tRNA-rRNA sequence matches from inter- and intraspecies comparisons suggest common origins for the two RNAs.

1. Comparisons were made of the results of searches within and among different species of organisms for sequence matches between transfer RNAs and ribosomal RNAs. The purpose was to determine whether the matching sequences might result from selection acting on the two RNAs within a common cellular environment. 2. The results indicate that most matches do not reflect such selection. The matches described were more frequent than those found in searches among randomized sequences and the frequency of intraspecific matches was not significantly higher than that of interspecific matches. 3. The matches are thought to identify conserved vestiges of a molecule or molecules ancestral to both classes of RNAs (Bloch, D.P., McArthur, B. and Mirrop, S. (1985). BioSystems, 17: 209-225). The matching sequences are interpreted as homologies.

tRNA rRNA sequence matches from inter- and intraspecies comparisons suggest common origins for the two RNAs

1. Comparisons were made of the results of searches within and among different species of organisms for sequence matches between transfer RNAs and ribosomal RNAs. The purpose was to determine whether the matching sequences might result form selection acting on the two RNAs within a common cellular environment. 2. The results indicate that most matches do not reflect such selection. The matches described we more frequent than those found in searches among randomized sequences and the frequency of intraspecific matches was not significantly higher than that of interspecific matches. 3. The matches ara thought to identify conserved vestiges of a molecule or molecules ancestral to both classes of RNAs (Bloch, D.P., McArthur, B. and Mirrop, S (1985), BioSystems, 17:209-225). The matching sequences are interpreted as homologies

Demonstrating evolutionary relationships between macromolecular sequences through mutual relationships with a third sequence.

Corresponding sites of the Euglena chloroplast and yeast small subunit ribosomal RNAs (rRNAs) show only an insignificant match with each other but show extensive matches with Euglena chloroplast tRNA(arg). The match with the tRNA extends farther toward the 5' end of the Euglena rRNA and toward the 3' end of the yeast rRNA. The expected number of such configurations given the number of RNAs searched is about 1 in 100,000. Comparison of two sequences with a third sequence frequently reveals relationships where pairwise comparisons fail to do so.

Evolution of E. coli tRNA(Trp).

Earlier studies (1) have shown there are direct correlations between the hydrophobicity ranking of most amino acids and their anticodonic nucleotides. However, four anticodonic assignments, i.e. those for Trp, Tyr, Ile and the XGA anticodons for Ser, did not correlate. It was our proposal that this failure to correlate was due to the fact that these assignments were made late, relative to the bulk of the assignments, in evolution through the mutation of existing tRNAs. We have shown (2) that E. coli tRNA(Ile 1) and tRNA(Ile 2) were likely derived from tRNA(Val 1) and tRNA(Lys) respectively and E. coli tRNA(Tyr) was possibly derived from E. coli 5s rRNA or a common precursor with 5s rRNA (3). The fact that quite high homologies were observed in these comparisons is consistent with the late evolution of the tRNAs in question. We now examine the evolution of E. coli tRNA(Trp) by comparing its homology with other E. coli tRNAs. The data suggest a possible evolutionary relationship with E. coli tRNA(Gly) or tRNA(Arg). The data support the idea of the late assignment of anticodons to Trp.

Cybernetic origins of replication.

An evolutionary progression leading toward replication is resolved into several phases; (a) the replication of RNA segments by self-priming and -templating, (b) the replication of single stranded molecules by elongation and controlled scission, (c) replication of complementary duplexes and (d) replication of DNA. The initial phase is suggested by evidence for the existence of tandem repeats in an early population of molecules presumed to be ancestral to today's structural RNAs. Relics of these repeats are seen in the positioning of sequence matches between transfer and ribosomal RNAs. Conservation of the positions of the matches is indicated by persistence of a periodicity in their spacings along the molecules. Selection is viewed as a vector, with a source and a focus. The evolutionary progression entails shifts in the source of selection, from external catalysts to the replicating molecule itself, and in its focus, from substrate to replicator, to the products of the replicator's activity. When the source and focus of selection are the same selection becomes internalized, and replication and Darwinian evolution follow. Catalytic specificity is regarded as an antecedent to natural selection. Shifting of the source and focus of selection and switches in evolution's 'vehicle', the most fundamental thing that evolves, result in profound changes in the modes of evolution. Control provides a conceptual framework within which entry into a Darwinian mode of evolution, and ultimately liberation from Darwinian evolution might be explained.

