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Biomedical subjects

P Leder

Publications and source records attributed to P Leder.

At least 163 records · Page 9Linked to original sources
PubMed1988

neu protooncogene fused to an immunoglobulin heavy chain gene requires immunoglobulin light chain for cell surface expression and oncogenic transformation.

The protein encoded by the neu protooncogene (human gene symbol NGL for neuro/glioblastoma-derived) is a member of the surface receptor/tyrosine kinase family. Though its structure suggests that it can transduce a transmembrane signal, neither its extracellular ligand nor its critical intracellular substrates are known. To explore the functional properties of the protein encoded by neu, we created a fusion gene that joins the cytoplasmic domain of neu to the extracellular portion of an immunoglobulin heavy chain. The localization of the fusion polypeptide can then be controlled by coexpression with immunoglobulin light chain. In the absence of light chain, the heavy chain-neu polypeptide is expressed intracellularly and has no transforming activity. By contrast, in the presence of light chain the fusion polypeptide is expressed at the cell surface and produces tumorigenic foci. Thus, transformation apparently requires expression at the cell surface, where the neu intracellular domain can interact with components that are localized to the plasma membrane. The fusion protein is active in cellular transformation when the transmembrane domain is derived either from neu or from immunoglobulin, indicating that the neu transmembrane domain is not specifically required for transformation, although neu activation in tumors is known to result from a point mutation in this region. The extracellular immunoglobulin heavy and light chain domains of the fusion protein form a functional binding site that allows antigen to modulate its activity, reversing the transforming effect.

Animals↗
PubMed1988

Human and rat mast cell high-affinity immunoglobulin E receptors: characterization of putative alpha-chain gene products.

We have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative alpha subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH2-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat alpha subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

Amino Acid Sequence↗
PubMed1987

The c-myc gene encodes superimposed RNA polymerase II and III promoters.

The first exon of the c-myc gene has unusual properties that suggest some further role in gene regulation. It encodes a large, evolutionarily conserved leader exon that is transcribed more frequently than the remaining exons of the c-myc gene. In what follows, we provide a possible explanation for these observations. We find that the major promoter of the c-myc gene is bifunctional; that is, it supports transcription by RNA polymerases II and III (pol II and III). Both enzymes initiate in vitro transcription from the major c-myc initiation site (P2), but pol III is completely blocked near the 3' end of the first exon while pol II, though partially blocked, transcribes through this region. These superimposed transcriptional activities suggest a potential regulatory mechanism by which one polymerase system could influence the activity of another.

Animals↗
PubMed1987

Parental legacy determines methylation and expression of an autosomal transgene: a molecular mechanism for parental imprinting.

We have created a transgenic mouse strain in which an autosomal transgene bearing elements of the RSV LTR and a translocated c-myc gene obeys very unusual rules. If the transgene is inherited from the male parent, it is expressed in the heart and no other tissue. If it is inherited from the female parent, it is not expressed at all. This pattern of expression correlates precisely with a parentally imprinted methylation state evident in all tissues. Methylation of the transgene is acquired by its passage through the female parent and eliminated during gametogenesis in the male. These observations provide direct molecular evidence that autosomal gene expression can depend upon the sex of the parent from which the gene is inherited. They also provide a plausible mechanism for understanding parental imprinting that may be relevant to the failure of parthenogenesis in mammals, the apparent non-Mendelian behavior of some autosomal genes, and the role of methylation in gene regulation.

Animals↗
PubMed1987

Coexpression of MMTV/v-Ha-ras and MMTV/c-myc genes in transgenic mice: synergistic action of oncogenes in vivo.

We have derived and mated separate strains of transgenic mice that carry either the v-Ha-ras or the c-myc gene driven by the mouse mammary tumor virus (MMTV) promoter/enhancer. Mice carrying the MMTV/v-Ha-ras transgene manifest two distinct disturbances of cell growth. The first, a benign hyperplasia of the Harderian lacrimal gland, is diffuse, involves the entire gland, and likely requires only the abnormal action of the v-Ha-ras gene. The second involves the focal development of malignancies of mammary, salivary, and lymphoid tissue and likely requires additional somatic events. When the MMTV/v-Ha-ras and MMTV/c-myc strains are crossed to yield hybrid mice, their joint action results in a dramatic and synergistic acceleration of tumor formation. Since these tumors arise stochastically and are apparently monoclonal in origin, additional somatic events appear necessary for their full malignant progression, even in the presence of activated v-Ha-ras and c-myc transgenes.

Adenocarcinoma↗
PubMed1987

Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin mu.

