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M Linial

Publications and source records attributed to M Linial.

At least 55 records · Page 3Linked to original sources
PubMed1993

Brain contains two forms of synaptic vesicle protein 2.

Molecular cloning of a cDNA encoding synaptic vesicle protein 2 (SV2) revealed that it is homologous to a family of proton cotransporters from bacteria and fungi and to a related family of glucose transporters found in mammals. The similarity to proton cotransporters raised the possibility that SV2 might mediate the uptake of neurotransmitters into vesicles, an activity known to require a proton gradient. To determine whether SV2 is a member of a family of vesicular proteins, we used the SV2 clone to screen for similar cDNAs in rat brain. We characterized 42 clones, 25 of which encode SV2 and 4 of which encode a protein, SV2B, that is 65% identical and 78% similar to SV2. The protein encoded by SV2B cDNA is recognized by the monoclonal antibody that defines the SV2 protein. When SV2B is expressed in COS cells, antibody labeling is reticular in nature, suggesting that SV2B, like SV2 (hence, SV2A), is segregated to intracellular membranes. The expression of SV2B is limited to neural tissue. While both forms of SV2 are expressed in all brain regions, SV2B is expressed at highest levels in the cortex and hippocampus, whereas the highest level of expression of SV2A is in subcortical regions. Therefore, the SV2 proteins, like other characterized synaptic vesicle proteins, comprise a small gene family.

Amino Acid Sequence↗
PubMed1993

VAT-1 from Torpedo is a membranous homologue of zeta crystallin.

VAT-1 is a major protein from Torpedo synaptic vesicles. A protein data-base search revealed a striking homology to zeta crystallin from guinea pig lens. The overall amino-acid identity is 27%, and 58% similarity is reached by including conserved substitutions. The highest similarity (60% to 85%) between the two proteins is observed in five discrete domains, which are also conserved in zinc-dependent dehydrogenases, particularly in the alcohol dehydrogenase family. The cofactor-binding domain of oxidoreductases is conserved in VAT-1 and in zeta crystallin. VAT-1 preferably binds NADPH in the presence of zinc. In contrast with its homologous proteins, VAT-1 is an integral membrane protein of synaptic vesicles.

Amino Acid Sequence↗
PubMed1993

VAT-1 from Torpedo synaptic vesicles is a calcium binding protein: a study in bacterial expression systems.

1. Calcium binding properties were examined in VAT-1, an abundant 41-kDa membrane protein expressed in the cholinergic cynaptic vesicles of Torpedo. 2. An overlay assay, using 45Ca2+ as a tracer, demonstrated the ability of a recombinant VAT-1 produced from the IPTG-inducible pKK223-3 expression vector to bind calcium. 3. A high yield of recombinant VAT-1 was obtained from the glutathione S-transferase (GST) expression system. The fusion product enabled VAT-1 purification via affinity chromatography. Subsequent cleavage by thrombin resulted in its separation from the GST carrier protein. 4. A direct Ca(2+)-binding study was performed with purified VAT-1 by a quick-spin column technique, in the presence of 45Ca2+. Quantitative analysis revealed a 1:1 molar stoichiometry for binding of Ca2+ to VAT-1, with a dissociation constant of 130 microM. 5. A GST-linked truncated protein consisting of 13 kDa from the VAT-1 carboxy-terminal domain was found to retain the capacity to bind Ca2+. 6. A data search for homologies between VAT-1 and known Ca(2+)-binding proteins revealed considerable similarity to members of the annexin family in a 140-amino acid region from the carboxy terminal of VAT-1, which overlaps two tandem Ca(2+)-binding domains of the annexin proteins.

Adenosine Triphosphatases↗
PubMed1992

Sharing of antigenic epitopes between synaptophysin and granulophysin.

The immunological crossreactivity between the two granule-specific membrane glycoproteins, synaptophysin and granulophysin, was studied using a series of site-specific monoclonal and polyclonal antibodies. The epitope relatedness of six monoclonal antibodies against granulophysin was examined by competitive ELISA. The antibodies are shown to recognize distinct, but overlapping epitopes within a compact region that is constructed by the three-dimensional configuration of the molecule. All these antibody clones also recognize rat neuronal synaptophysin. Two monoclonal antibodies against synaptophysin, of which one is the well-characterized SY38 antibody, directed against the carboxy terminal of the molecule, are also shown to react with granulophysin. Characterized polyclonal antibodies against different peptide antigens of synaptophysin failed to recognize granulophysin. Synaptophysin and granulophysin are distinctly recognized in brain cell (white matter) and the pituitary both qualitatively and quantitatively. Based on these and other observations, it is suggested that the repeat motif in the cytoplasmic tail of synaptophysin represents an immunodominant construct that is the target for the observed crossreactive antibodies and that a similar tertiary construct has been preserved in granulophysin and in other transmembrane proteins.

