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Biomedical subjects

J M Bishop

Publications and source records attributed to J M Bishop.

At least 19 recordsLinked to original sources
PubMed1992

Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes.

Interest in obtaining cell lines for use in studies on the development and biochemistry of the central nervous system has motivated efforts to establish cells from primary brain cultures by the use of oncogene-transfer techniques. In previous reports, cell lines derived from astrocytes in this way have had immature or abnormal phenotypes. We have explored the possibility of specifically "targeting" expression of exogenous oncogenes to differentiated astrocytes by using the promoter of the gene encoding glial fibrillary acidic protein, which is expressed almost exclusively in such cells. We report here that cell lines displaying the phenotypic characteristics of type 1 astrocytes can be established reproducibly in this manner. Given the heterogeneity of primary cultures, the availability of clonal cell lines displaying characteristics of type 1 astrocytes should greatly facilitate our understanding of the biology of these cells.

Animals↗
PubMed1992

A human gene (AHNAK) encoding an unusually large protein with a 1.2-microns polyionic rod structure.

We report here the identification and partial characterization of a human gene (designated AHNAK) that encodes an unusually large protein (approximately 700 kDa). AHNAK is expressed by means of a 17.5-kilobase mRNA in diverse cellular lineages but is typically repressed in cell lines derived from human neuroblastomas and in several other types of tumors. Unique-sequence domains at the two ends of the protein flank a large internal domain (approximately 4300 amino acids) composed of highly conserved repeated elements, most of which are 128 amino acids in length. The repeated elements in turn display a redundant motif, marked by the recurrence of proline at every seventh residue. Within these sequences, hydrophobic and hydrophilic residues alternate in a manner that is incompatible with a helical coiled-coil structure. Instead, we propose a structure resembling a beta-strand but with a periodicity of 2.33. The structure would engender a polyionic rod approximately 1.2 microns long. Preliminary evidence indicates that the protein resides predominantly within the nucleus, but no function has yet been discerned.

Adrenal Gland Neoplasms↗
PubMed1992

Specific association of the proto-oncogene product pp60c-src with an intracellular organelle, the PC12 synaptic vesicle.

The protein product of the proto-oncogene c-src is a membrane-associated tyrosine kinase of unknown function. Identification of pp60c-src target membranes may elucidate the function of the c-src protein. The available evidence indicates that pp60c-src associates with distinct membranes within single cell types and has different distributions in different cell types. Our experiments demonstrate targeting of pp60c-src to an isolatable and biochemically identified membrane fraction in the neuroendocrine cell line PC12. The c-src protein was found to be specifically associated with synaptic vesicles since: (a) the pp60c-src immunofluorescent pattern overlapped with a synaptic vesicle marker, synaptophysin; (b) a significant proportion (44%) of the pp60c-src from PC12 but not fibroblast postnuclear supernatants was recovered in a small vesicle fraction; (c) an anti-synaptophysin cytoplasmic domain antibody immunodepleted all of the pp60c-src vesicles in this fraction, and (d) pp60c-src copurified during a 100-fold purification of PC12 synaptic vesicles. These results suggest a role for the c-src protein in the regulation of synaptic vesicle function.

Animals↗
PubMed1992

Human protein-tyrosine kinase gene HCK: expression and structural analysis of the promoter region.

The vertebrate gene HCK encodes a protein-tyrosine kinase that is closely related to the product of the proto-oncogene SRC. HCK is expressed principally in monocytic and granulocytic hematopoietic cells, in coordination with differentiation of these cells. Here we report an initial description of the mechanisms by which expression of human HCK is controlled. Induction of the gene during differentiation was manifested by an increase in the steady-state levels of HCK RNA and protein product. The accumulation of RNA apparently resulted from modulation of transcription itself, since no change occurred in the stability of the transcripts. Transcription initiated at multiple sites, clustered c. 145 nucleotides upstream of the first intron of HCK. The sequence of 660 bp upstream of the major initiation site was determined, revealing candidate binding sites for Sp1 and AP-2 transcription factors, but neither TATA nor CAAT elements. Comparison to the same region of the mouse hck locus showed five small regions of similarity, only two of which were topographically analogous between the two sequences. It appears that expression of HCK is regulated primarily through control of transcription, but the mechanisms by which tissue-specific expression and increase of transcription during differentiation are achieved remain to be explored.

Base Sequence↗
PubMed1991

Premature termination of transcription from the P1 promoter of the mouse c-myc gene.

