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M I Simon

Publications and source records attributed to M I Simon.

At least 109 records · Page 6Linked to original sources
PubMed1995

Characterization of a human chromosome 22 enriched bacterial artificial chromosome sublibrary.

Selection of chromosomal sublibraries from total human genomic libraries is critical for chromosome-based physical mapping approaches. We have previously reported a method of screening total human genomic library using flow sorted chromosomal DNA as a hybridization probe and selection of a human chromosome 22-enriched sublibrary from a total human bacterial artificial chromosome (BAC) library (Nucleic Acids Res 1995; 23: 1838-39). We describe here further details of the method of construction as well as characterization of the chromosome 22-enriched sublibrary thus constructed. Nearly 40% of the BAC clones that have been mapped by fluorescence in situ hybridization (FISH) analysis were localized to chromosome 22. By screening the sublibrary using chromosome 22-specific hybridization probes, we estimated that the sublibrary represents at least 2.5 x coverage of chromosome 22. This is in good agreement with the results from FISH mapping experiments. FISH map data also indicate that chromosome 22-specific BACs in the sublibrary represent all the subregions of chromosome 22.

Animals↗
PubMed1995

Construction and utility of a human chromosome 22-specific Fosmid library.

We have previously demonstrated the capability of the Fosmid vector based on Escherichia coli F-factor replicon to stably propagate cosmid-sized human genomic DNA fragments. Using the Fosmid vector, we have constructed and arrayed a 10 x human chromosome 22-specific library, partly by picking human positive clones from a total Fosmid library constructed using DNA from human-hamster hybrid cell line containing human chromosome 22, and partly by using flow-sorted chromosomal DNA. The clones and physical contig maps of the clones in the library will serve as a valuable resource for detailed analysis of the chromosome by providing reliable materials for high resolution mapping and sequencing. In order to efficiently build physical maps for the chromosomal regions of interest spanning several hundred kilobases to a megabase, it is necessary to rapidly identify subsets of the Fosmid clones from the library that cover such regions. In this report, we describe a method of using random amplification products derived from YAC clones to rapidly identify a subset of Fosmid clones that cover a specific genomic subregion.

Animals↗
PubMed1995

Localized perturbations in CheY structure monitored by NMR identify a CheA binding interface.

Phosphotransfer between the autophosphorylating histidine kinase CheA and the response regulator CheY represents a crucial step in the bacterial chemotaxis signal transduction pathway. The 15N-1H correlation spectrum of CheY complexed with an amino-terminal fragment of CheA exhibits specific localized differences in chemical shifts when compared to the spectrum of uncomplexed CheY. When mapped onto the three-dimensional structure of CheY, these changes define a region distinct from the active site. A single amino-acid substitution within this binding region on CheY, alanine to valine at position 103, significantly decreases the affinity of CheY for CheA. The binding face described by these changes partially overlaps a flagellar switch binding surface previously defined by mutagenesis.

Bacterial Proteins↗
PubMed1995

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ.

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phosphorylated form of the protein allow counter-clockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephosphorylation rates of these mutants. CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of cheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephosphorylation rates. Interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of interaction with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.

Bacterial Proteins↗
PubMed1995

Multiple regions of G alpha 16 contribute to the specificity of activation by the C5a receptor.

The C5a chemoattractant factor receptor, when expressed in COS-7 cells, can stimulate phosphoinositide-specific phospholipase C activity through the activation of the G16 isoform of the heterotrimeric G protein, but not through the G11 isoform. To identify the regions of the G alpha 16 subunit protein that are responsible for its activation by the C5a receptor, a series of chimeras between G alpha 16 and G alpha 11 were constructed and tested for their ability to be activated by the C5a receptor. Co-transfection experiments with chimeras in which the carboxyl-terminal regions of G alpha 11 were replaced with the corresponding regions of G alpha 16 demonstrated that changes in the carboxyl terminus, e.g., replacement of 134 amino acids, were not sufficient to confer receptor specificity. An additional segment encompassing residues 220-240 of G alpha 16 was required to confer C5a-induced activation. Testing of a reciprocal series of chimeras composed of G alpha 16 sequences at the amino terminus and G alpha 11 sequences at the carboxyl terminus revealed that certain sequences extending from the amino terminus to amino acid 209 of G alpha 16 were sufficient to endow the chimera with much of the specificity for C5a-induced activation. These results suggest that receptor specificity may involve specific conformations of the G protein stabilized by concerted interactions of multiple amino acid sequences distributed throughout the G alpha protein.

