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Home , Bacterial conjugation, Horizontal gene transfer, Horizontal transmission

a. Genet., Vol. 75, Number 2, August 1996, pp. 219-232, (O~ Indian Academyof Sciences

REVIEW ARTICLE

Horizontal transfer

VIJI KRISHNAPILLAI Department of and Developmental , Monash University, Clayton 3168, Victoria, Australia

MS received22 August 1996

Abstract. This review explores examples of horizontal genetic transfer in and . The best understood of these involvesvarious conserved familiesof transposable elements, but examples of non-transposable-element-based movement of or gene clusters have also been identifiedin prokaryotic gcnomes. A unifying theme is the structural and DNA- of transposable elements from widely unrelated , suggesting evolutionarilyconserved mechanisms for horizontal transfer. This is reinforced by the fundamental similarity in the enzymaticmechanisms of retroviral integration (by integ- rases) and of transposition (by ). The reviewdeals with various types of horizontal transfer, the mechanismsavailable for such transfer, potential barriers, and the evolutionary significanceof horizontal genetictransfer.

Keywords. Horizontal transfer; eukaryotes;prokaryotes; transposable dements.

1. Introduction

The biological world is composed of a vast array of simple and complex , and current evolutionary ideas place all of them within two major biological classifications, namely the Prokarya ( and ) and the Eukarya (, fungi, plants and animals). Considerable evidence has been accumulated on the biological relation- ships between these two major groupings (Margulis 1996). However, revealed behind this panoramic view of the biological universe is the considerable complexity of individual genomes.

2. complexity

At one level of complexity are the huge size differences between the genomes of organisms. Starting with bacteria, but excluding the even smaller genomes of , genome sizes vary from 580 kb for Mycoplasma to 9500 kb for Myxococcus (Krawiec and Riley 1990). This range of about 15-fold is to be contrasted with the large genome-size differences observed in the eukaryotes. For example, in plants of the family Graminae (which includes the cereal crops) genome size varies from the rice genome of 430Mb to that of the hexaploid wheat of 16,000Mb, a range of about 37-fold (Moore et al. 1993). Other estimates show that among flowering plants the range is between 50,000 and 80,000 Mb (Bennett and Smith 1976). The largest genome known (102,000 Mb) is that of the primitive South American lungfish (Kornberg and Baker 1992). For comparison, the human genome is only 3000 Mb. One immediately striking and distinctive difference in the structure of prokaryotic and eukaryotic genomes is the incredible excess of noncoding over coding DNA in the 219 220 Viji Krishnapillai latter. For example, 97% of the human genome is noncoding DNA and only 3% has coding functions, e.g. for synthesis and regulation of and structural compo- nents of the (Bernardi t995). Among the genomes of barley (Hordeum vulgare), wheat (Triticum aestivum) and rice () 50-80% is noncoding and comprised of different types of repetitive DNA (Moore et al. 1993). There are a range of different types of repetitive DNA in eukaryotic genomes, including minisatellite (15-bp repeats) and microsatellite (2-5 bp) DNA and transposable elements. The former two types are composed of moderately and tandemly repeated , while the latter represent dispersed and mobile sequences (Charlesworth et al. 1994). Well-characterized repeats in the mammalian genome include the LINES (tong sequences) and SINES (short interspersed repeat sequences), which are present in 20,000-60,000 and 500,000 copies respectively (Passarge 1995). In comparison, less than about 2% of prokaryotic genomes is comprised of different types of repetitive DNA, either ribosomal RNA operons, transfer RNA genes, or insertion sequences and other transposable elements (transposons); the bulk of the genome (98 %), however, is coding DNA (Lupski and Weinstock 1992). A further distinctive feature of eukaryotic genomes is its compositional compartmen- talization into distinct isochores or 'islands' of varying GC content (Bernardi 1995). For example, in the human genome this can be identified physically by ultracentrifugation into three major fractions: GC-poor (<40%), GC-rich (40-50%) and GC-very-rich (> 55%) DNA. This last category makes up 3-4% of the genome, and contains all the coding sequences (Bernardi 1995). Within related genomes, such as those of the verte- brates, the GC compositional distribution is highly conserved but different fi'om that of the avians. Such compositional differences are also evident in plants. For example, 10-20% of the maize genome contains all the coding genes; the GC range is restricted to 1-2% and this isochore is called the 'gene space' (Bernardi 1995). Current major thrusts in genome research are identification and characterization of genes of medical, veterinary and agricultural interest (in human, animal and plant genome research respectively) and also identification and functional elucidation of the vast array of tandem and dispersed repetitive DNAs in genomes, These are being investigated in the large number of genome sequencing projects for various organisms, of which the Human Genome Sequence Project is paramount. The rapid pace of these sequencing projects is attested to by the achievement of high-density physical/genetic maps of the human and mouse genomes on the basis of use of microsatellite-DNA markers and their mapping correlation with genetic markers. The human genome is now delineated by markers about 700,000 bp apart and the mouse genome by markers 400,000 bp apart (Dib et al. 1996; Dietrich et al. 1996). Shnilar sequencing projects are also under way for a range of other organisms, including the plant Arabidopsis, the Caenorhabditis eIegans and the yeast , and, increasingly, also for other plants and animals of agronomic, agricultural and veterinary importance. With the completion of sequencing of the genomes of the prokaryotes HaemophiIus influenzae (1,830,137 bp; Fleischmann et al. 1995), Myeoplasma genitalium (580,070 bp; Fraser et aI. 1995), Methanococcus jannaschii (Bult et al. 1996), Synechocystis (Holden 1996), and of the lower Saccharomyces cerevisiae (Dujon 1996), a suite of others are being considered for sequencing on the basis of medical, industrial and scientific interest. Among those favoured are two extremophiles, Archaeoglobus fuIgidus (an archaebacterium) and Thermatoga maririma (a eubacterium), and either a Synechococcus sp. (a cyanobacterium) or Clostridium acetobutylicum (a eubactefium) (Holm,nan 1996). 221

