IUBMB 2003 Abstract Template

Publication Saga

Abstracts, and a Historical Note on Publication Difficulties

IUBMB 2003 Abstract Template
The following abstract (IUBMB03-3277) was received (13 January 2003) and accepted (3 April) for presentation at the July 8th International Congress of Biochemistry and Molecular Biology in Toronto. Unfortunately, the meeting was cancelled because of the SARS epidemic, and the 8th IUBMB Congress was held a year later in Boston.

Genes as Microisochores: Evidence from Muto-Osawa Plots

D.R.Forsdyke, S.J. Lee,

Queen's University, Kingston, Canada

Isochores are regions of uniform DNA base composition (GC%) found in many higher eukaryotes. Just as interspecies differences in genome GC% may be responsible for recombinational isolation, and hence the existence, of species, so intraspecies differences in GC% between genome sectors may be responsible for recombinational isolation, and hence the existence, of isochores (Forsdyke & Mortimer 2000; Gene 261, 127-137). Given a need for some intragenomic recombinational isolation between functional units, how is this achieved in prokaryotes? Muto-Osawa plots (1987; Proc. Nat. Acad. Sci. USA 84, 166-169) were used to compare the variation among genes of the GC% of the three codon positions in the cases of low, intermediate and high average GC% genomes. In each case, as the overall GC% of individual genes increases, the GC% of the three codon positions of those genes increases. However, at the extremes (low and high genome GC%), the third codon position, which is least concerned with specifying the encoded amino acid, adopts a nearly uniform GC% (i.e. the slope of the plot is very low). Thus, extreme GC% values, while achieving species isolation, leave the organism vulnerable to intragenomic recombination. In contrast, in organisms with intermediate GC% the third codon position shows the greatest change in GC% (i.e. the slope of the plot is greater than those for the first and second codon positions). Thus, the third codon position serves to amplify within genes both a downward pressure towards low GC% by exceeding the first and second codon positions in its AT-richness, and an upward pressure towards high GC% by exceeding the first and second codon positions in its GC-richness. In this way, each gene in an intermediate GC% prokaryote genome comes to occupy a discrete GC% niche, or "microisochore," amongst its fellow genes, which collectively span a wide GC% range.

Agreement

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8th IUBMB/ASBMB Abstract 2004

June Boston meeting FASEB.J (2004) 18, no. 8. page C27. Abstract 18.11.

GC% as a Recombinational Isolator both of Genes and of Genomes D. R. Forsdyke

Recombination can homogenize diverging genes within a genome, or the diverging genomes of members of an incipient species and the ancestral species, so impairing evolutionary advance. How is recombination avoided? Among species within a phylogenetic group, genomic GC% values can cover a wide range that is particularly evident at third codon positions. However, among genes within a genome, genic GC% values can also cover a wide range that is, again, particularly evident at third codon positions. Wada and coworkers noted that individual genes and genomes each have a "homostabilizing propensity" to adopt a relatively uniform GC% (Nature 1976, 263:439; FEBS Lett. 1985, 188:291). Each gene (a "microisochore") occupies a discrete GC% niche of relatively uniform base composition amongst its fellow genes, which can collectively span a wide GC% range. A "selfish" gene being defined as "that which segregates and recombines with appreciable frequency" (Williams, Adaptation and Natural Selection 1966), then homostabilization of GC% may serve to recombinationally isolate both genome sectors (facilitating gene duplication and differentiation) and genomes (facilitating genome duplication and differentiation; e.g. speciation). Although they may sometimes be in conflict (S-J. Lee, J. R. Mortimer & D. R. Forsdyke, unpublished work), the individualities of genomes, and of genes within those genomes, are separately sustained by a common mechanism, uniformity of GC%. The protection against inadvertent recombination afforded by GC% differentiation is, in the general case, a prerequisite for phenotypic differentiation. [An early version of this work was accepted for presentation at the nineteenth IUBMB meeting in Toronto in 2003, which was cancelled: see http://www.queensu.ca/academia/forsdyke/Toront03.htm.]

T. H. Huxley. March 5th 1852. Letter to his sister:

"Science is, I fear, no purer than any other region of human activity, though it should be. Merit alone is very little good; it must be backed by tact and knowledge of the world, to do very much. For instance, I know the paper I have just sent in is very original and of some importance, and I am equally sure that- - [here a professional rival who may be asked to review the paper is mentioned]. So I must manoeuvre a little to get my more memoire out of his hands. The necessity for these little stratagems utterly disgusts me."