Modulation of transcriptional elongation within the c-myc gene is thought to play a major role in determining levels of c-myc mRNA in both normal and tumor cells. A discrete site of blockage to transcriptional elongation has previously been localized at the 3' end of exon 1 of the mouse and human c-myc genes. We here identify an additional site of transcriptional attenuation that is located between the P1 and P2 promoters of the c-myc gene and that mediates premature termination of transcripts initiating from the P1 promoter. A 95-nucleotide DNA fragment derived from this region prematurely terminated transcription when placed downstream from the promoter of the H-2Kbm1 gene and assayed by expression in Xenopus oocytes. We also show that the previously identified attenuation signal in exon 1 of the mouse c-myc gene can mediate premature termination of P1-initiated transcripts. Premature termination of P1-initiated transcripts presumably increases transcription from the downstream P2 promoter; aberrant regulation of this termination may explain the increased use of the P1 promoter that is characteristic of certain tumors in which myc is overexpressed.

Animals↗
PubMed1991

Dominant male sterility in mice caused by insertion of a transgene.

While examining a series of transgenic mouse lines carrying the HCK protooncogene, we encountered one line in which males hemizygous for the transgene were sterile. The sterile males mated normally but failed to impregnate females. Light and electron microscopy revealed that spermatogenesis proceeds normally until nuclear condensation, which occurs but gives rise to a variety of abnormally shaped nuclei. Expression of the transgene was not detectable. Thus, the insertion itself probably caused the abnormal phenotype by disrupting a gene (or genes) important in spermatogenesis. The mutation is genetically dominant, causing an abnormal phenotype even though the sterile mice carry an ostensibly normal counterpart of the disrupted locus. The mutant phenotype is completely penetrant only in some genetic backgrounds, suggesting a modifying influence from a second locus. Junctions between the inserted transgene and adjoining cellular DNA were cloned, allowing us to confirm the heterozygous nature of the genetic disruption and to detect and associated deletion. We have designated the mutation Lvs (lacking vigorous sperm) and presume that it may define a previously undescribed locus important in spermatogenesis.

Animals↗
PubMed1991

Identification of platelet membrane proteins that interact with amino-terminal peptides of pp60c-src.

Platelets contain exceptionally high levels of pp60c-src and, thus, provide a convenient system for investigating the physiological function of this protein-tyrosine kinase. We have employed chemical cross-linking of myristylated amino-terminal peptides of pp60c-src to platelet membranes in order to identify platelet membrane components that interact with pp60c-src to regulate or mediate its activity. We detected specific binding of radioiodinated peptides to platelet membrane proteins of 32, 50, 92, and 105 kDa. The 32-kDa protein may be related to the putative src receptor component recently identified in fibroblast membranes. The most reactive platelet protein, however, is the 50-kDa protein, which is either absent or nonreactive in fibroblast membranes. Binding of src peptides to this protein was saturable, and we estimate the presence of approximately 1 x 10(6) of the 50-kDa binding sites per platelet. The specificity of the peptide binding to the 50- and 32-kDa platelet proteins was analyzed by competition with different peptides. The binding sites displayed an absolute requirement for an N-myristoyl moiety and a strong preference for pp60c-src amino-terminal sequences. The identification of these src-interacting proteins may help to decipher the biochemical pathways in which platelet pp60c-src is involved.

3T3 Cells↗
PubMed1991

Isolation of Legionella maceachernii from an immunocompromised patient with severe underlying lung disease.

OBJECTIVE: To present the first case in Australia in which Legionella maceachernii has been documented as a cause of infection. CLINICAL FEATURES: A 71-year-old retired carpenter with underlying autoimmune disease and pulmonary fibrosis was admitted to hospital with increasing shortness of breath and fever, and pneumonia was diagnosed. INTERVENTION AND OUTCOME: He was treated with ampicillin then gentamicin and flucloxacillin and later imipenem but his condition deteriorated with progression of the pneumonia and abscess formation and he died 25 days after admission. Subsequently, L. maceachernii was isolated from three sputum samples collected before the patient died. CONCLUSION: There is a need for clinical awareness of the value of sputum culture in the diagnosis of infection due to Legionella spp. other than L. pneumophila.

Aged↗
PubMed1991

Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma 1 by a kinase activity associated with the product of the trk protooncogene.