3. Horizontal gene transfer

It is in the context of organizational features of genomes that the issue of horizontal genetic has to be considered, as much of this, but not all, pertains to the repetitive DNA in genomes. This review will be restricted to natural examples of horizontal transfer and will not include those achieved in the laboratory by gene manipulation techniques. An example of the dramatic and frightening consequences of horizontal genetic transfer are the dreaded human immunodeficiency viruses (HIV), which, it is speculated, have been transferred from wild primates (chimpanzees and sooty mangabeys) to humans (Reines 1996). We have been aware traditionally that much of the genetic variability within is generated by homologous recombina- tion during , and the enzymology of this process is now welI understood (Leach 1996). This is what we understand by Mendelian inheritance as it simply involves the of alleles of genes from generation to generation, i.e. by germinal . However, it has become increasingly clear that an alternative mechanism of inheritance is at work in prokaryotes and eukaryotes, namely horizontal (or lateral) genetic transmission, and this is based not on (requiring extensive sequence homology) but on site-specific, transpositional or illegitimate recombination (not requiring sequence homology) (Leach 1996). It is such a mechanism that accounts for the movement of transposable elements fi'om genome to genome, between both related and unrelated genomes. There are three main types of transposable elements in eukaryotes, distinguished on the basis of their genetic and DNA/ sequence organization (Leach 1996). These are: (i) Transposons, which have perfect or imperfect terminal repeats, which transpose using a DNA intermediate and produce target-site duplications of 8 bp. Examples include the Ac element of maize (4565 bp) (Kunze 1996) and the P element of (2907bp) (Engels 1996); these resemble prokaryotic transposons (see below). Very promiscuous elements, found in organisms ranging from fungi to vertebrates, include TcI from C. elegans and mariner from Drosophila. (ii) , which are similar to in their genome organization, e.g. by having long terminal repeats (LTR) and a mode of transposition via an RNA intermediate, generation of 5-bp duplications at the target site, and in their gene products. Examples include the -type element Tyl of yeast (6000 bp) and copia of Drosophila. (iii) Retroposons, which are chiefly distinguished by the absence of LTR and the generation of target-site duplications of 12 bp. The enzymatic process of transposition by all of these elements is based on transposases or integrases. Table 1 shows a selection of eukaryotic transposable elements, which have been identified to be directly involved or as likely to be involved in horizontal genetic transmission. The list shows the ubiquity of these elements in terms of the variety of organisms in which they are found, and includes examples of retrotransposons, ~ransposons and retroposons. Table 2 lists similar elements from prokaryotic sources and, again, a range of elements have been identified, including classical insertion sequences, transposons, conjugative transposons and . Again their ubiquity is to be noted. Finally, table 3 shows examples of horizontal transmission involving gene 222 Viji Krishnapillai

