Gene replacement in Haloarcula marismortui: construction of a strain with two of its three chromosomal rRNA operons deleted.
Site-directed mutagenesis were done in Haloarcula marismortui using the strategy that Khorana and coworkers devised for deleting the bacteriorhodopsin gene from Halobacterium halobium [Krebs et al. Proc Natl Acad Sci USA 90:1987-1991 (1993)]. Strains have been prepared from H. marsimortui, which normally has three rRNA operons, that are missing either its rrnB operon or both its rrnB and rrnC operons. In rich media, both strains grow at about the same rate as wild type. The G2099 in the 23S rRNA gene of the single operon strain was changed to A, and a three amino acid deletion was introduced into the gene for ribosomal protein L22 of the wild-type organism. The structural consequences of these and other such mutations can be determined with unusual accuracy because crystals of the large ribosomal subunit of H. marismortui diffract to atomic resolution.
Tu
Daqi
D
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, P.O. Box 208114, New Haven, CT 06520-8114, USA.
Blaha
Gregor
G
Moore
Peter B
PB
Steitz
Thomas A
TA
D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004583:Electrons; D029968:Escherichia coli Proteins / Q000378:metabolism; D017353:Gene Deletion; D005821:Genetic Techniques; D019614:Haloarcula marismortui / Q000235:genetics*; D008956:Models, Chemical; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009876:Operon; D010957:Plasmids / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D012407:rRNA Operon
D017931:DNA Primers; D029968:Escherichia coli Proteins; D012335:RNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C489371:rplV protein, E coli; D013395:Sucrose; D004247:DNA