The above abstracted work was judged complete in January 2003, and a paper entitled "Bacterial Genes as Microisochores" was prepared and submitted to Nature. The choice of this high-profile journal reflected the senior author's belief that the work presented a major advance. The Editors decided that it was "unlikely to succeed in the competition for limited space" and it was declined without being sent for external view. The title was then changed to "Genes as Microisochores" and the paper was submitted to a succession of journals (see Table below).

After much bizarre feedback from reviewers, the senior author hypothesized that the "isochore" buzzword might be leading editors to choose hostile external reviewers. So the title was changed in December 2003 to "Genomic conflict settled in favour of the species rather than of the gene at extreme GC% values." The proposed revision of the isochore concept was down-played. After a strange negative review by BMC Evolutionary Biology (e.g. "Reject because scientifically unsound"), the paper found sane and fertile ground in July 2004 in the form of the reviewers for the new New Zealand-based journal Applied Bioinformatics, a product of Open Mind Journals Ltd., which was in the process of being taken over by a larger publishing house (Adis Data Information BV).

While the above battle was ongoing, the senior author also attempted to get the work out in the formal literature by writing reviews. Attempts to get reviews in BioEssays and Trends in Genetics (Click Here) were unsuccessful, although in the latter case the editor did send the "letter," which he insisted must be brief, out to referees (Click Here). The editor finally replied:

N39 Forsdyke Preservation more fundamental&

Dear Don

Once again I must apologise to you for the length of time this has taken, I had to approach nine referees before I would find two to look at it. I am afraid this is common with your papers - I am sure you find it as tiresome as I do.

This is not for us - the referees do not like it and neither do I - so as a result we are unable to publish it. I attach their comments.

Best Wishes

Robert Shields

However, happily, the Journal of Biological Systems (for which I happen to be an advisory editor) accepted a review entitled "Regions of relative GC% uniformity are recombinational isolators," without imposing any length limitations.

Another approach was to mention the work as part of a paper that the Journal of Theoretical Biology was not likely to decline, since it was a reply to a previous paper by some evolutionists who has questioned the senior author's hypothesis on the role of GC% in chromosomal speciation ("Chromosomal speciation: a reply").

The outcome of all this was that in the latter part of 2004 three papers giving different versions of the same message were scheduled to appear. This redundancy was forced on the senior author by defects in the peer-review process. Remarkably, the papers written after the original paper was written, appeared first.

From the viewpoint of the general scientific public, it can be noted that many scientists in the field had anonymous privileged access to the work (through acting, or being invited to act, as reviewers) for a year or two, prior to the public being able to access it. Whereas T. H. Huxley above implies adverse criticism fuelled by professional animosity, it seems likely in the present case that whatever merits the work had just were not recognizable in the intellectual climate prevailing among those asked to review papers in 2003-4. For example, the isochore community was preoccupied with defending itself against the strange assertion by E. S. Lander (International Human Genome Sequencing Consortium) that isochores did not exist at all (see Li et al. 2003. Comp. Biol. Chem. 27, 5-10)! Thank heavens for Open Mind Journals of Auckland! We lament that their life was so short, but are delighted that Applied Bioinformatics, while still "down-under", did not go-under so soon!

Journal	First Submitted	Revised version	Date Declined	External Review	Manu- script number
*Nature*	20 Jan 03		27 Jan 03	No	F01428
Genome Research	26 Feb 03		18 Apr 03	Yes	013029
Nature Genetics	20 Apr 03		25 Apr 03	No	A12992
J. Theor. Biol.	30 Apr 03	24 Aug 03	4 Nov 03	Yes	JTBI-227
Mol. Biol. Evol.	6 Nov 03		2 Dec 03	Yes	03-0561
J. Mol. Evol	2 Dec 03		11 Dec 03	No	00180
BMC Evol. Biol.	13 Dec 03		3 Mar 04	Yes	12118208- 27273907
Appl. Bioinform.	5 Mar 04	5 Jul 04	10 Jul 04	Yes

For readers not familiar with scientific publication etiquette, please note that it is not permissible to submit to more than one journal at a time. Thus, authors must be in receipt of a rejection letter from journal A before submitting to journal B. The senior author had to get round this using the "salami" approach of separately submitting, as reviews, different aspects of the same basic message.

