Classical neutral theory implies that absence of non-amino acid changing mutations in P. falciparum results from a recent population bottleneck. However, absent mutations can also reflect conservation of the genome phenotype.

Keywords: Plasmodium falciparum, synonymous mutation, positive selection, neutral hypothesis, low complexity segments, GC-pressure, AG-pressure, fold pressure

 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.

 Negative and positive selection are two, mutually exclusive, consequences of a base mutation in a nucleic acid sequence. The extreme imperative of negative selection is: if you mutate, you die. The extreme imperative of positive selection is: if you do not mutate, you die. Occupying the middle-ground between these extremes are mutations that may lead to either weak positive or weak negative selection. In these cases, there will be long-term effects on the number of progeny. The existence and size of a class of mutations that are truly neutral, in that there are no long-term effects on number of progeny, are subjects of much debate^1-8.

     Whether a base mutation will lead to negative or positive selection depends on its location. A mutation affecting the active site of an enzyme will usually disturb enzyme function and this may impair the function of an organism to the extent that it will produce fewer descendents than will organisms without the mutation (i.e. its fitness to reproduce its kind is impeded). In the extreme, this is ensured by the death of the organism that has the mutation. On the other hand, a mutation affecting an antigenic determinant at the surface of a pathogen may allow it to evade the immune defences of its host. A pathogen mutating in this way may produce more descendents than will pathogens without the mutation (i.e. its fitness to reproduce its kind is enhanced). In the extreme this is ensured by the death of pathogens that do not have the mutation. If selection is not balanced (e.g. in the case of major histocompatability complex polymorphism), this may generate a population bottleneck (i.e. decreased population diversity).

 Nucleic acid bases that are conserved among related organisms (i.e. are evolving slowly) are likely to affect functions subject to negative selection, and nucleic acid bases that are not conserved among related organisms (i.e. are evolving rapidly) are likely to affect functions subject to positive selection. Thus, a determination of evolutionary rate can assist the distinction between bases under positive selection, and bases under negative selection. For this, intraspecies or interspecies base differences between organisms can be determined and calibrated against some temporal scale. Accumulation of a large number of differences in a short period would indicate positive selection. However, accurate temporal calibration is difficult.

     An amino acid can usually be encoded by more than one nucleic acid codon (termed 'synonymous' codons). A mutation changing a codon to a non-synonymous codon will, by definition, change the encoded amino acid. To provide a relatively time-independent, internal, frame of reference for determining the form of selection, it has been found convenient locally to compare non-synonymous mutations per non-synonymous site (d_n) to synonymous mutations per synonymous site (d_s). This assumes that synonymous mutations are adaptively neutral, and hence reflect the 'background' rate of accepted (uncorrected) mutations in the genomic segment of interest³. Thus the d_n/d_s ratio within a nucleic acid segment would to provide an index of the rate at which that segment is evolving. A high ratio would suggest that the segment is under positive selection. A low ratio would suggest that the segment is under negative selection.

 In most cases d_s values are significantly above zero, and determinations of d_n/d_s agree with expectations. However, for certain proteins of the malaria parasite, Plasmodium falciparum, d_s values are at, or close to, zero. Rich and Ayala^9-13 interpreted this as revealing a population bottleneck following which there had been insufficient time for mutations to accumulate. Thus, at the extreme, existing species members could have derived from one recent 'Eve' . However, Arnot^14-15 and Saul^16-17 argued that zero d_s values could result from high conservation of bases that do not determine the nature of an encoded amino acid (a violation of the 'neutral' assumption). In this case, both the recent origin argument, and calculations based on d_n/d_s ratios could be invalid.

     The recent origin argument was also contested by Hughes and Verra^18-20. However, the finding of few mutations in certain non-protein-encoding regions (introns) was held by Volkman et al.²¹ to support the argument. Both parties concede conceptual problems and the situation is described as 'paradoxical'²². The purpose of the present communication is to introduce new evidence supporting the argument of Arnot and Saul. It is suggested that the genome of P. falciparum is more sensitive than other genomes to several non-classical selective factors, which collectively constitute the 'genome phenotype'.

    Several non-classical pressures on genome sequences are listed in Table 2. Saul¹⁷ rested his arguments primarily on GC-pressure and pressures acting at the translation level. GC-pressure, AG-pressure and fold pressure will be considered here. GC-pressure might be referred to as S-pressure, but GC-pressure has acquired popular usage and will be used here. Similarly, AG-pressure (purine-loading pressure) might be referred to as R-pressure but, for consistency, the two-letter designation is used here.

