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D C Prasher

Publications and source records attributed to D C Prasher.

16 recordsLinked to original sources
PubMed1997

Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant Arabidopsis thaliana, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains sequences similar to those required for recognition of normal plant introns. We have modified the codon usage of the gfp gene to mutate the intron and to restore proper expression in Arabidopsis. GFP is mainly localized within the nucleoplasm and cytoplasm of transformed Arabidopsis cells and can give rise to high levels of fluorescence, but it proved difficult to efficiently regenerate transgenic plants from such highly fluorescent cells. However, when GFP is targeted to the endoplasmic reticulum, transformed cells regenerate routinely to give highly fluorescent plants. These modified forms of the gfp gene are useful for directly monitoring gene expression and protein localization and dynamics at high resolution, and as a simply scored genetic marker in living plants.

Agrobacterium tumefaciens↗
PubMed1995

Using GFP to see the light.

Green fluorescent protein (GFP) is responsible for the green bioluminescence from the jellyfish Aequorea victoria. The intense fluorescence of GFP is due to the nature of a chromophore composed of modified amino acids within the polypeptide. Formation of the fluorescent chromophore is species independent and apparently does not require any additional factors. Hence, because the gene product is easily detectable by its intense fluorescence, the gfp cDNA has become a unique reporter system. The advantages of GFP are being exploited in a variety of experimental systems and this is illustrated in the accompanying articles on Dictyostelium, plants, Drosophila and mammalian cells.

Animals↗
PubMed1994

Wavelength mutations and posttranslational autoxidation of green fluorescent protein.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.

Amino Acid Sequence↗
PubMed1994

Green fluorescent protein as a marker for gene expression.

A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

Animals↗
PubMed1993

Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.

The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.

Amino Acid Sequence↗
PubMed1993

Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria.

Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) A. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis.

Aequorin↗
PubMed1992

Primary structure of the Aequorea victoria green-fluorescent protein.

Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated phosphoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated Mr of 26,888. Comparison of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the lambda GFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.

Amino Acid Sequence↗
PubMed1992

Evolutionary origins of bacterial bioluminescence.

In bacteria, most genes required for the bioluminescence phenotype are contained in lux operons. Sequence alignments of several lux gene products show the existence of at least two groups of paralogous products. The alpha- and beta-subunits of bacterial luciferase and the non-fluorescent flavoprotein are paralogous, and two antennae proteins (lumazine protein and yellow fluorescence protein) are paralogous with riboflavin synthetase. Models describing the evolution of these paralogous proteins are suggested, as well as a postulate for the identity of the gene encoding a protobioluminescent luciferase.

Amino Acid Sequence↗
PubMed1991

Borrowed proteins in bacterial bioluminescence.

A library of Photobacterium phosphoreum DNA was screened in lambda 2001 for the lumazine protein gene, using two degenerate 17-mer oligonucleotide probes that were deduced from a partial protein primary sequence. The lumazine protein gene was localized to a 3.4-kilobase BamHI/EcoRI fragment in one clone. The fragment contained an open reading frame, encoding a 189-residue protein, that had a predicted amino acid sequence that concurred with the partial sequence determined for lumazine protein. Considerable sequence similarity was detected between lumazine protein, the yellow fluorescence protein from Vibrio fischeri, and the alpha subunit of riboflavin synthetase (EC 2.5.1.9). A highly conserved sequence in lumazine protein corresponds to the proposed lumazine binding sites in the alpha subunit of riboflavin synthetase. Several secondary structure programs predict the conformation of this site in lumazine protein to be a beta-sheet. A minimal model with three interactions between the ligand and this beta-sheet structure is proposed, which is consistent with the results of NMR and ligand binding studies.

Amino Acid Sequence↗
PubMed1989

The enzymology and molecular biology of the Ca2+-activated photoprotein, aequorin.

Aequorin is a bioluminescent protein, isolated from the hydromedusan Aequorea victoria. A recombinant cDNA plasmid (pAEQ1) was shown to encode apoaequorin by detecting photoprotein activity in an extract of an E. coli strain containing pAEQ1 (Prasher et al., 1986, Biochem. Biophys. Res. Comm. 126, 1259-1268). The nucleotide sequence of the pAEQ1 insert has been determined and is shown to differ significantly from the aequorin cDNA (AQ440) isolated by Inouye et al. (1985, Proc. Natl. Acad. Sci. USA 82, 3154-3158). Comparisons of the coding regions of the two cDNAs show there are 52 nucleotide differences, 19 of which are responsible for 18 amino acid replacements. These differences explain the microheterogeneity observed at 17 positions during the sequencing of native apoaequorin. Five aequorin isotypes extracted from Aequorea tissue are observed on 2-dimensional gels and the E. coli-expressed apoaequorin is shown to co-migrate with one of these isotypes. The multiple isotypes could be caused by the presence of a multi-gene family since Southern blot analysis of Aequorea DNA suggests the presence of a minimum of four aequorin genes. Immunoblot analysis suggests that purified native aequorin is proteolytically cleaved during its purification from Aequorea. Comparison of the deduced cDNA translations and the protein sequence suggests the loss of seven residues from the amino terminal. Overexpression of the apoaequorin cDNA in E. coli now provides the means of obtaining gram quantities of a single isotype of the protein which can be converted to aequorin in the presence of coelenterate luciferin, oxygen and an appropriate thiol. Proper extraction procedures and a single chromatographic step provides apoaequorin which is greater than 95% homogeneous.

Aequorin↗
PubMed1988

Physical and biochemical characterization of cloned sbcB and xonA mutations from Escherichia coli K-12.

