Site-directed mutagenesis: Generation of an extracistronic mutation in bacteriophage Qβ RNA

208

Mouse thymidine kinase-negative (TK-) L cells were transformed with concatenates of cloned herpes simplex virus 1 TK DNA and different rabbit .-globin DNAs in which the globin genes were preceded by native flanking sequences of 14 66, 76, 425, and 1500 nucleotides. In all cases, selection for TKw cell lines led to a high yield of lines producing 5-1500 mature rabbit ,B-globin-specific RNA strands per cell. The 5' termini of the transcripts mapped to (i) the "cap" nucleotide, (ii) positions 42 to 48 nucleotides downstream from the cap site, or (iii) positions in the vector DNA preceding the gene. In the case of the gene with only 14 base pairs of 5' flanking sequence, a high level of rabbit ,t-globin RNA was produced, but none of the transcripts had the correct 5' end; most of them originated in the vector moiety. With 66 base pairs of 5' flanking sequence, 5% of the 5' termini were correct, and with 76 or more base pairs, 3045% were correct. The region between 14 and 66 base pairs preceding the cap site contains the Hogness box and appears to be essential for correct initiation of transcription. The region between 66 and 76 base pairs before the cap site contains a variant of the canonical sequence G-G-4-C-A-A-T-C-T found preceding many other genes at a similar location, and this region may modulate the efficiency of transcription. The sequence of 425 nucleotides preceding the rabbit ,8-globin gene is reported.It has been reported earlier that when thymidine kinase-negative (TK-) mouse L cells were transformed with cloned rabbit chromosomal 3-globin DNA (1, 2) linked to a herpes simplex virus type 1 (HSV1) DNA fragment containing the TK gene (3), 90% of the TK+ transformants also contained the 1-globin gene, frequently in multiple copies (4-6). In the experiments of Mantei et al. (6), three-quarters of the cell lines containing ,B-globin DNA produced rabbit ,B-globin-specific RNA, of which at least a large fraction was indistinguishable from authentic globin mRNA as regards mobility, poly(A) tail, absence of introns, and correct 5' terminus; the presence of cap was not ascertained (6).To determine sequences required for the correct initiation of the ,B-globin transcripts, we have transformed mouse L cells with hybrid plasmids in which the 5' flanking sequences of the f3-globin gene were deleted to various extents, and we have characterized and quantitated the 3-globin RNAs accumulating in the transformed cells. MATERIALS AND METHODSMaterials. Restriction enzymes Bsp I and EcoRI were gifts from A. Kiss and W. Boll, respectively; all others were purchased from New England BioLabs. Polynucleotide kinase was purchased from P-L Biochemicals, phage T4 DNA ligase from New England BioLabs, and calf intestine alkaline phosphatase from Boehringer, and terminal deoxyribonucleotidyl transferase and SI nuclease were gifts from W. Boll and A. Schambock, respectively.Construction of Plasmids. Z-pBR322/Rchr/3G-A425B and Z-pBR322/Rchrf3G-A425C: Z-pCR1/RchrfG-1 (1) was partially cleaved with Bgl II, and the 2070-base-pai...

Section: Resultsmentioning

confidence: 99%

DNA sequences preceding the rabbit beta-globin gene are required for formation in mouse L cells of beta-globin RNA with the correct 5' terminus.

Dierks¹,

Ooyen²,

Mantei³

et al. 1981

208

“…Numerous studies using various approaches have been done to elucidate the specific adducts that result in mutations. In vitro misincorporation studies (1)(2)(3)(4)(5)(6), in vivo site-directed mutagenesis studies (7)(8)(9), and in vivo studies comparing specific adducts and mutation frequency (10)(11)(12)(13) have all been reported. These studies have suggested that of the adducts examined, O4-alkylthymidine (04-alkyldT) and 06-alkylguanine (06-alkylG) are the principal promutagenic lesions.…”

mentioning

confidence: 99%

DNA base changes and alkylation following in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