    The pressures are not independent either of each other, or of classical phenotypic pressures. For example, as GC-pressure increases, base C can be acquired at the expense of base A, so AG-pressure decreases^26,27. Very small changes in S bases (assessed as the percentage of G + C in a segment of duplex DNA) can have a major impact on fold pressure²⁸. GC-pressure influences protein composition²⁹ and extreme values (low or high) can correlate with decreases both in observed synonymous mutations³⁰ and in protein complexity³¹.

     While the adaptive roles of pressures affecting the genome phenotype may be controversial, of their existence there is little doubt⁵. Thus, a sequence that is evolving slowly with respect to bases that do not determine amino acids could be under negative selection with respect to these pressures (i.e. in the extreme, an organism that mutates these bases dies, and in protein-encoding regions d_s values are kept low). That the genome of P. falciparum might be highly susceptible to such non-amino-acid-changing pressures was evident in a recent study by Pizzi and Frontali³² of low complexity (simple sequence) regions in P. falciparum proteins (see below).

 Long considered a manifestation of biases in cellular mutational mechanisms²⁹, the case for a selective role of GC-pressure is now stronger. Small differences in percentage GC should suffice to impede pairing at meiosis, and initiate species divergence^{5, 28, 33, 34}. Consistent with this, Saul¹⁷ has proposed that, in the genus Plasmodium, high substitution rates would characterize speciation events. Following this, species GC integrity would be sustained by selection. Thus, co-infection of a single mosquito by two species would be mutually inhibitory due to impaired meiosis, and eventually fewer and/or less fit sporozoites would emerge. In this way allied species exert a selection pressure on each other's percentage GC⁸.

 When compared with their homologs in other organisms, most proteins of P. falciparum are longer by virtue of their content of low complexity regions that include internally-repetitive runs of amino acids (tandem arrays, homopolymer runs). Such low complexity regions are in non-globular domains of 90% of the open reading frames in chromosomes 2 and 3^{35, 36}. The observed amino acid preferences of these regions correlate, not with the properties individual amino acids confer (e.g. hydrophilicity, volume, flexibility), but with the A-richness of their codons. Accordingly, Pizzi and Frontali³² concluded that there is 'an amino acid choice strongly conditioned by a genome property (i.e. A-richness in the coding strand)'.

     The pressure to purine-load the mRNA-synonymous strands of DNA was noted in a bacteriophage in 1966³⁷ and, apart from a few intriguing exceptions, is likely to be a general phenomenon (reviewed in Refs 5-8). Similar to GC-pressure, AG-pressure can sometimes dominate the amino acid content of proteins (i.e. the needs of the genome phenotype are served better than those of the classical phenotype^{26, 27}). Evaluation of AG-pressure as the purine-loading index (PLI) is outlined in Table 3.

The needs of secondary structure can sometimes dominate the sequence and composition of a nucleic acid^38-39. The well-known ability of single-stranded tRNA molecules to fold into complex structures is reflected in the potential of the corresponding duplex DNA sequences (tRNA genes) to adopt similar structures when the two strands of the duplex separate. The stability (folding energy) of such structures is significantly greater than that of the structures generated from the same sequences after base order is shuffled. In the course of evolution, the order of bases appears to have been arranged to maintain the stem-loop configurations that contribute to the overall structure. Thus, the cytoplasmic RNAs that function by virtue of their structure [e.g. tRNAs and ribosomal RNAs (rRNAs)] rather than by encoding information for amino acid sequences [messenger RNAs (mRNAs)], are transcribed from DNA sequences with a similar potential for structure when in single-stranded form.

     However, many mRNAs also show the ability to adopt significant higher ordered structures and this is reflected in the potential for structure of the corresponding DNA sequences. That the structure of mRNAs might not necessarily relate to their own functioning became evident when it was observed that a similar or greater potential for higher ordered structure is found in introns and intergenic regions. The potential to form stem-loop structures of significantly greater stability than that of the corresponding randomized sequences is a general property of biological DNA sequences^40-43. The evaluation of the relative contributions of base composition and base order to the folding energy of a DNA segment is outlined in Table 4.

 When the needs of the genome phenotype (e.g. GC-pressure, AG-pressure, fold-pressure) are not compatible with the needs of the classical phenotype (e.g. amino acid composition and sequence of a protein), there is a conflict which appears to be ameliorated by the insertion of introns^40,41,43 and/or of simple sequence elements. The gene encoding Epstein-Barr Nuclear Antigen-1 (EBNA-1) of Epstein-Barr virus (EBV) is instructive in this respect²⁷.

