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J D Marth

Publications and source records attributed to J D Marth.

52 records · Page 3Linked to original sources
PubMed1992

Tissue- and site-specific DNA recombination in transgenic mice.

We have developed a method of specifically modifying the mammalian genome in vivo. This procedure comprises heritable tissue-specific and site-specific DNA recombination as a function of recombinase expression in transgenic mice. Transgenes encoding the bacteriophage P1 Cre recombinase and the loxP-flanked beta-galactosidase gene were used to generate transgenic mice. Genomic DNA from doubly transgenic mice exhibited tissue-specific DNA recombination as a result of Cre expression. Further characterization revealed that this process was highly efficient at distinct chromosomal integration sites. These studies also imply that Cre-mediated recombination provides a heritable marker for mitoses following the loss of Cre expression. This transgene-recombination system permits unique approaches to in vivo studies of gene function within experimentally defined spatial and temporal boundaries.

Animals↗
PubMed1992

Purification and initial characterization of the lymphocyte-specific protein-tyrosyl kinase p56lck from a baculovirus expression system.

A baculovirus expression system has been used to express large quantities of the lymphocyte-specific protein-tyrosyl kinase p56lck. A series of chromatographic steps, including the novel application of metalchelate affinity chromatography for protein kinase purification, were employed to obtain p56lck in a highly active form. Recombinant p56lck was purified to apparent homogeneity as determined by polyacrylamide gel electrophoretic analyses and was found to migrate in SDS gels as two related species, both with apparent molecular masses close to 56 kDa. p56lck phosphorylated all assayed substrates exclusively on tyrosyl residues, and underwent autophosphorylation at one principal site, also on a tyrosyl residue. p56lck displayed a high affinity for a synthetic peptide corresponding to the cytoplasmic domain (residues 52-164) of the T-cell receptor zeta-chain (TCR-zeta) (Km approximately 6.5 microM) but a low affinity for a peptide corresponding to its own autophosphorylation site (Km approximately 900 microM). p56lck was also found to be highly active for a purified protein-tyrosyl kinase (Vmax greater than 400 pmol.min-1.micrograms-1 using the TCR-zeta (52-164) as a substrate). A variety of agents were tested for their ability to inhibit p56lck, with zinc ions (I50 approximately 1.7 mM) and staurosporine (I50 approximately 500 nM) proving the most potent.

Animals↗
PubMed1992

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I gene.

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.

Amino Acid Sequence↗
PubMed1991

Delayed thymocyte development induced by augmented expression of p56lck.

Accumulating evidence supports the contention that CD4 and CD8 receptor molecules play a critical signaling role during thymocyte development. The lymphocyte-specific protein tyrosine kinase (p56lck), by virtue of its physical association with these surface components, provides a likely candidate for the biochemical signal transducing element required for these effects. To investigate the function of p56lck in T lymphocytes, transgenic mice were produced that carry either the wild-type lck gene or a mutated lck gene encoding a constitutively activated form of p56lck (p56lckF505). Both transgenes were expressed in thymocytes under the control of the lck proximal promoter element. A large set of founder animals was obtained in which steady-state accumulation of lck transgene mRNA directly correlated with transgene copy number, suggesting that this transgene contains a dominant control region. Progeny of these founders exhibited a transgene-dependent dose-related decrease in the production of thymocytes bearing functional antigen receptors. This effect was strictly dependent on p56lck activity, in that both wild-type and mutated versions of the genes induced similar effects with differing efficiencies. Remarkably, even a twofold increase in p56lck abundance was sufficient to substantially disrupt the appearance of functional thymocytes. These results indicate that thymocyte maturation is regulated in part by signals derived from p56lck.

Animals↗
PubMed1991

Thymic tumorigenesis induced by overexpression of p56lck.

The lck gene encodes a membrane-associated protein tyrosine kinase (p56lck) that is believed to participate in lymphocyte-specific signal transduction pathways. To investigate the function of this molecule, transgenic mice were generated carrying the wild-type lck gene or a mutated lck gene encoding a constitutively activated form of p56lck (p56lckF505). Transgene expression in thymocytes was achieved in each case using the lck proximal promoter element. Mice expressing high levels of either p56lckF505 or p56lckY505 reproducibly developed thymic tumors. The sensitivity of thymocytes to p56lck-induced transformation suggests that disturbances in lck expression may contribute to the pathogenesis of some human neoplastic diseases.

