Molecules and : where's the ?


A few years ago l, molecular biologists have also been considered (at least early speculations centered on the were chastised for sloppy and con- implicitly). If doubt remained, se- possibility that and fusing use of the term 'homology'. quence data could be collected to share a conserved mechanism of Many treated homology as an objec- confirm that descend from - , even though this con- tive observation rather than an in- less . tradicted the conventional view that ference, and as a quantitative trait Now, suppose the molecular the last common ancestor of arthro- ('percentage homology') rather than mechanisms controlling develop- pods and vertebrates was not seg- a relationship of common evol- ment in and bats are examined. mented. However, the discovery of utionary origin that either does or Given the known conservation of clusters in unsegmented does not exist (see description of mechanisms in vertebrates, homolo- creatures like terminology in Box 1). There is gous molecules and conserved path- undermined these speculations, par- another source of confusion that ways would certainly be found oper- ticularly since analyses of expression threatens to become increasingly ating in the development of patterns in the worm confirmed that troublesome as the fascinating mol- in both groups. However, such simi- there is no relationship between ecular homologies that lie at the larities would not be interpreted homeobox and reiterated cell of developmental mechanisms are as supporting the surprising' con- lineages that might be regarded as a unraveled: there is not necessarily a clusion that the two sorts of wings are, primitive form of segmentation 8,9. simple relationship between hom- after all, homologous. Instead, the Homeobox genes are involved in ology of molecules (or even path- molecular similarities would be other divergent processes such as ways) and homology of the anatomi- recognized as reflecting homology at development in vertebrates 1° cal features in whose development a deeper level (). In other and gut differentiation in insects1( those components participate. In studies, the danger arises when Thus, the focus of interpreting other words, some recent suggestions evidence beating on homology is less homeobox function shifted notwithstanding v7, molecular simi- extensive or decisive (or less well progressively from segmentation to larities in the developmental mech- known to the average molecular or anterior-posterior polarity lz and to anisms that produce specific organs developmental biologist) than in this axial patterning in general 13. Hom- are not, by themselves, strong evi- example. ology at even deeper levels, such as dence for homology of those organs. positional information per se or Interpreting homeobox gene simply transcriptional regulation, may Levels of homology comparisons be most relevant to some homeobox The central point of this article is Turning to real molecular ex- gene comparisons. that questions of homology can be amples, the evolving interpretation of A cautious initial interpretation of examined at multiple levels and that comparisons between homeobox similarities among insects and ver- homology between a pair of struc- genes and clusters in different - tebrates would have considered all tures can simultaneously be present isms is instructive. When these were of these possibilities and recognized at some levels but absent at others. discovered in vertebrates following the need for additional information to The term 'levels of homology' refers their initial characterization in insects, distinguish between them. As in the to a nested series of progressively more ancient and inclusive ('deeper') relationships. The classic textbook Box 1. Addendum on terminology example of homology, the The terminology used in this article to describe rehtionships is that proposed by , conveniently illustrates the Fitch16, elaborated by Patterson17, and summarized in the instructions to authors point. Considered only as forelimbs, writing for Molecular and . Briefly, features (including mol- the wings of birds and bats are ecules) that are similar by virtue of common ancestry are homologous, while homologous; considered as wings, those that are similar by convergence are analogous. Among homologous mol- they are not. In other words, the last ecules, those produced by are paralogous and those separated common ancestor of these two by are orthologous. It is possible (and useful), as Patterson suggests, to groups had forelimbs but not wings. give precise definitions even when there are substantial practical difficulties in Note that this conclusion is partly deciding which relationship applies in particular cases. There is, however, one based on evidence other than that problem of definition not dealt with in the cited sources. When duplication pro- cluces a paralogous gene set in one species,- is the orthologous relationship to derived from the direct comparisons homologs in other lineages retained by both, one, or neither of the copies? If it "is of wings: the comparative of retained by only one c0py, to which the orthologous relationship be other vertebrate forelimbs; the assigned? This difficulty does not need to be resolved for the present purpose but record; and other anatomical com- it further highlights the complexities of using molecular similarities as evidence for parisons that reveal, for exaraple, the anatomical homology. relationship of bats to other- ,l~ammals

