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M Zerial

Publications and source records attributed to M Zerial.

89 records · Page 5Linked to original sources
PubMed1990

Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments.

A set of 11 clones encoding putative GTP binding proteins highly homologous to the yeast YPT1/SEC4 gene products have been isolated from an MDCK cell cDNA library. We localized three of the corresponding proteins in mammalian cells by using affinity-purified antibodies in immunofluorescence and immunoelectron microscopy studies. One, the MDCK homolog of rab2, is associated with a structure having the characteristics of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus. The second, rab5, is located at the cytoplasmic surface of the plasma membrane and on early endosomes, while the third, rab7, is found on late endosomes. These findings provide evidence that members of the YPT1/SEC4 subfamily of GTP binding proteins are localized to specific exocytic and endocytic subcompartments in mammalian cells.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line.

Molecular analysis of Saccharomyces cerevisiae secretion mutants has led to the identification of two Ras-like GTP-binding proteins, Ypt1p and Sec4p, which are essential for transport along the exocytic route. To study the regulation of membrane traffic in epithelial cells, a set of 11 clones encoding proteins similar to the YPT1/SEC4 products were isolated from an MDCK (Madin-Darby canine kidney) cell cDNA library. Four of these proteins, Rab8, -9, -10, and -11, are novel members of this subfamily of Ras-like proteins, and two of them are closely related to Ypt1p and Sec4p. The ratio of the number of clones isolated over the total number screened reveals a high level of complexity for this subfamily of GTP-binding proteins. This diversity supports their proposed function in controlling different steps in membrane traffic.

Amino Acid Sequence↗
PubMed1989

The product of a novel growth factor activated gene, fos B, interacts with JUN proteins enhancing their DNA binding activity.

We have identified a gene, fos B, encoding a nuclear protein of 338 amino acids presenting a 70% homology with c-fos, whose expression is activated during G0/G1 transition. Growth factor stimulation of quiescent cells leads to a rapid and transient accumulation of fos B mRNA, with kinetics similar to those of c-fos. The induction of fos B mRNA levels is in part due to a dramatic increase in the transcription of the gene. The half-life of fos B mRNA is in the order of 10-15 min. Both transcriptional activation and mRNA stability are substantially increased in the presence of protein synthesis inhibitors. Immunoprecipitation studies showed that fos B as c-fos protein, forms a complex in vitro with c-jun and jun B proteins in the absence of a target binding sequence. Gel retardation assays demonstrated that fos B protein positively influences the binding of c-jun and jun B proteins to an AP-1 binding consensus sequence, suggesting that fos B protein plays a role in control of gene expression.

Amino Acid Sequence↗
PubMed1989

Coordinate induction of fibronectin, fibronectin receptor, tropomyosin, and actin genes in serum-stimulated fibroblasts.

From a collection of more than 80 nonoverlapping clones, isolated by differential screening of a lambda cDNA library prepared from serum-stimulated cells in the presence of cycloheximide, we have identified four clones that encoded for components of the cytoskeleton and extracellular matrix. DNA sequencing of clones B2, V58, TT1, and P38 demonstrated that they corresponded to beta-actin, alpha-tropomyosin, fibronectin, and the beta-subunit of fibronectin receptor. All four mRNA levels showed a detectable increase 30 min after stimulation and remained at high levels for at least 8 h. The half-lives of these mRNAs were found to be very long in contrast to those of other growth factor-inducible genes. An increase in transcription was observed for the four genes. Actin and fibronectin showed nearly maximal increase at 15 min, while fibronectin receptor and tropomyosin reached their maximum transcription at 1 h. These results demonstrated that four interacting components of the cytoskeleton and extracellular matrix are rapidly induced in stimulated quiescent cells, possibly reflecting part of the coordinate changes in gene expression that occur during embryogenesis and wound healing.

Actins↗
PubMed1989

Expression, cloning and cDNA sequence of a fibroblast serum-regulated gene encoding a putative actin-associated protein (p27).

A cDNA clone for a basic putative actin microfilament-associated protein, p27, highly induced in serum-stimulated NIH 3T3 cells, has been isolated by polyclonal antibodies and sequenced. p27 mRNA is a 1.2-kb molecule which is very low in resting NIH 3T3 cells but can be induced at least 100 times after 8 h of fetal calf serum stimulation. In contrast to other inducible mRNAs, p27 mRNA is stable, and its levels can be superinduced by cycloheximide mainly by prolonging transcription. The lack of expression of this messenger in mouse tissues, as well as in all cell lines so far tested, suggests that p27 may be an fibroblast-specific protein. One major open reading frame found in p27 cDNA codes for a 201 amino acid polypeptide not related to any previously described actin-binding protein. Interestingly, it shows alternative hydrophilic and hydrophobic domains of amino acids symmetrically arranged from the middle of the protein. The coordinate induction of p27 and actin mRNAs suggest that p27 may be involved in the cytoskeletal rearrangements induced early in cell growth and proliferation.

