The nucleotide sequence of the luciferase gene from firefly Photinus pyralis was determined analysis cDNA and genomic clones. contains six introns, all less than 60 bases in length. 5' end mRNA by both S1 nuclease primer extension. Although clone lacked N-terminal codons open reading frame, we were able to reconstruct equivalent a full-length using as source missing sequence. full-length, intronless inserted into mammalian expression vectors introduced monkey (CV-1) cells which enzymatically...

The firefly luciferase protein contains a peroxisomal targeting signal at its extreme COOH terminus (Gould et al., 1987). Site-directed mutagenesis of the gene reveals that this consists COOH-terminal three amino acids protein, serine-lysine-leucine. When tripeptide is appended to cytosolic (chloramphenicol acetyltransferase), it sufficient direct fusion into peroxisomes. Additional experiments reveal only limited number conservative changes can be made in without abolishing activity. These...

Translocation of proteins across membranes the endoplasmic reticulum, mitochondrion, and chloroplast has been shown to be mediated by targeting signals present in transported proteins. To test whether transport into peroxisomes is also a peptide signal, we have studied firefly luciferase gene that encodes protein both insect mammalian cells. We identified two regions which are necessary for this peroxisomes. demonstrate one these, region II, represents peroxisomal signal because it...

DNA, isolated from Simian virus 40 (SV40), has been encapsulated in large (0.4-micrometer diameter) unilamellar phospholipid vesicles. The procedure used for liposome preparation the SV40 DNA at high efficiency (30 to 50% entrapment) and did not alter physical or biological properties of molecules. activity liposome-entrapped viral was determined by plaque assays on a permissive monkey cell line. infectivity enhanced least 100-fold over that free naked DNA. Importantly, vesicle-entrapped...

As part of an effort to understand how proteins are imported into the peroxisome, we have sought identify peroxisomal targeting signals in four unrelated proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and acyl-CoA oxidase. Using gene fusion experiments, identified a region each protein that can direct heterologous peroxisomes. In case, signal is contained at or near carboxy terminus protein. For located within COOH-terminal 27 amino acids carboxy-terminal...

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into unconventional secretion proteins are lacking. Acyl coenzyme A (CoA)–binding (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking (GRASP) for its secretion. We discovered, surprisingly, that processing, function AcbA-derived peptide, SDF-2, conserved between yeast Pichia pastoris D. discoideum....

A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 acid: (i) those that can be propagated as virions permissive cells and (ii) introduced into maintained stably various mammalian cells. Both types express by using signals supplied sequences. Moreover, plasmid carrying complement Chinese hamster ovary lacking reductase.

Human hepatitis B virus (HBV) causes chronic and is associated with the development of hepatocellular carcinoma. HBV infection alters mitochondrial metabolism. The selective removal damaged mitochondria essential for maintenance cellular homeostasis. Here, we report that shifts balance dynamics toward fission mitophagy to attenuate virus-induced apoptosis. induced perinuclear clustering triggered translocation dynamin-related protein (Drp1) by stimulating its phosphorylation at Ser616,...

By virtue of their synthesis in the cytoplasm, proteins destined for import into peroxisomes are obliged to traverse single membrane this organelle. Because targeting signal most peroxisomal matrix is a carboxy-terminal tripeptide sequence (SKL or its variants), these must remain competent until translation complete. We sought determine whether stably folded were substrates import. Prefolded stabilized with disulfide bonds and chemical cross-linkers shown be import, as mature...

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of required mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed vitro as substrates quantitate intramolecular cultured monkey Excision wild-type 40 by was scored viral plaque assay. Using series containing 0 243 base pairs homology, we shown that frequency decreases reduced, with...

Ciprofibrate, a hypolipidemic drug that acts as peroxisome proliferator, induces the transcription of genes encoding peroxisomal beta-oxidation enzymes. To identify cis-acting promoter elements involved in this induction, 5.8 kilobase pairs sequence from gene rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (EC 4.2.1.17/EC 1.1.1.35) was inserted upstream luciferase reporter gene. Transfection expression vector into hepatoma H4IIEC3 cells presence ciprofibrate resulted 5- to 10-fold,...

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of green fluorescent protein appended to peroxisomal targeting signal, Ser-Lys-Leu (SKL), label vacuolar membrane matrix, respectively, in living Pichia pastoris cells followed by fluorescence microscopy morphological kinetic intermediates degradation peroxisomes microautophagy macroautophagy. Structures corresponding were also identified electron...

Analysis of the Schizosaccharomyces pombe chromosomes by pulsed field gel electrophoresis showed that fission yeast has a very efficient DNA double-strand-break (dsb) repair system, which properly restores three after they are degraded gamma-irradiation. The radiation-sensitive mutant rad21-45 is deficient in this pathway but capable cell-cycle arrest G2 following damage. We cloned rad21 gene complementing radiation sensitivity mutant. plasmid-borne completely reestablished dsb pathway. was...