Evolution of E. coli tRNA(Ile): evidence of derivation from other tRNAs.

Two E. coli tRNA(Ile) sequences were compared against those of 36 other E. coli tRNAs. tRNA(Ile) 1 was found to bear high similarity with tRNA(Val) 1 (E = 1.11 X 10(-18] while tRNA(Ile) 2 had the greatest match (E = 3.40 X 10(-19] with tRNA(Lysl) (E is the expected number of such matches, per search, based on coincidence). These matches, which we consider to represent homologies, extend from base 7 to base 67 in the former and base 7 to the end (76) in the latter pair. These results coupled with others on the lower activity of isoleucine in reactions postulated to be important in primitive protein synthesis (i.e., esterification reactions and non-enzymatic activation by ATP [1-3]) lead us to propose that isoleucine was included among the proteinaceous amino acids, and received its anticodonic assignment, relatively late in evolution through mutation of tRNAs previously employed for other amino acids.

Detection of a fundamental modular format common to transfer and ribosomal RNAs: second-order spectral analysis.

Second-order spectral analysis is used to detect rigorously and to characterize the principal periodicities in the positions of conserved sequences common to tRNAs and rRNAs. It is shown that the shared periodicity having the largest spectral amplitude is 9, followed by 8 and 10, thus forming a closed triad of significant multiplets centered at 9 bases. This conclusion is proposed to reflect a closed triadic set of fundamental tandem repeat lengths in a class of ancestral macromolecules possessing a restricted sequence symmetry. The terms "remanent" and "archeomodular" are used to describe a relic modular format, traces of which are shown here to persist despite the changes that have occurred in the primary structures of ribonucleic acids during the course of their evolution.

tRNA-rRNA sequence homologies: evidence for an ancient modular format shared by tRNAs and rRNAs.

Homologies between tRNAs and rRNAs are identified in searches using various combinations of Escherichia coli, yeast, Halobacterium volcanii and bovine mitochondrial sequences. As in previously reported comparisons, the homologies are too frequent and long to be attributed to coincidence, and similar frequencies from inter- and intraspecies comparisons preclude evolutionary convergence as an explanation. In contrast to the earlier studies, patterns in the positioning of the homologies are now described. Graphing the positions of the homologies along orthogonal axes that represent numbers of bases in tRNA and rRNA shows recurring patterns in the alignments. Preferred spacings of integral multiples of 9 bases are found, suggesting a periodicity in the ancestral structure from which the tRNAs and rRNAs were derived. The periodicity also suggests persistence of a modular format in both classes of molecules that survived changes in sequence that occurred during evolution. A model is proposed for the generation of the ancestral molecule and the early evolution of the coding mechanism. Elongation by self-priming and self-templating gave a hairpin with a 9 base stem. Two additional cycles gave a 70-80 base tRNA-like structure. Additional cycles yielded a tandem repeat of this unit, roughly equivalent in size to the combined rRNAs of prokaryotes. The larger RNA would contain the information and materials for generating the smaller RNAs. It is proposed that multiple recombination among such molecules gave composite structures, presumed progenitors of today's t- and rRNAs. The distribution of the conserved domains among today's species argues for the existence of the ancestral molecule prior to divergence of lines leading to the various kingdoms. Their presence in the different nucleic acids suggests the existence of a nucleic acid with multiple functions prior to partitioning of these functions among the nucleic acids that exist today. The occurrence of overlaps, overlays and consensus alignments among the homologies provides the means for identifying contiguous and neighboring conserved regions and holds promise for the reconstruction of the sequence of an ancestral molecule.

tRNA-rRNA sequence homologies: evidence for a common evolutionary origin?

Many tRNAs of E. coli and yeast contain stretches whose base sequences are similar to those found in their respective rRNAs. The matches are too frequent and extensive to be attributed to coincidence. They are distributed without discernible pattern along and among the RNAs and between the two species. They occur in loops as well as in stems, among both conserved and non-conserved regions. Their distributions suggest that they reflect common ancestral origins rather than common functions, and that they represent true homologies.

A unique DNA found in post-mitochondrial fractions from Ehrlich ascites tumor cells.

