Showing papers by "Alexander N. Glazer published in 1992"

Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications

Abstract: The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.

Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection.

Abstract: Fluorescent intercalation complexes of certain polycationic ligands with double-stranded DNA provide a new class of multichromophore labels for fluorescence assays.

Phycocyanin alpha-subunit phycocyanobilin lyase

Abstract: Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separately, the surprising result is elimination of correct bilin attachment at only one site, that on the alpha subunit of phycocyanin. We have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to the alpha subunit of apophycocyanin at the appropriate site, alpha-Cys-84, to form the correct adduct. CpcE and CpcF also efficiently catalyze the reverse reaction, in which the bilin from holo-alpha subunit is transferred either to the apo-alpha subunit of the same C-phycocyanin or to the apo-alpha subunit of a heterologous C-phycocyanin. The forward and reverse reactions each require both CpcE and CpcF and are specific for the alpha-Cys-84 position. Phycocyanobilin is the immediate precursor of the protein-bound bilin.

A streptavidin-metallothionein chimera that allows specific labeling of biological materials with many different heavy metal ions.

Abstract: We have designed a streptavidin-metallothionein chimeric protein in which the streptavidin moiety provides a means of binding the metallothionein moiety tightly to specific biological targets. A gene fusion of streptavidin with mouse metallothionein I was efficiently expressed in Escherichia coli, and the expressed chimeric protein was purified to homogeneity by a simple procedure. The purified chimera, consisting of four identical subunits, bound one biotin and approximately seven Cd2+ ions per subunit (19.5 kDa). This indicates that both the streptavidin and the metallothionein moieties are fully functional. The high binding affinity of the chimera both for biotin and for heavy metal ions allows the specific labeling or conjugation of any biological material containing unhindered biotin with a variety of different heavy metal ions and their isotopes, thereby opening the way for simultaneous assay systems for a large number of biological targets.

Genes encoding the phycobilisome rod substructure are clustered on the Anabaena chromosome: characterization of the phycoerythrocyanin operon.

Abstract: The phycoerythrocyanin (pec) operon, cloned from Anabaena sp. strain PCC 7120, encodes four genes, pecBACE, located upstream of the C-phycocyanin (cpc) operon. This pec-cpc cluster includes all the genes for the structural components of the phycobilisome rod. Oligonucleotide probes based on the amino-terminal sequence of the phycoerythrocyanin beta subunit were used to clone an 8.0-kbp EcoRI fragment which was determined, by sequencing, to partially overlap the previously cloned cpc operon. A 5.0-kbp EcoRI-ClaI fragment corresponding to the region upstream of the cpc operon was subsequently subcloned and sequenced. Five open reading frames whose polarity of transcription is parallel to that of the cpc genes were identified. pecB and pecA encode the beta and alpha subunits of phycoerythrocyanin, respectively. pecC encodes the phycoerythrocyanin-associated linker polypeptide LR34.5,PEC. The identities of these genes are confirmed by agreement with amino-terminal sequences determined from purified phycobilisome components. A gene homologous to cpcE, found downstream of pecC, has been designated pecE. The cpcE gene product is involved in the attachment of the phycocyanobilin chromophore to the alpha subunit of phycocyanin. Three transcripts were observed by Northern (RNA) analyses. The most abundant of these transcripts, 1.35 kbp, corresponds to the beta and alpha subunit genes, whereas the less-abundant transcripts, 2.3 and 3.1 kbp, correspond to pecBAC and pecBACE, respectively. Phycoerythrocyanin is strongly induced in cells cultured under low light. In parallel, all three transcripts were present at much higher levels in cells cultured under low light. Images

Sequence comparison of two highly homologous phycoerythrins differing in bilin composition

Abstract: Genes encoding the α and β subunits of class II phycoerythrin from Synechococcus sp. strain WH8103 were cloned and sequenced. The deduced amino acid sequences were compared to class II phycoerythrin from Synechococcus sp. strain WH8020 and found to share 92% identity, yet the proteins differ in the bilin isomer (phycoerythrobilin versus phycourobilin) bound to two of the six chromophore attachment sites. Amino acid residues which might contact the bilin at each of the two variable sites were inferred by sequence alignment with phycocyanins. Putative bilin-contacting residues differing between the two phycocrythrins were identified which may determine bilin specificity.