     EBV has various forms of latency in its human hosts, a state associated with the transcription of only a few 'latency-associated transcripts'. All forms of latency have in common the transcript encoding the EBNA-1 protein. This contains a long simple-sequence element (diagonally striped box in Fig. 1) that can be removed without interfering with known protein functions²⁷. Because the EBV genome appears to be under pressure for compactness (i.e. there are few introns and little intergenic DNA), the simple-sequence element should only be present if, under some circumstance, it confers an adaptive advantage either at the protein level, or at the level of the encoding nucleic acid. One function of the element is to facilitate the purine-loading of the corresponding mRNA (Fig. 1a)²⁷. In the EBV genome (60% GC), purines are contributed both by A (measured as DW) and by G (measured as DS). In organisms with a very low GC%, such as P. falciparum, A is the major contributor to the PLI (see below).

    In the DNA encoding the EBNA-1 repetitive element the PLI approaches 800 bases per kilobase so that there are relatively few pyrimidines to pair with purines for formation of the stems of DNA stem-loop secondary structures. Thus, the overall potential for forming secondary structure is greatly decreased as measured by the FONS value (Fig. 1c). The latter value decomposes into a base composition-dependent component (FORS-M; Fig. 1c) and a base order-dependent component (FORS-D; Fig. 1b)²⁸. Compared with neighbouring regions, base order contributes poorly to the folding potential (assessed as negative kcal/mol) in the region of the repetitive element. Indeed, base order supplements base composition in constraining the folding potential (so most FORS-D values are slightly positive; Fig. 1b). Because secondary structure in DNA is likely to enhance recombination^5,8, this predicts decreased recombination between markers corresponding to the N-terminal and C-terminal ends of the protein.

     Apart from the low complexity repetitive element in the EBNA-1 protein, there are other non-repetitive low complexity elements (Fig. 1a). Similar elements are encoded in neighbouring genes, but are at a lower abundance. Thus, the immediate upstream gene (1614 bases) encodes two small elements. The two immediate downstream genes (414 and 768 bases) each have only one small element. In many respects (discussed in Ref 27) the EBNA-1-encoding gene is different from all other EBV genes. Indeed, there are more similarities with genes of P. falciparum.

 The circumsporozoite protein (CSP) of P. falciparum is expressed on sporozoites that are injected by infected mosquitoes and enter liver cells where there is the possibility of attack by cytotoxic T cells. The N-terminal and C-terminal segments correspond to polymorphic antigens that are evolving rapidly under positive selection³. There is a central low-complexity simple sequence element encoding octapeptide (NVDPNANP) or tetrapeptide (NANP) repeats, and one low-complexity, non-repetitive element (Fig. 2a). Downstream of the single exon CSP-encoding gene is a three exon gene encoding an unidentified 'hypothetical' protein. The upstream half of the small middle exon has a low complexity, non-repetitive, element.

    The codons of the low complexity segments in the CSP gene tend to be A-loaded, and contribute to the generally high level of purine-loading (Fig. 2a). In regions corresponding to the N- and C-terminal segments this is achieved mainly by A bases, with a small contribution from G bases. For the middle segment this is achieved entirely by A bases, and ∆S is negative (i.e. there is an excess of C compared with G). The inclusion of the low-complexity regions can be considered as a device to enhance purine-loading, as in the case of the EBNA-1 antigen (Fig. 1). However, the fact that ∆S works against this suggests other factors are in play.

     Figures 2b and 2c show the potential of windows in the segment to form secondary structure. The base order-dependent component (FORS-D) is impaired in regions of the CSP gene corresponding to the N- and C-terminal segments of the protein (values tend to be positive); this is characteristic of sequences under positive selection^34,41,44. Consistent with the high purine-loading of the repetitive middle segment, the base composition-dependent component of the folding potential (FORS-M) is greatly decreased in the middle segment (Fig. 2c); but this is compensated for by an increase in the base order-dependent component (Fig. 2b). Hence, the natural sequence shows a large increase in overall stem-loop potential (FONS) in this region (Fig. 2c). This suggests that, unlike the EBNA-1 encoding gene of EBV (Fig. 1), recombination might be enhanced between markers corresponding to the N- and C-terminal segments. Saul¹⁷ has speculated that the failure to find this to date is due to sample bias.

     In general, stem-loop potential in genes tends to be relegated to introns, especially in the case of genes under positive selection where introns may be more conserved than exons^40,41,43. In the case of the CSP gene, what might have been an intron appears to have been retained and expressed in the protein sequence, probably because of an adaptive value at the RNA level (i.e. purine-loading) and/or at the protein level. Remarkably, while showing a relatively poor degree of purine-loading, the gene encoding the small hypothetical protein shows a similar stem-loop pattern, with a large central contribution of base order (Fig. 2b), but not base composition (Fig.2c), to the total folding energy (Fig 2c). This correlates with the insertion of two introns and a small exon much of which encodes a low complexity segment.