Table 1. Selected examples ofeukaryotic transposable or mobile elements known or likely to be involved in horizontal genetic transmission.

Element(s) Species example(s) Reference

Retrotransposons gypsy~ 176, Insect Drosophila melanogaster Pimpinelli e~ al. 1995; 297, 412, copia, Alberola and de Frutos 1996 mdg-1, blood tom Insect D. ananassae Alberola and de Frutos 1996 Ulysses Insect D. virdis Alberola and de Frutos 1996 micropia Insect D. hydei Alberola and de Frutos 1996 TED Insect Trichoplusia ni Alberola and de Frutos 1996 dot1 Plant Lilium henryi Smyth etal. 1989 IFG7 Plant Pinus radiata Alberola and de Frutos 1996 Tal to Tat0 Flowering plants, Arabidopsis thaliana Kidwell 1992 (Tyl-copia insects, yeast, group) slime mould, herring Ty3 Yeast Saccharomyces cerevisiae Kidwell 1992 Tfl Yeast Schizosaccharomyces pomhe Kidwell 1992 C0e/ Plant pathogenic fungi Cladosporiumfidvum Kidwell 1992 SURL Marine invertebrate Tripreustes gratilla Kidwell 1992 R1, R2 (non-LTR) Many insects Bombyx mori Jakubczak etal. 1991 Zeon-I Maize Zea mays Hu etal. 1995 mctgellan Maize Z. mays Purugganan and Wessler 1994 (Ty3-gypsy) Cerl Nematode Caenorhabditis elegans Britten 1995 skippy Plant pathogenic fungi Fusm'iumoxysporttm Anaya and Roncero 1995 easel Chinook salmon, Oncorhy~Tchustsawytscha, Tristem etal. 1995 Atlantic salmon Salmo salar Transposons P Insects D. melanogaster, D. willistoni Engels 1996 mariner Insects D. mauritiana Plasterk 1996 TcI, To3 Nematode C. elegans Plasterk 1996 GandaIf Insect D. koepferae Marin and Fontdevila 1995 Phobo Insect D. melano~daster Handler and Gomcz 1995 En/Spm Maize Z. mays Gierl 1996 Ac Maize Z. mays Kunze 1996 Tc1-1ike Teleost fish (zebrafish, Brachydaniorerio, Salmo Radice et al. 1994 rainbowtrout, Atlantic 9airdneri, Salmo salar salmon) impah~ Plant pathogenic fungi Fusariumoxysporum Langin etal. 1995 TdrI Zebrafish Brachydan~o rerio lzsvak etal. 1995 humarI Human Homo sapiens Oosumi et ~d, 1995; (mariner-l~ke) Robertson etal. 1996 rigger1, rigger2 Human Homo sapiens Smith and Riggs 1996 Retroposons I, F, G, Doc Insect D. melanoqaster Pimpinelli etal. 1995 Eg-RI (SINES Plant pathogenic ffmgi Erysiphe gramil~is Wei etal. 1996 & LINES-like)

clusters that are not known to be associated with transposable elements; a possible exception is the putative rfb QRS associated with the reassortment of genes encoding surface polysaccharides important in the of . It Horizontal 9ene transfer 223

Table 2. Selected examples of prokaryotic transposable elements known or likely to be involved in horizontal genetic transmission.