Animals↗
PubMed1989

Lymphocyte activation provokes modification of a lymphocyte-specific protein tyrosine kinase (p56lck).

The protein tyrosine kinase p56lck is implicated in the control of lymphocyte growth by virtue of its overexpression in some lymphoid malignancies and its transforming activity in heterologous systems. Previous studies have demonstrated that levels of lck mRNA and of p56lck decline rapidly after T cell activation. The disappearance of p56lck results primarily from post-translational conversion of p56lck to more slowly migrating forms with apparent sizes of approximately 60 kDa. This modification can be provoked by treatment of lymphocytes with PMA, and has been associated with increased serine phosphorylation of the p56lck molecule. Here we demonstrate that conversion of p56lck to p60lck is a feature of the physiologic activation of T lymphocytes by antigen-presenting cells. In addition, we show that the PMA-induced modification of p56lck proceeds via a mechanism distinct from conventional protein kinase C activation. The rapid conversion of p56lck to p60lck after antigenic stimulation is consistent with the view that this membrane-associated protein tyrosine kinase regulates some aspects of the lymphocyte activation sequence.

Adult↗
PubMed1988

Translational activation of the lck proto-oncogene.

The lck proto-oncogene, a member of the src gene family, encodes a lymphocyte-specific protein tyrosine kinase (p56lck) that is implicated in the pathogenesis of lymphoid neoplasia. We report here that 5' lck sequence elements, containing AUG codons, significantly reduce the in vivo efficiency of p56lck translation from the normal messenger RNA. This result provides a quantitative explanation for the overexpression of p56lck in two retrovirally-induced murine lymphomas that express abnormal lck transcripts from which the physiological 5' translational control region has been deleted and non AUG-containing viral sequences substituted. In contrast to other mammalian genes, most proto-oncogenes contain AUG codons 5' to the authentic initiation codon, suggesting that cells can regulate translational start sites in these mRNAs. Abrogation of translational control may be a general mechanism of proto-oncogene activation in malignancy.

Animals↗
PubMed1988

Structure and expression of lck transcripts in human lymphoid cells.

The murine lck gene encodes a membrane-associated protein tyrosine kinase that has been implicated in lymphocyte oncogenesis. Here we report the structure of normal human lck transcripts and the pattern of expression of these transcripts in developing thymus and in peripheral T cell subsets. The human lck gene encodes a 509 amino acid polypeptide that is closely related to the murine lck-encoded protein throughout its length. Analysis of the deduced amino acid sequence of human p56lck demonstrates that an amino-terminal domain, widely divergent among the seven known src family members, has been conserved between murine and human p56lck, and thus probably includes sequences crucial to the lymphocyte-specific function of this molecule. Human lck transcripts were detected in CD4+ and CD8+ T cells, in partially purified B cells, and in Epstein-Barr virus-immortalized B cell lines, but not in monocytes, granulocytes, or in nonhematopoetic cell types. Human lck transcripts are readily detectable in fetal thymocytes at 70 days of gestation, but not at 57 days of gestation, indicating that lck expression appears coordinately with the appearance of lymphoid cells in the developing thymus. Thus lck gene expression is a marker for cells of the lymphocyte lineage in man. We conclude that the lck gene probably participates in a signal transduction pathway uniquely present in lymphoid cells.

Base Sequence↗
PubMed1988

p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen.

p56lck and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56lck from introduced DNA molecules to compare the subcellular localizations of p60c-src and p56lck and their abilities to bind polyomavirus middle T antigen (mT). p56lck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60c-src is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60c-src binds to mT, but p56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60c-src mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60c-src to bind mT and be soluble, we tested whether the different localization and mT binding properties of p56lck and p60c-src were dictated by their different carboxy termini. A protein consisting largely of p60c-src sequences but carrying a p56lck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60c-src not only require sequences common to the carboxy termini of p60c-src and p56lck, but also require sequences unique to the body of p60c-src.

Animals↗
PubMed1988

Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck).

The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck.