 Merozoites in erythrocytes are not subject to T cell attack. Figure 3 shows a segment of chromosome 2 of P. falciparum with two double-exon genes encoding merozoite surface proteins 4 and 5 (MSP-4; MSP-5) that are of equal length, upstream of the gene encoding merozoite surface protein 2 (MSP-2)³. Two vertical dotted lines divide the single MSP-2 exon into conserved 5' and 3' segments, and a polymorphic immunodominant middle segment with a highly variable simple-sequence repeat in its 5'-half.

    Consistent with Szybalski's transcription direction rule^5,37, all genes show purine-loading with A residues predominant, and intergenic regions tend to be negatively purine-loaded (i.e. pyrimidine-loaded; Fig. 3a). Purine-loading in the MSP-2-encoding gene is mainly contributed by A residues in the middle segment.

     Figures 3b and 3c show the potential of windows in the segment to form DNA secondary structure. Consistent with previous work^40,41,43, folding energy tends to be lower (low negative, or positive, values) in exons, and greater (high negative values) in introns and intergenic regions. While the base composition-dependent component of the folding energy (FORS-M) remains relatively constant, the base order-dependent component (FORS-D) usually makes a major contribution to the total folding energy (FONS).

     Consistent with being under positive selection³, the FORS-D component of the folding energy is considerably impaired (values become positive) in parts of the gene encoding MSP-2, especially at the 5'-end of the middle segment and in the 3'-segment (Fig. 3b). In the region of the repeats both base order and base composition contribute to the high overall folding energy value (FONS). Thus, this repeat region appears to be an adaptation for enhancing the potential for DNA secondary structure, hence facilitating recombination. In this respect, the region resembles an intron.

 Selective pressures conserving bases that, while not determining amino acids, determine GC%, purine-loading and fold potential, should be sufficient to account for low rates of synonymous mutations in P. falciparum. Thus, it may be incorrect to rest on such low rates the argument that existing species members are derived from a relatively recent ancestor. Distinctive features of its genome (low GC%, many low complexity segments) may be leading us to understand that the herald of the demise of so many human beings is now heralding that of the neutral theory and of the assumptions that rest upon it.

Acknowledgements

I thank Michael Zuker for his nucleic acid folding programs and Jim Gerlach for assistance with computer configuration. Queen's University supports my bioinformatics web-pages.

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End of paper

End-Note: In July 2002 Mu et al. reported 100,000 to 180,000 years to the most recent common ancestor, based on an analysis of single nucleotide polymorphisms from 204 genes of chromosome 3 (Nature 418, 323-326).

 Protein segments that contain few of the possible twenty amino acids, sometimes in tandem repeat arrays, are referred to as containing "simple" or "low complexity" sequence. Many P. falciparum proteins are longer than their homologs in other species by virtue of their content of such low complexity segments that have no known function; these are interspersed among segments of higher complexity to which function can often be ascribed. 

     If there is low complexity at the protein level there is likely to be low complexity at the corresponding nucleic acid level (departure from equifrequency of the four bases). Thus, low complexity may have been selected primarily at the nucleic acid level and low complexity at the protein level may be secondary. In this case the amino acid composition of low complexity segments should be more reflective than that of high complexity segments on forces operating at the nucleic acid level, which include GC-pressure and AG-pressure. 

     Consistent with this, for amino acid-determining first and second codon positions, open reading frames containing low complexity segments show increased contributions to downward GC-pressure (revealed as decreased percentage of G +C) and to upward AG-pressure (revealed as increased percentage A + G). 

     When not countermanded by high contributions to AG-pressure, low complexity segments can contribute to base order-dependent fold potential; in this respect they resemble introns. Thus, in P. falciparum low complexity segments appear as adaptations primarily serving nucleic acid level functions.

 Many eukaryotic genes consist of segments that are expressed as a mature transcript (exons), and of segments that are removed from the initial transcript (introns). Among both types of segment are segments of low complexity with biases towards individual bases or groups of bases, and segments of high complexity in which the four bases tend to be equally present. Exons have a narrow size range, with a peak at about 100 bases, and the sum of intron lengths tends to increase proportionately as the sum of exon lengths increases [1-5]. Notable exceptions are genes of the malarial parasite, Plasmodium falciparum. Here, as exon length increases there is a proportionate increase in low complexity sequence within exons. This is evident at the protein level with segments biased towards individual amino acids or groups of amino acids (usually less conserved, hydrophilic, sequences associated with non-globular protein domains) being dispersed among segments in which the 20 amino acids tend to be more evenly present (usually corresponding to conserved, globular, domains of defined function). Thus, Pizzi and Frontali noted that proteins of P. falciparum are often longer than their homologs in other species that are composed mainly of domains of defined function [6].