In Escherichia coli K-12, sbcB/xonA is the structural gene for exonuclease I, an enzyme that hydrolyzes single-stranded DNA to mononucleotides in the 3'-to-5' direction. This enzyme has been implicated in the DNA repair and recombination pathways mediated by the recB and recC gene products (exonuclease V). We have cloned several sbcB/xonA mutant alleles in bacterial plasmids and have partially characterized the cloned genes and their protein products. Two of the mutations (xonA2 and xonA6) retain no detectable exonucleolytic activity on single-stranded DNA. The xonA6 allele was shown to harbor an insertion of an IS30-related genetic element near the 3' end of the gene. Two other mutations, sbcB15 and xonA8, exhibited significantly reduced levels of exonuclease I activity as compared to the cloned wild-type gene. A correlation was observed between levels of exonuclease I activity and the ability of the sbcB/xonA mutations to suppress UV sensitivity in recB and recC strains. Also, recombinant plasmids bearing either the sbcB15 or xonA6 allele exhibited a high degree of instability during growth of their bacterial hosts. The results suggest that the sbcB/xonA gene product is a bi- or multifunctional protein that interacts with single-stranded DNA and possibly with other proteins in the suppression of genetic recombination and DNA-repair deficiencies in recB and recC mutants.

Alleles↗
PubMed1987

Sequence comparisons of complementary DNAs encoding aequorin isotypes.

Aequorin is the Ca2+-activated photoprotein which participates in the bioluminescence from the circumoral ring of the hydromedusa Aequorea victoria. The nucleotide sequences of five aequorin cDNAs have been compared and shown to code for three aequorin isoforms. The cDNA AEQ1 contains the entire protein coding region of 196 amino acids. The other four cDNAs contain only 70-90% of the coding region and apparently code for at least two other isoforms whose amino acid sequences differ significantly from that encoded by AEQ1. The nucleotide sequences coding for the three isotypes differ at a minimum of 54 positions out of a total of 588 nucleotides necessary to code for apoaequorin. Of these nucleotide differences, 24 account for 23 amino acid replacements, substantiating the microheterogeneity observed during sequencing of purified native aequorin [Charbonneau, H., Walsh, K.A., McCann, R.O., Prendergast, F.G., Cormier, M.J., & Vanaman, T.C. (1985) Biochemistry 24, 6762-6771]. Comparison of the deduced cDNA translations with the native protein sequences suggests the loss of seven residues from the amino terminus during purification of aequorin from Aequorea. Aequorin rapidly extracted from the jellyfish using conditions to minimize proteolysis is shown to have a larger molecular weight than that of purified native aequorin. Escherichia coli expressed aequorin encoded by AEQ1 is shown to have the same molecular weight and isoelectric point as those of one of the isotypes rapidly extracted from Aequorea.

Aequorin↗
PubMed1983

Amplification and purification of exonuclease I from Escherichia coli K12.

Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities. The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M. (1974) J. Biol. Chem. 249, 5379-5381). Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.

DNA↗
PubMed1982

Nucleoside phosphotransferase from barley. Characterization and evidence for ping pong kinetics involving phosphoryl enzyme.

A nucleoside phosphotransferase has been purified to homogeneity from barley seedlings. Its Mr is about 50,000 and it consists of two subunits of equal size. A tightly bound metal ion required for activity can be replaced by Mg, Ba, or Co ions, but not by divalent Cu, Ca, Cd, or Hg. The enzyme is capable of catalyzing the transfer of phosphate from nucleoside monophosphates to the 5'-hydroxyl of any other nucleoside, but shows a decided preference for purine deoxynucleoside phosphate acceptors. A short lived phosphoryl enzyme intermediate has been trapped by rapid denaturation in presence of [32P]AMP. The steady state kinetics of this enzyme is found to be fully consistent with a branched ping pong pathway involving a compulsory phosphoryl enzyme intermediate from which phosphate may be transferred to nucleoside or to water. With appropriately chosen substrates, parallel line reciprocal plots were obtained, provided that both products of the branched pathway were taken into account in the rate measurements. Conversely, when only transphosphorylation was measured, converging reciprocal plots were obtained. Accordingly, assays were devised for the measurement of three velocity components: transphosphorylation, hydrolysis, and the sum of both reactions. Competitive inhibition of the transferase reaction by higher concentrations of either substrate is attributed to deadend interactions. Instead of being inhibited by the formation of a putative E-P.AMP complex, however, the phosphatase reaction is accelerated almost 2-fold.

Adenosine Monophosphate↗
PubMed1979

Chiral [18O]phosphorothioates. The stereochemical course of thiophosphoryl group transfer catalyzed by nucleoside phosphotransferase.

Nucleoside phosphotransferase from barley seedlings was used to catalyze the equilibration of adenosine-5'-[18O]phosphorothioate having the S configuration at phosphorus with [adenine-8-14C]adenosine to produce [adenine-8-14C]adenosine-5'-[18O]phosphorothioate and adenosine. The configuration of the chiral phosphorus in adenosine-5'-[18O]phosphorothioate which was used as the donor substrate was then compared with that of the [adenine-8-14C]adenosine-5'-[18O]phosphorothioate isolated from the reaction mixture. They were found to be the same, showing that the reaction proceeds with 99.7% retention of configuration of the [18O]phosphorothioate. This is interpreted to be indicative of the involvement of a thiophosphoryl-enzyme intermediate in the nucleoside phosphotransferase reaction. The synthesis of adenosine-5'-[18O]phosphorothioate having the R and S configurations at the phosphorus atoms is described.

Adenosine Monophosphate↗