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Biomedical subjects

M I Simon

Publications and source records attributed to M I Simon.

At least 199 records · Page 11Linked to original sources
PubMed1988

Cone cell-specific genes expressed in retinoblastoma.

Retinoblastoma, an intraocular tumor that occurs in children, has long been regarded, on the basis of morphological criteria, as a malignancy of the photoreceptor cell lineage. Here it is shown that when this tumor is grown in vitro, the cells express highly specialized photoreceptor cell genes. Transcripts for the transducin alpha subunit, TC alpha, which is specific to the cone cell, as well as transcripts for the red or green cone cell photopigment, were found in seven out of seven low-passage retinoblastoma cell lines. No marker genes specific to rod cell were expressed, suggesting that retinoblastoma has a cone cell lineage.

DNA↗
PubMed1988

Mapping and gene order of U1 small nuclear RNA, endogenous viral env sequence, amylase, and alcohol dehydrogenase-3 on mouse chromosome 3.

Linkage was established between a number of genes that map on chromosome 3 by studying the distribution patterns of DNA polymorphisms and protein electrophoretic mobility polymorphisms in recombinant inbred (RI) strains of mice. This analysis resulted in the following suggested gene order between the newly assigned genes and previously mapped genes: gamma-fibrinogen (Fgg), Xmmv-22 of mink cell focus-inducing (MCF) virus, U1b small nuclear RNA gene cluster (Rnu-1b), amylase (Amy-1,2), cadmium resistance (cdm), alcohol dehydrogenase-3 (Adh-3), alcohol dehydrogenase-1 (Adh-1). In situ hybridization to chromosome spreads confirmed the assignment of the Ulb small nuclear RNA (snRNA) gene cluster and the gamma-fibrinogen gene to the center of chromosome 3.

Alcohol Dehydrogenase↗
PubMed1988

Isolation of the gene encoding the Hin recombinational enhancer binding protein.

In vitro DNA inversion mediated by the protein Hin requires the presence of a recombinational enhancer sequence located in cis relative to the recombination sites and a protein, Fis, which binds to the enhancer. We have cloned and determined the primary sequence of the gene encoding Fis. The deduced amino acid sequence of Fis indicates that the protein is 98 amino acids long and contains a potential helix-turn-helix DNA binding motif at its carboxyl terminus. The gene encoding Fis maps at 72 min on the Escherichia coli chromosome. The construction of mutant strains of E. coli that lack a functional fis gene demonstrates that Fis is not essential for cell growth under laboratory conditions but is required for high rates of Hin-mediated site-specific inversion in vivo.

Amino Acid Sequence↗
PubMed1988

Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin.

Recent molecular cloning of cDNA for the alpha subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called Tr and Tc, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, we have identified a G-protein alpha subunit, which we refer to as Gz alpha, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine Tr alpha-subunit cDNA. The deduced amino acid sequence of Gz alpha is 41-67% identical with those of other known G-protein alpha subunits. However, the 355-residue Gz alpha lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein alpha subunits. We suggest that Gz alpha, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

Adenosine Diphosphate Ribose↗
PubMed1988

Phase variation in Salmonella: analysis of Hin recombinase and hix recombination site interaction in vivo.

The bacteriophage P22-based challenge phase selection was used to characterize the binding of Salmonella Hin recombinase to the wild-type hixL and hixR recombination sites, as well as to mutant and synthetic hix sequences in vivo. Hin recombinase binds to the hixL or hixR recombination sites and represses transcription from an upstream promoter in the challenge phage system. Hin-mediated repression results from Hin associating into multimers either prior to binding or during the binding process at the hix operator sites (cooperativity). The ability of Hin multimers to repress transcription is eliminated when the hix 13-bp half-sites are rotated to opposite sides of the DNA helix by inserting 4 bp between them. Insertion of 1 bp between half-sites reduces overall repression. Hin also binds one of the hixL half-sites to repress transcription, but only when high levels of Hin protein are present in the cell. Mutations have been identified in the hix sites that impair Hin binding. Five of the 26 bp in the hix sites are critical; sites with base-pair substitutions at these five positions show greatly reduced binding. Three additional base pairs make minor contributions to binding. These results are consistent with the results of binding studies between Hin and the hix sites in vitro.

Base Sequence↗
PubMed1988

Purification and characterization of the wild-type and mutant carboxy-terminal domains of the Escherichia coli Tar chemoreceptor.