Animals↗
PubMed1994

Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes.

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.

Adenylate Cyclase Toxin↗
PubMed1994

Binding of beta gamma subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase.

Bruton tyrosine kinase (Btk) has been implicated as the defective gene in both human and murine B-cell deficiencies. The identification of molecules that interact with Btk may shed light on critical processes in lymphocyte development. The N-terminal unique region of Btk contains a pleckstrin homology domain. This domain is found in a broad array of signaling molecules and implicated to function in protein-protein interactions. By using an in vitro binding assay and an in vivo competition assay, the pleckstrin homology domain of Btk was shown to interact with the beta gamma dimer of heterotrimeric guanine nucleotide-binding proteins (G proteins). A highly conserved tryptophan residue in subdomain 6 of the pleckstrin homology domain was shown to play a critical role in the binding. The interaction of Btk with beta gamma suggests the existence of a unique connection between cytoplasmic tyrosine kinases and G proteins in cellular signal transduction.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

A fifth member of the mammalian G-protein beta-subunit family. Expression in brain and activation of the beta 2 isotype of phospholipase C.

The beta-subunits of the heterotrimeric GTP-binding proteins (G-proteins) are important regulators of G-protein alpha-subunits as well as of certain signal transduction receptors and effectors. We have identified a fifth member of the G-protein beta-subunit family, G beta 5, by molecular cloning. Sequence analysis of recombinant clones revealed an open reading frame of 1,059 base pairs, potentially encoding a protein of 353 amino acids having 53% identity to previously identified G beta-subunit proteins. Northern blot analysis of poly(A)-selected mRNA prepared from different mouse tissues demonstrated that G beta 5 is predominantly expressed in adult brain as two transcripts of 2.3 and 1.75 kilobases. A G beta 5-specific antiserum detected a band of 39.3 kilodaltons on immunoblots of crude membrane proteins isolated from normal mouse brain but not from other tissues. Using a transient cotransfection assay, we showed that a plasmid construct expressing the G beta 5 open reading frame, when coexpressed with different G-protein gamma (G gamma)-subunits, can stimulate the activity of the beta 2 isoform of phosphoinositide-specific phospholipase C (PLC beta 2). The stimulation of PLC beta 2 was most pronounced when G beta 5 was co-expressed with G gamma 2, although activation was observed when G beta 5 was cotransfected with either G gamma 1, G gamma 3, G gamma 5, or G gamma cone. These results prove that G beta 5 can functionally associate with G gamma proteins and is therefore a bona fide member of the G beta protein family. The rather low level of identity between G beta 5 and the other mammalian G beta-subunits may shed light on structural/sequence elements necessary for G beta protein function.

Amino Acid Sequence↗
PubMed1994

Selection of chromosome 22-specific clones from human genomic BAC library using a chromosome-specific cosmid library pool.

A new approach to rapidly identify chromosome-specific subsets of clones from a total human genomic library is described. We report here the results of screening a human bacterial artificial chromosome (BAC) library using the total pool of clones from a chromosome 22-specific cosmid library as a composite probe. The human BAC library was gridded on filters at high density and hybridized with DNA from the pooled chromosome 22-specific Lawrist library under suppressive conditions. In a single hybridization, we picked 280 candidates from the BAC library representing over 30,000 clones (or 1.2x coverage of human genome). This subset contained more than 60% of the chromosome 22-specific BAC clones that were previously found to be present in the original BAC library. In principle, this approach can be applied to select a subset of clones from other global libraries with relatively large inserts using a pool from a regional library as a composite probe. It is important to note that the target and probe libraries must be based on vectors that share no homology with each other.

Chromosomes, Bacterial↗
PubMed1994

The human blue opsin promoter directs transgene expression in short-wave cones and bipolar cells in the mouse retina.

Transgenic mouse lines were generated using either 3.8 or 1.1 kb of 5' upstream flanking sequence from the human blue opsin gene fused to the lacZ or human growth hormone reporter gene. Mice were analyzed for appropriate cell-specific and developmental expression patterns. In 13 independently derived lines of animals, transgene expression was limited to photoreceptor and inner nuclear layer cells. Photoreceptors were identified as cone cells based on morphological criteria and colocalization of transgene expression with the cone-associated marker, peanut agglutinin lectin. More specifically, transgene-positive photoreceptors were identified as short-wave cone cells (S-cones) by using the short-wave color opsin-specific antibody, OS-2. Reporter-gene-positive cells of the inner nuclear layer were identified as bipolar cells based on morphological criteria. Transgenes and the endogenous mouse short-wave opsin gene were transcriptionally coactivated at embryonic day 13. These results show that 3.8 or 1.1 kb of human blue opsin upstream flanking sequences are capable of directing expression in short-wave cone cells in a spatially and temporally appropriate fashion and that the human blue opsin gene is the homologue of the short-wave-sensitive pigment, S-opsin, in the short-wave cones of the mouse retina. Expression in the bipolar cells may reflect regulatory mechanisms that are common to these cells and to the cone photoreceptors.

