Effects Of Nucleotide Substitutions Research Articles

Global analysis of single nucleotide polymorphisms in the exons of human deoxyribonuclease I-like 1 and 2 genes

Several SNPs in the deoxyribonuclease I-like 1 (DNase 1L1) and DNase 1L2 were investigated. In the present study, the genotype distributions of three synonymous SNPs (V59V, rs1050095; P67P, rs1130929; A277A, rs17849495) in the DNase 1L1 gene and four non-synonymous SNPs, V122I (rs34952165), Q170H (rs6643670), and D227A (rs5987256) in the DNase 1L1 gene, as well as D197A (rs62621282) in the DNase 1L2 gene were investigated in 13 populations. In all the populations, no variation was found in four SNPs (V59V, Q170H, D227A, and A277A) in DNASE1L1 or in D197A in DNASE1L2. As for V122I, only the German population showed a low degree of polymorphism. The SNP V122I in DNASE1L1 was monoallelic for the G-allele in all of the Asian and African populations examined, with no polymorphism being evident. Since the A-allele in SNP V122I was distributed in only the Caucasian populations, not in the other ethnic groups, it was confirmed that the A-allele in SNP V122I was Caucasian-specific. On the other hand, only P67P in DNASE1L1 was polymorphic among three synonymous SNPs. The effect of nucleotide substitution corresponding to polymorphic SNP P67P on DNase 1L1 activity was examined: the corresponding nucleotide substitution in polymorphic SNP P67P has little effect on the DNase activity.

Molecular biology of hepatitis B virus: effect of nucleotide substitutions on the clinical features of chronic hepatitis B

Hepatitis B virus (HBV) is a major cause of liver disease worldwide. It is covered with envelope (surface antigen) proteins with the nucleocapsid (core antigen) inside. In the nucleocapsid, there is an incomplete double-stranded DNA and a DNA polymerase. Four genes, S, C, X, and P, are encoded, and these partially overlap. Mutations have been reported in each gene and in their promoter regions, and these mutations can change the efficiency of HBV replication and the clinical course of patients. In this article, we review the relationship between the molecular biology of HBV and its clinical outcome.

Deletion of a Conserved, Central Ribosomal Intersubunit RNA Bridge

Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.

Effects of nucleotide substitution and modification on the stability and structure of helix 69 from 28S rRNA

The helix 69 (H69) region of the large subunit (28S) rRNA of Homo sapiens contains five pseudouridine (Psi) residues out of 19 total nucleotides (26%), three of which are universally or highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine. The role of a loop nucleotide substitution from A in bacteria (position 1918 in Escherichia coli 23S rRNA) to G in eukaryotes (position in 3734 in H. sapiens) was also examined. The thermodynamic parameters were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by 1H NMR and circular dichroism spectroscopy. In addition, a [1,3-15N]Psi phosphoramidite was used to generate H69 analogs with site-specific 15N labels. By using this approach, different Psi residues can be clearly distinguished from one another in 1H NMR experiments. The effects of pseudouridine on H. sapiens H69 are consistent with previous studies on tRNA, rRNA, and snRNA models in which the nucleotide offers stabilization of duplex regions through PsiN1H-mediated hydrogen bonds. The overall secondary structure and base-pairing patterns of human H69 are similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved. Nonetheless, pseudouridine-containing RNAs have subtle differences in their structures and stabilities compared to the corresponding uridine-containing analogs, suggesting possible roles for Psi such as maintaining translation fidelity.

Effect of nucleotide substitutions in N-acetyltransferase-1 on N-acetylation (deactivation) and O-acetylation (activation) of arylamine carcinogens: implications for cancer predisposition

Genetic polymorphism in N-acetyltransferase-1 ( NAT1) is associated with increased risk of various cancers, but epidemiological investigations are compromised by poor understanding of the relationship between NAT1 genotype and phenotype. Human reference NAT1 ∗4 and 12 known human NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region were cloned and expressed in yeast ( Schizosaccharomyces pombe). Large reductions in the N-acetylation of 4-aminobiphenyl and the O-acetylation of N-hydroxy-2-aminofluorene were observed for recombinant NAT1 allozymes encoded by NAT1 ∗14B , NAT1 ∗15 , NAT1 ∗17 , NAT1 ∗19 , and NAT1 ∗22 . Each of these alleles exhibited substantially lower expression of NAT1 protein than the reference NAT1 ∗4 and the other NAT1 alleles. These results show an important effect of the NAT1 genetic polymorphism on the N- and O-acetylation of arylamine carcinogens, suggesting modification of cancer susceptibility following exposures to arylamine carcinogens.

Tracking Changes in HIV-1 Envelope Quasispecies Using DNA Heteroduplex Analysis

A DNA heteroduplex tracking assay (HTA) using single-stranded probes is described. This assay provides a rapid means of resolving genetic variants coamplified by PCR and of measuring the level of particular variants in complex populations. To confidently detect minor quasispecies changes, the importance of maximizing template input into nested PCR (nPCR) and of duplicating nPCR and HTA to ensure correct population sampling is highlighted. The sensitivity of detection of rare variants within a genetically mixed population using single-stranded DNA probes is shown to be 1:500. The effects of nucleotide substitution at different locations on heteroduplex electrophoretic mobility are used to illustrate the limits of HTA for mutation detection. This simple assay may be used to track the evolution of HIV as well as to address issues of contamination and transmission.

Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA.

The gene start (GS) and gene end (GE) transcription signals of human respiratory syncytial virus (RSV) strain A2 were analyzed in helper-dependent monocistronic and dicistronic minireplicons which were complemented by a standard RSV strain. The GS signal, which is the start site for mRNA synthesis, is highly conserved for the first nine genes: 3'-CCCCGUUUA(U/C) (negative sense). This conserved version of the signal was analyzed by "saturation" mutagenesis, in which all 10 positions, as well as one downstream and one upstream position, were changed one at a time into each of the other three nucleotides. Most of the positions appear to contribute to the signal: positions 1, 3, 6, 7, and, in particular, 9 were the most sensitive, whereas position 5 was relatively insensitive. The effect of nucleotide substitution in the first position of the signal was examined further by cDNA cloning and sequence analysis of the residual mRNA which was produced. For the two mutants examined (1C to U, and 1C to A), the site of initiation was unchanged. However, the mRNAs were dimorphic with regard to the assignment of the 5'-terminal nucleotide: two-thirds contained the predicted mutant substitution, and one-third contained the parental assignment. Intracellular minigenome contained only the mutant assignment, indicating that the heterogeneity was at the level of transcription by the RSV polymerase. This suggests that the templated mutant assignment at position 1 can sometimes be overridden by an innate preference for the parental assignment, a phenomenon which we dubbed quasi-templated initiation. The GS signal of the L gene, encoding the 10th RSV mRNA, contains three differences (3'-CCCUGUUUUA) compared to the conserved version. It was shown to be equal in efficiency to the conserved version. This was unexpected, since the saturation mutagenesis described above indicated that U in place of A at position 9 should be highly inhibitory. Instead, the A at position 10 of the L GS signal was found to be critical for activity, indicating that an essential A residue indeed was present in both versions of the GS signal but that its spacing differed. The GE signal, which directs termination and polyadenylation, has more sequence diversity in nature than does the GS signal. The naturally occurring GE signals of strain A2 were compared by their individual incorporation into a dicistronic minigenome. They were similar in the ability to produce translatable mRNA except in the cases of NS1 and NS2, which were approximately 60% as efficient.

Strength of translation initiation signal sequence of mRNA as studied by quantification method: effect of nucleotide substitutions upon translation efficiency in rat preproinsulin mRNA.

Concerning the translation initiation signals in vertebrate mRNAs, both the ATG initiation codon and the sequences flanking the initiation codon are required to direct the position of initiation. A consensus sequence for the signal, (GCC)GCC(A or G)CCATGG, has been proposed, but actual initiation sequences differ from it to a greater or lesser degree. In the present report, the translation initiation signal sequences of rat preproinsulin and its mutant mRNAs were analyzed using a quantification method proposed previously. In this method, each 16 nt sequence in the mRNA was characterized by its sample score, which shows strength of the signal. So far, Kozak has constructed a number of preproinsulin mutant mRNAs in which nucleotides flanking the ATG codon are systematically varied, and measured the translation initiation efficiency in terms of the proinsulin product. Her experimental results were well understood on the basis of the strength of the translation initiation signal sequence.

Both Codon Context and Leader Length Contribute to Efficient Expression of Two Overlapping Open Reading Frames of a Cucumber Necrosis Virus Bifunctional Subgenomic mRNA

The importance of codon context and leader length in the translational regulation of p20 and p21 from the bifunctional 0.9-kb subgenomic mRNA of cucumber necrosis virus was investigated. Nucleotide substitutions introduced into the −3 and +4 positions of the p21 AUG codon (where the A of the AUG is +1) verified that purines in these positions are favored and demonstrated the similar contribution of the −3 and +4 positions to the efficiency of initiation codon selection in plants. The effect of nucleotide substitutions in the +5 position, most clearly demonstrated when pyrimidines occupy the −3 and +4 positions, also provided direct insight into the influence of the +5 position in plants. The codon context of the upstream p21 initiation codon affected expression from the downstream p20 AUG codon. In addition, an increase in the length of the subgenomic mRNA leader decreased expression from the downstream p20 initiation site. These latter observations are in accordance with the “Kozak rules” for accession of internal AUG codons by leaky ribosomal scanning and provide the first example of an effect of leader length on the efficiency of translation initiation in a plant (viral) mRNA.

Effects of nucleotide substitutions within the T-loop of precursor tRNAs on interaction with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and yeast.