The carboxy-terminal half of the Escherichia coli Tar chemoreceptor protein was cloned into an overproducing plasmid with the transcription of the insert under the control of the strong hybrid tac promoter. Two dominant mutations in the tar gene, which result in "tumble-only" (tar-526) or "swim-only" (tar-529) phenotypes and which are postulated to produce proteins locked in specific signalling modes, were introduced separately onto the overproducing plasmid. After induction with isopropyl-beta-D-thiogalactopyranoside, cells containing the plasmids produced about 10% of their soluble cellular protein as the carboxy-terminal fragments. A scheme to purify the overproduced fragments was developed. Typical yields of pure fragment were 5, 30, and 20 mg per liter of induced culture for the wild type, 526 mutant, and 529 mutant, respectively. Fast-protein liquid chromatography-gel filtration analysis of the pure fragments showed that they all existed as oligomers (ca. 103,000 daltons), possibly trimers or tetramers (monomer size is 31,000 daltons). However, the 529 mutant fragment showed an additional oligomeric form (240,000 daltons) corresponding approximately to an octamer. When chromatographed in the presence of 1% octylglucoside, all three fragments showed an identical single oligomeric size of about 135,000 daltons. Further differences between the fragments such as ion-exchange behavior and susceptibility to degradation were found. Taken together, these results suggest that conformational differences between the 529 mutant fragment and the other fragments exist and that these differences may correlate with the phenotypic effects of the tar-529 mutation.

Bacterial Proteins↗
PubMed1988

Cloning of the C-terminal cytoplasmic fragment of the tar protein and effects of the fragment on chemotaxis of Escherichia coli.

A gene encoding only the C-terminal portion of the receptor-transducer protein Tar of Escherichia coli was constructed. The gene product was detected and localized in the cytoplasmic fraction of the cell by immunoblotting with anti-Tar antibodies. The C-terminal fragments from wild-type and mutant tar genes were characterized in vivo. The C-terminal fragment generated from tar-526, a mutation that results in a dominant "tumble" phenotype, was found to be deamidated and methylated by the CheB and CheR proteins, respectively. The C-terminal fragment derived from a wild-type gene was poorly deamidated, and the C-terminal fragment derived from tar-529, a dominant mutant with a "smooth swimming" phenotype, was not apparently modified. Cells carrying the C-terminal fragment with the tar-526 mutation as the sole receptor-transducer protein showed a high frequency of tumbling and chemotaxis responses to changes in intracellular pH. These results suggest that the cytoplasmic C-terminal fragment of Tar retains some of the functions of the whole protein in vivo.

Aspartic Acid↗
PubMed1987

Synthesis of a sequence-specific DNA-cleaving peptide.

A synthetic 52-residue peptide based on the sequence-specific DNA-binding domain of Hin recombinase (139-190) has been equipped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. In the presence of Fe(II), this synthetic EDTA-peptide cleaves DNA at Hin recombination sites. The cleavage data reveal that the amino terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin recombination sites. This work demonstrates the construction of a hybrid peptide combining two functional domains: sequence-specific DNA binding and DNA cleavage.

Bacterial Proteins↗
PubMed1987

Synthesis of a site-specific DNA-binding peptide.

The Hin recombinase binds to specific sites on DNA and mediates a recombination event that results in DNA inversion. In order to define the DNA-binding domain of the Hin protein two peptides 31 and 52 amino acids long were synthesized. Even though the 31mer encompassed the sequence encoding the putative helix-coil-helix-binding domain, it was not sufficient for binding to the 26-base pair DNA crossover site. However, the 52mer specifically interacted with the site and also effectively inhibited the Hin-mediated recombination reaction. The 52mer bound effectively to both the 26-base pair complete site and to a 14-base pair "half site." Nuclease and chemical protection studies with the 52mer helped to define the DNA base pairs that contributed to the specificity of binding. The synthetic peptide provides opportunities for new approaches to the study of the nature of protein-DNA interaction.

Amino Acid Sequence↗
PubMed1987

Assignment of the prealbumin (PALB) gene (familial amyloidotic polyneuropathy) to human chromosome region 18q11.2-q12.1.

The assignment of the human prealbumin (PALB) gene to chromosome region 18q11-q12.1 has been achieved using a human genomic probe in the study of human-mouse somatic cell hybrids and by in situ hybridization. Because familial amyloidotic polyneuropathy was reported previously to be due to a mutation in prealbumin, it can be inferred that the gene for this disorder also maps to 18q11.2-q12.1.

Amyloidosis↗
PubMed1987

Distinct forms of the beta subunit of GTP-binding regulatory proteins identified by molecular cloning.

Two distinct beta subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as beta 1 and beta 2 subunits. The bovine transducin beta subunit (beta 1) has been cloned previously. We have now isolated and analyzed cDNA clones that encode the beta 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue Mr 37,329 beta 2 protein is 90% identical with beta 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine beta 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than beta 1 subunit mRNA in all tissues examined. The beta 1 and beta 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that beta 1 and beta 2 are encoded by separate genes. The amino acid sequences for the bovine and human beta 2 subunit are identical, as are the amino acid sequences for the bovine and human beta 1 subunit. This evolutionary conservation suggests that the two beta subunits have different roles in the signal transduction process.

