Biomedical subjects
J V Ravetch
Publications and source records attributed to J V Ravetch.
Organization of chromosomes in Plasmodium falciparum: a model for generating karyotypic diversity.
Chromosomes of the human malaria parasite, Plasmodium falciparum, are compartmentalized into conserved and polymorphic domains. The conserved domains are transcribed and localized within the central region o f a chromosome, whereas the polymorphic domains are transcriptionally silent and situated at chromosome ends. Unlike the central chromosomal domains, chromosome ends contain repetitive sequence elements. In this review, Michael Lonzer, Derik de Bruin, Samuel Wertheimer and Jeffrey Ravetch propose that the chromosome ends play a functional role in generating genetic diversity by promoting meiotic and mitotic recombination and chromosomal rearrangement events.
Atopy and Fc receptors: mutation is the message?
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Myelin protein zero gene mutated in Charcot-Marie-tooth type 1B patients.
Autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells. In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family. The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate. The same MPZ locus cosegregates with the CMT1B disease gene in a second CMT1B family [total multipoint logarithm of odds (lod) = 11.4 at theta = 0.00] with a splice junction mutation. Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago. MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes > 50% of myelin protein. These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.
Human interferon-inducible protein 10: expression and purification of recombinant protein demonstrate inhibition of early human hematopoietic progenitors.
Human interferon-inducible protein 10 (IP-10), a member of the family of the small secreted proteins called intercrine cytokines or chemokines, is secreted by interferon gamma-stimulated T cells, monocytes, endothelial cells, and keratinocytes. We have begun to explore the biological properties of IP-10 by cloning and overexpression in baculovirus and in bacterial protein expression systems. A 9.9-kD protein was secreted by infected insect cells, which on sodium dodecyl sulfate-polyacrilamide gel electrophoresis comigrated with keratinocyte IP-10 and with f(22-98), a bacterial recombinant fragment lacking the signal sequence but containing all other residues of IP-10. All three reacted with antibodies recognizing residues 10-98 (alpha IP-10) and 77-98 of IP-10 (alpha 22), demonstrating that it is secreted by keratinocytes and insect cells after removal of the signal sequence but without proteolysis of the COOH-terminal end. Purified rIP-10 suppresses in vitro colony formation by early human bone marrow progenitor cells which need r-steel factor (rSLF) and rGM-CSF or rSLF and r-erythropoeitin (rEPO). The inhibition is dose dependent, is complete at concentrations > or = 50 ng/ml, is prevented by preincubation of rIP-10 with alpha IP-10, but not by alpha 22, and is seen with highly purified CD34+ cells, suggesting direct effect of rIP-10 on the progenitors. Combination of rIP-10 and other chemokines at inactive concentrations inhibited colony formation in a synergistic manner. rIP-10 did not affect colony formation in the absence of any growth factors or in the presence of rEPO or rGM-CSF but in absence of rSLF. The effects of IP-10 may be relevant to normal marrow function and might be harnessed to protect human hematopoietic progenitors from the cytotoxic effects of chemotherapy.
Interferon gamma-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3.
A 39-nt DNA sequence, the interferon gamma (IFN-gamma) response region (GRR), is necessary for the IFN-gamma-induced transcription of the high-affinity Fc receptor for IgG (Fc gamma RI) and sufficient for the IFN-gamma-induced transcription of transfected plasmids. By using extracts from IFN-gamma-treated cells, three protein complexes will assemble in vitro on a 9-nt core region in the 3' domain of the GRR. The sequence of this core resembles the IFN-gamma-activated sequence (GAS) described for the GBP gene. Mutations in this GAS core region prevent complex assembly and result in the loss of IFN-gamma induction of reporter constructs containing the mutation. In addition to the GAS core region, a 5' region of the GRR is necessary for optimal IFN-gamma induction and for formation of one of the DNA-protein complexes. By antibody reactivity, we show that a 91-kDa protein, first identified as a component of ISGF3, the IFN-alpha-induced transcription complex, is present in at least two of the DNA-protein complexes. IFN-alpha can induce the formation of the faster-migrating 91-kDa protein-GAS complex but not the slower-migrating complex. Furthermore, IFN-alpha does not result in appreciable transcriptional activation of Fc gamma RI or constructs containing the GRR. Thus, these data demonstrate that the IFN-gamma-activated 91-kDa protein is required for IFN-gamma induction of Fc gamma RI and suggest that an additional complex may be required for optimal expression and specificity.
