The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S-23S intergenic spacer region (ISR) for tRNA(Glu-2) and three (rrnA, D, and H) contain genes for tRNA(Ile-1) and tRNA(Ala-1B). To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.
VKORC1 and CYP2C9 polymorphisms are used to predict the safe dose of oral anticoagulant therapy. A new variant of CYP4F2 (V433M) has recently been related to the required warfarin dose. We evaluated its influence in earliest response to acenocoumarol in 100 selected men who started anticoagulation (3 mg for 3 consecutive days). V433M genotype exerted a gene dosage-dependent effect on the decrease of factors II, VII, IX, and X in the earliest response to acenocoumarol, with homozygous 433V subjects being the most sensitive. Similarly, after the initiation of therapy, international normalized ratio also experienced a gene dosage-dependent effect (P ؍ .015), and 433V subjects needed 4 mg/week less than 433M carriers to achieve a steady anticoagulation (P ؍ .043). Multivariate linear regression analysis revealed a significant contribution of V433M polymorphism to variability of both early international normalized ratio value (R 2 ؍ 0.14) and dose requirements (R 2 ؍ 0.19). Our data underline the relevant role of CYP4F2 V433M polymorphism in the pharmacogenetics of coumarin anticoagulants. (Blood. 2009;113:4977-4979)
Sequence heterogeneities of variable positions located a t regions V1 and V6 of 56 cloned 16s rRNA genes were determined from six Escherichia coli strains. These nucleotides were involved in secondary structure base-pairing of stem-loops. Compensatory and single mutations have occurred but secondary structure was conserved. Eight different sequences were found in the stem a t region V1 indicating that in these sites mutation rates are higher than those of homogenization processes. Region V6 showed two different structures (V6-I and V6-ll) although heterogeneities were determined in nine sites. Strains ECOR52 and ECOR56 only showed the V6-I sequence, ECOR35 showed V6-ll, whereas clones from ECOR42 and ECOR49 showed both types of V6 structures. Results were confirmed b y PCR using V6 sequence-specific probes. Stem V6-ll was also found in 165 rRNA sequences deposited in the RDP (Ribosomal Database Project) belonging to distantly related taxa; ancestral sequence V6-II seems to be homogenized in all rm operons of the multigene family of strain ECOR35 producing effects of distortion in the molecular clock, similar to those that homoplasies could produce. V6 sequence-specif ic probes were applied to the 72 ECOR strains: half showed both V6-I and V6-ll, and the rest had one or another. Only strain ECOR24 did not yield products in the PCR test and sequencing of 12 cloned 165 rRNA genes revealed a third form, V6-Ill, also found in the RDP. Concerted evolution by homogenization of the rRNA family may induce chronometric distortions responsible for a loss of ultrametricity in phylogenetic trees, particularly, of very closely related micro-organisms.
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