Adrenal Glands↗
PubMed1987

Protein phosphorylation is involved in bacterial chemotaxis.

The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis.

Bacterial Proteins↗
PubMed1987

Fis binding to the recombinational enhancer of the Hin DNA inversion system.

The recombinational enhancer of the Hin inversion system in Salmonella stimulates recombination in vitro 150-fold in the presence of the Escherichia coli host factor Fis. To gain an understanding of the roles of the enhancer and Fis in stimulating the Hin-mediated inversion reaction, we have used nuclease and chemical protection/interference studies and gel retardation assays to examine the interactions between Fis and the recombinational enhancer. These studies combined with mutational analysis defined the enhancer sequences required for Fis binding and function. Fis binds with different affinities to two domains within the enhancer sequence. The binding of Fis at each domain is independent of the occupancy of the other domain and appears to be to opposite faces of the DNA helix. These results support a model for the role of the recombinational enhancer in Hin-mediated inversion in which the interaction between Hin bound at recombination sites and Fis bound to each domain of the recombinational enhancer results in a structure with the proper alignment and topology to promote DNA inversion.

Base Sequence↗
PubMed1986

Host protein requirements for in vitro site-specific DNA inversion.

Flagellar phase variation is mediated by a recombination event that occurs at specific sites leading to inversion of a chromosomal segment of DNA. The presence of a 60 bp recombinational enhancer sequence on the DNA substrate molecule results in a 150-fold stimulation in the initial rate of inversion. The protein components required for inversion have been purified. They include the 21,000 dalton recombinase (Hin), a 12,000 dalton host protein (Factor II), and one of the major histone-like proteins of E. coli HU. The dependence of the initial rate of recombination on HU varies with respect to the location of the recombinational enhancer. The role of HU, Factor II, and the enhancer in facilitating site-specific recombination is discussed.

Bacterial Proteins↗
PubMed1986

Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA.

Injection of poly(A)+ RNA from rat brain into Xenopus oocytes caused the appearance of Cl currents in response to serotonin (5-HT) and acetylcholine (ACh). Both neurotransmitters evoked two-component currents similar in their time course to the oocyte's endogenous cholinergic muscarinic response, which was shown in previous studies to be mediated by IP3 synthesis leading to Ca release from intracellular stores. The responses to ACh and 5-HT exhibited self- and cross-desensitization, i.e., application of either ACh or 5-HT inhibited the subsequent response to either one of the two transmitters. Intracellular injection of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) mimicked the 5-HT and ACh response, and also completely suppressed the response to the subsequent application of either ACh or 5-HT. Treatment of the oocytes with pertussis toxin (PTX) caused a 50% attenuation of ACh and 5-HT responses. In the membranes of both control and mRNA-injected oocytes, PTX catalyzed the ADP-ribosylation of a single Mr = approximately 40,000 protein. Injection of the purified beta gamma-subunits of transducin enhanced the 5-HT response. The 5-HT and GTP-gamma-S responses were inhibited by intracellular injection of the Ca2+ chelator, EGTA, as previously shown for the ACh response. These data suggest that ACh and 5-HT receptors, synthesized in the oocytes on the template of brain mRNA, act through a common pathway that involves (a) a guanine nucleotide binding protein and (b) IP3 production leading to Ca mobilization.

Acetylcholine↗
PubMed1986

Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs.

Retinal transducin, a guanine nucleotide regulatory protein (referred to as a G protein) that activates a cGMP phosphodiesterase in photoreceptor cells, is comprised of three subunits. We have identified and analyzed cDNA clones of the bovine transducin beta subunit that may be highly conserved or identical to that in other G proteins. From the cDNA nucleotide sequence of the entire coding region, the primary structure of a 340-amino acid protein was deduced. The encoded beta subunit has a Mr of 37,375 and is comprised of repetitive homologous segments arranged in tandem. Furthermore, significant homology in primary structure and segmental sequence exists between the beta subunit and the yeast CDC4 gene product. The Mr 37,375 beta subunit polypeptide is encoded by a 2.9-kilobase (kb) mRNA. However, there exists in retina other beta-related mRNAs that are divergent from the 2.9-kb mRNA on the basis of oligonucleotide and primer-extended probe hybridizations. All mammalian tissues and clonal cell lines that have been examined contain at least two beta-related mRNAs, usually 1.8 and 2.9 kb in length. These results suggest that the mRNAs are the processed products of a small number of closely related genes or of a single highly complex beta gene.

Amino Acid Sequence↗