It should also be noted that, in the senior author's experience, the struggle described here is not unusual. If every paper included in these web-pages were accompanied by an account of the corresponding publication saga, sometimes enduring over several years, their lengths would be prohibitive.

Although most papers of the senior author have eventually found a journal, this is largely due to his persistence. The tragedy is that many authors, especially young authors, are likely to be discouraged by the frequent incompetence, and sometimes ferocity, of peer-review, and so have given up on science altogether. Some cannot afford the luxury of persistence since their careers are at a stage where rejection means end of career. For more on this please see Forsdyke's peer-review web-pages.

Donald Forsdyke 21st December 2004

Subject: JME-2003-00181 Date: Thu, 11 Dec 2003

Dear Dr. Forsdyke,

Thank you for submitting your manuscript entitled, "Genes as Microisochores" to the Journal of Molecular Evolution.

Unfortunately, we cannot consider your submission for publication in JME. Generally we return manuscripts if the results presented are not novel. I have published a fair amount on base composition and on codon bias, and after reading this paper I don't think it adds anything that is unknown. One of our Associate Editors, who has expertise on genome base composition also recommends returning the manuscript.

I hope that you will continue to consider JME for publication of your work.

Sincerely,
Martin Kreitman,
Editor in Chief

Hello Robert,

Thank you for agreeing to consider my proposal as a letter. I include as email attachment the text condensed down to 819 words plus 26 references. Hope this length will be OK. There are no figures or tables.

Donald Forsdyke, Department of Biochemistry,

Queen's University, Canada

Preservation more fundamental than function: GC% preserves both selfish genes and genomes

Donald R. Forsdyke

In most definitions of the "gene" there is a loose or explicit reference to function (e.g. "a gene is defined biochemically as that segment of DNA - - that encodes the information required to produce a functional biological product" [1]). However, before it can function, information must be preserved. Classical Darwinian theory states that function, through natural selection, is itself the preserving agent. Thus, function and preservation go hand-in-hand, but function is more fundamental than preservation. Can preservation be more fundamental than function? If so, is an agency other than natural selection involved?

Four decades have passed since William Hamilton and George Williams presented a concept popularized as the "selfish gene"[2]. Seeming to argue that preservation was more fundamental than function, Williams [3] proposed that a gene be defined by its property of remaining intact as it passes from generation to generation. For Williams, "gene" meant any DNA segment that has the potential to persist for enough generations to serve as a unit of natural selection.

"Socrates' genes may be with us yet, but not his genotype, because meiosis and recombination destroy genotypes as surely as death. - - I use the term gene to mean 'that which segregates and recombines with appreciable frequency'. - - A gene is one of a multitude of meiotically dissociable units that make up the genotypic message."

Thus, recombination is a threat to genotypes and genes. Recombination is a potential hazard as well as a benefit, and has to be regulated.

"Sexual reproduction is as old as life, in that the most primitive living systems were capable of fusion and combination and recombination of their autocatalytic particles. Modern organisms have evolved elaborate mechanisms for regulating this primitive power of recombination and for maximizing the benefits to be derived from it."

However, for Williams, the ultimate agency was "natural selection of alternative alleles." No other agency facilitated the preservation of genes. His seminal text Adaptation and Natural Selection made a compelling case for natural selection (i.e. function) "as the primary or exclusive creative force" in evolution [3]. The controversial "higher levels of selection" that later won the advocacy of Stephen Jay Gould, were "impotent and not an appreciable factor in the production and maintenance of adaptation." This view of the power of natural selection was shared by those whom Gould came to characterize as "ultraDarwinists" [4]. The conflict between Darwinists and supporters of hierarchical levels of selection [5] did not fade with the Gould's death in 2002. Although neither Williams nor Gould were aware of it, over four decades a potential preserving agency, internal to the organism, had been fleshed out in chemical terms as a component of the base composition of DNA - GC%.

The works of Akiyoshi Wada were prominent among those suggesting a link between GC% and recombination [6-13]. Recombination between two sequences usually follows a successful search for similarity. Allelic genes can recombine because they have homologous sequences. More than sequence non-similarity per se, Wada proposed that GC% non-similarity would prevent genes blending by recombination, thus losing their individualities. Indeed, bioinformatic analyses revealed a mechanism by which GC% differences could impair the initial similarity search [14].

GC% has the potential to protect not only genes from blending with allied genes in the same genome (so facilitating an origin of genes), but also genomes from blending with allied genomes in the same phylogenetic group (so facilitating an origin of species; [15-17]). Thus, individual genes within a genome and individual species within a phylogenetic group should be preserved, both by virtue of functions (the classical phenotype), and by a distinct agency, GC% (the genome phenotype).