Physical and functional association of p56lck with Fc gamma RIIIA (CD16) in natural killer cells.
The transmembrane receptor for immunoglobulin G immune complexes on natural killer (NK) cells and macrophages, Fc gamma RIIIA (CD16), mediates cellular activation through a tyrosine kinase-dependent pathway. We show that Fc gamma RIII crosslinking results in activation of the src-related kinase p56lck in NK cells and demonstrate a physical association of p56lck with Fc gamma RIIIA in immunoprecipitates from NK cells obtained using anti-Fc gamma RIII antibodies or immune complexes. Our studies show that the zeta chain, the signal transducing subunit of Fc gamma RIIIA and of T cell receptor, associates with p56lck and, in NK cells, is a substrate for this kinase. Such direct association of p56lck with the zeta subunit as confirmed by demonstrating the interaction in heterologous cells transfected with cDNA expressing p56lck and zeta. Our findings demonstrate both functional and physical association of p56lck with Fc gamma RIIIA, through direct interaction of the kinase with the zeta and/or the gamma signal transducer subunits of the receptor. These data suggest a possible mechanism by which activation via Fc gamma RIIIA occurs.
Transcriptional differences in polymorphic and conserved domains of a complete cloned P. falciparum chromosome.
Classical genetic studies on the human malaria parasite Plasmodium falciparum have been hampered by a complex life cycle which alternates between vertebrate and invertebrate hosts. Consequently, only a few genetic crosses have been performed so far. In addition, molecular genetics has provided only limited access to the genes of this pathogen, a consequence of an unusually high A + T content. To overcome these limitations we have constructed an ordered telomere-to-telomere contig map of P. falciparum chromosome 2 by isolating overlapping yeast artificial chromosome clones. This approach was used to examine the strain-dependent polymorphisms commonly observed for P. falciparum chromosomes. Our analysis reveals that polymorphisms of chromosome 2 are restricted to regions at either end, representing 20% of the chromosome. Transcription mapping of the entire chromosome suggests a compartmentalization of chromosome 2 into a transcribed central domain and silent polymorphic ends.
Plasmodium: control of gene expression in malaria parasites.
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A sequence element associated with the Plasmodium falciparum KAHRP gene is the site of developmentally regulated protein-DNA interactions.
The Plasmodium falciparum gene encoding the knob associated histidine-rich protein (KAHRP) is shown to be transcriptionally regulated during its expression in the intraerythrocytic cycle as demonstrated by stage specific nuclear run-on analysis. The genomic organization of the KAHRP gene was determined and the structural basis for the stage specific transcription investigated. A sequence motif with two-fold symmetry was found 160 bp upstream of the RNA initiation site. This sequence element interacts with parasite derived nuclear extracts in a stage specific manner that correlates with the transcriptional activity of the KAHRP gene. These studies suggest a functional role for this structural element in the developmental regulation of a P. falciparum erythrocytic gene.
Evidence for the participation of the Ssp-3 antigen in the invasion of nonphagocytic mammalian cells by Trypanosoma cruzi.
Trypomastigotes of Trypanosoma cruzi have to invade mammalian cells in order to multiply. They bear on their plasma membrane a sialic acid-containing epitope (Ssp-3) defined by a series of monoclonal antibodies (mAbs). Previous investigations have shown that Fab fragments of these mAbs inhibit the attachment of trypomastigotes to 3T3 fibroblasts. To further define the role of Ssp-3 in invasion, here we use, as targets for infection, L cells and CHO cells stably transfected with cDNA coding for the mouse Fc receptors genes. When the trypomastigotes are incubated with small, nonagglutinating amounts of antibodies to Ssp-3, their attachment to the transfected cells is greatly enhanced, without a parallel increase in invasion. The enhancement in attachment is Fc mediated, since it is abolished by treatment of the transfected cells with mAbs to Fc receptors. In contrast, both attachment to, and invasion of, the transfected cells are increased if the parasites are incubated with polyclonal or monoclonal antibodies against T. cruzi surface membrane antigens other than Ssp-3. If, however, antibodies to Ssp-3 are added to the incubation mixtures containing any of the other anti-T. cruzi antibodies, the enhancement of invasion (but not of attachment) is reversed. These results suggest that Ssp-3-bearing molecules participate in the process of parasite internalization.