Element(s) Host Species example(s) Reference

Insertion sequence families IS1, IS3, IS4, IS630-TC1, Many bacterial Escherichia coil Ohtsubo and Sekine Unusual ISs, IS1t?71/Tn3, species 1996 I830,ISI5(IS6), IS91, IS256

Conjugative transposons Tn916, Tni545 and others Many bacterial Enterococcus fi~ecalis, Clewell and Flannagan species pneumoniae, 1993;ClewelI eta[. 1995; Lactococcus lactis, Salycrs et al. 1995a, b; IJacteroides spp. Scott and Churchward 1995

Tansposons Tn3, TnT, TnlO and others Many bacterial E. coli Berg and Howe 1989 species

Integrons In(?, InT, In2, [n 3, I n6, I n 1, ln4 Members of aeruginosa, Hall and Collis 1995 families E. coIi Enterobacteriaceae and Pseudomonadaceae

should be noted that some of the examples involve chromosomal genes while others involve a -borne gene or gene cluster. A further complex example of horizontal transfer, on the basis of the high homology between the respective genes, is of the isopenicillin N synthetase genes between the Strepwmyces lipmanii and the lower eukaryotes Cephalosporium acremonium, Penicillium chrysogenum and Aspergil- tus nidulans (Weigel et al. 1988). While these are extensive examples of horizontal genetic transmission, others are being recognized, such as the group I and group II introns. These elements can invade related or unrelated genomes by ~ endonuc- lease functions that are functionally similar to those ofretrotransposons (Mueller et al. 1993; Belfort and Perlman 1995; Curcio and Belfort 1996; Grivell 1996). Group I introns are characterized by homing endonucleases that have the conserved amino acid sequence motif LAGLIDADG. Such introns occur in a wide range of genomes, including those of yeast, mitochondria, archaebacteria, , algal chloro- plasts, and mitochondria and nuclei of protists (Grivell 1996). Similarly, group II introns are widely distributed in the genomes of mitochondria, , cyanobac- teria and proteobacteria (Grivell 1996).

4. Evidencefor horizontal genetic transfer in eukaryotes

It is clear from the above that a large number of examples exist which suggest horizontal gene transfer. But what exactly is the evidence for such transmission? Direct 224 Viji Krishnapittai

Table 3. Prokaryotic non-traasposable-element-based DNA segments postulated to be involved in horizontal genetic transmission.

Genes Bacterial species Reference

Chromosomal ~Jb cluster O-antigen surface enterica Reeves 1993, 1994; polysaccharides Lan and Reeves 1996 nod cluster Nodulation of Rhizobium loti Sullivan etal. 1995 leguminous plants gnd 6-Phosphogluconate S. enterica, E. coli Nelson and Selander dehydrogenase and others 1994

1~ QRS O-antigen surface Vibrio choIerae Manning etal. 1994; (insertion sequence?) polysaccharides Bik et al. 1995 cob cluster Cobalamin E. coil, S. typhimurium Lawrence and Roth 1996 1'1"1(IVSs) 23S ribosomal RNA S. typhimurium, Skurnik and Toivanen subunit Yersinia enterocoIitica 1991; Mattatall and Sanderson 1996

Plasmid r./b O-antigen surface S. emerica Keenleyside and polysaccharides Whitfield 1995