Evidence on how an agency could affect preservation independently of function came from many quarters, and included studies of base compositions at different codon positions. Third positions of codons play little role in the encoding of amino acids, and so are freer to vary than first and second codon positions. Thus, a third codon position difference between potentially recombining alleles should, without affecting protein function, serve to impair a similarity search and so militate against recombination. However, differences in GC% being a major factor in the impairment of a search [14], it is not just any third codon position difference, but a difference that changes GC%, that is likely to be important. Indeed, early differences in third codon position GC% precede the functional differences that characterize duplicating genes [18-22]. Early differences in third codon position GC% are observed when prokaryotes, which when so inclined can engage in sexual recombination [23], duplicate into two species [24-25]. There is much, albeit indirect, evidence that this also applies to eukaryotes [15-17]. Studies of hybrid sterility could provide critical evidence [16]. Indeed, while not envisaging a role for GC% differences, Horacio Naviera and Xulio Maside found the results of such studies "unexpected" and suggested that "a new paradigm is emerging, which will force us - - to revise many conclusions of past studies" [26].

Acknowledgements

I thank J. R. Mortimer for Perl programs, and S.-J. Lee for bioinformatic analyses. Queen's University hosts my web-pages where full text versions of several of the cited references may be found (http://www.queensu.ca/academia/forsdyke/homepage.htm).

References

1. Lehninger, A. L. et al. (1993) In Principles of Biochemistry, 2^nd Edition, pp. 789. Worth Publishers, New York

2. Dawkins, R. (1976) The Selfish Gene, Oxford University Press

3. Williams, G. C. (1966) Adaptation and Natural Selection, pp. 1, 8, 22-24, 56, 138. Princeton University Press

4. Gould, S. J. (2002) The Structure of Evolutionary Theory, pp. 595-744. Harvard University Press, Cambridge, MA

5. Morris, R. (2001) The Evolutionists. W. W. Norton, New York

6. Skalka, A. et al. (1968) Segmental distribution of nucleotides in the DNA of bacteriophage lambda. J. Mol. Biol. 34, 1-16

7. Vizard, D. L. and Ansevin, A. T. (1976) High resolution thermal denaturation of DNA thermalites of bacteriophage DNA. Biochemistry 15, 741-750

8. Wada, A. et al. (1976) Long-range homogeneity of physical stability in double-stranded DNA. Nature 263, 439-440

9. Bibb, M. J. et al. (1984) The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene 30, 157-166

10. Wada, A. and Suyama, A. (1985) Third letters in codons counterbalance the (G + C) content of their first and second letters. FEBS Lett. 188, 291-294

11. Suyama, A. and Wada, A. (1983) Correlation between thermal stability maps and genetic maps of double-stranded DNAs. J. Theor. Biol. 105, 133-145

12. Wada, A. and Suyama, A. (1986) Local stability of DNA and RNA secondary structure and its relation to biological functions. Prog. Biophys. Mol. Biol. 47, 113-157

13. Forsdyke, D. R. (2004) Regions of relative GC% uniformity are recombinational isolators. J. Biol. Sys. (in press)

14. Forsdyke, D. R. (1998) An alternative way of thinking about stem-loops in DNA. J. Theor. Biol. 192, 489-504

15. Forsdyke, D. R. and Mortimer, J. R. (2000) Chargaff's legacy. Gene 261, 127-137

16. Forsdyke, D. R. (2001) The Origin of Species, Revisited. McGill-Queen's University Press, Montreal

17. Forsdyke, D. R. (2003) William Bateson, Richard Goldschmidt, and non-genic modes of speciation. J. Biol. Sys.11, 341-350

18. Matsuo, K. et al. (1994) Short introns interrupting the Oct-2 POU domain may prevent recombination between the POU family genes without interfering with potential POU domain 'shuffling' in evolution. Biol. Chem. Hoppe-Seyler 375, 675-683

19. Zhang, Z. et al. (2003) Evolutionary history and mode of the amylase multigene family in Drosophila. J. Mol. Evol. 57, 702-709

20. Zhang, Z. and Kishino, H. (2004) Genomic background drives the divergence of duplicated amylase genes at synonymous sites in Drosophila. Mol. Biol. Evol. 21, 222-227