Amino Acid Sequence↗
PubMed1989

Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.

We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway.

Amino Acid Sequence↗
PubMed1988

A gene encoding a protein with zinc fingers is activated during G0/G1 transition in cultured cells.

Zinc fingers are DNA-binding domains present in several eukaryotic regulatory proteins. We have identified a mouse gene, Krox-20, encoding a protein with three zinc fingers and whose expression is activated during G0/G1 transition in cultured cells. Serum stimulation of quiescent cells leads to rapid and transient accumulation of Krox-20 mRNA, with kinetics similar to those of the c-fos proto-oncogene. The induction does not require de-novo protein synthesis. In the mouse, Krox-20 is expressed at low levels in tissues which contain rapidly dividing cells. These properties suggest that Krox-20 encodes a transcription control factor, possibly involved in the modulation of cell proliferation.

Amino Acid Sequence↗
PubMed1987

Nonrandom distribution of MMTV proviral sequences in the mouse genome.

Integrated sequences of mouse mammary tumor virus (MMTV) have been localized in the genomes of five inbred mouse strains (Balb/c, C3H, DBA/2, A.TH, 129-SV) and one mammary tumor cell line (GR). Two major classes of MMTV sequences have been detected in mouse DNA fractions as obtained by Cs2SO4/BAMD (3,6-bis-(acetatomercurimethyl)dioxane) density gradient centrifugation. The first one corresponds to previously described endogenous sequences (Mtv loci), whereas the second one corresponds to endogenous sequences not previously known, and/or recently acquired; in the case of GR cells exogenous sequences may also be present in this class. The genome distribution is somewhat different for the two classes of sequences, the first one being practically only present in the lightest DNA segments of the mouse genome (GC congruent to 38%); the second one being also represented in heavier segments (GC congruent to 43%). This integration pattern suggests that "ancient" endogenous sequences are practically only localized in genome segments of roughly matching composition, whereas exogenous and recently acquired endogenous MMTV sequences may also be present in heavier fractions.

Animals↗
PubMed1987

Foreign transmembrane peptides replacing the internal signal sequence of transferrin receptor allow its translocation and membrane binding.

Each subunit of the human transferrin receptor (TR) dimer is inserted into the ER membrane as a transmembrane polypeptide having its N-terminus in the cytoplasm. The transmembrane segment of the molecule serves both as a signal for chain translocation and as a membrane anchor. To study which structural features of this segment are required for its dual function, we have essentially replaced the transmembrane peptide with the C-terminal membrane-spanning segment of two proteins having a separate N-terminal translocation signal and with an artificial uncharged peptide. In each case the mutant TR molecules are efficiently translocated in vitro. In contrast, substitution of the transmembrane peptide of TR with a hydrophilic peptide results in no detectable translocation activity of the mutant TR. This suggests that the hydrophobic character of the transmembrane peptide of TR, rather than its actual amino acid sequence, is important for chain translocation and membrane binding.

Biological Transport↗
PubMed1987

Phosphorylation of the human transferrin receptor by protein kinase C is not required for endocytosis and recycling in mouse 3T3 cells.

We have investigated the role of phosphorylation in the endocytosis of the human transferrin receptor (TR) by replacing its phosphorylation site, Ser24, with Ala through site-directed mutagenesis of the TR cDNA. The TR Ala24 mutant expressed in mouse 3T3 cells was not phosphorylated, even following stimulation of protein kinase C by phorbol ester. However, in spite of this defect the mutant was efficiently endocytosed and recycled back to the plasma membrane with kinetics similar to those of TR and a control mutant TR Ala63. Thus, these results confirm earlier results by Davis et al. (1986, J. Biol. Chem., 261-9034-9041) that Ser24 of human TR is the phosphorylation site for protein kinase C but do not support a role of this modification as a signal for TR endocytosis and recycling.

Animals↗
PubMed1986

Genomic localization of hepatitis B virus in a human hepatoma cell line.

