Construction of a cDNA for the human c-fes protooncogene protein-tyrosine kinase and its expression in a baculovirus system.

Previous studies have established that the 93-kDa protein-tyrosine kinase (PTK) encoded by the human c-fes protooncogene plays an active role in the induction of terminal myeloid differentiation. However, this enzyme is expressed at very low levels in myeloid cells, making isolation of sufficient quantities for detailed biochemical analysis difficult. To overcome this problem, we used the polymerase chain reaction to construct a full-length c-fes cDNA from overlapping 5' and 3' partial cDNA sequences. The c-fes cDNA was expressed at high levels in a baculovirus system, and the catalytically active recombinant c-fes gene product p93c-fes was partially purified by DEAE-Sepharose and tyrosine-agarose chromatography. Recombinant p93c-fes was indistinguishable from the native protein in terms of its apparent molecular weight following SDS-PAGE, catalytic activity, Km for poly(Glu,Tyr)4:1, antigenicity, and phosphopeptide pattern generated with Staphylococcus aureus protease.     

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.     

Selective disruption of energy flow from phycobilisomes to Photosystem I

Efficient production of ATP and NADPH by the light reactions of oxygen-evolving photosynthesis demands continuous adjustment of transfer of absorbed light energy from antenna complexes to Photosystem I (PS I) and II (PS II) reaction center complexes in response to changes in light quality. Treatment of intact cyanobacterial cells with N-ethylmaleimide appears to disrupt energy transfer from phycobilisomes to Photosystem I (PS I). Energy transfer from phycobilisomes to Photosystem II (PS II) is unperturbed. Spectroscopic analysis indicates that the individual complexes (phycobilisomes, PS II, PS I) remain functionally intact under these conditions. The results are consistent with the presence of connections between phycobiliproteins and both PS II and PS I, but they do not support the existence of direct contacts between the two photosystems.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - NEM N-ethylmaleimide - PBS phycobilisome - PS photosystem     

Genes of the R-phycocyanin II locus of marine Synechococcus spp., and comparison of protein-chromophore interactions in phycocyanins differing in bilin composition

R-phycocyanin II (RPCII) is a recently discovered member of the phycocyanin family of photosynthetic light-harvesting proteins. Genes encoding the and subunits of RPCII were cloned and sequenced from marine Synechococcus sp. strains WH8020 and WH8103. The deduced amino acid sequences of RPCII were compared to two other types of phycocyanin, C-phycocyanin (CPC) and phycoerythrocyanin (PEC). These three types vary in the composition of their covalently bound bilin prosthetic groups. In terms of amino acid sequence identity RPCII is highly homologous to CPC and PEC, suggesting that the known three-dimensional structures of the latter two are representative of RPCII. Thus the amino acid residues contacting the three bilins of RPCII could be inferred and compared to those in CPC and PEC. Certain residues were identified among the three phycocyanins as possibly correlating with specific bilin isomers. In overall sequence RPCII and CPC are more homologous to one another than either is to PEC. This probably reflects functional homology in the roles of RPCII and CPC in the transfer of light energy to the core of the phycobilisome, a function not attributed to PEC. The genomes of Synechococcus sp. strains WH8020, WH8103 and WH7803 share homologous open reading frames in the vicinity of RPCII genes. The nucleotide sequence extending 3 from RPCII genes in strain WH8020 revealed two open reading frames homologous to components of an CPC phycocyanobilin lyase. These open reading frames may encode a lyase specific for the attachment of phycoerythrobilin to RPCII.     

Cryptomonad biliproteins — an evolutionary perspective

Each cryptomonad strain contains only a single spectroscopic type of biliprotein. These biliproteins are isolated as 50000 kDa '₂ complexes which carry one bilin on the and three on the subunit. Six different bilins are present on the cryptomonad biliproteins, two of which (phycocyanobilin and phycoerythrobilin) also occur in cyanobacterial and rhodophytan biliproteins, while four are known only in the cryptomonads. The subunit is encoded on the chloroplast genome, whereas the subunits are encoded by a small nuclear multigene family. The subunits of all cryptomonad biliproteins, regardless of spectroscopic type, have highly conserved amino acid sequences, which show > 80% identity with those of rhodophytan phycoerythrin subunits. In contrast, cyanobacteria and red algal chloroplasts each contain several spectroscopically distinct biliproteins organized into macromolecular complexes (phycobilisomes). The data on biliproteins, as well as several other lines of evidence, indicate that the cryptomonad biliprotein antenna system is primitive and antedates that of the cyanobacteria. It is proposed that the gene encoding the cryptomonad biliprotein subunit is the ancestral gene of the gene family encoding cyanobacterial and rhodophytan biliprotein and subunits.Abbreviations Chl chlorophyll - CER chloroplast endoplasmic reticulum - SSU rRNA small subunit ribosomal RNA     

