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M C Kiefer

Publications and source records attributed to M C Kiefer.

51 records · Page 3Linked to original sources
PubMed1991

The molecular biology of heparan sulfate fibroblast growth factor receptors.

Two distinct classes of cell surface FGF-binding proteins have been identified. These receptors differ in both mode of interaction and in affinity for the FGFs. cDNAs that encode the low-affinity receptor were isolated from a hamster kidney cell line cDNA library by expression cloning. Transfected cells that contained these heparan sulfate proteoglycan FGF receptor cDNAs were enriched for by panning on basic FGF-coated plates. The analogous human cDNA was isolated from a hepatoma cell line cDNA library. The homology of our hamster cDNAs to the previously described murine integral membrane proteoglycan syndecan, together with an exact amino acid sequence match of our human-cDNA-encoded product to human syndecan, clearly indicates the identity of these independently isolated proteoglycans. Further confirmation that the expressed molecule serves as a proteoglycan core protein was achieved by immunoprecipitation of 35SO4-labeled material from solubilized transfected cells. Nitrous acid treatment and chondroitinase digestion revealed that 77% of the label was associated with heparan sulfate chains and 22% with chondroitin sulfate chains. These heparan sulfate chains contributed to the fivefold increase in the total heparan sulfate found to be present on the surface of the transfected cells compared with cells transfected with a vector lacking the cDNA insert.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of a human basic fibroblast growth factor receptor cDNA and expression of a biologically active extracellular domain in a baculovirus system.

A cDNA clone encoding a human fibroblast growth factor (FGF) receptor was isolated from a hepatoma cell line cDNA library. The cDNA encodes a three immunoglobulinlike-domain FGF receptor that is similar to a human placental FGF receptor cDNA but lacks two amino acids. The variation observed at these two amino acids, also seen in the two immunoglobulinlike-domain FGF-receptors, can be explained by an alternate splicing mechanism. We have used a baculovirus expression system to produce high levels of a soluble, extracellular domain form of the FGF receptor (EC-FGF receptor). Spodoptera frugiperda (Sf9) insect cells infected with recombinant EC-FGF receptor viruses synthesized and secreted an EC-FGF receptor of apparent Mr = 58,000. The EC-FGF receptor purified from conditioned media of infected Sf9 cells by lentil lectin affinity chromatography was shown to bind basic FGF with high affinity (Kd = 1-5 nM), to inhibit the binding of radioiodinated basic FGF to its high affinity receptor and to inhibit endothelial cell proliferation. Furthermore, binding of basic FGF to the EC-FGF receptor was shown to be significantly enhanced by heparin. The availability of biologically active FGF receptors will allow an analysis of their interaction with members of the FGF family of proteins and viruses of the herpes family that have been shown to use the FGF receptor system for cell entry.

Amino Acid Sequence↗
PubMed1990

Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor.

Expression cloning of cDNAs encoding a basic fibroblast growth factor (FGF) binding protein confirms previous hypotheses that this molecule is a cell-surface heparan sulfate proteoglycan. A cDNA library constructed from a hamster kidney cell line rich in FGF receptor activity was transfected into a human lymphoblastoid cell line. Clones expressing functional basic FGF binding proteins at their surfaces were enriched by panning on plastic dishes coated with human basic FGF. The amino acid sequence deduced from the isolated cDNAs revealed several interesting features, including hydrophobic signal and transmembrane domains that flank an extracellular region containing six potential attachment sites for glycosaminoglycan side chains. The structure also contains a short hydrophilic cytoplasmic tail sequence homologous to previously reported actin binding domains. Binding of basic FGF to cells expressing the binding protein could be inhibited by heparin and heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. In addition to binding basic FGF, this protein or related surface proteins may function as an initial cellular attachment site for other growth factors and for viruses, such as herpes simplex virus.

Amino Acid Sequence↗
PubMed1990

Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site.

Intracellular proteolytic processing of precursor polypeptides is an essential step in the maturation of many proteins, including plasma proteins, hormones, neuropeptides, and growth factors. Most frequently, propeptide cleavage occurs after paired basic amino acid residues. To date, no mammalian propeptide processing enzyme with such specificity has been purified or cloned and functionally characterized. We report the isolation and functional expression of a cDNA encoding a propeptide-cleaving enzyme from a human liver cell line. The encoded protein, called PACE (paired basic amino acid cleaving enzyme), has structural homology to the well-characterized subtilisin-like protease Kex2 from yeast. The functional specificity of PACE for mediating propeptide cleavage at paired basic amino acid residues was demonstrated by the enhancement of propeptide processing of human von Willebrand factor when coexpressed with PACE in COS-1 cells.

Amino Acid Sequence↗
PubMed1990

Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.

Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS; also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme.

Acyltransferases↗
PubMed1989

Genetic and molecular mapping of the Hmt region of mouse.

We have mapped a new region of the mouse major histocompatibility complex (MHC) that contains the nuclear gene, Hmt, for the maternally transmitted antigen, Mta. The Hmt region of chromosome 17 lies between a recombinational breakpoint distal to Tla and another proximal to Tpx-1, thus including Pgk-2. A novel MHC class I gene fragment, R4B2, was cloned and mapped to this region as was another new class I gene, Thy19.4. Both lie proximal to Pgk-2, within the distal inversion in t-haplotypes. The presence of several other MHC class I genes in the Hmt region is predicted from analysis of the recombinants that define the region.

Amino Acid Sequence↗
PubMed1987

Structure function relationships for the IL 2 receptor system. III. Tac protein missing amino acids 102 to 173 (exon 4) is unable to bind IL 2: detection of spliced protein after L cell transfection.