Nerve growth factor (NGF) promotes the survival and differentiation of specific populations of neurons. The molecular mechanisms by which cells respond to NGF are poorly understood, but two clues have emerged recently. First, NGF rapidly stimulates tyrosine phosphorylation of several unidentified proteins in the NGF-responsive pheochromocytoma cell line PC12 [Maher, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6788-6791]. Second, the protein-tyrosine kinase encoded by the protooncogene trk (p140trk), a member of the receptor class of tyrosine kinases, becomes activated and phosphorylated on tyrosine after NGF treatment of PC12 cells [Kaplan, D. R., Martin-Zanca, D. & Parada, L. F. (1991) Nature (London) 350, 158-160]. We now report that NGF rapidly induces tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), and we present evidence that the responsible tyrosine kinase is either p140trk or a closely associated protein. Treatment of responsive cells with NGF elicited phosphorylation of PLC-gamma 1 on tyrosine and serine. PLC-gamma 1 immunoprecipitated from NGF-stimulated cells was phosphorylated in vitro by coprecipitating protein kinase activity, and the phosphorylations occurred principally on tyrosine. The responsible kinase could be depleted from cellular lysates by antibodies specific for p140trk. This procedure also depleted a 140-kDa protein that normally coprecipitated with PLC-gamma 1 and became phosphorylated on tyrosine in vivo in response to NGF. Analysis of tryptic peptides from PLC-gamma 1 indicated that the residues phosphorylated in vitro by p140trk-associated kinase activity were largely congruent with those phosphorylated in vivo after NGF treatment. Our findings identify PLC-gamma 1 as a likely substrate for the trk-encoded tyrosine kinase, and they provide a link between NGF-dependent activation of p140trk and the stimulation of intracellular second messenger pathways.

Adrenal Gland Neoplasms↗
PubMed1991

The MYC protein activates transcription of the alpha-prothymosin gene.

The proto-oncogene MYC encodes a nuclear protein whose biochemical and physiological functions remain uncertain. We used an estrogen-regulated version of the MYC protein to explore these functions. Activation of MYC in quiescent rat and mouse fibroblasts elicited re-entry into and progression through the cell cycle, bypassing early events that would follow stimulation of the cells with serum. Activation of MYC led to a rapid increase in transcription of the alpha-prothymosin gene, even in the absence of protein synthesis. We conclude that the product of MYC acts directly on transcription, in accord with inferences based on the structure of the MYC protein. The function of alpha-prothymosin is not known, but our results suggest that the protein may play a role in the proliferation of mammalian cells.

Actins↗
PubMed1991

Expression during Drosophila development of DER, a gene related to erbB-1 and neu: correlations with mutant phenotypes.

We used in situ hybridization to study expression of the DER gene during Drosophila development. DER encodes a transmembrane cell-surface receptor with a cytoplasmic protein-tyrosine kinase domain, and resembles the vertebrate genes that encode the EGF receptor and the neu protein. We examined most stages of development in the Drosophila life cycle and found a substantial correlation between DER expression and the phenotypes associated with a variety of mutant alleles. Of particular note were high levels of expression in the primordia of the mouth parts, which are the embryonic tissues most sensitive to reductions in DER activity; discrete expression in a subset of neural cells essential for construction of the axonal scaffold, a structure that is deformed in mutant embryos; uneven expression in the eye disc, the development of which is abnormal in both mild hypomorphs and hypermorphs; and expression in the follicular epithelial cells of the ovary, which are responsible for producing the eggshell of developing oocytes and do so aberrantly in the mildest hypomorphs. However, DER transcripts were also detected in a subset of tissues that have not been reported to be abnormal in mutant organisms. Our findings agree with and extend recently reported results for the DER protein, indicating that DER is regulated primarily at the level of transcription, in contrast to previous suggestions. We conclude that the phenotypes displayed by recessive mutants can be attributed to loss of DER function in the affected tissues.

Animals↗
PubMed1991

A gene related to the proto-oncogene fps/fes is expressed at diverse times during the life cycle of Drosophila melanogaster.

The proto-oncogene fps/fes encodes a distinctive type of protein-tyrosine kinase. We identified a Drosophila gene (dfps85D) whose product resembles the proteins encoded by vertebrate fps/fes and the closely related gene fer. dfps85D is located at chromosomal position 85D10-13 and is unlikely to correspond to any previously defined genetic locus in Drosophila melanogaster. Expression of the gene is entirely zygotic in origin and occurs throughout the life cycle. But hybridization in situ revealed that the pattern of expression is specialized and evolves in a provocative manner. The most notable feature of expression is the diversity of developmental periods, tissues, and cells in which it occurs. In some tissues, expression is transient; in others, it is continuous. Expression occurs in both mitotic and terminally differentiated tissue and, at various times in development, is prominent in imaginal disks, gut, muscle, testes, ovaries, retina, and other neural tissues. It appears that the use of dfps85D is more diversified than that of other Drosophila protein-tyrosine kinases reported to date and contrasts sharply with the restricted expression of fps itself in vertebrates. The detailed description of expression provided here will help guide the search for mutants in dfps85D.

