Donald R. Forsdyke

Relative roles of primary sequence and (G + C)% in determining the hierarchy of frequencies of complementary trinucleotide pairs in DNAs of different species

To an approximation Chargaffs rule (%A = %T; %G = %C) applies to single-stranded DNA. In long sequences, not only complementary bases but also complementary oligonucleotides are present in approximately equal frequencies. This applies to all species studied. However, species usually differ in base composition. With the goal of understanding the evolutionary forces involved, I have compared the frequencies of trinucleotides in long sequences and their shuffled counterparts. Among the 32 complementary trinucleotide pairs there is a hierarchy of frequencies which is influenced both by base composition (not affected by shuffling the order of the bases) and by base order (affected by shuffling). The influence of base order is greatest in DNA of 50% G + C and seems to reflects a more fundamental hierarchy of dinucleotide frequencies. Thus if TpA is at low frequency, all eight TpA-containing trinucleotides are at low frequency. Mammals and their viruses share similar hierarchies, with intra- and intergenomic differences being mainly associated with differences in base composition (percentage G + C). E. coli and, to a lesser extent, Drosophila melanogaster hierarchies differ from mammalian hierarchies; this is associated with differences both in base composition and in base order. It is proposed that Chargaffs rule applies to single-stranded DNA because there has been an evolutionary selection pressure favoring mutations that generate complementary oligonucleotides in close proximity, thus creating a potential to form stem-loops. These are dispersed throughout genomes and are rate-limiting in recombination. Differences in (G + C)% between species would impair interspecies recombination by interfering with stem-loop interactions.

Purine loading, stem-loops and Chargaff’s second parity rule: a discussion of the application of elementary principles to early chemical observations

DNA base compositions were determined chemically long before sequencing technologies permitted the direct counting of bases. Some recent observations made using modern sequencing technologies could have been deduced by application of elementary principles to early chemical observations. This paper draws attention to the fact that the potential for significant stem-loop structure is a general property of single-stranded DNA (genic and nongenic) and hence for any corresponding transcripts, whether they function by virtue of their structure (eg rRNA) or as mRNA. Furthermore, there is Chargaff’s second parity rule: in single strands, the percentage of purines approximately equals the percentage of pyrimidines. Since, in stems, purines match pyrimidines, Szybalski’s rule that transcripts violate the second parity rule in favour of purines, must apply to loops. Since purine loading occurs in both mesophilic and thermophilic species, genes with transcripts that need stable secondary structures for functioning at high temperatures must achieve this by selectively increasing the GC percentage (GC%) of stems, while retaining purine loading of loops. Arguments that purine loading is specific for the loops of RNA-synonymous strands of genes whose transcripts function by virtue of their secondary structure (ie rRNAs, not mRNAs) need to take into account, as controls, the loop regions of mRNA-synonymous strands. Entire genes, or entire genomes where gene orientation is not considered, are not appropriate controls.

Are introns in-series error-detecting sequences?

Abstract The probability of the accurate transmission of a message sequence can be increased by the addition of non-message sequences which permit errors in the message sequence to be detected and corrected. It is proposed that sequences in introns (or in other non-message genomic regions) serve this function with respect to the transmission of genetic information.

Selective pressures that decrease synonymous mutations in Plasmodium falciparum.

Rich and Ayala propose that the zero rate of non-amino-acid-changing (synonymous) mutations in some proteins of Plasmodium falciparum reflects a recent population bottleneck. Alternatively, Arnot and Saul propose sequence conservation in response to selective pressures other than the pressure to encode protein. Among these are fold pressure and purine-loading pressure. Genomes adapt to these by acquisition of introns and/or low-complexity (simple-sequence) segments in proteins. Adaptive explanations include facilitation of intragenic recombination (and hence diversification of the encoded protein) by DNA stem-loop secondary structures.

The origins of the clonal selection theory of immunity as a case study for evaluation in science

Evaluation of high achievement forms part of an evaluation continuum operating throughout the scientific enterprise. Correct evaluation of individuals and their work means that the best work is published and the most able individuals obtain researchgrants and awards. It is important that evaluations be carried out fairly and objectively. This paper reviews evaluations of the roles of Ehrlich, Jerne, Talmage, and Burnet in the conception and development of the clonal selection theory. These evaluations show varying degrees of bias; in particular, the major role of Ehrlich tends to be overlooked. The objective record shows that Talmage first conceived clonal selection. The proclivity of evaluation in science toward error suggests that our evaluation processes should be redesigned to take this into account.

SENSE IN ANTISENSE

A correspondence between open reading frames in sense and antisense strands is expected from the hypothesis that the prototypic triplet code was of general form RNY, where R is a purine base, N is any base, and Y is a pyrimidine. A deficit of stop codons in the antisense strand (and thus long open reading frames) is predicted for organisms with high G + C percentages; however, two bacteria (Azotobacter vinelandii, Rhodobacter capsulatum) have larger average antisense strand open reading frames than predicted from (G + C)%. The similar Codon frequencies found in sense and antisense strands can be attributed to the wide distribution of inverted repeats (stem-loop potential) in natural DNA sequences.

Reciprocal relationship between stem-loop potential and substitution density in retroviral quasispecies under positive Darwinian selection.