n/f cluster Enterobacter Stein et al. 1995 agglomerans

proof is lacking and may never be demonstrable because of the of the genetic events involved. However, there is a substantial body of circumstantial evidence which is consistent with the hypothesis of horizontal genetic transmission. One line of evidence comes from the and spread of transposable elements in Drosophila species (Kidwel11992; Engels 1996). In 19. melanogaster, P elements have existed in the genome for less than 100 years and are postulated to have been inherited by horizontal transfer fiom another Drosophila species (D. willistoni). This idea is supported by dysgenesis experiments (Engels 1996). A more convincing example is the distribution of the Tcl-mariner superfamily of transposons in genetically unrelated organisms repre- sentative of nearly all animal phyla, including vertebrates, , arthropods, planarians and ciliates, and even fungi. These organisms clearly have heterologous DNA sequences, yet the transposases that are responsible for transposition of the respective Tcl elements are highly conserved in amino acid sequence in all the cases (Robertson 1995; Plasterk 1996). Of course, an alternative explanation is that a pro- genitor of these elements had evolved in an ancestral genome prior to the evolutionary separation of these organisms. However, this is made highly unlikely by the fact that homologues of these elements are also found in bacteria, e.g. IS630 from ShigeIla is highly homologous to Tcl (Robertson 1995). A second line of evidence for horizontal transmission comes from a comparison of the inverted repeat (IR) sequences at the termini of 23 elements from animal and plant genomes (Kunze 1996; Gierl 1996). Two families of IRs are recognized on the basis of the conserved nucleotide sequences: one, Horizontal gene transfer 225 referred to as Ac-like, produces 8-bp target-site duplications and the other, the 'CACTA' family, terminates with a CACTA motif and produces 3-bp target-site duplications. Members of these two families occur in both the plant and animal kingdoms (Kunze 1996), and furthermore, the transposases of four of the elements (Ac, Tam3, hobo and P) have an exceptionally conserved and colinear sequence of about 600 amino acids. An example of interreplicon transmission, involving nine fragments and representatives of all three classes of nuclear retrotransposons (Tyl/copia, Ty3/.qypsy and non-LTR/LINE families), is the postulated transmission to the mitochondrial genome fi'om the nuclear genome of these fragments in Arabidopsis thaliana during its evolutionary history (Knoop et al. 1996). Of course not all cases of transposable elements in a genome need to be explained by horizontal genetic transmission; as an example the distribution of 17 non-LTR retrotransposons (Ta12- Ta28) in the mitochondrial and nuclear genomes of A. thaliana is adequately explained by vertical transmission (Wright et al. 1996).

5. Evidence for horizontal genetic transfer in prokaryotes

A variety of evidences exist for horizontal genetic transfer in prokaryotes: (i) The very large and extensive families of insertion sequences (IS) display sequence similarity both within and between families. At least for the IS1 family, there is evidence for horizontal transfer because of the sequence similarity of homologous elements found among different members of the bacterial family Enterobacteriaceae (Ohtsubo and Sekine 1996). (ii) The conjugative transposons of the Tn916/Tni545 family have an extremely promis- cuous host range, extending across the two major subdivisions of the eubacteria (Grarn-positive and Gram-negative bacteria) and including a wide range of species within the genera Bacillus, Clostr~dium, Enterococcus, Streptococcus and Mycoplasma as well as E. coli, Neissieria gonorrhoeae, Citrobacter freundii and many others (Clewell and Flannagan 1993; Salyers and Shoemaker 1994; Clewell et al. 1995; Salyers et al. 1995a,b; Scott and Churchward 1995). It is difficult to explain the existence of such a range of related transposons in such widely differing bacterial genomes other than by ancient and more recent horizontal transmission. This also applies to the families of integrons, which are located on and have a unique and different molecular structure from that of conjugative transposons. The plasmids themselves are widely distributed in bacteria, even though some of the plasmids are conjugative and others are nonconjugative but mobilizable between bacteria by conjugation (Hall and Collis 1995). (iii) Another line of evidence comes from analysis of the DNA sequence variation that exists within the cluster of 15 contiguous genes that encode the major O-antigen lipopolysaccharides of the human and animal Salmonella enterica (Reeves 1993, 1994; Lan and Reeves 1996). Detailed analysis by quantitative subtractive hybridization has shown that 16 out of 39 fragments tested within this 7fb cluster have a GC content of 45% or lower whereas the overall GC content of the genome of the species is 52% (Lan and Reeves 1996); the latter is symptomatic of the relative homogeneity of DNA within species (Sueoka 1992). A similar DNA sequence heterogeneity has been found within the cluster of genes encoding the surface polysaccharides of the human pathogen V. cholerae, but with the difference that the alien sequence is associated with a putative insertion sequence rfbQRS (Manning 226 Viii KrishnapiIIai etal. 1994; Bik e~ at. 1995). This modular nature of DNA sequence variation cannot be explained by convergent evolution, genetic drift or the accumulation of base-substitu- tion mutations; it is however consistent with horizontal transmission of some of these genes to Salmonella from alien genomic sources (Reeves 1993, 1994; Lan and Reeves 1996). Another example is in H. influenzae, which has a Mu--like region of 50 % GC compared to the 38 % GC of the rest of the genome (Fleischmann e r al. 1995). It has been speculated that horizontal transmission of genes is particularly significant for bacterial to environmental niches, including those within a vertebrate host. In these situations the accumulation of genes that modify the surface polysacchar- ide structure of the bacterium could result in genetic superiority through the selective advantage of evading or minimizing the effects of the defense arsenal of the host (Reeves 1993, 1994; Lan and Reeves 1996). (iv) An example similar to those discussed in (iii) above is the loss or acquisition of the vitamin cobalamin biosynthetic genes (cob operon) in E. coli and S. typhimurium. Here the gone cluster has a higher GC content (59%) than the genome average of 52% (Lawrence and Roth 1996). Horizontal transmission has also been postulated for the emergence and spread of the nodulation (nod) genes in Rhizobium loti strains where, despite the genome heterogeneity of the strains as identified by pulsed field gel electrophoresis of SpeI digests, growth rate and RFLP, there is near identity of the nod sequences (Sullivan etal. 1995). A different type of example is seen with the intervening sequences (IVSs) of the rrl genes, which encode the 23S ribosomal units, of S. typhimurium, S. arizonae and Yersinia enterocolitica (Skurnik and Toivanen 1991; Mattatal and Sanderson 1996). Here the ~ 90-bp IVSs in helix-45 are more than 80% identical in nucleotide sequence among these species, even though the overall chromo- somal homology between Salmonella and Yersinia is only about 20%. This and another IVS comparison (that of ~ 110 bp in helix-25) have led to the suggestion that three independent horizontal transfer events have led to the capture of these IVSs by S. typhimurium (Mattatal and Sanderson 1996). Other examples include horizontal transfer of the 6-phosphogluconate dehydrogenase gene (gnd) among enteric bacteria (Nelson and Selander 1994), the postulated transfer of nitrogen fixation (n!f) genes within dissimilar plasmids of Enterobacter aggtomerans (Steibl et al. 1995), and the mobility of 7fb genes between a plasmid and the of S. enteriea (Keenley- side and Whitfield 1995).