Amino Acid Sequence↗
PubMed1991

Expression of CD44 is repressed in neuroblastoma cells.

We have used cDNA subtractive cloning to identify a group of human genes that are expressed in diverse differentiated derivatives of neural crest origin but not in neuroblastoma cell lines. One of these genes was identified as CD44, which encodes an integral membrane glycoprotein that serves as the principal receptor for hyaluronate and participates in specific cell-cell and cell-extracellular matrix interactions. The repression of CD44 expression in neuroblastoma cell lines might be relevant to their high metastatic potential. We have cloned full-length cDNAs corresponding to CD44 trancscripts and identified a novel splice variant of CD44 lacking 31 amino acids of the extracellular domain. As a first step toward analysis of CD44 downregulation in neuroblastoma cells, we have mapped the CD44 RNA initiation site and analyzed the structure of the upstream regulatory region. We constructed a series of plasmids containing different amounts of CD44 upstream regulatory region linked to the bacterial chloramphenicol acetyltransferase gene and then analyzed their ability to promote transcription in neuroblastoma and melanoma cells. We found that a DNA segment including about 150 bp of the CD44 upstream region and the 5' end of the gene itself was sufficient to induce substantial transcription of the chloramphenicol acetyltransferase gene in both neuroblastoma and melanoma cells. Several upstream cis-acting elements contribute to the downregulation of CD44 in neuroblastoma cells, the most prominent being a 120-bp DNA fragment located 450 bp upstream to the RNA initiation site. Our data suggest that multiple factors might be involved in downregulation of CD44 in neuroblastoma cells.

Amino Acid Sequence↗
PubMed1991

The amino-terminal 14 amino acids of v-src can functionally replace the extracellular and transmembrane domains of v-erbB.

The retroviral oncogene v-erbB encodes a truncated form of the receptor for epidermal growth factor, an integral membrane protein-tyrosine kinase. By contrast, the oncogene v-src encodes a protein-tyrosine kinase that is a peripheral membrane protein. The morphologies and spectra of cells transformed by these two oncogenes differ. In an effort to identify the functional determinant(s) of these differences, we constructed and tested first deletion mutants of v-erbB and then chimeras between v-src and v-erbB. As reported previously, the absence of any membrane anchorage eliminated transformation by v-erbB. Anchorage of the cytoplasmic kinase domain of v-erbB to membranes with amino-terminal portions of the v-src protein permitted transformation. The phenotype and spectrum of transformation were those expected for v-erbB rather than for v-src. The transforming chimeras lost their biological activity if the signal for myristylation at the amino terminus of v-src was compromised by mutation. Biochemical fractionations revealed a correlation between transforming activity and the association of chimeric gene products with the membrane fraction of the cell. For reasons not yet apparent, the combined presence of membrane anchorage domains of v-src, and the transmembrane domain of v-erbB in the same chimera typically (but not inevitably) impeded transformation. Our results suggest that the specificity of transformation by v-erbB resides in the selection of substrates by the cytoplasmic domain of the gene product. The protein retains access to those substrates even when anchored to the membrane in the manner of a peripheral rather than a transmembrane protein.

Alleles↗
PubMed1991

A 3' truncation of myc caused by chromosomal translocation in a human T-cell leukemia is tumorigenic when tested in established rat fibroblasts.

We have previously identified in human T-leukemia cells a myc gene with an unusual 3' rearrangement, and we have shown that expression of the gene is deregulated by stabilization of mRNA. Here we report that the rearranged gene transforms established rat fibroblasts to a tumorigenic phenotype. In hybrid genes, the transforming capability segregates with the 3' rearrangement. Transformation is apparently due to a more than fivefold enhancement in myc expression, attributable to stabilization of mRNA. The rearranged allele of myc also contains a point mutation in a region upstream of the gene, identified previously as a potential negative regulator of myc expression. The mutation may increase expression of myc, but not sufficiently to cause cellular transformation. Our findings enlarge the variety of genetic lesions that may activate myc to an oncogene and sustain the view that augmented expression of an otherwise normal allele of myc can be pathogenic.

Alleles↗