Efficient correction of a mutation by use of chemically synthesized DNA.

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An efficient method for oligonucleotide-directed mutagenesis was developed to construct a set of sitespecific mutations by using a mixture of oligodeoxyribonucleotides. With this method, as high as 40% of the tested clones turned out to be desired mutants. Seven single-point mutants were isolated in the "TATA" box region of the fibroin gene. In vitro transcription experiments showed that single-base transversions at the TATA box (A -> T at position -29, T --A or G at -28, A --T at -27, and A --T at -26) resulted in decreased promoter activities, whereas A -+ G transitions at positions -24 and -23 had no effect. The initiation site of transcription was normal in three down-promoter mutants at positions -28 and -26, but the A -* T transversions at positions -29 and -27 induced an additional transcription start from position +4. Using 10 single-point mutants obtained as above or by nitrous acid-induced mutagenesis, we have prepared a pair of heteroduplex DNAs consisting of a mutant strand and the wild-type one. The molecules heterozygous at positions -30, -21, and -20 showed reduced transcription activities when the noncoding strand bears the mutation, whereas that at position -26 gave a low activity when the coding strand carries the mutation. Both types of heteroduplex at positions -29, -28, -27, and -17 exhibited decreased activities. These results suggest a transcription machinery contact to a major groove of the DNA helix at the TATA box and region of position -20.Transcriptional analysis of eukaryotic protein-coding genes has revealed a set of promoter elements (1-11). Core promoter consists of the "TATA" box and its flanking sequence, which is required for fixing the start site as well as efficient transcription. Another element, located at a variable position upstream from the TATA box, appears to modulate the utilization of the core promoter. We believe it is important to fully characterize these elements in order to explore underlying mechanisms of gene expression in eukaryotes. In an attempt to pursue such an approach, we have isolated single-base transition mutants by nitrous acid-induced mutagenesis within the fibroin gene core promoter and have shown that not only the TATA box but also the sequence around position -20 are important for faithful and efficient transcription in vitro (9). However, whether the sequence between the TATA box and the -20 region is essential or not was left unknown because no mutant was available in this area.In study reported here, we used a modified method of oligonucleotide-directed mutagenesis to produce a series of defined point mutants at or near the TATA box of the fibroin gene. Using these mutants, we demonstrated (i) the presence of a silent space between the TATA box and the -20 region substitutable by other bases and (it) the role of the second and the fourth bases of the TATA box in pinpointing the start site. As a next step, we asked which strand of the DNA helix is responsible for the promoter function. For this purpose, we constructed heteroduplex templ...

Section: Methodsmentioning

confidence: 99%

Contact points between transcription machinery and the fibroin gene promoter deduced by functional tests of single-base substitution mutants.

Hirose¹,

Takeuchi²,

Hori³

et al. 1984

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“…A single-base change (see Fig. 1A) was introduced in the Drosophila tRNAT~, gene (13) contained in the 0.9-kilobase EcoRI-Sal I fragment of the plasmid p22F91Rs (gift of Michael Hoffman, Harvard University) by site-directed mutagenesis (14)(15)(16) …”

Section: Methodsmentioning

confidence: 99%

Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.

Laski

Ganguly²,

Sharp³

et al. 1989

Drosophila melanogaster strains with a stably incorporated amber suppressor tRNA gene have been generated. A tRNATY gene was site specifically mutated to produce an anticodon sequence that recognizes the amber codon and then introduced into Drosophila by using P-element-mediated transformation. Transformants from four integration events were recovered. Two integrations resulted in both male and female sterility, whereas the other two resulted in male sterility but female fertility. Strains derived from the two female-fertile integration events were shown to have a low level of ambersuppressing activity by their ability to suppress an amber mutation in a chloramphenicol acetyltransferase gene.Nonsense suppressors are useful tools for the genetic analysis of organisms. In Escherichia coli (1), yeast (1), and Caenorhabditis elegans (2) nonsense suppressors were isolated as second-site mutations able to suppress mutations in many other genes. However, no nonsense suppressors have been identified in mammals or Drosophila. Suppressor tRNA genes constructed in vitro and transformed into mammalian tissue culture cells were shown to have nonsense-suppressing activity (3-11). In this paper we describe the activity of a Drosophila amber-suppressor tRNATIr gene after introduction into flies by P-element-mediated transformation. METHODSConstruction of P[iy, DtT(Su+)]. A single-base change (see Fig. 1A) was introduced in the Drosophila tRNAT~, gene (13) contained in the 0.9-kilobase EcoRI-Sal I fragment of the plasmid p22F91Rs (gift of Michael Hoffman, Harvard University) by site-directed mutagenesis (14)(15)(16)