Richardson¹,

Richardson²,

Crosby³

et al. 1987

196

Dideoxy chain-termination DNA sequencing was used to determine the specific DNA base changes induced after in vivo exposure of Escherichia coli to N-methyl-Nnitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. The resultant mutation spectra were compared with the levels of O6-alkylguanine and 04-alkylthymidine in genomic DNA immediately after exposure. All (39/39) of the MNU-induced mutations were G-C-+A-T transitions. In contrast, 24/33 point mutations isolated following ENU treatment were G-C-+AT transitions, 7/33 were A-T-*GC transitions,

“…12 for enzyme and in ref. 13 Qfl minus strands substituted in position 15 from their 5' terminus with N4-hydroxycytidine monophosphate (HOCMP) were synthesized, purified, and used as templates for RNA synthesis by Qj3 replicase as described by Flavell et al (14).…”

Section: Methodsmentioning

confidence: 99%

“…14. Analysis of 32p Plus Strands. Purified plus strands were digested with T1 RNase and the oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis following the procedure of De Wachter and Fiers (16), as described elsewhere (14).…”

Section: Methodsmentioning

confidence: 99%

Site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA.

Flavell¹,

Sabo²,

Bandle³

et al. 1975

It has been proposed that the nucleotide sequences of the 3' terminal extracistronic regions of phage RNA plus and minus strands have been strictly conserved during evolution because they are stringently required for recognition by the viral replicase. We The 5'-and 3'-terminal regions of Q13 RNA comprise nontranslatable sequences of 61 and >61 nucleotides, respectively (1-3). A similar situation obtains in the case of the RNA of the group I phages MS2, f2 and R17 (4-7). Although about 3% of the nucleotide positions in the coat cistron differ from one group I RNA to another (7-9), the terminal extracistronic regions, comprising more than 150 nucleotides, are invariant. This observation led to the proposal that the replication of these phages depends strictly on the primary structure of the extracistronic sequences (9); in particular it was suggested that the specific interaction of phage RNA with its cognate replicase is mediated by these regions (6) Mmol of Tris-HCl (pH 7.5), 4 Mmol of KCl, and 20 nmol of EDTA were added, giving a final volume of 20 A. After 15 min at 370, 10 IA were transferred to 10 M1 of a mixture of the same composition but containing no RNA template. After 15 min at 370, this procedure was repeated, for a total of nine transfers. Ten microliters of the 10th incubation and 2 units of replicase were added to 20 Al of fresh reaction mixture and incubation was continued for 30 min. Ten microliters of fresh mixture and 4 units of replicase were added, and after an additional 30 min another 30 pl of mixture were added and incubation was continued for a further 90 min. After extraction with an equal volume of phenol at 20°the aqueous layer was filtered through a column (7-mm diameter) of Sephadex G-50 (7 cm) and Chelex-100 Na+ (0.5 cm). The pooled excluded fractions were adjusted to 0.05 M Tris * HCl (pH 7.5), 0.1 M NaCl, 5 mM EDTA, and the RNA was precipitated with 2 volumes of ethanol at -10°and recovered by centrifugation (10,000 rpm in a Sorvall HB-4 rotor, 10 min at -10°).The RNA was dissolved in 150 MAl of 0.5 mM EDTA and centrifuged through .a 5-23% sucrose gradient in 50 mMTris-HCl (pH 7.5)-5 mM EDTA. The fractions containing 30S RNA were pooled and precipitated with ethanol and the RNA was dissolved in 20 Ml of water. The yield of RNA was 4 Mg. Two micrograms of this product were denatured and used as template to initiate a series of nine further transfers, as described above. A 2-Ml sample of each of the incubations (except for the 1st, 5th, 10th, and 21st) was taken to determine acid-insoluble radioactivity.Serial replication of wild-type Q# RNA (extracted from phage) was carried out in a similar fashion, except that incuba-