Signal transduction by Fc gamma RIII (CD16) is mediated through the gamma chain.
To determine the functional role of the two isoforms of Fc gamma RIII (CD16) (IIIA, IIIB), the signal transduction capabilities of wild-type and mutant forms of these receptors were analyzed in transfected lymphoid, myeloid, and fibroblastic cell lines. Functional reconstitution of receptor signalling was observed in hematopoietic T and mast cells, and was absent in nonhematopoietic (CHO) cells. Fc gamma RIIIA, a hetero-oligomeric receptor composed of a ligand-binding subunit alpha and dimeric gamma chains, generated both proximal and distal responses in Jurkat and P815 cells, typical of what is seen in natural killer cells and macrophages upon receptor activation. In contrast, Fc gamma RIIIB, which is normally attached to the cell surface via a glycosyl-phosphatidylinositol anchor, was incapable of transducing signals. After crosslinking, Fc gamma RIIIA signalling was dependent only upon the gamma chain. Fc gamma RIIIA chimeras in which the alpha subunit transmembrane and cytoplasmic domains were substituted with the corresponding gamma chain sequences functioned as well as wild-type hetero-oligomeric receptors. These data indicate that the ability of the Fc gamma RIIIA complex to activate the appropriate pathways for cell activation is cell-type restricted and independent of the transmembrane and cytoplasmic domains of the alpha subunit. The presence of the gamma chain is responsible for the assembly of, as well as the signal transduction by, the functional cell surface complex.
The beta subunit of the Fc epsilon RI is associated with the Fc gamma RIII on mast cells.
Fc epsilon RI is a tetrameric receptor, composed of a ligand recognition subunit, alpha, a beta chain, and dimeric gamma chains. Previous studies have indicated that the dimeric gamma chain is associated with Fc gamma RIIIA (CD16) on natural killer cells and macrophages as well as the clonotypic T cell receptor. Here we show that in mast cells, in addition to the dimeric gamma chains, the beta subunit is associated not only with Fc epsilon RI, but also with Fc gamma RIIIA. Functional reconstitution studies with a mastocytoma cell line indicate that Fc gamma RIIIA composed of alpha, beta, and gamma subunits has the capacity for signal transduction. These studies suggest that through the association of alternative ligand recognition subunits (alpha epsilon, alpha gamma), a common signal transduction complex (beta gamma 2) mediates similar biochemical and effector functions in response to immunoglobulin G (IgG) and IgE.
Transcription mapping of a 100 kb locus of Plasmodium falciparum identifies an intergenic region in which transcription terminates and reinitiates.
We have mapped Plasmodium falciparum erythrocytic stage transcription units on chromosome 10 in the vicinity of the gene encoding the glycophorin binding protein (GBP130) using yeast artificial chromosomes (YACs). Three erythrocytic stage transcription units are clustered in a 40 kb region. Two of these genes are closely linked, separated by less than 2 kb. Nuclear run-on data demonstrate that transcription of these two genes, though unidirectional, is monocistronic. Within this intergenic region are the sites at which transcription of the upstream gene terminates and the GBP130 gene initiates. These studies represent the first description of the minimal and necessary cis-acting elements for transcription termination and initiation in this protozoan parasite.
Characterization of yeast artificial chromosomes from Plasmodium falciparum: construction of a stable, representative library and cloning of telomeric DNA fragments.
