virus-producing erythroblasts, though this MAb was found to react with myeloid cells which produce a helper virus of the same class.
The transforming protein erbB of avian erythroblastosis virus (AEV) has considerable sequence homology with the epidermal growth factor (EGF) and appears to represent a truncated form of this receptor. The sequence of the erbB gene is furthermore related to that of other viral transforming genes such as src, fps, yes or abl. The transforming proteins of these src-related oncogenes as well as receptors for EGF, platelet-derived growth factor (PDGF), and insulin are associated with tyrosine-specific protein kinases. It has been difficult to demonstrate this activity for the erbB protein. To analyze the erbB gene product, we prepared polyclonal antibodies against a bacterially expressed erbB DNA restriction fragment (BamHI/BamHI). The antiserum is shown to immunoprecipitate the erbB protein from AEV-transformed chicken fibroblasts and also recognizes the EGF receptor protein. Both proteins become phosphorylated in vitro on tyrosine residues upon the addition of [gamma-32P]ATP. The protein kinase activity is low compared to other oncogene-specific kinases. This is not due to kinase blocking by the serum, because erbB carboxyterminal synthetic peptide antibodies give rise to low levels of protein kinase activity as well indicating that this may be a characteristic property of erbB in vitro.
Two hundred and eighteen cases of hemorrhagic endovasculitis (HEV), a recently recognized abnormality of human placentas, were identified from placentas submitted to the Michigan Placental Tissue Registry over a 2.5-year period. HEV appears to focus on fetal placental blood vessels with resultant fragmentation and destruction of fetal RBCs, hemorrhage into villous stroma, microthrombi in villous capillaries and non-exudative necrosis of medium sized chorionic vessels. Placentas without HEV submitted for evaluation from the same hospitals were selected for comparison. Women with HEV placentas were of similar age, race and parity as control women. The sex of the fetus of HEV cases was somewhat more often female (57% vs. 48%, P = 0.05). The presence of HEV appeared to have a deleterious effect on the outcome of pregnancy: 52% (112/218) of the HEV placentas were associated with stillborn infants, in contrast to only 22% (89/400) of control placentas. The difference in the proportion of stillbirths was greatest when the gestational age was 25 weeks or greater. Other significant positive associations with HEV included the presence of meconium staining, intrauterine growth retardation, smaller placentas, and maternal hypertension or toxemia. Pathologic findings associated with HEV were chronic villitis, erythroblastosis, thrombosis and cord abnormalities. No association was found with infant Apgar score or fetal anomalies. The association of HEV with a high proportion of stillbirths in the registry suggests that further understanding of this lesion might shed light on the problem of unexplained stillbirth.
The response to infection of chicken hemopoietic cells derived from the early stages of embryogenesis by avian myeloblastosis virus (AMV) and avian erythroblastosis virus (AEV) was investigated. It was found that erythroid progenitor cells were present in the blastoderm at a higher frequency than that of myeloid progenitor cells. These results correlate with the observation that target cells for AEV were found to be more numerous than those for AMV. Therefore, blastoderm cells are of potential value in understanding the mechanisms of oncogenesis at the level of the target cells.
The genome of avian erythroblastosis virus (AEV), a defective acute leukemia retrovirus, carries two distinct cell-derived oncogenes in the structure 5' delta gag-erbA-erbB-delta env3'. The nucleotide sequence of the v-erbA gene was recently reported. In order to determine the boundary between the two adjacent oncogenes, the sequence of the v-erbA/v-erbB junction of AEV was compared to that of a recombinant lambda phage containing a chicken cellular sequence representing the 5' part of c-erbB. The four C-terminal amino acids of v-erbA are in fact encoded by a c-erbB intron-derived sequence thus demonstrating that the virus acquired a truncated c-erbA gene. Furthermore the 7 to 10 amino acid residues upstream from the 4 C-terminal amino acids mentioned above appeared to be derived from env-related sequences. The splice acceptor site at the beginning of the only open reading frame for v-erbB is also present and functional in c-erbB when expressed to generate a truncated EGF (epidermal growth factor) receptor. Thus AEV joins a truncated erbA gene to a truncated erbB gene through env-derived sequences and intronic sequences from c-erbB.