Animals↗
PubMed1992

Structure and chromosomal localization of the mammalian agrin gene.

Agrin, a component of the synaptic basal lamina, has been shown to induce clustering of ACh receptors on the surface of muscle fibers. Analysis of cDNAs isolated from a rat embryonic spinal cord library demonstrated that agrin contains domains similar to regions of protease inhibitors, laminin and epidermal growth factor. The domain structure of agrin is further revealed here in an analysis of the agrin gene. Two additional internal repeated sequences are defined: one rich in cysteine residues with no homology to other proteins, and another similar to the laminin G domain, which is involved in heparin binding. Alternative RNA splicing at two positions in the gene predicts up to eight possible forms of the agrin protein. The gene (symbol AGRN/Agrn) has been assigned to chromosome 1 region pter-p32 in human and to mouse chromosome 4.

Agrin↗
PubMed1991

Specificity of retroviral RNA packaging.

Encapsidation of retroviral RNA has been shown to be dependent on specific cis-acting signals, in particular, the packaging region (psi) located near the 5' end of the retroviral genome. In this report, we show that a 683-base avian extended packaging sequence (psi+) derived from Rous sarcoma virus will direct packaging of heterologous hygromycin mRNA into avian virions when present at the 3' end of the transcript in the sense orientation. However, this packaging is not as efficient as the packaging of RNA encoded by a standard avian retroviral vector. A quail cell line containing a Rous sarcoma virus mutant, SE21Q1b, produces virions which will package endogenous cellular mRNAs randomly, roughly in proportion to their intracellular concentrations. We found that viral particles from SE21Q1b retain the capacity to specifically encapsidate hygromycin mRNAs containing the avian psi+. To determine whether packaging of cellular mRNA would occur in other retroviral packaging lines, we assayed virion RNA isolated from the retroviral particles produced by avian and murine packaging lines for the presence of endogenous cellular mRNAs. Endogenous cellular mRNAs were not found randomly packaged into virions produced by any of the packaging lines examined except SE21Q1b. Some specific sequences, however, were found packaged into avian virions. Endogenous retrovirus-related mink cell focus-inducing murine leukemia virus RNAs and 30S viruslike RNAs were found to be efficiently packaged into murine virions even in the presence of RNAs containing all cis-acting retroviral sequences.

Animals↗
PubMed1990

Torpedo synaptophysin: evolution of a synaptic vesicle protein.

Synaptophysin is an integral membrane protein of synaptic vesicles found in neurons and endocrine cells. Synaptophysin monomers associate into hexamers forming a large conductance channel. We present an analysis of synaptophysin from the nervous system of the marine ray Torpedo californica. Analysis of cDNA clones reveals a 62% amino acid similarity between the Torpedo and rat sequences. The 4 hydrophobic membrane spanning domains and the glycosylation site are conserved. In contrast, the two intravesicular loops connecting the membrane spanning regions, show varying degrees of sequence conservation, suggesting that portions of these domains may play critical functional roles. The carboxyterminal tail has been proposed to bind calcium and is a major site for tyrosine phosphorylation. The precise sequence of this region has almost completely diverged while the proline-tyrosine rich nature is maintained. Blotting studies reveal the RNA and the protein in nervous system tissues and demonstrate that the molecule copurifies with cholinergic synaptic vesicles.

Amino Acid Sequence↗
PubMed1990

A unique neurofilament from Torpedo electric lobe: sequence, expression, and localization analysis.

A set of cDNA clones encoding a protein highly homologous to the mammalian middle-size class of neurofilaments (NF-M) was characterized. The amino acid similarity between the Torpedo and rat NF-M approaches 90% in the amino-terminal "rod-like" domain and is significantly lower in the carboxy-terminal tail. The Torpedo protein contains 13 tandem repeats of a unique six amino acid core, containing a Lys-Ser-Lys putative phosphorylation site. Surprisingly, the 3' untranslated region contains stretches of 80-90% nucleic acid homology with the mammalian, but not with the chicken sequences. This homology is greater than much of the coding region, suggesting that the 3' untranslated region of the message has an important functional role, perhaps governing RNA stability or localization. This Torpedo NF-M mRNA is expressed specifically in the electric lobe and was not detected in other tissues, including brain and spinal cord. A polyclonal antibody generated against a fusion protein synthesized in E. coli detects a 150-kDa protein in the electric lobe and organ, as well as a small amount of material in the brain. Cytochemical studies reveal immunoreactivity in electromotor neuron axons and terminals. Specific expression of neurofilament genes in subsets of central neurons may be important in determining the morphology and functional characteristics of specific neuronal subtypes.