Animals↗
PubMed1988

Structure of the murine lck gene and its rearrangement in a murine lymphoma cell line.

The lck gene encodes a lymphocyte-specific protein-tyrosine kinase that is implicated in neoplastic transformation. We have determined the germ line organization of the murine lck gene and have isolated and characterized a rearranged lck allele in the murine lymphoma cell line LSTRA. The overall exon-intron organization of the normal lck gene is almost identical to that of avian c-src. In LSTRA DNA, an internally rearranged Moloney murine leukemia virus genome is interposed between two distinct promoters that normally generate lck transcripts differing only in 5' untranslated regions. The rearrangement appears to have been selected to permit splicing of transcripts that initiate from the Moloney virus promoter to an acceptor site located within the first exon 3' to the downstream promoter, thus generating an lck mRNA with a novel 5' untranslated region that may be more efficiently translated.

Animals↗
PubMed1987

Regulation of pp56lck during T-cell activation: functional implications for the src-like protein tyrosine kinases.

The lymphocyte-specific protein tyrosine kinase pp56lck, encoded by a member of the src gene family, is implicated in the control of T-cell growth and differentiation. Purified resting human T lymphocytes contain appreciable levels of lck mRNA and of pp56lck. Upon activation of these T cells, levels of lck mRNA and of pp56lck promptly decline. These reductions in lck mRNA and protein expression are closely correlated with the induction of lymphokine production. Both require identical stimuli and follow a similar time course of response. Down-regulation of lck expression, however, is not correlated with proliferation. Our results provide an example of regulation of a src-like protein tyrosine kinase in a normal fully differentiated cell population and suggest that modulation of lck RNA and protein expression is an important feature of the lymphocyte activation sequence leading to lymphokine production.

Adult↗
PubMed1987

Novel protein-tyrosine kinase gene (hck) preferentially expressed in cells of hematopoietic origin.

Protein-tyrosine kinases are implicated in the control of cell growth by virtue of their frequent appearance as products of retroviral oncogenes and as components of growth factor receptors. Here we report the characterization of a novel human protein-tyrosine kinase gene (hck) that is primarily expressed in hematopoietic cells, particularly granulocytes. The hck gene encodes a 505-residue polypeptide that is closely related to pp56lck, a lymphocyte-specific protein-tyrosine kinase. The exon breakpoints of the hck gene, partially defined by using murine genomic clones, demonstrate that hck is a member of the src gene family and has been subjected to strong selection pressure during mammalian evolution. High-level expression of hck transcripts in granulocytes is especially provocative since these cells are terminally differentiated and typically survive in vivo for only a few hours. Thus the hck gene, like other members of the src gene family, appears to function primarily in cells with little growth potential.

Amino Acid Sequence↗
PubMed1986

Localization of a lymphocyte-specific protein tyrosine kinase gene (lck) at a site of frequent chromosomal abnormalities in human lymphomas.

The murine lck gene is closely related to a family of cellular protooncogenes and encodes a lymphocyte-specific, membrane-associated protein tyrosine kinase. We and others have demonstrated that the lck gene is rearranged and overexpressed in the murine lymphoma LSTRA, most likely as a result of the insertion of Moloney murine leukemia virus DNA immediately adjacent to the gene. We now report that the lck gene is located at the distal end of murine chromosome 4 and on human chromosome 1 at position 1p32-35 near a site of frequent structural abnormalities in human lymphomas and neuroblastomas. These results raise the possibility that structural alteration of the lck gene through chromosomal rearrangement may contribute to transformation in human malignant disease.

Animals↗
PubMed1985

A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA.

Protein-tyrosine kinases are implicated in the control of normal and neoplastic cell growth. We have used molecular cloning strategies to characterize a lymphocyte-specific protein-tyrosine kinase gene distinct from but closely related to src and yes. This gene, encoded by a genetic locus defined here as lskT, is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Thus alterations in the structure or expression of this protein-tyrosine kinase gene may in some cases mediate neoplastic transformation. In addition, transcription of the normal lskT gene is restricted to cells of lymphoid origin. We infer that the lskT-encoded protein-tyrosine kinase may aid in transducing proliferative or differentiative signals unique to lymphocytes.

Animals↗