Animals↗
PubMed1994

Activation of phospholipase C beta 4 by heterotrimeric GTP-binding proteins.

Transient transfection assays were used to determine how the activity of phospholipase C beta 4, which is preferentially expressed in retina, was regulated. An expression vector carrying the full-length cDNA corresponding to phospholipase C beta 4 was constructed and co-transfected into COS-7 cells together with cDNA encoding the alpha subunits of the Gq class and various beta and gamma subunits corresponding to the heterotrimeric GTP-binding proteins. We found that all the alpha subunits of the Gq class, including G alpha q, G alpha 11, G alpha 14, G alpha 15, and G alpha 16 could activate PLC beta 4 and that none of the G beta gamma subunits that we tested including G beta 1 gamma 1, G beta 1 gamma 2, G beta 1 gamma 3, or G beta 2 gamma 2 activated phospholipase C beta 4. In control experiments, cotransfection with cDNA encoding the alpha subunit of transducin or Gi2 gave no activation of PLC beta 4. These results indicate that phospholipase C beta 4 is activated by G alpha subunits that are members of the Gq class, and, like the phospholipase C beta 1 isoform, it is refractory to activation in the transfection assay by many of the combinations of beta and gamma subunits found in the heterotrimeric G-proteins.

Amino Acid Sequence↗
PubMed1994

Protein histidine kinases and signal transduction in prokaryotes and eukaryotes.

During the past several years, it has become apparent that prokaryotic organisms process much of their sensory information through families of similar proteins that make up two-component signal transducing systems. Biochemical and genetic analyses have clarified our understanding of how these proteins mediate the signaling circuitry necessary to allow an appropriate response. Recent advances in this field suggest that 'primitive' signaling systems may involve molecular strategies and mechanisms that are conserved in more complex organisms.

Bacterial Proteins↗
PubMed1994

Histidine and aspartate phosphorylation: two-component systems and the limits of homology.

Autophosphorylating histidine kinase and response-regulator domains constitute the building blocks of two-component signaling systems. These systems use a unique phosphotransfer chemistry to regulate many aspects of bacterial physiology. Homologous systems are now being found in eukaryotes. Despite their common mechanism of phosphotransfer, the two-component systems display an extensive diversity in the arrangement of their domains, and flexibility in their roles in different signal transduction circuits.

Animals↗
PubMed1994

Downregulation of cGMP phosphodiesterase induced by expression of GTPase-deficient cone transducin in mouse rod photoreceptors.

PURPOSE: Photoexcitation of vertebrate retinal rod photoreceptors stimulates GTP binding to the transducin alpha subunit. Like other GTP-binding proteins, transducin restores itself to an inactive form by hydrolyzing its bound GTP. The role of GTP hydrolysis in phototransduction was investigated. METHODS: A mutant form of cone transducin alpha deficient in its ability to hydrolyze bound GTP was expressed in mouse rod photoreceptors. RESULTS: Expression of the mutant cone transducin alpha at levels threefold to sixfold higher than endogenous rod transducin alpha led to a specific depletion of the transducin target, cGMP phosphodiesterase, and a decrease in the cGMP level. Suction electrode recordings revealed abnormally prolonged flash responses, decreased maximal response amplitudes, and a shift in the stimulus-response relation to higher flash strengths. CONCLUSIONS: Rods expressing high levels of GTPase-deficient cone transduction alpha have reduced levels of phosphodiesterase catalytic subunits and cGMP. These changes are associated with prolonged flash responses, reduced dark current, and decreased sensitivity to light.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Random mutagenesis of G protein alpha subunit G(o)alpha. Mutations altering nucleotide binding.

Nucleotide binding properties of the G protein alpha subunit G(o)alpha were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [35S]GTP gamma S (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G(o)alpha cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G(o)alpha for GTP gamma S or Mg2+ were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTP gamma S binding but retained the capacity to interact with the beta gamma subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G(o)alpha increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype.

Animals↗