Tuesday, May 28, 2013

Chargaff's Second Parity Rule is Broadly Violated

Erwin Chargaff, working with sea-urchin sperm in the 1950s, observed that within double-stranded DNA, the amount of adenine equals the amount of thymine (A = T) and guanine equals cytosine (G = C), which we now know is the basis of "complementarity" in DNA. But Chargaff later went on to observe the same thing in studies of single-stranded DNA, causing him to postulate that A = T and G = C more generally (within as well as across strands of DNA). The more general postulation is known as Chargaff's second parity rule. It says that A = T and G = C within a single strand of DNA.

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.

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


  1. Was Chargaff's original 2nd law just a guess or did he make this law on observation, suggesting that there are some organisms with equal amounts of purines to pyrimidines?

    1. He worked on a particular single stranded DNA probably in some bacteria. Now Chargaff's second rule seems to be an exception rather than a rule. Violation of this rule is a norm in invertebrate mitochondrial DNAs.

  2. This comment has been removed by the author.

  3. I think Chargaff's parity rule-2 is completely misinterpreted here.
    This blog talks about the A-T skew which Chargaff has acknowledged very much as a specfic feature. Violation of the rule in nature especially in the mitochondrial proteins is true however this explanation is not convincing.


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