Mutations That Affect the Efficiency of Translation of mRNA for the cII Gene of Coliphage Lambda

Starting with the lambda pRE-strain lambda ctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, lambda dya2 ctr1 cy3008 and lambda dya3 ctr1 cy3008, that form plaques with very slightly turbid centers. The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations in cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene. Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3'. --The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII-mutations that are characterized by inefficient translation of cII mRNA. These are cII3088, an A----G mutation four bases before the initial AUG codon, and cII3059, a GUU----GAU (Val2----Asp) second codon mutation. It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects. The dya3 mutation has no effect on translation efficiency under any condition tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse)

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.     

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.     

Mutation spectrum of o-aminoazotoluene in the cII gene of lambda/lacZ transgenic mice (MutaMouse)

The o-aminoazotoluene (AAT) has been evaluated as a possible human carcinogen by the International Agency for Research on Cancer. In rodents, it is carcinogenic mainly in the liver, and also in lung following long term administration. We previously examined in lambda/lacZ transgenic mice for the induction of lacZ mutations in liver, lung, urinary bladder, colon, kidney, bone marrow, and testis. AAT induced gene mutations strongly in the liver and colon. In the present report, we reveal the molecular nature of mutations induced by AAT in the lambda cII gene (the cII gene, a phenotypically selectable marker in the lambda transgene, has 294bp, which makes it easier to sequence than the original target, the 3kb lacZ gene). The cII mutant frequency in liver and colon was five and nine times higher, respectively, in AAT-treated mice than in control mice. Sequence analysis revealed that AAT induced G:C to T:A transversions, whereas spontaneous mutations consisted primarily of G:C to A:T transitions at CpG sites.     

Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cII protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cII protein caused by its decreased stability, since cII product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (lambda c1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion+ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.     

Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice

The lacI gene in Big Blue transgenic rodents has traditionally been used as a surrogate gene for in vivo mutations. Recently, a more efficient and less expensive assay involving direct selection in the smaller lambda cII gene has been developed. Little is known, however, about the comparative sensitivity of the two loci or their influence on the recovered mutation spectrum following mutagen treatment. We have compared the mutation frequency (MF) and mutational spectrum (MS) of lacI and cII from the same DNA samples isolated from the liver of control and dimethylnitrosamine (DMN)-treated mice. A three-fold (p<0.01) increase in the MF was observed at both loci in the DMN-treated group compared to the corresponding control groups. While the DMN-induced mutation spectrum at lacI was significantly different from its corresponding spontaneous mutation spectrum (p<0.001), the mutation spectrum at cII (p>0.28) was not. The mutation spectra at the two loci from the DMN-treated mice resembled each other but the 4, 2.5 and 12-fold increase in the mutation frequency of A:T>T:A transversions, single base deletions and deletions of more than four base pairs, respectively, at lacI, altered the spectra significantly (p<0.007). The number of mutations of these classes at cII was also increased, but the fractions were lower than at lacI. The spontaneous mutation spectra at the cII and lacI loci resembled each other except for the seven-fold increase in G:C相似文献   

Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation

Using hydroxylamine mutagenesis in vitro, mutations were introduced into a short DNA fragment containing the two overlapping promoters of the Escherichia coli galactose operon and the start of the first gal gene, galE. The mutagenised fragment was inserted into a lac expression plasmid. In such a vector, lac expression is controlled by the gal promoter region. Amongst eighteen candidates in which expression was reduced due to mutations in the gal fragment, twelve contained promoter mutations and six carried mutations that reduce the initiation of galE translation. The candidates in which promoter activity was reduced contained mutations affecting the promoter P1, which is dependent on the cyclic AMP-receptor protein complex (cAMP-CRP) for activation. All carried mutations in the sequence 5'GTGA3' at the CRP binding site. One of the twelve also contained a second mutation affecting the second promoter, P2, which normally functions in the absence of cAMP-CRP. Amongst the six candidates affecting galE translation, two contained a mutation that changes the initiator codon from AUG to AUA and almost completely suppresses galE expression. The mutations in the other four candidates affect the ribosome binding sequence, 5'GGAG3'. However, multiple mutations that abolish this sequence do not totally suppress galE expression, showing that there must be another way to guide ribosomes to the correct initiation site.     

Accurate selection of a 5' splice site requires sequences within fibronectin alternative exon B.

Inclusion of fibronectin alternative exon B in mRNA is developmentally regulated. Here we demonstrate that exon B contains two unique purine-rich sequence tracts, PRE1 and PRE2, that are important for proper 5' splice site selection both in vivo and in vitro. Targeted mutations of both PREs decreased the inclusion of exon B in the mRNA by 50% in vivo. Deletion or mutation of the PREs reduced removal of the downstream intron, but not the upstream intron, and induced the activation of cryptic 5' splice sites in vitro. PRE-mediated 5' splice selection activity appears sensitive to position and sequence context. A well characterized exon sequence enhancer that normally acts on the upstream 3' splice site can partially rescue proper exon B 5' splice site selection. In addition, we found that PRE 5' splice selection activity was preserved when exon B was inserted into a heterologous pre-mRNA substrate. Possible roles of these unique activities in modulating exon B splicing are considered.     

Non-AUG initiation of AGAMOUS mRNA translation in Arabidopsis thaliana

The MADS box organ identity gene AGAMOUS (AG) controls several steps during Arabidopsis thaliana flower development. AG cDNA contains an open reading frame that lacks an ATG triplet to function as the translation initiation codon, and the actual amino terminus of the AG protein remains uncharacterized. We have considered the possibility that AG translation can be initiated at a non-AUG codon. Two possible non-AUG initiation codons, CUG and ACG, are present in the 5' region of AG mRNA preceding the highly conserved MADS box sequence. We prepared a series of AG genomic constructs in which these codons are mutated and assayed their activity in phenotypic rescue experiments by introducing them as transgenes into ag mutant plants. Alteration of the CTG codon to render it unsuitable for acting as a translation initiation site does not affect complementation of the ag-3 mutation in transgenic plants. However, a similar mutation of the downstream ACG codon prevents the rescue of the ag-3 mutant phenotype. Conversely, if an ATG is introduced immediately 5' to the disrupted ACG codon, the resulting construct fully complements the ag-3 mutation. The AG protein synthesized in vitro by initiating translation at the ACG position is active in DNA binding and is of the same size as the AG protein detected from floral tissues, whereas AG polypeptides with additional amino-terminal residues do not appear to bind DNA. These results indicate that translation of AG is initiated exclusively at an ACG codon and prove that non-AUG triplets may be efficiently used as the sole translation initiation site in some plant cellular mRNAs.     

Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5' leader sequence

It has previously been proposed that Escherichia coli ribosomal protein S1 is required for the translation of highly structured mRNAs. In this study, we have examined the influence of structural features at or near the start codon of different mRNAs. The requirement for ribosomal protein S1 for translation initiation was determined when (i) the ribosome-binding site (RBS) was either preceded by a 5' non-translated leader sequence; (ii) the RBS was located 5' proximal to a mRNA start codon; and (iii) the start codon was the 5' terminal codon as exemplified by leaderless mRNAs. In vitro translation studies revealed that the leaderless lambda cl mRNA is translated with Bacillus stearothermophilusribosomes, naturally lacking a ribosomal protein S1 homologue, whereas ompA mRNA containing a 5' leader is not. These studies have been verified by toeprinting with E. coli ribosomes depleted for S1. We have shown that S1 is required for ternary complex formation on ompA mRNA but not for leaderless mRNAs or for mRNAs in which the RBS is close to the 5' end.