Abundant expression of ras proteins in Aplysia neurons.

We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.

Genetic structure of the replication origin of bacteriophage lambda.

A fragment of bacteriophage lambda DNA produced by the restriction endonuclease Eco RI and extending from the immunity region to a point inside gene O is found to have a fully functional origin of replication. Seven ori- mutations of lambda cluster in a small region just to the left of the Eco RI cleavage site which defines the right end of this fragment. These mutations lie within gene O.

The receptor for ciliary neurotrophic factor.

Although neurotrophic factors were originally isolated on the basis of their ability to support the survival of neurons, these molecules are now thought to influence many aspects of the development and maintenance of the nervous system. Identifying the receptors for these neurotrophic factors should aid in identifying the cells on which these factors act and in understanding their precise mechanisms of action. A "tagged-ligand panning" procedure was used to clone a receptor for ciliary neurotrophic factor (CNTF). This receptor is expressed exclusively within the nervous system and skeletal muscle. The CNTF receptor has a structure unrelated to the receptors utilized by the nerve growth factor family of neurotrophic molecules, but instead is most homologous to the receptor for a cytokine, interleukin-6. This similarity suggestes that the CNTF receptor, like the interleukin-6 receptor, requires a second, signal-transducing component. In contrast to all known receptors, the CNTF receptor is anchored to cell membranes by a glycosyl-phosphatidylinositol linkage.

Neurotrophin-3: a neurotrophic factor related to NGF and BDNF.

The development and maintenance of the nervous system depends on proteins known as neurotrophic factors. Although the prototypical neurotrophic factor, nerve growth factor (NGF), has been intensively studied for decades, the discovery and characterization of additional such factors has been impeded by their low abundance. Sequence homologies between NGF and the recently cloned brain-derived neurotrophic factor (BDNF) were used to design a strategy that has now resulted in the cloning of a gene encoding a novel neurotrophic factor, termed neurotrophin-3 (NT-3). The distribution of NT-3 messenger RNA and its biological activity on a variety of neuronal populations clearly distinguish NT-3 from NGF and BDNF, and provide compelling evidence that NT-3 is an authentic neurotrophic factor that has its own characteristic role in vivo.

Construction of chimeric phages and plasmids containing the origin of replication of bacteriophage lambda.

Segments of the replication control region of bacteriophage lambda (lambda) and lambda mutants defective in replication were attached in vitro to the phi80 phage vector Charon 3 and to the plasmid vector mini Col El (pVH51). The chimeric phages and plasmids have been used to localize the origin of lambda DNA replication and to facilitate a structural analysis of the lambda replicator.

Physical structure of the replication origin of bacteriophage lambda.

The nucleotide sequence of part of the replication region of wild-type bacteriophage lambda and of four mutants defective in the origin of DNA replication (ori-) has been determined. Three of the ori- mutations are small deletions, and one is a transversion. The sequence of the origin region, defined by these mutations, contains a number of unusual features.

Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo.

Adult rat sciatic nerve is known to express high levels of ciliary neurotrophic factor (CNTF) mRNA and protein. Here we examine the cellular localization of CNTF protein and mRNA in peripheral nerve and the regulation of CNTF expression by peripheral axons. In intact nerve, CNTF immunoreactivity is found predominantly in the cytoplasm of myelin-related Schwann cells. After axotomy, CNTF immunoreactivity and mRNA levels fall dramatically and do not recover unless axons regenerate. This behavior is similar to the pattern of myelin gene expression in these nerves. We conclude that the expression of CNTF in Schwann cells depends on axon-Schwann cell interactions.

Identification of functional receptors for ciliary neurotrophic factor on neuronal cell lines and primary neurons.

The lack of reagents or molecular probes specific for the ciliary neurotrophic factor (CNTF) receptor has hindered characterization of the molecular mechanism(s) by which CNTF influences the proliferation, survival, and differentiation of cells of the vertebrate nervous system. We have developed methods for the detection and separation of cells expressing CNTF receptors by using a variety of binding assays based on a genetically engineered CNTF molecule containing an "epitope tag" at its C-terminus. These assays have allowed us to identify several neuronal cell lines, as well as embryonic and adult neurons in primary cultures, that bind CNTF and functionally respond to CNTF by rapidly activating the transcription of immediate early primary response genes.

NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.

Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein.

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.

Expression of ras proto-oncogene proteins in normal human tissues.

The expression of ras proto-oncogenes in normal human tissues was studied by immunohistochemical staining and by immunoblotting using monoclonal antibodies. We detected p21ras protein in almost every fetal and adult tissue, but the level varied significantly among cell types. In some cell lineages, immature cells capable of proliferation contain more p21ras than do mature cells. By contrast, certain fully differentiated cells, such as neurons and the epithelial cells of endocrine glands, express abundant p21ras. Among mammalian tissues the highest level of ras protein was detected in brain. Crude synaptosomal membrane preparations from rat brain contain substantially more p21ras than do plasma membranes from rat liver. The observed distribution of p21ras suggests a role for these proteins both in cellular proliferation and in certain specialized cellular functions.

Expression of normal and mutant ras proteins in human acute leukemia.

The expression of normal and mutant ras genes in human acute leukemias was assessed by the direct analysis of p21ras polypeptides, using immunoprecipitation with monoclonal antibodies. High-resolution two-dimensional gel electrophoresis permits the identification of a wide array of activated ras alleles encoding proteins with single amino acid substitutions at any of several positions. The products of three ras genes, H-ras, N-ras, and K-ras, were detected in each of 33 specimens of fresh leukemic cells. The normal K-ras and N-ras polypeptides were substantially more abundant than H-ras p21 in all samples. In over three-fourths of the cases the total amount of p21ras exceeded that seen in control hematopoietic cell lines. The level of ras expression did not correlate simply with clinical parameters, although the two samples with the most abundant p21ras were obtained from patients with relapsed T-cell acute lymphocytic leukemia (ALL). Abnormal p21ras, consistent with oncogenic activation, was found in eight patients. Six of 11 samples from acute myelocytic leukemia (AML) patients displayed a mutant N-ras p21, while only one of 20 ALL specimens had abnormal N-ras, and one had a mutant H-ras. In every case the mutant protein comprised a minority of total p21ras. In two T-cell ALL cell lines both normal and activated N-ras gene products were expressed at equal levels. By contrast, in five fresh AML samples the abnormal N-ras protein was several-fold less abundant than the normal N-ras p21. This finding implies that only a proportion of leukemic cells in an individual patient may carry the mutant ras oncogene.

Antisera to the variable region of ras oncogene proteins, and specific detection of H-ras expression in an experimental model of chemical carcinogenesis.

Antisera were prepared in mice, rats and rabbits by immunization with peptides corresponding to regions of highest variability, located near the C-termini of four ras proteins. Two of these, H-ras (171-189) and K-rasB (171-186), react uniquely with H-ras and K-rasB gene products in immunoblots and immunoprecipitation reactions. Affinity-purified rabbit H-ras (171-189) antibody detects H-ras p21 in tissue culture cells and in tissue sections. Epithelial cells in normal mouse skin and cells in papillomas and carcinomas, in a mouse model system of chemical carcinogenesis in which mutational activation of H-ras occurs with high frequency, express high levels of H-ras p21 protein. These results suggest an hypothesis to explain the mechanism and preferential activation of particular ras loci in certain neoplasia.

Clinical evaluation of a DNA probe assay for the Philadelphia (Ph1) translocation in chronic myelogenous leukemia.