The integration of hepatitis B viral sequences in the human hepatoma Alexander cell line has been investigated after fractionation of the cell line DNA by centrifugation in a Cs2SO4/BAMD (3,6-(bis-acetato mercurimethyl) dioxane) density gradient. Eight out of nine integrated viral sequences were localized in DNA component H3, which only represents 4% of the human genome and matches the base composition of HBV sequences. These results indicate a targeting and/or a higher stability of the latter in a specific, small compartment of the host genome.

Carcinoma, Hepatocellular↗
PubMed1986

Gene distribution and nucleotide sequence organization in the mouse genome.

Mouse DNA was fractionated by preparative centrifugation in density gradients of Cs2SO4 containing 3,6-bis(acetatomercurimethyl)dioxane (BAMD). The effects of temperature, BAMD/nucleotide molar ratio and solvent on the fractionation were explored. The fractions so obtained were investigated by analytical centrifugation in CsCl density gradient and by hybridization with a number of gene probes. These approaches led to the definition of satisfactory conditions for the rapid fractionation of mouse DNA; to the localization of a number of genes in mouse DNA fractions; and to a better understanding of the mosaic organization of the mouse genome and, more specifically, to a better estimate of both the intermolecular and intramolecular compositional heterogeneity of mouse DNA in the (75-150) X 10(3)-base size range.

Actins↗
PubMed1986

Gene distribution and nucleotide sequence organization in the human genome.

Human DNA was fractionated by centrifugation in Cs2SO4 density gradients containing 3,6-bis(acetatomercurimethyl)dioxane (BAMD). Fractions were investigated in their analytical CsCl profiles and a number of specific sequences were localized in them. The results so obtained led to an improved understanding of the organization of nucleotide sequences in the human genome, as well as to the discovery that a class of DNA having a very high G + C content and not represented in the mouse genome, is particularly rich in genes and interspersed repetitive sequences.

Base Sequence↗
PubMed1986

The transmembrane segment of the human transferrin receptor functions as a signal peptide.

The human transferrin receptor (TR) is a protein comprising 760 amino acid residues that spans the membrane once with its N terminus towards the cytoplasm. It is synthesized without a cleavable signal peptide. We have tested whether the signal responsible for its membrane insertion is present within its transmembrane peptide using a combined recombinant DNA/in vitro translation approach. The complete TR coding region was first reconstructed from overlapping TR cDNA clones and then engineered into an SP6-based transcription vector. In vitro transcription and subsequent translation in the presence of rough microsomes yielded TR molecules that were glycosylated and correctly inserted into the membrane. Two kinds of experiments demonstrated that the spanning region of the TR polypeptide contained the signal for translocation across the membrane of the rough endoplasmic reticulum. First, we deleted the spanning region of TR and showed that this deletion mutant could not be inserted. Second, we showed that two cytoplasmic proteins (the mouse dihydrofolate reductase and the chimpanzee alpha-globin) could be inserted into the microsomal membrane in the expected orientation when the TR transmembrane segment was added to their N termini. Thus, the spanning peptide was shown to be both necessary and sufficient for chain translocation. Further analyses demonstrated that the translocation event was dependent on the signal recognition particle.

Cell Membrane↗
PubMed1985

The mosaic genome of warm-blooded vertebrates.

Most of the nuclear genome of warm-blooded vertebrates is a mosaic of very long (much greater than 200 kilobases) DNA segments, the isochores; these isochores are fairly homogeneous in base composition and belong to a small number of major classes distinguished by differences in guanine-cytosine (GC) content. The families of DNA molecules derived from such classes can be separated and used to study the genome distribution of any sequence which can be probed. This approach has revealed (i) that the distribution of genes, integrated viral sequences, and interspersed repeats is highly nonuniform in the genome, and (ii) that the base composition and ratio of CpG to GpC in both coding and noncoding sequences, as well as codon usage, mainly depend on the GC content of the isochores harboring the sequences. The compositional compartmentalization of the genome of warm-blooded vertebrates is discussed with respect to its evolutionary origin, its causes, and its effects on chromosome structure and function.

Animals↗
PubMed1984

Mobilization of heparan sulfate induced by immunostimulation in a patient with mucopolysaccharidosis IIIA.

Unspecific immunostimulation by bacterial vaccines of a patient with mucopolysaccharidosis IIIA (Sanfilippo A) syndrome induces a marked increase in the urinary excretion of heparan sulfate and of uronate-containing oligosaccharides. This event is presumably linked to an increased vascular permeability and exocytosis of storage material, elicited by mediators of inflammation, as well as to enhanced degradation of stored polymers in activated macrophages and surrounding tissue.

Bacterial Vaccines↗