Molecular genetic studies of the human malaria parasite Plasmodium falciparum have been hampered in part due to difficulties in stably cloning and propagating parasite genomic DNA in bacteria. This is thought to be a result of the unusual A+T bias (>80%) in the parasite's DNA. Pulsed-field gel electrophoretic separation of P. falciparum chromosomes has shown that large chromosomal polymorphisms, resulting from the deletion of DNA from chromosome ends, frequently occur. Understanding the biological implications of this chromosomal polymorphism will require the analysis of large regions of genomic, and in particular telomeric, DNA. To overcome the limitations of cloning parasite DNA in bacteria, we have cloned genomic DNA from the P. falciparum strain FCR3 in yeast as artificial chromosomes. A pYAC4 library with an average insert size of approximately 100 kb was established and found to have a three to fourfold redundancy for single-copy genes. Unlike bacterial hosts, yeast stably maintain and propagate large tracts of parasite DNA. Long-range restriction enzyme mapping of YAC clones demonstrates that the cloned DNA is contiguous and identical to the native parasite genomic DNA. Since the telomeric ends of chromosomes are underrepresented in YAC libraries, we have enriched for these sequences by cloning P. falciparum telomeric DNA fragments (from 40 to 130 kb) as YACs by complementation in yeast.
Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: transcriptional induction by gamma-interferon is mediated through common DNA response elements.
Expression of the high-affinity receptor for IgG (Fc gamma RI) is restricted to cells of myeloid lineage and is induced by gamma-interferon (IFN-gamma) but not by IFN-alpha/beta. The organization of the human Fc gamma RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-gamma induction are reported here. A 39-nucleotide sequence (IFN-gamma response region, or GRR) is defined that is both necessary and sufficient for IFN-gamma inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class II genes: i.e., X, H, and gamma-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-gamma indicates that the presence of these elements is a general feature of IFN-gamma-responsive genes. Our studies suggest that the combination of X, H, and gamma-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.
A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions.
The zeta subunit is a component of the Fc gamma receptor of natural killer cells (Fc gamma RIII or CD16), as well as the multimeric T-cell receptor/CD3 complex, and is required for assembly of both native receptors. The role of the zeta subunit in human Fc gamma RIIIA assembly differs from its role in T-cell receptor/CD3 complex assembly. The transmembrane domain of the Fc gamma RIIIA alpha subunit forms noncovalent interactions with the comparable domain of the zeta subunit and is sufficient for surface expression of the Fc gamma RIIIA complex. In the absence of these interactions, sequences in the transmembrane domain of the Fc gamma RIIIA alpha subunit signal its degradation. Leu-46, present in the transmembrane domain of the human zeta subunit, is important for assembly with the Fc gamma RIIIA alpha subunit. Substitution of this leucine with an isoleucine, as found in the mouse zeta subunit, significantly reduces this interaction. In contrast, the mouse and human zeta subunits interact with the pentameric T-cell receptor/CD3 complex, resulting in surface expression of this receptor.
Cloning and molecular characterization of the murine macrophage "68-kDa" protein kinase C substrate and its regulation by bacterial lipopolysaccharide.
We have isolated and characterized a cDNA clone encoding the murine macrophage 68-kDa protein kinase C substrate, which is homologous to the 80- to 87-kDa protein identified by the acronym MARCKS (myristoylated alanine-rich C kinase substrate). The murine MARCKS cDNA clone encodes an acidic protein of 309 amino acids with a calculated molecular weight of 29,661. Transfection of the murine MARCKS gene into TK-L fibroblasts produced a myristoylated protein kinase C substrate that migrated on SDS/PAGE with an apparent molecular mass of 68 kDa. Peptide mapping studies indicated that MARCKS produced by the transfected gene was indistinguishable from the endogenous murine macrophage protein. Comparison of the murine macrophage sequence with the previously published chicken and bovine brain sequences revealed two conserved domains: an N-terminal membrane-binding domain and a phosphorylation domain that also contains calmodulin and actin binding sites. In murine peritoneal macrophages, bacterial lipopolysaccharide increased MARCKS mRNA levels by greater than 30-fold. Multiple MARCKS transcripts were observed and could be accounted for by differential polyadenylylation and incomplete processing. Genomic Southern blot analysis suggested a single MARCKS gene per haploid genome.