The second parity rule seemed to make sense, because there was and is no a priori reason to think that DNA or RNA, whether single-stranded or double-stranded, should contain more purines than pyrimidines (nor vice versa). All other factors being equal, nature should not "favor" one class of nucleotide over another. Therefore, across evolutionary times frames, one would expect purine and pyrimidine prevalences in nucleic acids to equalize.
What we instead find, if we look at real-world DNA and RNA, is that individual strands seldom contain equal amounts of purines and pyrimidines. Szybalski was the first to note that viruses (which usually contain single-stranded nucleic acids) often contain more purines than pyrimidines. Others have since verified what Szybalski found, namely that in many organisms, DNA is purine-heavy on the "sense" strand of coding regions, such that messenger RNA ends up richer in purines than pyrimidines. This is called Szybalski's rule.
In a previous post, I presented evidence (from analysis of the sequenced genomes of 93 bacterial genera) that Szybalski's rule not only is more often true than Chargaff's second parity rule, but in fact purine-loading of coding region "message" strands occurs in direct proportion to the amount of A+T (or in inverse propoertion to the amount of G+C) in the genome. At G+C contents below about 68%, DNA becomes heavier and heavier with purines on the message strand. At G+C contents above 68%, we find organisms in which the message strand is actually pyrimidine-heavy instead of purine-heavy.
I now present evidence that purine loading of message strands in proportion to A+T content is a universal phenomenon, applying to a wide variety of eukaryotic ("higher") life forms as well as bacteria.
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| According to Chargaff's second parity rule, all points on this graph should fall on a horizontal line at y = 1. Instead, we see that Chargaff's rule is violated for all but a statistically insignificant subset of organisms. Pink/orange points represent eukaryotic species. Dark green data points represent bacterial genera. See text for discussion. Permission to reproduce this graph (with attribution) is granted. |
To create the accompanying graph, I did frequency analysis of codons for 58 eukaryotic life forms (pink data points) and 93 prokaryotes (dark green data points) in order to derive prevalences of the four bases (A, G, C, T) in coding regions of DNA. Eukaryotes that were studied included yeast, molds, protists, warm and cold-blooded animals, flowering and non-flowering plants, alga, and insects and crustaceans. The complete list of organisms is shown in a table further below.
It can now be stated definitively that Chargaff's second parity rule is, in general, violated across all major forms of life. Not only that, it is violated in a regular fashion, such that purine loading of mRNA increases with genome A+T content. Significantly, some organisms with very low A+T content (high G+C content) actually have pyrimidine-loaded mRNA, but they are in a small minority.
Purine loading is both common and extreme. For about 20% of organisms, the purine-pyrimidine ratio is above 1.2. For some organisms, the purine excess is more than 40%, which is striking indeed.
Why should purines migrate to one strand of DNA while pyrimidines line up on the other strand? One possibility is that it minimizes spontaneous self-annealing of separated strands into secondary structures. Unrestrained "kissing" of intrastrand regions during transcription might lead to deleterious excisions, inversions, or other events. Poly-purine runs would allow the formation of many loops but few stems; in general, secondary structures would be rare.
The significance of purine loading remains to be elucidated. But in the meantime, there can be no doubt that purine enrichment of message strands is indeed widespread and strongly correlates to genome A+T content. Chargaff's second parity rule is invalid, except in a trivial minority of cases.