We report the clinical evaluation of an improved DNA probe assay for the characteristic genetic marker of human CML, observed by cytogenetics and designated the Philadelphia chromosome (Ph1). The Ph1 chromosome results from the fusion of c-abl proto-oncogene sequences from chromosome 9 to phl gene sequence on chromosome 22. (The phl gene is often referred to as bcr. However, for clarity we prefer to reserve the designation "bcr" for the region within the phl gene in which translocation breakpoints have been found to occur. We also find it useful to distinguish between two such regions in phl, bcr-210 and bcr-190, named after the 210- and 190-kDa phl/abl fusion proteins resulting from translocations with breakpoints in the respective regions. We refer to the corresponding chromosomal translocations as Ph1(bcr-210) and Ph1(bcr-190).) DNA, extracted from peripheral blood (PB) or bone marrow (BM) and digested with restriction endonuclease BglII, is hybridized with a probe (phl/bcr-3) spanning a breakpoint cluster region within phl. Rearrangements are revealed by the presence of one or two novel junction fragments. Clinical specimens from leukemic patients with active disease were compared by cytogenetic and DNA probe analysis at seven centers in the United States and Europe. The probe assay identified the phl rearrangement in 190 of 191 cases of Ph1-positive CML, as well as in 12 of 27 clinically diagnosed CML specimens lacking a typical Ph1 chromosome. DNA rearrangements also were seen in two of six cases of Ph1-positive ALL. No false positive results were obtained among 93 non-leukemic controls. Mixing experiments showed that the DNA probe assay can detect as few as 1% leukemic cells in a specimen. A preliminary study of CML patients in remission after allogeneic BM transplantation revealed a small fraction of residual Ph1-positive leukemic cells in a significant number of such patients.

The neurotrophin family of NGF-related neurotrophic factors.

The recent molecular cloning of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) has established the existence of an NGF-related family of neurotrophic factors - the neurotrophins. Purification and recombinant production of BDNF and NT-3 has allowed the initiation or extension of in vitro studies of the neuronal specificity of each of these factors. We have found that NT-3, like NGF and BDNF, promotes survival and neurite outgrowth from certain populations of sensory neurons. There appear to be both distinct and overlapping specificities of the 3 neurotrophins towards peripheral neurons - sympathetic neurons and subpopulations of neural crest and neural placode-derived sensory neurons. Using cultures of central nervous system neurons, we have recently established that BDNF: (i) promotes the survival and phenotypic differentiation of rat septal cholinergic neurons, a property consistent with the discovery of high levels of BDNF mRNA expression within the hippocampus; (ii) promotes the survival of rat nigral dopaminergic neurons and furthermore protects these neurons from two dopaminergic neurotoxins, 6-hydroxydopamine (6-OHDA) and MPTP. Thus the neurotrophic effects of these factors towards peripheral neurons and neuronal populations known to degenerate in two of the major human neurodegenerative diseases - Alzheimer's and Parkinson's disease - provokes the question of whether neurotrophic factors may have therapeutic potential in halting the progression and ameliorating the symptoms of devastating neurological disorders of the CNS or PNS, or improving regeneration of neurons of CNS or PNS after traumatic injury.

Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product.

The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.

Cellular ras oncogene expression and cell cycle measured by flow cytometry in hematopoietic cell lines.

Human hematopoietic malignancies provide an excellent model for the study of the activity of cellular oncogenes in a context of known defects in cell proliferation and differentiation. A flow cytometric immunofluorescence assay was developed to quantitate the expression of the cellular ras oncogenes in relation to the cell cycle in individual leukemic cells. Specific binding of a monoclonal antibody to the 21-kd protein (p21ras) encoded by the Ha-ras, Ki-ras, and N-ras genes was measured by flow cytometry and confirmed by fluorescence microscopy. P21ras was detected in 416B, a murine hematopoietic precursor cell characterized by a high level of Ki-ras expression, and in the human leukemic cell lines P-12 and KG-1. The presence of p21ras in the cell lines was also shown by immunoprecipitation. Cellular DNA content was determined simultaneously to define cell cycle phases. There was an equal distribution of p21ras in G1, S, and G2M, with considerable heterogeneity of ras gene expression in the G1 compartment. The assay allows oncogene expression to be studied in populations of intact single cells in which cell heterogeneity is maintained, requires very few cells per sample, and directly correlates oncogene expression to cell kinetic data.