Recognition of tRNA and tRNA-like substrates by the enzyme ATP/CTP:tRNA nucleotidyltransferase requires chemically intact nucleotides within the T-loop, especially at positions 57 and 58, which are invariant purines among naturally occurring tRNAs. To test the effects of base substitutions at these positions, which are distant from the site of catalysis, we synthesized mutant tRNA(Glu) molecules. These in vitro-synthesized RNAs also contained an extra 33 bases at the 5' end and lacked post-transcriptionally modified bases. The precursor tRNAs were used as substrates for nucleotidyltransferases from Escherichia coli and yeast. Substitution of cytidines at either position 57 or 58 had dramatic inhibitory effects on recognition by both enzymes, including raising the apparent Km and lowering the apparent Vmax.; substitution of an adenosine at position 57 or a uridine at position 58 inhibited the reaction only slightly by comparison. Our results demonstrate that the identities of nucleotides at positions 57 and 58 are relevant to recognition by nucleotidyltransferase, and that a purine is required at position 57. The extra bases at the 5' end and the lack of post-transcriptionally modified bases did not substantially inhibit interaction with the enzyme, as judged by the wild-type precursor tRNA(Glu) acting as an effective substrate for both enzymes in the presence of equal concentrations of appropriate tRNA substrates isolated from E. coli.

Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA.

In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal; few shifts in the 19F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29.A41 base-pair in the anticodon stem with C29.G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA. Nucleotide substitution at the 3' end of the anticodon (position 36) reduced the aminoacylation efficiency (Vmax/Km) of tRNAVal by three orders of magnitude. Base replacement at the 5' end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency. Substitution of the FU29.A41 base-pair increased the Km value 20-fold, while Vmax remained almost unchanged. The FU4.A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E. coli tRNAVal, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.

Fine-structure mapping ofcis-acting control sites in thelysCoperon ofBacillus subtilis

Mutations at the aecA locus of Bacillus subtilis lead to derepression of the lysC operon, which encodes aspartokinase II, and analysis of three independent aecA mutations has shown them to be nucleotide substitutions in the lysC leader region (Y. Lu, N.Y. Chen and H. Paulus (1991) J. Gen. Microbiol. 137, 1135–1141). DNA sequence analysis of the lysC control region of nine other mutants with derepressed levels of aspartokinase II revealed each of the mutations to be associated with changes in one or a few nucleotide residues. The nucleotide substitutions were clustered at two sites in the lysC leader: in a region of imperfect dyad symmetry about 40 base pairs from the transcription start site, and in the open reading frame for a putative leader peptide, which starts about 40 residues further downstream. The effect of nucleotide substitutions at the two sites differed in that those at the upstream site gave twice the degree of derepression. A mutant with a small deletion in the leader peptide coding region potentially affecting RNA secondary structure also had a higher level of lysC derepression. These results suggest that the lysC leader region contains at least two cis-acting control sites that play important and perhaps independent roles in the repression of the lysC operon by lysine.

Fine-structure mapping of cis-acting control sites in the lysC operon of Bacillus subtilis

Mutations at the aecA locus of Bacillus subtilis lead to derepression of the lysC operon, which encodes aspartokinase II, and analysis of three independent aecA mutations has shown them to be nucleotide substitutions in the lysC leader region (Y. Lu, N.Y. Chen and H. Paulus (1991) J. Gen. Microbiol. 137, 1135–1141). DNA sequence analysis of the lysC control region of nine other mutants with derepressed levels of aspartokinase II revealed each of the mutations to be associated with changes in one or a few nucleotide residues. The nucleotide substitutions were clustered at two sites in the lysC leader: in a region of imperfect dyad symmetry about 40 base pairs from the transcription start site, and in the open reading frame for a putative leader peptide, which starts about 40 residues further downstream. The effect of nucleotide substitutions at the two sites differed in that those at the upstream site gave twice the degree of derepression. A mutant with a small deletion in the leader peptide coding region potentially affecting RNA secondary structure also had a higher level of lysC derepression. These results suggest that the lysC leader region contains at least two cis-acting control sites that play important and perhaps independent roles in the repression of the lysC operon by lysine.

Effect of nucleotide substitution on the peptidyltransferase activity of 2'(3')-O-(aminoacyl) oligonucleotides

Seven 2'(3')-O-(aminoacyl) trinucleotides with structures derived from the 3'-terminal C-C-A sequence of aa-tRNA via nucleotide substitutions were investigated as acceptor substrates in the peptidyltransferase reaction and as inhibitors of substrate binding to the peptidyltransferase A site. It was found that all tested compounds were active in both systems, although substitution in the first and second nucleotide position results in some decrease of acceptor activity. Remarkably, replacement of natural cytidylic acid residues in C-C-A-Phe with guanylic acid moieties resulted only in a small decrease of acceptor or binding activity. The results indicate that the acceptor sequence of aa-tRNA is not probably engaged in base pairing with a sequence of 23S RNA during its interaction with the peptidyltransferase A site.

The effect of nucleotide substitution on DNA denaturation profiles.

The melting profiles were obtained for DNA restriction fragments of approx. 1150 bp with deletion of one, five or six base pairs making them different from each other. In all cases the deletions caused a shift of one melting peak without affecting the positions of the other three peaks. The effect amounted to 0.28 +/- 0.03C upon the deletion of one GC pair. The melting of DNA fragments was also studied by electrophoresis in denaturing gradient gels. The deletion of one GC pair was shown to cause an appreciable shift of the electrophoretic denaturation profile.