Amino Acid Sequence↗
PubMed1989

VAT-1: an abundant membrane protein from Torpedo cholinergic synaptic vesicles.

Expression screening was used to isolate cDNA clones encoding a synaptic vesicle membrane protein, VAT-1, which is specifically expressed in the electric lobe of marine rays. The predicted protein has a molecular weight of 41,572 daltons and contains several hydrophobic regions. An antibody raised against a fusion protein synthesized in E. coli recognizes an abundant 42 kd protein that copurifies largely with synaptic vesicles. Trypsin digestion of intact and lysed vesicles as well as membrane extractions suggests that VAT-1 is an integral membrane protein. The VAT-1 RNA is localized to the electromotor nucleus, and the fusion protein antibody stains the electric organ, demonstrating that the protein is transported to nerve terminals. These studies define a novel synaptic vesicle protein that is likely to play a central role in the functions mediated by specific classes of synaptic vesicles.

Acetylcholine↗
PubMed1988

Bent DNA structures associated with several origins of replication are recognized by a unique enzyme from trypanosomatids.

Sequence-directed bending of the DNA double helix is a conformational variation found in both prokaryotic and eukaryotic organisms. The utilization of bent DNA structures from various sources as specific signals recognized by an enzyme is demonstrated here using a unique endonuclease purified from trypanosomatid cells. Crithidia fasciculata nicking enzyme was previously shown to recognize specifically the bent structure found in kinetoplast DNA minicircles. The binding constant measured for this specific interaction is of two orders of magnitude higher than that measured for the binding of the enzyme to a non-curved sequence. As determined by binding competition and mobility shift electrophoresis analyses, this enzyme recognizes the sequence-directed bends associated with the origins of replication of bacteriophage lambda and simian virus 40 (SV40), as well as that located within the autonomously replicating sequence (ARS1) region of the yeast S. cerevisiae.

Animals↗
PubMed1988

A unique endonuclease from Crithidia fasciculata which recognizes a bend in the DNA helix. Specificity of the cleavage reaction.

The introduction of a single nick in DNA circles by Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) requires the presence of a bent structure in the DNA helix. However, the sequence directing the local bending of the DNA helix is not per se a preferred site for nicking by the enzyme. No extensive sequence specificity is involved in defining the cleavage site for C. fasciculata nicking enzyme in the duplex circular DNA substrate. However, the abundance of A and T residues is significantly high at both the 3' and the 5' termini generated at the nicked site. Nicking of the sequence-directed bent fragment from C. fasciculata kinetoplast DNA minicircles correlates with the periodicity determined by the unique nucleotide distribution in the bent sequence, reflected in its thermodynamic parameters. Occurrence of nicking is best correlated with the predicted minima of the melting temperature and delta G profiles, as well as with A and T dinucleotide sequences at the nicked site, in both the supercoiled and the relaxed sequence-directed bent DNA substrates. The potential role of the bend-dependent nicking reaction in the replication of kinetoplast DNA minicircles is discussed.

Animals↗
PubMed1988

Lability of leukosis virus enhancer-binding proteins in avian hematopoeitic cells.

Bursal lymphomas induced by avian leukosis virus (ALV) are characterized by integration of long terminal repeat (LTR) enhancer sequences next to the myc proto-oncogene and by subsequent myc hyperexpression. Nuclear runoff transcription analyses have shown that protein synthesis inhibition specifically decreases transcription of LTR-enhanced genes in bursal lymphoma cell lines (M. Linial, N. Gunderson, and M. Groudine, Science 230:1126-1132, 1985). Here, we show that LTR-enhanced transcription is also labile in nontransformed bursa, bone marrow, and spleen but not in other ALV-infected tissues from lymphoma-susceptible chickens. The bursal cells demonstrated this lability of LTR-enhanced transcription only at an early stage of development, when chickens are susceptible to ALV-induced lymphomagenesis. Mature bursal cells show stable LTR transcription enhancement (unaffected by inhibition of protein synthesis) and are not susceptible to lymphomagenesis. In lymphoma-resistant chicken strains, LTR-enhanced transcription was stable in all tissues during development. These data suggest that lability of LTR transcription enhancement in hematopoietic cells is involved in susceptibility to lymphomagenesis, and we propose a model for the action of these labile enhancing factors. Gel shift analysis of nuclear proteins from lymphoma cells indicated that four or more binding proteins specifically interact with the three LTR enhancer regions. These proteins can be separated by their differential sensitivity to heat treatment or protein synthesis inhibition. The lability of a subset of these binding proteins correlates with lability of LTR-enhanced transcription in certain lymphoid cell types, suggesting that these proteins are essential for LTR transcription enhancement.

