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Biomedical subjects

M I Simon

Publications and source records attributed to M I Simon.

At least 163 records · Page 9Linked to original sources
PubMed1991

G alpha 16, a G protein alpha subunit specifically expressed in hematopoietic cells.

Signal-transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins) determine many of the responses of hematopoietic cells. A recently identified gene encoding a G protein alpha subunit, G alpha 16, is specifically expressed in human cells of the hematopoietic lineage. The G alpha 16 cDNA encodes a protein with predicted Mr of 43,500, which resembles the G q class of alpha subunits and does not include a pertussis toxin ADP-ribosylation site. In comparison with other G protein alpha subunits, the G alpha 16 predicted protein has distinctive amino acid sequences in the amino terminus, the region A guanine nucleotide-binding domain, and in the carboxyl-terminal third of the protein. Cell lines of myelomonocytic and T-cell phenotype express the G alpha 16 gene, but no expression is detectable in two B-cell lines or in nonhematopoietic cell lines. G alpha 16 gene expression is down-regulated in HL-60 cells induced to differentiate to neutrophils with dimethyl sulfoxide. Antisera generated from synthetic peptides that correspond to two regions of G alpha 16 specifically react with a protein of 42- to 43-kDa in bacterial strains that overexpress G alpha 16 and in HL-60 membranes. This protein is decreased in membranes from dimethyl sulfoxide-differentiated HL-60 cells and is not detectable in COS cell membranes. The restricted expression of this gene suggests that G alpha 16 regulates cell-type-specific signal-transduction pathways, which are not inhibited by pertussis toxin.

Amino Acid Sequence↗
PubMed1991

Diversity of G proteins in signal transduction.

The heterotrimeric guanine nucleotide-binding proteins (G proteins) act as switches that regulate information processing circuits connecting cell surface receptors to a variety of effectors. The G proteins are present in all eukaryotic cells, and they control metabolic, humoral, neural, and developmental functions. More than a hundred different kinds of receptors and many different effectors have been described. The G proteins that coordinate receptor-effector activity are derived from a large gene family. At present, the family is known to contain at least sixteen different genes that encode the alpha subunit of the heterotrimer, four that encode beta subunits, and multiple genes encoding gamma subunits. Specific transient interactions between these components generate the pathways that modulate cellular responses to complex chemical signals.

Amino Acid Sequence↗
PubMed1991

Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis.

We describe a technique that uses reverse transcription and the polymerase chain reaction (pcr) to rapidly quantitate numbers of specific mRNA transcripts from nanogram quantities of total cellular RNA. Linearity of input molecules to output signal was maintained by limiting the cycle number and the amount of input RNA and by minimizing the number of manipulations. Absolute levels of specific transcripts were determined by the inclusion of a separate standard curve composed of serially diluted in vitro transcribed RNA run alongside the experimental samples. This allowed rapid quantitation of many samples simultaneously. We applied this technique to measuring the expression of phosphoglycerate kinase 2 (Pgk-2) transgenes in the mouse testis during development. A human PGK-2 transgene, a PGK-2/CAT transgene, and the endogenous mPgk-2 gene all displayed similar patterns and levels of expression, consistent with the conclusion that peak RNA accumulation occurs in pachytene spermatocytes. Mouse protamine 2 (mP2) is expressed at a level approximately tenfold higher than Pgk-2 and displays a different pattern of expression consistent with initiation of transcription occurring in haploid round spermatids.

Animals↗
PubMed1991

Transposon-facilitated DNA sequencing.

We describe here a transposon-based DNA sequencing strategy that allows the introduction of sequencing priming sites throughout a target sequence by bacterial mating. A miniplasmid was designed to select against transposon insertions into the vector. Sites of transposon insertion are mapped by the polymerase chain reaction with bacterial overnight cultures providing the templates. A small set of plasmids with transposons spaced several hundred base pairs apart can then be sequenced. Sequencing primers corresponding to the transposon ends allow sequencing in both directions. Thus, the entire sequence of both strands can be easily determined.

Base Sequence↗
PubMed1991

Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice.

Chimeric gene fusions between 4.4 kb of rod opsin 5' flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5' flanking sequence fused to the E. coli IacZ gene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-IacZ transgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression.

Animals↗
PubMed1991

Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.

Amino Acid Sequence↗
PubMed1990

The dynamics of protein phosphorylation in bacterial chemotaxis.

