Nuclear encoding of a chloroplast RNA polymerase sigma subunit in a red alga

Science

Volumn 272, Issue 5270, 1996, Pages 1932-1935

  Tanaka, Kan ^a   Oikawa, Kosuke ^a   Ohta, Niji ^b   Kuroiwa, Haruko ^a   Kuroiwa, Tsuneyoshi ^a   Takahashi, Hideo ^a  

^a UNIVERSITY OF TOKYO   (Japan)

^b WASEDA UNIVERSITY   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

RNA POLYMERASE; SIGMA FACTOR;

ARTICLE; CHLOROPLAST; CYANOBACTERIUM; GENE TRANSFER; GENOME; NONHUMAN; PRIORITY JOURNAL; RED ALGA; SEQUENCE HOMOLOGY;

ALGAE; CYANIDIUM CALDARIUM; CYANOBACTERIA; RHODOPHYTA;

EID: 0030038746     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5270.1932     Document Type: Article

References (38)

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24. To produce SigA in E. coli we inserted a 1733-bp Nde I-Bam HI fragment containing the sigA open reading frame into the linker site of the expression plasmid pET15b (Novagen) (15). The resultant plasmid, pETCA, was introduced into the host BL21(DE3) strain and the bacterial cells were incubated at 37°C in 300ml of Luria broth (LB) medium until the optical density of the culture at 660 nm reached 0.5. Isopropyl-β-D-thiogalactopyranoside was then added to a final concentration of 0.5 mM, and after incubation for an additional 2 hours cells were collected by centrifugation. The recombinant protein was recovered as inclusion bodies, solubilized in TGED buffer [10 mM tris-HCl (pH 8.0), 5% (w/v) glycerol, 0.1 mM EDTA, and 0.1 mM dithiothreitol] containing 6 M guanidine hydrochloride, and renatured by dialyzing against TGED buffer. The SigA protein was further purified in a fast protein liquid chromatography (FPLC) system (Pharmacia) on a POROS HQ column (4.6 mm × 100 mm; PerSeptive Biosystems, Cambridge, MA). Elution was performed with a linear gradient of 0 to 1.0 M NaCl in the same buffer, and the peak fraction was precipitated with ammonium sulfate, dialyzed against storage buffer [TGED containing 0.5 M NaCl and 50% (w/v) glycerol], and stored at -30°C. The calculated molecular weight of the protein is 46,794 (440 amino acids).
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32. 32P by the random priming method.
33. Nuclear and chloroplast DNAs were enriched by density gradient centrifugation, first on ethidium bromide-CsCl and then on Hoechst 33258-CsCl (5). Mitochondrial, chloroplast, and nuclear DNAs banded separately in this order from the top.
34. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
35. 70 or SigA and then incubated the mixture for 30 min at 37°C. Single-round transcription reactions were performed under standard conditions (17). Transcripts separated by electrophoresis through a 7% polyacrylamide gel containing 8 M urea were analyzed with a Bioimage analyzer (BAS1000, Fuji Photo Film). The positions and the nucleotide length of transcripts initiated from the tac and RNA I promoters are indicated.
36. Rabbit antiserum to recombinant SigA was prepared as described (18). The antiserum was passed over a protein A column and the immunoglobulin G fraction was further passed over an affinity column prepared from SigA (10 mg) conjugated with HiTrap NHS-activated (1 ml; Pharmacia) as suggested by the supplier. C. caldarium RK-1 cells were ground in liquid nitrogen, and an amount corresponding to 10 μg of protein was fractionated by SDS-polyacrylamide gel electrophoresis in a 9.4% gel. Other procedures for immunoblot analysis were as described (19).
37. Immunogold electron microscopy was performed basically as described (20). Cyanidium cells were fixed in 2% glutaraldehyde, embedded in LR White resin, cut with an ultramicrotome, and treated with the primary antibody. The mouse monoclonal antibodies to human double-stranded DNA were from Chemicon. After the grids were washed, immune complexes were detected through incubation for 2 hours at room temperature with antibodies to rabbit or mouse immunoglobulin G labeled with 10-nm colloidal gold particles (BioCell Research Laboratories, Cardiff, UK). The grids were then stained with 3% aqueous uranyl acetate for 20 min and examined under an electron microscope.
38. We thank H. Hirokawa for encouragement and H. Takahashi for technical assistance. Supported in part by grants for scientific research from the Ministry of Education, Science, and Culture of Japan (05454069, 07760074. and 06101002), and by the Biodesign Research Program of RIKEN