Animals↗
PubMed1987

The sequence-directed bent structure in kinetoplast DNA is recognized by an enzyme from Crithidia fasciculata.

Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) interrupts a single phosphodiester bond in duplex DNA circles from various sources, only in their supercoiled form, but not following their relaxation by DNA topoisomerases. However, this requirement for DNA substrate supercoiling was not observed using the natural kinetoplast DNA as a substrate. Relaxed kinetoplast DNA minicircles, either free or topologically linked, were efficiently nicked by the enzyme. Furthermore, bacterial plasmids, containing a unit length kinetoplast DNA minicircle insert, were used as substrates for nicking in their relaxed form. This capacity to activate a relaxed DNA topoisomer as a substrate for nicking is an intrinsic property of the sequence-directed bend, naturally present in kinetoplast DNA. The 211-base pair fragment of the bent region from C. fasciculata kinetoplast DNA could support the nicking of a relaxed DNA substrate in a reaction dependent upon the DNA helix curvature.

Animals↗
PubMed1987

Creation of a processed pseudogene by retroviral infection.

We have previously characterized a cell line transformed by a Rous sarcoma virus mutant, SE21Q1b, which contains a mutation preventing encapsidation of genomic RNA. A unique property of this mutant is that cellular RNAs are packaged into virions, even in the presence of replication-competent virus. In the current study, SE21Q1b quail cells were transfected with the plasmids pRSVneo or pCMVneo. Virions produced by SE21Q1b neoR clones contained neo RNA, and when virus from some SE21Q1b neoR clones was used to infect a chemically transformed quail cell line, QT35, neoR QT35 clones were obtained that contained single integrated copies of the neo gene. An intron inserted into pRSVneo was removed during gene transfer. These data are consistent with transfer of neo mRNAs by a pathway involving reverse transcription of mRNA encapsidated within SE21Q1b virions, and integration of resultant cDNAs into the genome of infected QT35 cells.

Animals↗
PubMed1987

Sequence-directed bent DNA helix is the specific binding site for Crithidia fasciculata nicking enzyme.

The sequence-directed bent structure of kinetoplast DNA minicircles specifies a unique binding site for Crithidia fasciculata nicking enzyme. Binding of the purified enzyme to the bent structure results in the formation of a tight enzyme-DNA complex that is highly specific to curved DNA. Recognition of the binding site is not determined by the nucleotide sequence at the site of binding per se but through the specific local variation in the DNA helix geometry. Both dynamic curved structures, which are generated by supercoiling, and static ones, which are sequenced-directed, could support and efficient enzyme-DNA complex formation. Binding interactions are dependent upon the degree of the helix curvature and decrease with the straightening of the binding site. DNase I protection experiments identify distinct domains of enzyme binding within the bent structure and suggest the induction of structural changes within these regions as a result of protein-DNA interactions.

Animals↗
PubMed1987

Retention or loss of v-mil sequences after propagation of MH2 virus in vivo or in vitro.

During propagation of the defective avian retrovirus MH2 in the presence of replication-competent helper virus, deletion of portions of the viral genome occurred frequently. After transformation of quail cells in vitro, v-mil sequences were lost, leading to populations of MH2 viruses which were highly deficient for mil gene expression but which could transform macrophage and fibroblast cells in vitro with high efficiency. In contrast, after induction of tumors in quail with mil-deficient MH2 viral stocks, a majority of the tumor DNAs contained mil+ proviruses, suggesting that there is selection for retention of the v-mil gene in vivo and that the mil protein may play a role in the oncogenicity of MH2 virus. We also isolated MH2-transformed cell lines which contained deleted proviruses arising from packaging and subsequent integration of the subgenomic v-myc-encoding mRNA. Some of these cell lines produced viruses which encoded abnormal v-myc proteins and had altered in vitro transforming properties. These altered phenotypes may be caused by mutations within the v-myc gene.

Alpharetrovirus↗