      Since low complexity at the nucleic acid level correlates with low complexity at the protein level (e.g. poly(dT) encodes polyphenylalanine), it is possible that a primary selective influence operating at one level is the cause of low complexity at both levels. Indeed, Wan and Wootton [7] noted for bacteria, archaea, and eukaryotes that GC-rich genomes and GC-poor (AT-rich) genomes have more low complexity segments in their proteins than genomes in which the quantity of G + C is approximately equal to the quantity of A + T. Thus, it is likely that "GC-pressure" (pressure to depart from equifrequency of these base combinations) is a primary cause of a bias in amino acid composition of proteins towards certain amino acids, which would tending to generate low complexity segments in proteins. It is intuitively evident, and follows from information theory (see Methods), that sequences without base equifrequency (e.g. TTTTTTTT) have less potential to transmit information than sequences with base equifrequency (e.g. ACGGCTTA). Thus, assuming a need to optimize information-carrying capacity (i.e. there is not a redundancy of information-carrying capacity), deviations from base equifrequency due to one pressure should constrain the ability of a sequence to respond to other pressures. Genomes under extreme GC-pressure, such as that of P. falciparum, should be particularly informative in this respect.

   While the cause of GC-pressure is controversial [8,9; see 10 for review], there is agreement that it operates genome-wide both on genes and on intergenic DNA. On the other hand, the pressure to encode a protein operates only on exons. If the base composition of a segment of an open reading frame (ORF) primarily serves a nucleic acid level function, then the amino acid composition of the encoded protein (and hence the base composition of first and second codon positions) might reflect that fact. Third codon positions are usually independent of the amino acid they encode and hence should be able to serve nucleic acid level functions both in segments of ORFs that primarily serve protein level functions, and in segments of ORFs that primarily serve nucleic acid level functions.

     GC-pressure is but one of a variety of pressures appearing to operate directly on nucleic acid sequences, and is a major contributor to what has been referred to as the "genome phenotype" [10-12]. Among these pressures, "AG-pressure" (purine-loading pressure; [13]) can account for some prominent low complexity elements in herpesvirus proteins [14]. Evidence that this pressure might also generate low complexity segments in proteins of P. falciparum came from the observation that amino acid biases in low complexity segments do not correlate with properties of individual amino acids (hydrophobicity, volume, flexibility), but do correlate with the A content of their codons [6]. 

    We here further examine the role of low complexity segments in the AT-rich P. falciparum genome where the third codon position, least related to amino acid composition, tends to be dominated by GC-pressure. The results assist our understanding of the roles of low complexity segments and of the introns that they appear to replace. Some biological implications of this work are reviewed elsewhere [15; see above paper].

2.1. Sequences

P. falciparum sequences of chromosome 2 (AE001362; 209 predicted ORFs [16]) and of chromosome 3 (NC_000521; 215 predicted ORFs [17]), and an Epstein-Barr virus (EBV) sequence (V01555; 84 predicted ORFs [18]), were obtained from GenBank (National Center for Biotechnological Information, Bethesda, MD). A few genes that were difficult to evaluate (e.g. non-canonical start codons) were eliminated generating totals of 205  (chromosome 2), 206 (chromosome 3), and 65 (EBV). For detailed studies of individual genes, updated sequences were obtained from The Institute for Genomic Research or from the Sanger Centre.

2.2. Low complexity segments

If the four bases of a nucleic acid sequence were distributed randomly, but with equal probability, along a sequence (i.e. at a particular position the uncertainty as to which base was coming next was maximum), then, by definition, each base would add 2 binary digits (bits) of information (4 = 2²). If there were only one base, there would be no uncertainty as to which base was coming next, and each base would add 0 bits of information (1 = 2⁰). Thus variation in nucleic acid sequences can be assessed in terms of their information content on a scale from 0 to 2. Similar considerations apply to protein sequences, where the scale is from 0 (1 = 2⁰) to 4.322 (20 = 2^4.322). The quality measured can be referred to as "uncertainty," "entropy," or "complexity" [19].  This also relates to "compressibility," since a simple sequence is easier to characterize in terms that allow a decrease in total symbol number (e.g. "AAAAAA" can be written "6A").