High affinity receptors for interleukin 2 (IL 2) contain the Tac protein as one ligand-binding subunit. Localization of the IL 2-binding site on this molecule, as well as localization of the complementary site on IL 2, should provide insight into the design of IL 2 analogs. In this report, we examine the ability of normal and modified Tac protein to bind IL 2 and several antibodies that recognize the native Tac molecule. Using a transient L cell expression system, we have determined that transfection with cDNA-missing Tac exon 4 resulted in expression of spliced protein that had no measurable binding to IL 2 or the monoclonal anti-receptor antibodies, anti-Tac, and 7G7/B6. This protein was detected, however, by rabbit polyclonal antibodies prepared against synthetic Tac peptides. Thus, one or more amino acids encoded by exon 4 is important, either for direct ligand contact or for the proper folding of critical segments of the Tac molecule. In addition, insertion of stop codons at a unique restriction enzyme site near the beginning of exon 5 resulted in cellular secretion of truncated Tac molecules that were capable of binding IL 2, anti-Tac, and 7G7/B6. Amino acids encoded by exons 5 to 8 thus play no critical role in IL 2 binding. The ligand association demonstrated for truncated Tac protein produced by exons 2 to 4 should guide attempts to define the IL 2-binding segment of the Tac molecule.

Amino Acid Sequence↗
PubMed1984

RNA and capsid accumulation in cowpea protoplasts that are resistant to cowpea mosaic virus strain SB.

Leaf protoplasts from the Arlington line of cowpea (Vigna unguiculata) support only a limited increase of cowpea mosaic virus strain SB (CPMV-SB), whereas cowpea severe mosaic virus, another member of the comovirus group, replicates efficiently in Arlington cowpea protoplasts. CPMV-SB replicates efficiently in protoplasts of cowpea line Blackeye 5. Some characteristics of the virus-specific resistance of Arlington protoplasts to CPMV-SB are reported. Differences between progeny CPMV-SB from Arlington and Blackeye 5 protoplasts were not detected. Inoculation with CPMV-SB RNA, rather than virions, did not make Arlington protoplasts fully susceptible. These results favor, for likely involvement in the CPMV-SB restriction phenomenon, events in the virus life cycle that occur after exposure of virion RNA to the cytoplasm and before assembly of particles is completed. The accumulation of CPMV-SB RNAs of both polarities was found to be depressed in inoculated Arlington protoplasts. However, (+)RNA (virion RNA polarity) accumulated to no lesser extent, per unit of (-)RNA, in Arlington protoplasts than in Blackeye 5 protoplasts. Capsid antigen accumulation, per unit of (+)RNA, was reduced in Arlington protoplasts as compared to Blackeye 5 protoplasts. A working hypothesis consistent with the above and other observations is that Arlington protoplasts have an inhibitory substance that interferes with the production or/and function of CPMV-SB specified proteins.

Capsid↗
PubMed1983

Construction of infectious potato spindle tuber viroid cDNA clones.

Contiguous restriction fragments from two cloned partial-length potato spindle tuber viroid (PSTV) cDNAs were used to construct recombinant DNAs containing full-length monomeric and dimeric PSTV cDNA. When five different PSTV cDNA plasmids and RNA isolated from E. coli cells harboring these plasmids were tested for infectivity on tomato, plasmid DNAs containing PSTV cDNA dimers were infectious. RNA transcripts containing the sequence of PSTV from these plasmids were also infectious. The sequences of the viroid progeny and the cloned DNA were identical. In vitro mutagenesis of infectious PSTV cDNAs will allow systematic investigation of the role of specific sequences in viroid replication and pathogenesis.

Base Sequence↗
PubMed1983

Structural similarities between viroids and transposable genetic elements.

The primary structures of the tomato planta macho and tomato apical stunt viroids have been determined, and probable secondary structures are proposed. Both viroids can assume the rodlike conformation with extensive base-pairing characteristic of all known viroids. Sequence homologies between the two viroids (75%) and with members of the potato spindle tuber viroid group (73-83%) indicate that they both belong to this group. Comparative sequence analysis of all members of the group reveals striking similarities with the ends of transposable genetic elements. These similarities, the presence of inverted repeats often ending with the dinucleotides U-G and C-A, and flanking imperfect direct repeats suggest that viroids may have originated from transposable elements or retroviral proviruses by deletion of interior portions of the viral (or element) DNA.

Base Sequence↗
PubMed1975

Specificity of ATP-dependent and GTP-dependent protein kinases with respect to ribosomal proteins of Escherichia coli.

Two protein kinases differing in substrate specificity were used to phosphorylate the 30-S and the 50-S ribosomal subunits of Escherichia coli. The catalytic subunit from the rabbit skeletal muscle protein kinase phosphorylates proteins S1, S4, S9, S13 and S18 of the 30-S subunit and proteins L2, L4, L5, L16, L18 and L23 of the 50-S subunit with (gamma-32P)ATP as phosphoryl donor. A second protein kinase isolated from rabbit reticulocytes, formerly shown to phosphorylate preferentially acidic proteins and to use GTP as well as ATP, strongly phosphorylated protein S6, an acidic protein of the small ribosomal subunit, and to a lesser extent proteins L7 and L12 or the large subunit. Evidence is presented showing different phosphorylation patterns when either whole subunits or the extracted proteins were used as substrate for the protein kinase. Kinetic studies showed proteins S1 and S4 to become most rapidly phosphorylated. Although most proteins incorporated less than stoichiometric amounts of phosphate, it is shown that with a high excess of ATP L2 bound 1 mol phosphate/mol protein.

Adenosine Triphosphate↗