A novel murine retrovirus complex was derived from the in vivo passage of a molecularly cloned Friend ecotropic helper virus. The virus isolate, myeloproliferative leukemia virus (MPLV), causes an acute (2-3 weeks) and generalized myeloproliferative disorder in adult mice. All strains of mice examined, including the C57BL/6J strain, developed the acute syndrome. This syndrome is characterized by a rapid hepatosplenomegaly, no thymus or lymph node involvement, granulocytosis, thrombocytosis, and erythroblastosis leading to polycythemia. The most prominent feature at the terminal phase of the disease is a granulocytic hyperplasia. The MPLV isolate replicates in vitro on NIH 3T3 fibroblasts but does not induce foci of transformed cells. Thus, MPLV exhibits unique biological properties that distinguish it either from the Friend virus complexes or from acutely transforming sarcomatogenic murine retrovirus which also induced a rapid splenomegaly.
The transmembrane domain was deleted from within the v-erb B protein coding region of avian erythroblastosis virus. The mutant oncogene encoded a shortened, apparently soluble form of the normally membrane bound v-erb B protein. Despite this alteration in subcellular distribution, the mutant polypeptide retained the ability to induce fibroblast transformation by several parameters, including the ability to display anchorage-independent growth. It appears that the transmembrane domain, although important for full manifestation of the transformed phenotype, is not essential for v-erb B-mediated oncogenic transformation.
Chicken DNA segments homologous to the ets region from the transforming gene of avian erythroblastosis virus, E26, were molecularly cloned and shown to be almost identical to v-ets by sequence analysis. The transforming gene acids from two cell-derived sequences, myb and ets. Whereas the mammalian ets genes are present on two chromosomes, the chicken ets sequence is present as a single locus with v-ets homologous sequences found in nine regions over about 60 kb of genomic DNA. The major sequence difference between the v-ets and c-ets is found at the 3' end, resulting in different carboxy termini of p135 and the chicken proto-ets product. The chicken locus is primarily expressed in normal thymus cells as a 7.5-kb mRNA. The first two viral homologous regions are not found in this c-ets transcript or any other minor species, suggesting that they may not be true exons. Thus, the v-ets region of E26 demonstrates that two major structural differences may have occurred during the transduction of proto-ets sequences by the virus: (1) Truncation of sequences present at the 5' and 3' ends of the gene; and (2) Acquisition of noncoding proto-ets sequences into the virus. Either or both of these differences may be, in part, responsible for the oncogenic potential of this retrovirus.
We have constructed retroviral vectors derived from the genome of avian erythroblastosis virus ES4 (AEV ES4). The neo selectable gene was substituted for the original v-erbA or v-erbB oncogenes of AEV, either in the same or in a different reading frames. Recombinant retrovirus were rescued and used to infect chicken embryo fibroblasts or quail QT6 cells. When the neo gene was inserted in the same reading frame as the original oncogene, we obtained (1) a high level of expression of the neo gene, (2) a balanced ration of both genomic and subgenomic RNAs, and (3) high titer recombinant viruses. Conversely, when the neo gene was inserted in a reading frame different from that of the original oncogene, we observed (1) a very low level of expression of the neo protein, (2) a predominance of the viral transcript used as translational template for the neo protein synthesis, and (3) low titer recombinant viruses. One of the vectors was used to transfer a human delta-globin gene into avian cells in culture without detectable rearrangement of this gene, but exhibited a deletion within the conserved noncoding region located between the two original oncogenes. Our data provide information for further construction of double expression vectors. Furthermore, three of the vectors would provide helpful tools to identify genetic elements of the virus genome involved in splicing regulation.
Hematopoietic malignant cells of various types were isolated from ICFW mice inoculated as newborn or adult with F-MuLV and grafted into recipient mice. After repeated in vivo cell transplants, several recombinant polytropic viruses (also termed MCF or dualtropic viruses) were isolated from tumors by limiting dilution. Two virus isolates designated RA1-17 and EA1-17 were recovered from the spleen and the omentum, respectively, of the same grafted animal. When inoculated into newborn mice, RA1-17 induced erythroblastosis similar to that induced by other Friend recombinant polytropic viruses. Remarkably, EA1-17 induced T-cell leukemia after a short latency. To our knowledge, this is the first description of a polytropic recombinant virus with a T-cell tropism isolated after inoculation of F-MuLV.