Antibody-producing cells display a special form of regulation whereby each cell produces immunoglobulin from only one of its two sets of antibody genes. This phenomenon, called allelic exclusion, is thought to be mediated by the product of one heavy chain allele restricting the expression of the other. Heavy chains are synthesized in two molecular forms, secreted and membrane bound. In order to determine whether it is specifically the membrane-bound form of the immunoglobulin M (IgM) heavy chain (mu) that mediates this regulation, transgenic mice were created that carry a human mu chain gene altered so that it can only direct the synthesis of the membrane-bound protein. The membrane-bound form of the human mu chain was made by most of the B cells in these animals as measured by assays of messenger RNA and surface immunoglobulins. Further, the many B cells that express the human gene do not express endogenous mouse IgM, and the few B cells that express endogenous mouse mu do not express the transgene. Thus, the membrane-bound form of the mu chain is sufficient to mediate allelic exclusion. In addition, the molecular structures recognized for this purpose are conserved between human and mouse systems.

Alleles↗
PubMed1987

Cloning of the mast cell protease, RMCP II. Evidence for cell-specific expression and a multi-gene family.

We have isolated, cloned, and characterized cDNA and genomic DNA corresponding to the mRNA and gene for the rat mast cell protease, RMCP II. The amino acid sequence deduced from the cDNA provides evidence that this protease is synthesized as a precursor, with a signal peptide and additional residues at the N and C termini. RNA homologous to the cDNA is expressed only in mast cells. Analysis of RNA from the two subclasses of mast cell, mucosal and serosal, indicates subclass specific expression of the different proteases found in each of these two subclasses. S1 protection analysis and the sequence of the genomic clone indicate that the serosal mast cell protease, RMCP I, is likely to be coded for by a separate, highly homologous gene. A comparison of the exon/intron structure of the RMCP II gene with genes of related serine proteases further indicates that RMCP II is a member of a family of proteases distinct from those found in the pancreas. We have also isolated a third gene, highly homologous to RMCP II but different from it and from the gene for RMCP I. Analysis of the 5' transcriptional control region of both genes showed striking homology to the TATA box and enhancer regions of the pancreatic proteases.

Amino Acid Sequence↗
PubMed1987

Evolutionarily conserved regions of the human c-myc protein can be uncoupled from transforming activity.

The myc family of oncogenes contains coding sequences that have been preserved in different species for over 400 million years. This conservation (which implies functional selection) is broadly represented throughout the C-terminal portion of the human c-myc protein but is largely restricted to three clusters of amino acid sequences in the N-terminal region. We have examined the role that the latter three regions of the c-myc protein might play in the transforming function of the c-myc gene. Several mutations, deletions and frameshifts, were introduced into the c-myc gene, and these mutant genes were tested for their ability to collaborate with the EJ-ras oncogene to transform rat embryo fibroblasts. Complete elimination of the first two N-terminal conserved segments abolished transforming activity. In contrast, genes altered in a portion of the second or the entire third conserved segment retained their transforming activity. Thus, the latter two segments are not required for the transformation process, suggesting that they serve another function related only to the normal expression of the c-myc gene.

Animals↗
PubMed1986

Consequences of widespread deregulation of the c-myc gene in transgenic mice: multiple neoplasms and normal development.

We have constructed a transgenic mouse strain in which a mammary tumor virus LTR/c-myc fusion gene is anomalously expressed in a wide variety of tissues. The deregulated c-myc transgene, now glucocorticoid inducible, contributes to an increased incidence of a variety of tumors, including those of testicular, breast, lymphocytic (B cell and T cell), and mast cell origin. The deregulated gene does not, however, otherwise disturb cell proliferation, nor does it interfere with normal development in these animals. Moreover, since not all tissues that express the transgene develop neoplasms, these results begin to define the transforming spectrum of the c-myc oncogene. They also extend to several organ systems the notion that elements in addition to an activated c-myc gene are required to induce malignancy in the living organism.

Adenocarcinoma↗
PubMed1986

Structure and expression of a human subgroup II immunoglobulin kappa gene.

We have determined the entire nucleotide sequence of an active immunoglobulin V kappa gene of the human subgroup II. Comparison of the main coding sequence of this gene to another member of this subgroup reveals germline sequence differences that occur not only in complementarity determining regions but also in framework regions. We find that at least two regions commonly associated with active immunoglobulin genes--the TATAA box and the decanucleotide TGATT-TCAT--are represented in this gene. The gene was expressed by permanent and transient DNA-mediated transfection in various cell lines. We have determined the precise site of transcriptional initiation using S1 analysis of gene transcripts and show that this site is the same in the transfected cell lines and in the cell line from which the gene was isolated.

Animals↗
PubMed1986

Identification of three new Ig lambda-like genes in man.

Three new human lambda L chain-like Ig genes are identified by restriction enzyme and nucleotide sequence analysis. Two genes, 14.1 and 16.1, have intact J and C regions, and are potentially functional, with open reading frames. A third gene, 18.1, is a pseudogene. The evolutionary lineage of these genes compared to the known functional locus lambda C1-lambda C6 can be surmised from Southern blot and nucleotide homologies. This study demonstrates that the human lambda gene family is more complex than previously recognized.