The chemotaxis signal transduction pathway allows bacteria to respond to changes in concentration of specific chemicals (ligands) by modulating their swimming behavior. The pathway includes ligand binding receptors, and the CheA, CheY, CheW, and CheZ proteins. We showed previously that phosphorylation of CheY is activated in reactions containing receptor, CheW, CheA, and CheY. Here we demonstrate that this activation signal results from accelerated autophosphorylation of the CheA kinase. Evidence for a second signal transmitted by a ligand-bound receptor, which corresponds to inhibition of CheA autophosphorylation, is also presented. We postulate that CheA can exist in three forms: a "closed" form in the absence of receptor and CheW; an "open" form that results from activation of CheA by receptor and CheW; and a "sequestered" form in reactions containing ligand-bound receptor and CheW. The system's dynamics depends on the relative distribution of CheA among these three forms at any time.

Bacterial Proteins↗
PubMed1990

Importance of minor-groove contacts for recognition of DNA by the binding domain of Hin recombinase.

Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein.

Amino Acid Sequence↗
PubMed1990

Orientation of the putative recognition helix in the DNA-binding domain of Hin recombinase complexed with the hix site.

On the basis of sequence similarity with other known DNA-binding proteins, the DNA-binding domain of Hin recombinase, residues 139-190, is thought to bind DNA by a helix-turn-helix motif. Two models can be considered that differ in the orientation of the recognition helix in the major groove of DNA. One is based on the orientation of the recognition helix found in the 434 repressor (1-69) and lambda repressor-DNA cocrystals, and the other is based on the NMR studies of lac repressor headpiece. Cleavage by EDTA.Fe attached to a lysine side chain (Ser183----Lys183) near the COOH terminus of Hin(139-184) reveals that the putative recognition helix is oriented toward the center of the inverted repeats in a manner similar to that seen in the 434 and lambda repressor-DNA cocrystals.

Amino Acid Sequence↗
PubMed1990

The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels.

We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.

Base Sequence↗
PubMed1990

Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis.

A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was used quantitatively to measure accumulated levels of RNA transcripts in total mouse RNAs derived from male germ cells at various spermatogenic stages. RNA levels for two X-linked enzymes, phosphoglycerate kinase (PGK-1) and hypoxanthine phosphoribosyl transferase (HPRT), both decrease during spermatogenesis, although the transcript levels decrease much more rapidly for PGK-1. RNA for the Y-linked ZFY (zinc finger protein) is elevated in all spermatogenic cell fractions tested, being particularly high in leptotene/zygotene spermatocytes and round spermatids. RNA for adenine phosphoribosyltransferase (APRT) increases 5-fold to a peak during late pachynema. RNA for PGK-2, undetectable in spermatogonial cells, increases at least 50-fold by the round spermatid stage. DNA (cytosine-5-)-methyltransferase (MTase) transcript levels are over an order of magnitude higher throughout spermatogenesis than in non-dividing liver cells.

Adenine Phosphoribosyltransferase↗
PubMed1990

Gz, a guanine nucleotide-binding protein with unique biochemical properties.

Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.

Amino Acid Sequence↗
PubMed1990

Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase.

Stimulation of the T lymphocyte antigen receptor-CD3 complex (TCR-CD3) causes T cell activation by a process associated with increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Evidence exists suggesting that GTP-binding (G) proteins, particularly the pertussis toxin (PT)-sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR-CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte-specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL-2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck-PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT-sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.

Animals↗
PubMed1990

Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY.

The CheY protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY protein is to stabilize a transient conformational change that can generate tumbling behavior.

Aspartic Acid↗
PubMed1990

Alternative splicing produces transcripts encoding two forms of the alpha subunit of GTP-binding protein Go.

The alpha subunit of the guanine nucleotide-binding protein Go ("o" for other) is believed to mediate signal transduction between a variety of receptors and effectors. cDNA clones encoding two forms of Go alpha subunit were isolated from a mouse brain library. These two forms, which we call GoA alpha and GoB alpha, appear to be the products of alternative splicing. GoA alpha differs from GoB alpha over the C-terminal third of the deduced protein sequence. Both forms are predicted to be substrates for ADP-ribosylation by pertussis toxin. GoA alpha transcripts are present in a variety of tissues but are most abundant in brain. The GoB alpha transcript is expressed at highest levels in brain and testis. It is possible that GoA alpha and GoB alpha have different functions.

Amino Acid Sequence↗