TIG APPalL 1995 VOL. 11 NO. 4

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example of and wings men- u'aced backwards from the current As with other developmental regu- tioned above, more detailed analyses examples under consideration. Such lators, these factors belong to a lim- of the relevant systenxs would not, in an analysis would identify the level at ited number of families and typically isolation, have resolved the question. which the contemporary functions function in a variety of contexts. The progressive interpretation sum- and contexts could usefully be said to Again, coincidental similarities be- marized above depended on in- be homologous. tween analogous systems are to be fom~ation about additional species expected. This case is also confused (e.g.C. elegans) and other contexts of Some questionable cases by the seemingly interchangeable use expression within species (e.g. limbs Molecular similarities have some- of the terms 'homology' and '' and guts); in turn, those comparisons times not been interpreted in an in file discussion. depend, at least implicitly, on ad- appropriately cautious manner. Laufer and Marigo 4 summarize ditional data of various kinds (such as Based on comparisons of function additional examples in which con- that relevant to phylogeny). and expression of the orthodenticle nections between molecular and gene in Drosophila and of homologs anatomical homology have been Homologous molecules in in vertebrates, Finkelstein and considered. The issues raised in this analogous orgam Boncinelli 2 suggest that, contrary to article have not always been given A second example highlights prevailing opinion, head specializ- adequate attention. It is noteworthy the classic problem of convergence, ation may have occurred before the that the majority of 'surprising' ana- with the deceptive twist that truly ancestral lineages separated. How- tomical homologies thus far pro- homologous molecules may be ever, the facts permit hypotheses posed on the basis of molecular data involved in processes that are only similar to those proposed for inter involve comparisons between insects analogous. Products of the hedgehog preting analyses of homeobox genes and vertebrates. This certainly reflects gene in Drosophila and of an avian mentioned above: these ortho- the intense effort devoted to molecular homolog serve strikingly similar func- denticle homologs could be deeply analyses of development in these par- tions in wing development 14. Quite conserved components involved in ticular systems. As other groups re- properly, their roles in that context axial patterning (or another aspect of ceive more attention, the incidence of are recognized as analogous, not positional information) not specifi- convergent examples will surely in- homologous. Again, hedgehog hom- cally related to cephalization. crease, reinforcing the importance of ologs play comparable roles in inter- Defects caused by eyeless in caution and precision in the interpret- cellular signaling in various other Drosophila and a homolog, Small eye, ation of molecular similarities. developmental contexts in both in mice have prompted speculation insects and vertebrates. Undoubtedly, that and vertebrate eyes Conclusions there is deep and interesting hom- are homologous despite fundamental In no case am I arguing that ology here but the wing is not the differences in organizationS,7. This suggested inferences about organ- level at which it should be sought. situation may be comparable to that level homology are definitely wrong; The probability of encountering of the hedgehog gene in wing de- I claim only that the molecular such convergence is greatly increased velopment. The roles of these genes evidence alone is weak and that some by three well-established features of in eye development should be temled authors have been vague or ambigu- : (1) even within homologous only if other evidence ous with respect to the level of hom- a single species the same molecule suggests that an orthologous ante- ology suggested. It must be recognized can assume functions in quite differ- cedent of both eyeless and Small eye that molecular similarities could re- ent developmental pathways; (2) functioned in the development of an flect homology at any of several levels, gene duplication generates paral- eye in a common ancestor of arthro- that other data must be evaluated to ogous gene families whose members pods and vertebrates. decide which level is most likely in a can encompass an even wider range Kispert et al. 3 suggest homology particular case, and that the level of roles; (3) domain shuffling gener- between the vertebrate notochord under discussion must be carefully ates molecules with clear homology and the hindgut because the specified in reports of hypotheses in some regions but potentially with Bracbyury (T) gene and a T-related and conclusions. Anatomical hom- quite different overall fi2nctions. All of gene (Trg), respectively, are required ology will become a useless concept these can be subsumed within the for nomlal development of those if it is inferred in all organs in which idea of 'levels of homology' adopted organs. In this case, the molecular homologous molecules are found to here. Molecules with multiple func- homology is confined to a DNA-bind- have similar functions. tions could presumably be traced ing region. This region could have back to some primordial function combined with other domains to Acknowledgements while paralogous sequences, whether generate molecules with distinct A stimulatingdiscussion with D. Feener entire molecules or domains, could functions either before or after the and participants in the Biology 601 sem- be traced to a molecule (and func- separation of vertebrate and arthro- inar focused my thoughts and prompted tion) that existed before some rel- pod lineages. the writing of this article. J. Seger and evant duplication occurred. In either Finally, homologous transcription G. Herrick made useful comments. case, the molecular biologist's job factors seem to play similar roles in (not necessarily simple) would be to regulating some genes in the of References determine the context in which an mammals and the fat body of 1 Reeck, G.R. etal. (1987) Cell50, 667 ancestral molecule functioned at the Drosophila, leading to speculations 2 Finkelstein, R. and Boncinelli, E. point where paths merge when about the homology of these organs6,15. (1994) Trends Genet. 10, 310-315 TIG APRIL 1995 VOL. 11 NO. 4 120 COMMENT