Amino Acid Sequence↗
PubMed1986

Trans-acting elements modulate expression of the human c-myc gene in Burkitt lymphoma cells.

We have used a competition assay to identify the targets of trans-acting elements that modulate the expression of the human c-myc gene (designated MYC in human gene nomenclature). For this purpose, a c-myc hybrid indicator gene was formed by joining the c-myc promoter region, first noncoding exon, and intron to the bacterial gene for chloramphenicol acetyltransferase (CAT). The test assay consisted of cotransfecting the indicator gene with competing fragments of DNA derived from suspected control regions of the c-myc gene. Such experiments test the hypothesis that control regions are often targets for the binding of trans-acting regulatory factors that can be diverted to competing fragments of DNA. A negatively acting element will be diverted from the indicator gene, allowing the gene's enhanced expression, whereas a positively acting element will behave oppositely. Control indicator genes driven by non-myc promoters assess the specificity of the effect. Using this approach, we find three c-myc regions that are capable of enhancing the expression of the indicator gene in competition assays (i.e., putative sites of negative modulation). In addition, we find sequences near the c-myc promoters that suppress expression in competition assays (i.e., putative binding sites of positively acting factors). These results, with appropriate controls, suggest the existence of target sites near the c-myc gene that specifically modulate its expression both positively and negatively. Their locations fit well with regions damaged or lost in many Burkitt lymphoma and murine plasmacytoma translocations.

Acetyltransferases↗
PubMed1986

A translocated human c-myc oncogene is altered in a conserved coding sequence.

We have cloned and characterized a c-myc (now designated MYC) oncogene that had been translocated into the mu switch region of the immunoglobulin heavy chain locus in a Burkitt lymphoma cell line. The breakpoint of the translocation occurs within the first intron of the c-myc gene, thereby separating the untranslocated first exon from the two coding exons. Transcription from the translocated gene arises from a cryptic promoter within the first intron, which produces a 438-nucleotide untranslated 5' region. The amino acid sequence of the protein encoded by the c-myc gene has been substantially altered. In particular, a compensating set of frameshift mutations alters a string of 24 amino acids in a region of the protein tightly conserved in human, mouse, and chicken c-myc genes as well as in the human N-myc and L-myc oncogenes. Despite this, the mutated gene retains a reduced transforming ability in a rat embryo fibroblast focus-formation assay.

Amino Acid Sequence↗
PubMed1985

Structural analysis of both products of a reciprocal translocation between c-myc and immunoglobulin loci in Burkitt lymphoma.

The balanced translocations that occur between the c-myc and immunoglobulin loci in Burkitt lymphoma provide an unusual opportunity to analyze both products of a reciprocal recombination. Accordingly, we have determined the structure of the two reciprocal products of a translocation that joins the 5' portion of the c-myc gene on chromosome 8 to the immunoglobulin mu switch recombination signal on chromosome 14. By determining the nucleotide sequences at the translocation crossover points of both product chromosomes, we precisely locate these points with respect to nearby genes. This determination allows us to conclude that translocation involves nonhomologous recombination, is highly conservative of c-myc sequences (deleting only 16 bp at the crossover point), but deletes over 2 Kb of immunoglobulin sequences from the mu switch signal. The mu constant and c-myc genes are joined head-to-head about 3 Kb apart, while the IgH enhancer and an aberrantly rearranged D/J region are linked to sequences 5' of c-myc on the reciprocal product.

Burkitt Lymphoma↗
PubMed1985

Role of an RNA cleavage/poly(A) addition site in the production of membrane-bound and secreted IgM mRNA.

The switch from membrane-bound to secreted IgM is accomplished by producing alternative forms of mRNA from a single mu heavy-chain gene. This process might be controlled at any of three steps--transcription termination, RNA splicing, or RNA cleavage/poly(A) addition. To distinguish between these possibilities, we have constructed a model human mu gene and observed its expression in early- and late-stage murine B cells. In each case, expression of the model gene reflects the state of development of the host cell; i.e., more of the mRNA for membrane-bound IgM is made in early B cells and more of the secreted form is made in late B cells. Using systematic deletions and analyses of RNA products of the model gene, we implicate RNA cleavage/poly(A) addition as the governing reaction. Removal of the cleavage/poly(A) signal for secreted mu mRNA by a series of BAL-31 deletions produces not only a decrease in secreted mu mRNA but also a compensatory increase in the membrane-bound form. Further, transcripts that do not terminate to the 5' side of the membrane anchor exons are found in cells producing only secreted IgM. As defined by these deletions, we establish that the RNA cleavage signal spans at least 35 bases and speculate that it forms an RNA stemloop that may be important in 3' end formation.

Animals↗