The age of the molecular organization of life as expressed in the genetic code can be estimated from experimental data. Comparative sequence analysis of transfer RNA by the method of statistical geometry in sequence space suggests that about one-third of the present transfer RNA sequence divergence was present at the urkingdom level about the time when archaebacteria separated from eubacteria. It is concluded that the genetic code is not older than, but almost as old as our planet. While this result may not be unexpected, it was not clear until now that interpretable data exist that permit inferences about such early stages of life as the establishment of the genetic code.

Some 200 different SS rRNA sequences from eubacteria, chloroplasts, mitochondria, archaebacteria, and eukaryotes were analyzed for evolutionary kinship relationships and associated sequential features. Group-specific occupation schemes for the 149 positions of an overall alignment were established. Eubacterial, archaebacterial, and intermediate occupation schemes all yield a strongly biased base triplet pattern in one of the three possible reading frames strongest for eubacterial, chloroplastic, and archaebacterial, but still detectable for mitochondrial and eukaryotic cytoplasmic sequences. The frequency of triplets decays in the order RNY > RNR > YNY > YNR; R being a purine (guanine or adenine), Y is a pyrimidine (cytosine or uracil), and N is any base. A strong preference for guanine or cytosine was found in all triplet positions. The effects show no exceptions and are clearly above the level of statistical fluctuations.In this paper, we report a comparative study of the "200 5S rRNA sequences known today. Preliminary analysis of some mainly eubacterial 5S rRNA sequences (1) revealed a clear bias for the presence of a triplet pattern 5' RNY 3' where R is a purine (guanine or adenine), Y is a pyrimidine (cytosine or uracil), and N is any nucleotide. A similar phenomenon was found previously for tRNA sequences (2, 3). While the reading frame for tRNAs is defined through the position of the anticodon and the common assignment of the 5' terminus, 5S rRNA sequences vary in length and therefore had to be tested for the three possible reading frames. For each individual sequence, the RNY bias shows up in only one of the frames, varying with respect to the 5'-terminal position; in the two corresponding alternative frames, a weaker YNR bias always appears. We conjectured and proved with this study that the variable reading frames can be synchronized through proper alignment.Our analysis is essentially based on data from two sources. Most of the sequences are compiled in an alignment produced by Erdmann et al. (4), of which we used only nondegenerate sequences in order' to avoid statistical bias.These comprise 115 eukaryotic, 37 eubacterial, 9 chloroplastic, and 1 mitochondrial sequence. In addition, 17 archaebacterial sequences were kindly provided by G. E. Fox, C. R.Woese, and K. R. Luehrsen (personal communication).All data were filed and processed on a Philips P2000 M computer so as to yield alignment, common reading frames, tree topology, base composition, and periodic patterns. Alignment and Common Reading FramesAny comparative analysis of base composition and pattern structures is critically dependent on proper alignment of the sequences. There are sufficient homologies and invariances distributed along the entire sequence that assignment of positions does not pose any serious problem for an overwhelming majority of sequences. Minor uncertainties remain for only a few positions in the mitochondrial and some archaebacterial sequences.Alignment was greatly aided by the determination of master sequences, wh...

The emergence of viral escape mutants is usually a highly undesirable phenomenon. This phenomenon is frequently observed in antiviral drug applications for the treatment of viral infections and can undermine long-term therapeutic success. Here, we propose a strategy for evaluating a given antiviral approach in terms of its potential to provoke the appearance of resistant virus mutants. By use of Q␤ RNA phage as a model system, the effect of an antiviral gene therapy, i.e., a virus-specific repressor protein expressed by a recombinant Escherichia coli host, was studied over the course of more than 100 generations. In 13 experiments carried out in parallel, 12 phage populations became resistant and 1 became extinct. Sequence analysis revealed that only two distinct phage mutants emerged in the 12 surviving phage populations. For both escape mutants, sequence variations located in the repressor binding site of the viral genomic RNA, which decrease affinity for the repressor protein, conferred resistance to translational repression. The results clearly suggest the feasibility of the proposed strategy for the evaluation of antiviral approaches in terms of their potential to allow resistant mutants to appear. In addition, the strategy proved to be a valuable tool for observing virus-specific molecular targets under the impact of antiviral drugs.Ideally, antiviral strategies should be highly virus specific and should not affect the host organism. In addition, they should exhibit a minimal potential to allow viral escape mutants to appear. In this paper, we propose a general strategy to evaluate a given antiviral approach with regard to resistance phenomena that may occur due to the emergence of escape mutants. Utilizing RNA phage Q␤ as a model system, we investigated the potential of virus-specific translational repressor proteins to suppress viral propagation. Among the mechanisms that control gene expression, translational regulation is certainly one of the most specific and therefore should be a valuable target mechanism for antiviral strategies.First observed in the RNA phages (29), translational repression has since been discovered in many bacteriophages (47), prokaryotes (11,16,45), and eukaryotes (46) as well as in several mammalian viruses (14,17,35,44). Within the closely related RNA phage species R17, MS2, F2, fr, and Q␤, two proteins, namely, the coat protein and the replicase, are translational regulators of each other. Shortly after single-stranded viral RNA enters the host cell, replicase protein is translated from the proximal end of the phage genome, leading to high levels of replicated phage RNA.However, it has been shown that replication cannot occur when phage RNA is being translated (25,47). Therefore, strict translational regulation of the remaining cistrons, namely, A1/ coat protein and A2, from the distal portion of the Q␤ RNA genome is essential for the replication. Hence, blocking the translation of the coat protein, which is produced abundantly during the late phase of the viral infection cycl...

A novel transcription system was constructed that allows trimming of 3' termini of RNA transcripts in E. coli by endogenous RNase P. Here, the sequence of tRNASer from E. coli fused downstream of the target sequence directs posttranscriptional cleavage 3' of the target sequence. As a first-target MNV11(+), a self-replicating RNA from the QB system was subjected to transcription in vivo. Northern blotting experiments revealed that the primary transcript was indeed successfully processed to an RNA of expected length. The RNA released proved to function as an active template for QB replicase. Moreover, E. coli cells producing these short-chain replicator molecules no longer supported multiplication of QB phages upon infection. Since the novel transcript-trimming system utilizes the endogenous RNase P activity and does not depend on any particular 3'-terminal RNA sequence of target molecules, it may have wide applications for a number of different targets in prokaryotes. Further applications, including those in eukaryotes, are discussed.

Lytic coliphage Qbeta was grown in continuously cultured host bacteria using a cascade of stirred flow reactors. The apparatus was constructed so that the steady stream of exponentially growing bacterial cells passing through the stirred flow reactors served to prevent coevolution brought about by host-parasite interactions. Wall growth was the primary cause for deviation from ideal continuous culture conditions and is largely dependent on the surface structure of the host bacteria. Using an Escherichia coli strain deficient in adhesive type I pili expression, the desynchronization of single burst events could easily be followed over the course of four infection latency periods. Computer simulations based on a two-stage model for the Qbeta infection cycle were in perfect agreement with the experimental data. Applications of the optimized system to strategies of molecular evolution are discussed.