    By defining appropriate window sizes and cut-off points, sequences can be partitioned into low and high complexity segments. Thus, the SEG algorithm [7] as adapted for the GCG software suite (Accelrys Inc., San Diego, CA), was employed for determining low complexity segments in proteins using recommended default settings (window, 12 bases; trigger segment complexity 2.2 bits/residue; extension segment complexity 2.5 bits/residue). These settings are more stringent than those employed by Pizzi and Frontali [6] and no ORFs scored as 100% low complexity.

2.3. Base compositions

Programs for Microsoft Excel were written in Microsoft Visual Basic to calculate base compositions (GC%, AG%, AC%) from species codon usage tables [20].  From four bases there are six combinations of two different bases, consisting of three mutually-exclusive pairs (GC, AT; AG, CT; AC, GT). Thus, implicit in values of GC% for a DNA segment are values for the reciprocating base pair, AT%. When AG% is 50% there are equal quantities of purines and pyrimidines (R and Y, respectively), and a parameter employed in previous work, the "purine-loading index," is then zero (reviewed in [10]).

    Base counts in windows of fixed size were carried out with the GCG program WINDOWS. Although window sizes of around 1000 nt were found optimal in previous work with various species [21], satisfactory results were obtained in the present work with a window size of 400 nt, which is close to that used for fold potential (see below). However, although moved along sequences in 25 nt steps, this window size does not give good resolution when exons and introns are small (e.g. first four exons of Fig. 6).

    "Pressure" is a useful intuitive concept implying some force or tendency with a directional component. As indicated in the text, the terms "GC-pressure" and "AG-pressure" are used here to indicate evolutionary forces for departures from base equifrequencies. However, currently there is no strictly defined usage in the literature. Thus, in some contexts, the effect of "GC-pressure" (i.e. a change in GC%) is considered to increase over the range 0% GC to 100% GC, while the effect of "AT-pressure" is considered to decrease correspondingly from 100% AT to 0% AT. Here we avoid the latter term, which is implicit in the former. Thus, in the present context the effect of "GC-pressure" is considered to increase both from around 50% GC to 100% GC ("upward GC-pressure") and from around 50% GC to 0% GC ("downward GC-pressure"). 

2.4. Base Order-Dependent Fold Potential

Potential for single-stranded DNA at 37oC to form higher ordered structure at physiological salt concentrations (150 mM Na⁺ and 5 mM K⁺), was evaluated using version 3.1 of MFold [22]. Values were calculated for 200 nt windows moving along sequences in steps of 25 nt. Each value for the total folding energy ("folding of natural sequence" = FONS) was decomposed into a base composition-dependent component ("folding of randomized sequence mean" = FORS-M), and a base order-dependent component ("folding of randomized sequence difference" = FORS-D), as described previously [23-27].

3.1. Gene and intron lengths are not correlated

 Unlike many eukaryotic genomes, there is no correlation between gene length and total intron length in chromosomes 2 and 3 of the P. falciparum genome (Figs. 1a, b). However, as noted by Pizzi and Frontali [6], many P. falciparum genes have low complexity segments and there is a close correlation between gene length and the combined lengths of these segments in a gene. Linear regression plots cross the abscissa at 323 bases (chromosome 2; Fig. 1c) and 680 bases (chromosome 3; Fig. 1d), indicating a gene length threshold below which low complexity segments do not occur.

     The low complexity segments seem to replace introns. However, introns and low complexity segments are not reciprocally interchangeable in that, as absolute intron length increases, there is no significant decline in length of low complexity segments (Figs. 1e, f). Thus, the relationship appears additive rather than reciprocal. It is only when the lengths are expressed as a proportion of gene length that a reciprocal relationship emerges (Figs. 1g, h).

3.2. Low-complexity correlates with base composition of first and second codon positions

 For each gene of chromosomes 2 and 3, the percentage content of low complexity segment was plotted against percentage G+C (GC%) for each of the three codon positions in the ORF. Essentially the same results were obtained with both chromosomes (Fig. 2). Average GC% values become progressively less as codon position changes from first position (Figs. 2a,b) to third position (Figs. 2e,f) [28]. The extreme value of the third position (18 %) indicates that it has the most flexibility to respond to downward GC-pressure. This is largely independent of the percentage of low complexity segment in ORFs (low adjusted r² values in Figs. 2e, f). Thus, whatever their content of low complexity sequence, on the basis of their third codon position GC percentages ORFs contribute to downward GC-pressure.

     On the other hand, data points for first and second codon positions fit to second order linear regressions as quadratic parabolas (y = y0 + ax + bx²) with minima corresponding to ORFs with low percentages of low complexity segment (Figs. 2a-d). Most data points relate to the leftward limbs of the parabolas (especially in the case of second codon positions; Figs. 2c,d). For first and second codon positions, the parabolic relationships show that the acquisition by certain ORFs of GC percentages lower than the GC percentages corresponding to minimum low complexity content, correlates with an increased content of low complexity segment. 