The prokaryotic organisms used in this study were presented in a table previously. The eukaryotic organisms are shown in the following table:
| Organism | Comment | G+C% | Purine ratio |
| Chlorella variabilis strain NC64A | endosymbiont of Paramecium | 68.76 | 1.1055181128896376 |
| Chlamydomonas reinhardtii strain CC-503 cw92 mt+ | unicellular alga | 67.96 | 1.0818749999999997 |
| Micromonas pusilla strain CCMP1545 | unicellular alga | 67.41 | 1.1873268193087356 |
| Ectocarpus siliculosus strain Ec 32 | alga | 62.74 | 1.2090728330510347 |
| Sporisorium reilianum SRZ2 | smut fungus | 62.5 | 0.9776547360094916 |
| Leishmania major strain Friedlin | protozoan | 62.47 | 1.0325 |
| Oryza sativa Japonica Group | rice | 54.77 | 1.0668412348401317 |
| Takifugu rubripes (torafugu) | fish | 54.08 | 1.0655094027691674 |
| Aspergillus fumigatus strain A1163 | fungus | 53.89 | 1.013091641490433 |
| Sus scrofa (pig) | pig | 53.77 | 1.0680595779892428 |
| Drosophila melanogaster (fruit fly) | 53.69 | 1.0986989367655287 | |
| Brachypodium distachyon line Bd21 | grass | 53.32 | 1.0764746703677999 |
| Selaginella moellendorffii (Spikemoss) | moss | 52.83 | 1.1014492753623195 |
| Equus caballus (horse) | horse | 52.29 | 1.0844453711426192 |
| Pongo abelii (Sumatran orangutan) | orangutan | 52 | 1.0929015146227405 |
| Homo sapiens | human | 51.97 | 1.0939049081896255 |
| Mus musculus (house mouse) strain mixed | mouse | 51.91 | 1.0827720297201582 |
| Tuber melanosporum (Perigord truffle) strain Mel28 | truffle | 51.4 | 1.0836820083682006 |
| Phaeodactylum tricornutum strain CCAP 1055/1 | diatom | 51.06 | 1.0418452745458253 |
| Arthroderma benhamiae strain CBS 112371 | fungus | 50.99 | 1.0360268674944024 |
| Ornithorhynchus anatinus (platypus) | platypus | 50.97 | 1.1121909993661525 |
| Taeniopygia guttata (Zebra finch) | bird | 50.81 | 1.1344717182497328 |
| Trypanosoma brucei TREU927 | sleeping sickness protozoan | 50.78 | 1.106974784013486 |
| Danio rerio (zebrafish) strain Tuebingen | fish | 49.68 | 1.1195053003533566 |
| Gallus gallus | chicken | 49.54 | 1.1265418970650787 |
| Monodelphis domestica (gray short-tailed opossum) | opossum | 49.07 | 1.0768110918544194 |
| Sorghum bicolor (sorghum) | sorghum | 48.93 | 1.046422719825232 |
| Thalassiosira pseudonana strain CCMP1335 | diatom | 47.91 | 1.1403183213189638 |
| Hyaloperonospora arabidopsis | mildew | 47.75 | 1.053039546400631 |
| Daphnia pulex (common water flea) | water flea | 47.57 | 1.058036633052068 |
| Physcomitrella patens subsp. patens | moss | 47.33 | 1.1727134477514667 |
| Anolis carolinensis (green anole) | lizard | 46.72 | 1.113765477057538 |
| Brassica rapa | flowering plant | 46.29 | 1.1056659411640803 |
| Fragaria vesca (woodland strawberry) | strawberry | 46.02 | 1.1052853232259425 |
| Amborella trichopoda | flowering shrub | 45.88 | 1.0992441209406494 |
| Citrullus lanatus var. lanatus (watermelon) | watermelon | 44.5 | 1.0855134984692458 |
| Capsella rubella | mustard-family plant | 44.37 | 1.1041257367387034 |
| Arabidopsis thaliana (thale cress) | cress | 44.15 | 1.109853013573388 |
| Lotus Japonicus | lotus | 44.11 | 1.0773228019122847 |
| Populus trichocarpa (Populus balsamifera subsp. trichocarpa) | tree | 43.7 | 1.1097672456226706 |
| Cucumis sativus (cucumber) | cucumber | 43.56 | 1.0823847862298719 |
| Caenorhabditis elegans strain Bristol N2 | worm | 42.96 | 1.106320224719101 |
| Vitis vinifera (grape) | grape | 42.75 | 1.0859833393697935 |
| Ciona intestinalis | tunicate | 42.68 | 1.158652461848546 |
| Solanum lycopersicum (tomato) | tomato | 41.7 | 1.1177 |
| Theobroma cacao (chocolate) | chocolate | 41.31 | 1.1297481860862142 |
| Medicago truncatula (barrel medic) strain A17 | flowering plant | 40.78 | 1.093754366354618 |
| Apis mellifera (honey bee) strain DH4 | honey bee | 39.76 | 1.216042543762464 |
| Saccharomyces cerevisiae (bakers yeast) strain S288C | yeast | 39.63 | 1.1387641650630744 |
| Acyrthosiphon pisum (pea aphid) strain LSR1 | aphid | 39.35 | 1.1651853457619772 |
| Debaryomyces hansenii strain CBS767 | yeast | 37.32 | 1.1477345930856775 |
| Pediculus humanus corporis (human body louse) strain USDA | louse | 36.57 | 1.2365791828213537 |
| Schistosoma mansoni strain Puerto Rico | trematode | 35.94 | 1.0586902800658977 |
| Candida albicans strain WO-1 | yeast | 35.03 | 1.1490291609944834 |
| Tetrapisispora phaffii CBS 4417 strain type CBS 4417 | yeast | 34.69 | 1.17503805175038 |
| Paramecium tetraurelia strain d4-2 | protist | 30.03 | 1.2494922903347117 |
| nucleomorph Guillardia theta | endosymbiont | 23.87 | 1.1529462427330803 |
| Plasmodium falciparum 3D7 | malaria parasite | 23.76 | 1.4471365638766511 |
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