The replication-defective Friend spleen focus-forming virus (F-SFFV) induces acute erythroblastosis in adult mice. The envelope-related (env) gene and LTR are the only functional elements of the viral genome. The env-coded glycoprotein gp55 has been shown to be responsible for target cell specificity and for the short latency of the disease caused by SFFV. This molecule closely resembles the env coded proteins gp70 + p15E of mink cell focus inducing viruses (MCFV). The only substantial differences between these two env genes are a large deletion spanning 585 nucleotides in the middle of the F-SFFV gene and a frameshift mutation near the 3' end leading to a modified and shortened membrane anchor in the mature protein. To determine if the large deletion and/or the frameshift mutation are capable of changing the properties of a nonpathogenic MCFV into those of an acutely pathogenic SFFV we introduced these changes into the env gene of an MCFV. The results show that the mutated MCFV is as acutely pathogenic as F-SFFV. We therefore conclude that the modified membrane anchor of gp55 and the change caused by the large deletion are the essential determinants of the high pathogenicity of SFFV.
We previously isolated an avian erythroblastosis virus, AEV-GEE35, in which the complete extracellular and transmembrane domains of the v-erbB oncoprotein were replaced with sequences from the gag and env proteins. The GEE35 virus was capable of transforming both fibroblasts and erythroblasts as efficiently as wild-type v-erbB. Analysis of the v-erbB proteins encoded by GEE35 revealed two proteins of similar molecular weights of approximately 130,000 Da. One of these proteins was an N-linked glycosylated membrane protein, whereas the other was a cytoplasmic protein. Biochemical characterization of these two proteins revealed that the transmembrane protein has the v-erbB domain outside the cell, such that it no longer had access to its tyrosine kinase substrates. This implies that it is the cytoplasmically located v-erbB-encoded protein that is responsible for the efficient transforming ability of this virus.
Pure primary mesenchymal cells from definitive streak stage chick embryos have been prepared free of epiblast and hypoblast cells. These cells have the potential in culture to differentiate into erythroid cells, beating heart muscle tissue, chondrocytes and epithelial cells. Transformation in vitro of pure primary mesenchymal cells by avian erythroblastosis virus (wt-AEV) and a temperature-sensitive mutant (ts34-AEV) gave rise to rapidly growing cells which remained largely undifferentiated, could be cloned in semi-solid medium and could be maintained for up to 3 months in culture. The majority of mesenchymal cells transformed by wt-AEV (MAE cells) are benzidine-negative. Gel electrophoresis of radioactively labeled cell proteins, immunoprecipitated with specific antisera against chicken hemoglobin, showed that MAE cell clones synthesize the alpha D, pi (or pi') and some unidentified "globin" polypeptide chains. Treatment of MAE cell clones with 1.0 mM n-butyrate stops cell proliferation reversibly and causes an increased synthesis of alpha D and pi (or pi') globin polypeptide chains. In certain clones of mesenchymal cells transformed by a temperature-sensitive mutant of the virus, ts34-AEV (MAE-ts34 cells), benzidine-positive cells can be induced by a shift from 37 degrees to 41 degrees C. The ability of the clone to undergo an increase in benzidine-positivity by temperature shift is decreased with the age of the clone. Different clones show a variable proportion of cells which are positive by immunofluorescence for both globin and chicken-specific histone H5. The alpha A and alpha D globin chains are synthesized in MAE-ts34 clones, but the ratios and quantities of these chains vary for different clones. Temperature shift made little difference in the types and quantities of globin chains synthesized; the increase in benzidine positivity is probably due to an increase in heme biosynthesis.
The putative transforming protein of avian myelocytomatosis virus MC29 is a 110,000 dalton (P110gag-myc) polyprotein comprised of sequences derived from both the gag region and the MC29-specific myc region. Two approaches have been taken to determine the location of the MC29 gag-related proteins in transformed cells: subcellular fractionation and immunofluorescence. Analysis of subcellular fractions of MC29-transformed cells by immunoprecipitation indicates that the majority of the gag-myc polyprotein is found in the nuclear fractions of Q8 cells (a nonproducer line of MC29-transformed quail embryo fibroblasts) and nonproducer cells derived from a liver tumor of MC20-infected quail. This is in contrast to the distribution of gag-related helper virus proteins lacking myc, which are found only in nonnuclear fractions of superinfected Q8 cells. The purity of unlabeled nuclei was assessed by electron microscopy and enzyme assays, revealing little contaminating material from other subcellular fractions. Immunofluorescence experiments using monospecific anti-gag serum showed specific, intense immunofluorescence in the nuclei of fixed Q8 cells. In contrast, the majority of P75gag-erb, a candidate transforming protein produced by avian erythroblastosis virus (AEV), is absent from the nuclei of nonproducer AEV-transformed chick embryo fibroblasts. The nuclear association of the MC29 transforming protein may be related to some of the unique properties of MC29-transformed cells.