21. Montoya-Burgos, J. I. et al. (2003) Recombination explains isochores in mammalian genomes. Trends Genet. 19, 128-130

22. Iwase, M. et al. (2003) The amelogenin loci span an ancient pseudoautosomal boundary in diverse mammalian species. Proc. Natl. Acad. Sci. USA 100, 5258-5263

23. Gratia, J. P. and Thiry, M. (2003) Spontaneous zygogenesis in Escherichia coli, a form of true sexuality in prokaryotes," Microbiology 149, 2571-2584

24. Belgard, M. and Gojobori, T. (1999) Inferring the direction of evolutionary changes in genomic base composition. Trends Genet. 15, 254-256

25. Belgard, M. et al. (2001) Early detection of G + C differences in bacterial species inferred from the comparative analysis of the two completely sequenced Helicobacter pylori strains. J. Mol. Evol. 53, 465-468

26. Naveira, H. F. and Maside, X. R. (1998) The genetics of hybrid male sterility in Drosophila. In Endless Forms. Species and Speciation, pp. 330-338. Oxford University Press

This is a strange paper. The form is very attractive. The manuscript is clear, well-written, concise, and addresses a fundamental issue that has to do with major paradigms of genetics and evolution. The content of the paper, unfortunately, seems to me highly criticable. The author argues that variation in GC-content between genes/genomes is selected to prevent recombination and allow functional divergence/speciation. There are several reasons why I think this should not be published in TIG (or in any journal, to tell the truth).

First, this hypothesis is not supported. The bibliographic survey about base composition variation and gene/genome divergence is fragmentary, and highly biased toward examples that might, anecdotally, appear to be in accordance with the proposed scenario (but are actually not, see below). It should be noted that none of the cited references (18-26), but those from the author himself, even refer to the hypothesis of a selected divergence of base composition. It should also be noted that these few examples in which duplicated genes (or diverging genomes) undergo an early divergence of GC-content are in no way the general case, as it is spuriously suggested in the manuscript.

Let us go for a rapid review of the actual content of these papers - at least the ones I know. Zhang found that the amylase gene was duplicated independently in two Drosophila lineages, and that, in the two lineages, one duplicate used to undergo a decrease in GC-content (19), apparently in agreement with the author's hypothesis. But Zhang then published (ref. 20) that this GC-decay was explained by the genomic context: in the two lineages, the GC-decreasing duplicate is located near the centromere, in a little-recombining region (there is a well-known correlation between local recombination rate and GC-content in many species). Not any selection for diverging GC here. The same explanation applies to the Fxy gene in the mouse (ref 21), who moved from the little recombining X chromosome to the highly recombining pseudoautosomal region. This (not selection) resulted in a rapid increase of GC%, as everybody agrees (Perry & Ashworth 1999 Curr Biol, Yi et al 2004 Genome Res). Iwase et al (ref 22) finally, did not even comment on variations in GC-content in their paper (which, by the way, has very little to do with speciation or duplication). Marais & Galtier (2004, Curr Biol), commenting on this paper, showed how it illustrates the role of local recombination as a force driving GC-content evolution.

The main message of these papers, therefore, is that recombination determines GC-content evolution, not the reverse - a scenario for which there is a compelling body of evidence not cited in the manuscript.

Secondly, this hypothesis is highly dubious on general grounds. Speciation often occurs between populations/subspecies showing a relatively low level of sequence divergence (eg less than 1%), and virtually the same base composition. Recombination between such taxa is possible, and actually occurs in hybrids. What prevents gene flow between subspecies are a relatively low number of loci that confer a reduced survival/fertility on hybrids. GC-content has just nothing to do with speciation, at least in plants and animals, as any searcher in the field, I am sure, would agree. About duplications and functional divergence, I could imagine that a duplicate quickly departing by chance the GC-content of the ancestral copy would have an increased probability of achieving functional divergence - although I don't know of any empirical evidence for this process. Even if it was proved, this hypothesis is weaker than the scenario proposed in this ms, where GC divergence is selected (not just a favourable contingency) for preventing recombination.

The only merit I see in this manuscript is to point out the challenging result of Bellgard et al (ref 25), who detected an emerging shift in the GC-content of a group of genes from two recently separated bacterial strains. Although I agree that this result deserves an explanation, it can certainly not be considered as a support for the very ambitious hypothesis proposed here.

In short, I recommend rejection because of biased and spurious bibliographic survey, and unsupported and implausible claim.

Anonymous Reviewer for TIG