AN UNUSUAL PHYCOCYANOBILIN-CONTAINING PHYCOERYTHRIN OF SEVERAL BLUISH-COLORED,ACROCHAETIOID, FRESHWATER RED ALGAL SPECIES1

The reproductive biology and phycobiliproteins of four different culture isolates of the freshwater algae Audouinella and‘Chantransia’were investigated.‘Chantransia’sp. (3585/UTEX 2623) and Audouinella macrospora (Wood) Sheath et Burkholder (3394,3395) from California and Minnesota reproduced only by monospores. However, A. macrospora (3603/Necchi 1) reproduced by monosporangia that formed successive generations of the Audouinella phase, and Batrachospermum shoots developed from the basal and erect systems. The major light-harvesting phycobiliprotein in all of these isolates was a phycocyanobilin-containing phycoerythrin not previously detected in red algae or cyanobacteria. As in the commonly found R- and B-phycoerythrins, Audouinella phycoerythrin had a native molecular mass of ～ 240,000 and was made up of α, β, and γ subunits. Audouinella phycoerythrin carried two phycoerythrobilins on the α subunit; one phycourobilin, one phycoerythrobilin, and one phycocyanobilin on the β subunit; and one phycourobilin and two phycoerythrobilins on the γ subunit. With excitation at 495, 563, or 603 nm, the fluorescence emission peak of Audouinella phycoerythrin was at 626 nm, showing that phycocyanobilin was the terminal energy acceptor.     

Test systems for biomonitoring based on membrane-bound enzyme complexes. IV. Assessment of genotoxic effects in an Ames test system with metabolic activation by the microsomal mono-oxygenases from fish livers

The study of mutagenic effect of 2-aminoantracene and benz(alpha)pyrene on Salmonella triphimurium TA 100 in the Ames test-system in the presence of postmitochondrial fractions S-9 from carp liver with 3-methylcholantrene induced by microsomal oxidation system has been carried out. The metabolic activity and cytochrome P450 contence in carp liver microsomes have been shown to concede considerably those in rats liver. But these characteristics are sufficient for the use of fraction S-9 from carp liver for the study of genotoxic effect of these xenobiotics in the Ames test-system. Several regimes of storage of S-9 preparations from carp liver have been compared. S-9 preparations frozen immediately after isolation preserve their metabolic activity with respect to 2-aminoantracene and benz(alpha)pyrene well.     

Molecular architecture of a light-harvesting antenna. Isolation and characterization of phycobilisome subassembly particles

Synechococcus 6301 mutant, strain AN112, produces phycobilisomes containing two major biliproteins, phycocyanin and allophycocyanin, and two major linker polypeptides of 27 and 75 kilodaltons (27K and 75K). These phycobilisomes have a molecular weight of approximately 2.5 X 10(6) and are the smallest of these particles known to date. Sucrose density gradient centrifugation of AN112 phycobilisomes partially dissociated in 50 mM N-[tris(hydroxymethyl)methyl]glycine, 5 mM CaCl2, 10% (w/v) glycerol, pH 7.8, separated three distinct fractions: (1) free trimeric biliproteins, (2) hexameric complexes of phycocyanin with 27K (11 S particles), and (3) phycobilisome subassemblies equivalent in mass to approximately 25% of the intact phycobilisome (18 S particles). The 18 S particles contained equimolar amounts of phycocyanin and allophycocyanin, which represented approximately 30 and 50%, respectively, of the content of these biliproteins in the AN112 phycobilisome. The 18 S particles also contained 75% and 100%, respectively, of 27K and 75K polypeptides; i.e. 75K was present in a 2-fold higher amount than in the intact phycobilisome. The absorption spectrum (lambda max 648 nm) of the 18 S particles was similar to that of allophycocyanin. Upon excitation at 580 nm, these particles exhibited a fluorescence emission spectrum consisting of 680 and 660 nm components, identical with that of intact phycobilisomes. The circular dichroism spectra of AN112 phycobilisomes and of the 18 S particles, in the region between 650 and 700 nm, were also very similar. Allophycocyanin B, which fluoresces at 680 nm, was found in fraction 1, and was totally absent from the 18 S particle. Thus, the long wavelength emission of the 18 S particle must have arisen from another terminal energy acceptor. The most probable candidate is the 75K polypeptide, which has been shown to carry a bilin chromophore and emit near 680 nm (Lundell, D. J., Yamanaka, G., and Glazer, A. N. (1980) J. Cell Biol. 91, 315-319). The 27K polypeptide, present in both fractions 2 and 3, was a component of different complexes in the two fractions. Fraction 2 displayed the physical and spectroscopic properties characteristic of the phycocyanin-linker complex, (alpha beta)6.27K. However, in the 18 S particle, 27K functioned in the assembly and attachment of phycocyanin trimers to a core domain. Based on the analysis of the components in fractions 1-3, a model is proposed which describes the structure of the AN112 phycobilisome, with emphasis on the roles of the linker polypeptides in the assembly of the core.     

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.