Nucleic acids have the potential to form in trastrand stem-loops if complementary bases are suitably located. Computer analyses of poliovirus and retroviral RNAs have revealed a reciprocal relationship between “statistically significant” stem-loop potential and “sequence variability.” The statistically significant stem-loop potential of a nucleic acid segment has been defined as a function of the difference between the folding energy of the natural segment (FONS) and the mean folding energy of a set of randomized (shuffled) versions of the natural segment (FORS-M). Since FONS is dependent on both base composition and base order, whereas FORS-M is solely dependent on base composition (a genomic characteristic), it follows that statistically significant stem-loop potential (FORS-D) is a function of base order (a local characteristic). In retroviral genomes, as in all DNA genomes studied, positive FORS-D values are widely distributed. Thus there have been pressures on base order both to encode specific functions and to encode stem-loops. As in the case of DNA genomes under positive Darwinian selection pressure, in HIV-1 specific function appears to dominate in rapidly evolving regions. Here high sequence variability, expressed as substitution density (not indel density), is associated with negative FORS-D values (impaired base-order-dependent stem-loop potential). This suggests that in these regions HIV-1 genomes are under positive selection pressure by host defenses. The general function of stem-loops is recombination. This is a vital process if, from among members of viral “quasispecies,” functional genomes are to be salvaged. Thus, for rapidly evolving RNA genomes, it is as important to conserve base-order-dependent stem-loop potential as to conserve other functions.

Further implications of a theory of immunity

Abstract A theory of immunity presented previously showed that many immune phenomena could be explained in terms of a simple model involving interactions between (i) the receptors on immunologically competent cells, (ii) antigenic determinants, (iii) natural antibody and (iv) complement. Several features of the theory subsequently gained experimental support. The present paper extends the analysis and predicts that two mechanisms operate in the induction of immunological tolerance to self antigenic determinants. High specificity anti-self cells are destroyed by a mechanism involving complement. In contrast, low specificity anti-self cells are stimulated by self determinants and increase in numbers. This increases the concentration of the natural antibody secreted by these cells which acts as a “blocking” antibody preventing their continued stimulation by self determinants. The expanded clones of low specificity anti-self cells, which may be of high specificity for “near-self” determinants, (i) are responsible for the greater immunological responsiveness between non-identical members of the same species than between members of different species (“alloaggression”), and (ii) provide a barrier opposing the progressive evolution of the surface determinants of a pathogen into forms identical with the surface determinants of its host. Following exposure to a given self or not-self antigenic determinant, the distribution curve for cells of varying specificities for the determinant shows a sharp cut-off point between high and low specificity cells. The position of this cut-off point is critical in determining the subsequent response of the organism to the determinant. Variables affecting the cut-off point include, antibody present prior to the exposure of cells to the determinant, complement, complement inhibitors, the cell membrane and certain drugs. Autoimmune diseases are improved by drugs (e.g. chloroquine) which move the position of the cut-off point towards cells of low specificities for self determinants.

Stem-loop potential in MHC genes: a new way of evaluating positive Darwinian selection?

The domains of polymorphic major histocompatibility complex (MHC) proteins which interact with peptides and T-cell receptors are considered to have been under positive evolutionary selection pressure. Evidence for this is a high ratio of non-synonymous to synonymous mutations in the corresponding genomic domains. By this criterion snake venom phospholipaseA2 genes have also been under positive selection pressure. Recent studies of the latter genes indicate that positive selection has overridden an evolutionary pressure on base order which normally promotes the potential to extrude single-strand stem-loops from supercoiled duplex DNA (“fold pressure”). This has resulted in base order-dependent stem-loop potential being shifted to introns, which are highly conserved between species. It is now shown that, like snake venom phospholipaseA2 genes, the domains of polymorphic MHC genes which appear to have responded to positive selection pressure have decreased base order-dependent stem-loop potential. The evolutionary pressure to generate stem-loop potential (believed to be important for recombination) has been overridden less in exons under positive Darwinian selection. Thus, base order-dependent stem-loop potential shows promise as an independent indicator of positive selection.

ENTROPY-DRIVEN PROTEIN SELF-AGGREGATION AS THE BASIS FOR SELF/NOT-SELF DISCRIMINATION IN THE CROWDED CYTOSOL

Cytotoxic T cells recognize cell surface complexes of MHC class I proteins with peptides derived from proteins synthesized within the recognized cell. A mechanism permitting some intracellular discrimination between self proteins and not-self proteins (encoded by a foreign species) would allow the preferential loading of MHC proteins with peptides derived from not-self proteins. This would decrease competition with peptides derived from self proteins and decrease gaps in the T cell repertoire. A possible mechanism has been derived from studies of the specificity of self-aggregation of erythrocytes and of virus coat protein. It is postulated that genes whose products occupy a common cytosol have co-evolved such that product concentrations are fine-tuned to a maximum consistent with avoiding self-aggregation. Cytosolic proteins collectively generate a pressure tending to drive protein molecules into self-aggregates. Each individual protein species both contributes to, and is influenced by, this pressure. The aggregation involves a liberation of bound water and an increase in entropy. The concentrations of proteins encoded by viral genes (not-self) readily exceed the solubility limits imposed by the crowded host cytosol. This leads to their preferential degradation to peptides which associate with MHC proteins. The intracellular and extracellular self/not-self discrimination systems complement each other to ensure that there is no immunological reaction against normal self tissue components.