3 Kispert, A., Herrmann, B.G., 8 Kenyon, C. and Wang, B. (1991) 13 McGinnis, W. and Kmmlauf, R. Leptin, M. and Reuter, R. (1994) Science 253, 516-517 (1992) Cell 68, 283-302 Genes Dev. 8, 2137-2150 9 Salser, S.J. and Kenyon, C. (1994) 14 Ingham, P.W. (1994) Curr. Biol. 4, 4 Laufer, E. and Marigo, V. (1994) Trends Genet. 10, 159-164 347-350 Trends Genet. 10, 261-263 10 Oliver, G. et al. (1998) Cell55, 15 Abel, T., Bhatt, R. and Maniatis, T. 5 Quiring, R. et al. (1994) Science 265, 1017-1024 (1992) Genes De~'. 6. 466-480 785-789 11 Bienz, M. (1994) Trends Genet. 19, 16 Fitch, W.M. {19"{}} .~l'st. Zool. 19. 6 Sondergaard, L. (1993) D'ends 22-26 99-113 Genet. 9, 193 12 Akam, M. (1989) Cell57, 17 Patterson. C. {1988} .lh,I. Biol. Evol. 7 Zuker, C.S. (1994) Science265, 742z43 347-349 5, 603--625

Gatecrashers at the catalytic party



DNA transposition and site-specific 21* plasmid recombinase FLP, and In a thought experiment, one could recombination reactions are mediated was thought to comprise a second 'Int covalently link an active subunit to by assemblies containing several ' of related recombinases. its binding site with a chemical identical subunits. How can However, the relatedness of some crosslinking agent. In the cleaved we establish which subunits are members of the group has now been intermediate, this subunit would be doing what, and where? Attempts to questioned. attached to the DNA at the cleaved answer these questions in the field phosphate and also at its binding of site-specific recombination have Recombinases may use a shared site, and the connectivity could be come up with surprising and provoca- active site established by conventional electro- tive answers. Given the above information, a phoresis techniques. However, the Catalysis of DNA breakage and simple (but naive) question c,,n be development of efficient methods for rejoining in site-specific recombi- asked. There are four recombinase site-specific protein-DNA crosslink- nation is achieved by an assembly of subunits, and four bonds in the ing is at an early stage. The published four recombinase subunits. Two sub- recombining sites that must be broken. work to date has used more indirect units bind at a specific 'core' region Which subunits catalyse which methods. of about 30 bp in each of the two cleavage reaction (see Fig. lc)? The Much interest in these questions recombining sites; these core regions question should actually be asked has been generated by complemen- usually have identical sequences. much more carefully because cleav- tation experiments with the There are at least two different ways age and rejoining at a particular phos- recombinase FLP2. Four amino acid of exchanging the DNA strands to phate might require the intervention residues (two Arg residues, a His, and make recombinants (Fig. la,b) 1. of more than one subunit, and a par- the nucleophile Tyr) are conserved One large family of related recom- ticular subunit might interact with through the whole Int family of binases (exemplified by Tn3 and ",/8 more than one segment of the DNat in enzymes. of any of these resolvases) catalyses the breakage the complex. Nevertheless, the pri- residues in FLP abolishes recombi- ('cleavage') of all four strands before mary binding site for a recombinase nation activity. However, in a suicide concertedly swapping the ends and subunit can be defined using in vitro substrate assay (see below), the rejoining them (Fig. la). Cleavage assays such as footprinting, and the mutant Y343F, in which Tyr had been occurs when a DNA phosphodiester functions of specific amino acid changed to Phe, was inactive on its bond is attacked by a hydroxyl group residues can also be characterized by, own but could be complemented by on a Ser sidechain from the recombi- for example, analysing mutant pro- FLP mutant in any of the other three nase, forming a transient protein- teins. Therefore, experiments can residues3. This led to the idea of a DNA covalent linkage. Other recon: potentially be devised to establish 'shared active site' in which the binases break, exchange and rejoin connections between (1) a DNA bond Tyr343 nucleophile was provided by one pair of homologous strands to that is broken, (2) a particular cata- one subunit and a 'triad' (Arg-His- make a fourway junction intermedi- lytic residue, (3) the subunit of which Arg) of activating residues by a ate (Holliday junction); they then it is part, and (4) a particular binding second subunit (Fig. ld). complete recombination by breaking, site in the reactive complex. Suicide substrates 4 have been exchanging and rejoining the other The nucleophilic residue (Tyr or central to the analysis of intermedi- pair of strands (Fig. lb). These Ser) of the recombinase is an obvious ates in Int family reactions. Many vari- enzymes use a Tyr sidechain to pro- first choice for this sort of analysis, ations on the theme have been used vide the hydroxyl nucleophile that particularly because intermediates but the principle is the same; the cleaves the DNA. This group includes with the DNA covalently linked to the reversal of the DNA cleavage reaction phage ~. integrase (Int) and the yeast recombinase can be trapped in vitro 1. is inhibited because the leaving group TIG APmL 1995 VOL. 11 No. 4

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