     Thus, the low complexity segments could be evolutionary adaptations allowing ORFs, through the compositions of first and second codon positions, to contribute to extreme base compositional demands (i.e. downward GC-pressure) that could not be met by high complexity segments alone.

     Similar results were obtained when AG-pressure was evaluated. Departure from equifrequency of bases (50% purine: 50% pyrimidine) is greatest in the case of first codon positions. Consistent with the "RNY rule" (purine for first codon base, pyrimidine for third codon base; [29]), average AG% values become progressively less as codon position changes from first position (Figs. 3a, b) to third position (Figs. 3e, f). Values for first and second codon positions can best be fitted to second order linear regressions; most data points relate to rightward limbs of parabolas (Figs. 3a-d), indicating a positive correlation between purine content (i.e. upward AG-pressure) and ORF content of low complexity segment. Values for third codon positions show poor correlations (adjusted r² values of 0.062 and 0.130; Figs. 3e, f) indicating that distinction between ORFs with low and high percentages of low complexity segment length is least for this position.

3.3. Low complexity regions in EBV

 In contrast to P. falciparum (low genome GC%; high ORF AG%) the more compact EBV genome is 60% GC and has low AG% in ORFs (i.e. pyrimidine-loading). An exception to the latter is the gene encoding Epstein-Barr Nuclear Antigen 1 (EBNA1), which is highly purine-loaded, mainly by virtue of a long low complexity segment [14,15]. In general, ORFs of EBV encode less low complexity amino acid sequences than those of P. falciparum. Thus, on average, ORFs of EBV are 13.3%"1.9% low complexity, whereas the percentages in P. falciparum are 17.6%"0.9% (chromosome 2) and 18.1%"0.8% (chromosome 3).

3.4. Low-complexity segments countermand or support fold potential

 The long low complexity sequence in the EBNA1 gene appears to countermand the potential of the encoding DNA duplex to extrude single-stranded DNA that adopts stable stem-loop secondary structure [14,15]. Similar studies were carried out on P. falciparum genes, and we show here results from a multiexon gene (Fig.6), a two exon gene (Fig. 7) and a single exon gene (Fig. 8).

    The genes shown in Figures 6 and 7 have so far no assigned function; our data support a revised exon-intron assignment (see legends to figures), which has replaced that originally proposed [17]. Figure 8 shows a similar analysis of the long single exon gene encoding liver stage antigen 1 (LSA1), a major target for malaria immunotherapy [31]. Here there is a long region containing a 17 amino acid repeat (white box with slashed lines) that required a slight adjustment of the SEG program settings (see figure legend) for recognition as low complexity. Immunogenic epitopes T1, LSA-J, Is6, T3 and T5 correspond mostly to the non-repeat 5' and 3' regions, and tend to be separate from low complexity segments. In this respect the low complexity segments, as expected, do not function at the protein level.

    The entire region encoding the 17 amino acid repeat makes a major contribution both to purine-loading (AG%; Fig 8a), and to fold potential (Figs. 8b, c). The latter is largely by virtue of the contribution to base order-dependent fold potential (Fig. 8b). Similar analyses were carried out on several other genes of various functional classes, with results consistent with those shown here (e.g. circumsporozoite protein and merozoite surface proteins, 2, 4 and 5; see Ref. [15]).

4.1. Do low complexity segments replace introns?

 This question is prompted by the observation that, whereas increase in total low complexity segment length is directly related to gene length (Figs. 1c,d), in P. falciparum this does not apply to intron length (Figs. 1a,b). The present work shows that quantitatively (with respect to absolute lengths) introns and low complexity segments are not mutually exchangeable (Figs. 1e, f). Furthermore, the low complexity segments tend to be of high AG% (purine-loading of ORFs), whereas introns tend to be of low AG% (i.e. tending towards pyrimidine-loading [34,35]). The question might be further addressed by comparing intron locations in regular eukaryotic genes (small exons and multiple introns), with the positions of low complexity segments in homologous genes of P. falciparum that have no, or few, introns. However, intron locations often show no relationship to defined protein functional domains [ 32,33], whereas low complexity segments predominate between defined protein functional domains (i.e. proteins with these superfluous segments are longer than their homologues [6]).