“…Based on similar stretches of amino acids in known transmembrane proteins (27), these residues are suspected to comprise the membrane-anchoring site of the mT polypeptide. Here we use the method of site-specific mutagenesis with synthetic oligonucleotides (28)(29)(30)(31)(32)(33)(34) to explore the consequences of removing the presumptive membrane-anchoring sequence of mT antigen. The oligonucleotide used to create this mutant introduces an ochre codon (UAA) in place of a glutamine codon (CAA) at residue 384 ofmiddle T antigen and causes an alanine (GCA) to valine (GUA) change in the "minus one" overlapping large T-antigen frame (Fig.…”

mentioning

confidence: 99%

Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation.

Carmichael¹,

Schaffhausen²,

Dorsky³

et al. 1982

131

We have constructed a transformation-defective polyoma virus mutant (Py 1387-T) that directs the synthesis of a normal small tumor antigen, a functional large tumor antigen, and a truncated (51,000-dalton) middle-sized tumor (mT) antigen that lacks 37 amino acids at its COOH terminus. The shortened mT polypeptide is missing the hydrophobic "tail" thought to be responsible for the anchorage of this protein into the plasma membrane and is in fact in cytosol fractions. This truncated mT polypeptide is inactive in an in vitro protein kinase assay and is altered in its phosphorylation in viva Mutant 1387-T differs from wildtype virus in having a T-A base pair instead of a C-G base pair at nucleotide position 1387. This change was introduced into viral DNA by using a synthetic undecanucleotide as a specific mutagen. Wild-type polyoma DNA was rendered single stranded by molecular cloning into coliphage M13. The oligonucleotide, which hybridizes with a mismatch at the site to be altered, was used to prime the synthesis ofdouble-stranded closed circular DNA. Progeny recombinant phages were screened by DNA sequence analysis for the desired base change. The polyoma mutant was reconstructed from recombinant phage replicative form DNA molecules containing the mutation.The early region of polyoma virus encodes three proteins, the small [22-kilodalton (kDal)], the middle-sized (56-kDal), and the large (100-kDal) tumor (T) antigens. The large T antigen is required for transformation by intact virus (1-3) and may function by promoting stable association ofthe viral genome with cellularDNA. An intact large T protein is not essential for maintenance ofthe transformed phenotype (4)(5)(6)(7)(8)(9). Small T and middle T (mT) antigens have been implicated in maintenance of the transformed state and in tumorigenicity by the virus. The nontransforming hr-t mutants (10-12) have lesions affecting both the small T and mT polypeptides but not large T protein (4,(13)(14)(15).The importance of the mT polypeptide in transformation has been demonstrated directly; DNA coding only for mT antigen can transform rat cells (16). Mutants in which mT, but not small T, antigen is altered are affected in their transformation properties (17-19). When deletions affecting the COOH-terminal region of mT antigen are created in cloned polyoma DNA, the ability of that DNA to transform cells is reduced (20).Phosphorylation is important to mT antigen function. A protein kinase activity that phosphorylates tyrosine residue 315 of mT antigen is present in T-antigen immunoprecipitates (21-25). Middle T antigen can be resolved into two species, 56 kDal and 58 kDal, that differ strikingly in activity in the in vitro kinase reaction (24,25). These species also differ in their patterns of phosphorylation in vivo. The high specific activity 58-kDal form is phosphorylated in vivo at different serine or threonine residues than is the 56-kDal form (24, 25). These results suggest that the activity of mT antigen may be regulated by cellular protein kinase(s).mT antig...