Apresenta-se a caracterizacao parcial de um DNA encontrado em sedimentos pos-mitocondriais de celulas do tumor ascitico de Ehrlich. Esse DNA compoe 0,2% do DNA celular total e tem tamanho de 14,6 S, em homogenatos citoplasmicos. Sua eluicao de hidroxiapatita ocorre em tampao de fosfato de sodio 0,24 M.Sua densidade de flotacao em CS2SO4 e menor que a do DNA nuclear do tumor ascitico de Ehrlich e ele tem baixo conteudo de dG + dC, conforme determinado por cromatografia de hidrolisados apos marcacao por 32P. Ele e rico em sequencias que reassociam rapidamente na presenca de DNA nuclear em excesso. Ele pode ser usado como promotor de sintese de DNA por uma DNA polimerase dependente de DNA que e encontrada em associacao com os sedimentos pos-mitocondriais. Apos incubacao in vitro com UTP marcado, o DNA e encontrado associado com a radioatividade recem-incorporada. Nao se procedeu, ainda, a localizacao in situ do DNA. Postula-se que ele possa representar DNA associado a particulas viroticas tipo A ou DNA associado a membrana plasmatica

DNA and histone synthesis rate change during the S-period in Ehrlich ascites tumor cells.

Data from flow-cytometric analysis of DNA of Ehrlich ascites tumor cells were fitted using non-linear least squares curve fitting routines. Analysis of rates of synthesis from the derived S-period profiles revealed a pattern of changing rates of DNA synthesis during the S-period. Three main peaks are seen whose trough to through periods range from 0 to 16%, 16 to 65%, 65 to 100% of the DNA synthesized during S. The differences between the peak rates and rates in the intervening troughs are small, about 10% of the maximum, but these occur reproducibly. Some differences in the DNA distribution profiles, hence rate profiles, can be seen among samples taken at different times during the day. These are thought to reflect the effects of circadian rhythms, but they are not large enough to obscure the general pattern of rate shifts that occur during the S-period. Analyses of radioactivity of 3H-thymidine pulse labelled cells, sorted across the S-period, were in accord with the results obtained from the DNA distributions. A parallel analysis of DNA and histones showed a correspondence in the timing and direction of shifts in rate for both during the middle part of the S-period.

Spectra of cells in flow cytometry using a vidicon detector.

A TV type vidicon detector was interfaced to a flow cytometer (FCM) to obtain spectra of fluorophores in cells during flow. The normal operations of the FCM are undisturbed. A spectrograph spreads 320 nm of the fluorophore fluorescence emission across the 500 channels of the detector. Spectra of fluorescamine (a surface labeling agent) and of propidium iodide (a nuclear stain) were obtained from Balb 3T3 cells, and the chlorophyll and phycobilin peaks were resolved from flowing blue-green algae in the FCM. Under typical flow conditions, operation of the vidicon in the continuous mode gives for these fluorophores a S/N of several hundred to one in approximately 3 sec. The vidicon was also gated to obtain spectra of single cells and of cells in selected portions of the cell cycle. For example, the spectrum of fluorescamine was obtained from cells in the G1 phase of the growth cycle by using as a gate trigger the FCM discriminator output derived from the propidium iodide signal.

Isolation and characterization of chromatin from Neurospora crassa.

Different preparations of chromatin isolated from mycelia of Neurospora crassa were analyzed for DNA-associated RNA and proteins. The UV absorption spectra, the ultrastructure of chromatin, and the amino acid composition of the acid-extractable proteins were studied. The protein:DNA ratios range from 1.5 to 2.8; the RNA:DNA ratios range from 0.5 to 1.24. UV absorption shows a macimum at 259 mmicro and a minimum at 238-239 mmicro. The E280/E260 ranges from 0.59 to 0.70. Electron microscopy reveals a fibrous structure with individual fibers of 120-150 A average diameter. Attempts were made to study the protein by polyacrylamide gel electrophoresis and amino acid analysis. The results indicate that Neurospora chromatin does not contain basic proteins comparable to calf thymus histone. The ratios of basic to acidic amino acids range from 0.93 to 1.19. On electrophoresis, no bands are seen whose positions correspond to those of histones. Staining for basic proteins with fast green or eosin Y at pH 8.2 also shows a negative reaction, suggesting the absence of histones.