     Introns appear to facilitate base order-dependent fold potential, a function likely to be of importance for recombination [23]. This is particularly evident in genes under positive Darwinian selection, and with overlapping genes [24,25,36]. When contributing to AG-pressure (i.e. deviations from equifrequencies of A + G and C + T), low complexity segments can countermand fold potential (Figs. 6, 7) [14,15]. This is because the stems in stem-loop structures should be favoured optimally when AG is at 50% (i.e. when there are likely to be equal quantities of the Watson-Crick pairing bases [26]). Thus, the combined demands of purine-loading and protein-encoding may have been so severe as to instigate the generation of sequence segments that do not make these demands (i.e. the generation of introns). Here the potential to form secondary structure would be less constrained [11,12,23].

     However, when the contribution to AG-pressure is not too high, a low complexity segment can support fold potential by appropriately ordering bases (base order-dependent stem-loop potential; Fig. 8b). This also applies in the case of the single chromosome 3 ORF encoding the P. falciparum circumsporozoite protein which, like the LSA1-encoding gene, has a long low complexity segment which contributes both to AG-pressure and to base order-dependent fold potential (see Ref. [15]). Furthermore, of three merozoite surface protein ORFs tandemly arranged on chromosome 2 (MSP2, MSP4, MSP5, which may have arisen from the replication of a single original gene), two have introns which accommodate increased base order-dependent fold potential; the third (MSP-2) has no intron, but appears to accommodate the potential by virtue of a low complexity segment that also makes a small contribution to AG-pressure (see Ref. [15] on this page above).  


4.2. Low complexity correlates with base composition of amino acid-determining codon positions

 We have evaluated the relationship between low complexity and the frequencies of various pairs of bases, - GC%, AG% and AC%. Of these GC% and AG% have recognized (albeit sometimes controversial) roles at the nucleic acid level (reviewed in Ref. [10]). AC% completes the triad, but is currently an abstract entity of uncertain significance. Since low complexity is defined as a departure from equifrequencies of bases, it is not surprising that proteins with high percentages of low complexity segments correspond to the extremes of base pair percentages (Figs. 2-4). The issues are whether low complexity results from selective forces, and if so whether these forces operate primarily at the nucleic acid level or at the protein level.

     Low complexity minima would be expected to be close to the 50% values for base compositions. However, a segment which is 50% for A + G may not also be 50% for G + C, so precise correspondence is unlikely. The minima in plots of complexity against percentage base compositions (Figs. 2-5) may depart from 50% for this reason, and also because of pressures on individual codon positions. For example, tRNA anticodons are flanked by invariant 5' pyrimidine and 3' purines which participate in the stacking interactions involved in codon-anticodon recognition [37,38]. This can predispose first codon positions to be purine-rich and third codon positions to be pyrimidine-rich [29]. Thus, a preference in P. falciparum for asparagines (codons AAY) over lysine (codons AAR) would not necessarily imply a protein level adaptation, as has been suggested [6]. A recent comparison between the genomes of Plasmodium vivax and Plasmodium falciparum also indicates primary selective forces acting at the nucleic acid level [39].

      Unlike the first and second codon positions, the base composition of third codon positions tends not to correlate with the percentage of low complexity segment in proteins, especially in the case of GC% (Figs. 2-4). This suggests that, for this position, low and high complexity segments are equally free to respond to base compositional pressures. First and second positions would be constrained by other pressures. In high complexity protein segments, mainly corresponding to conserved globular domains, these pressures would include pressures to retain specific protein function. On the other hand, the protein-encoding of low complexity segments would be largely determined by the base compositional pressures, and accordingly such segments would mainly correspond to non-globular hydrophilic surface domains which do not play a critical role in protein structure and function [6]. For example, the long low complexity element in EBNA1 protein encodes a glycine-alanine repeat that can be removed without impairing known EBNA1 functions [40].

      We conclude that many low complexity segments exist in proteins of P. falciparum because of evolutionary forces acting at the nucleic acid level. The nature of the functions served by these forces is discussed elsewhere [10-12]. Any functions at the protein level, while not excluded [41,42], would have been derived secondarily. The results obtained here with P. falciparum (Figs. 2-4), are similar to those obtained with certain ORFs of herpesvirus genomes (Fig. 5 [14,15]).  Hence, our conclusion may apply to low complexity segments in other genomes. Further implications relating to the possibility of a recent P. falciparum population bottleneck and the neutral theory [9] are discussed elsewhere [15; see above]. 

Acknowledgements

We thank Chris Madill for programs that display base compositions in the context of other sequence features, and James Mortimer for programs that extract base compositions from codon usage tables. Michael Zuker (Rensselaer Institute) kindly provided programs that fold DNA. Access to the GCG suite of programs was provided by the Canadian Bioinformatics Resource (Halifax, Nova Scotia). Jim Gerlach assisted with computer configuration.

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