6. What mechanisms are available for horizontal genetic transfer?

Unlike the well-established mechanisms of vertical gene transmission of Mendelian inheritance based on gamete formation in eukaryotes involving meiosis and homo- logous recombination, and similar recombination following conjugation, and transformation in prokaryotes, the mechanisms available for nonhomologous recombination are not always so obvious; but they are increasingly being identified. The most obvious are the retroviruses, which are that have evolved the machinery for incorporation into the target genome by nonhomologous recombination. Other avenues for integration also exist. It has been speeulated that for the P elements of Drosophila viruses and parasitic (Proctolaelaps regatis) might be the delivery vehicles (Engels 1996). Direct evidence for this comes from the demonstra- tion that the gypsy element encodes a retroviral envelope protein, which suggests that Horizontal gene transfer 227 the element may be a degenerate insect and hence transmissible by insect feeding (Alberola and de Frutos 1996). Although examples are limited, it is plausible that mechanisms such as those involving a retrovirus-like route may indeed be responsible for the vast majority of horizontal genetic transmissions among eukaryodc genomes. The picture for prokaryotic genomes is much easier to comprehend because well- established mechanisms exist for . These are conjugation (including the mobilization of nonconjugative plasmids) mediated by conj ugative plasmids, transduc- tion mediated by bacteriophages, and transformation by naked DNA. One or more of these mechanisms can transmit mobile elements located on plasmid, phage or chromo- somal genomes. This would readily explain horizontal genetic transmission of prokaryotic mobile genetic elements (transposons and integrons) and acquisition of chromosomal DNA segments not necessarily associated with transposable elements fi'om alien sources. Quite apart from these mechanisms, the conjugative transposons have evolved novel, unique and independent conjugational transmission mechanisms for their promiscuous mobility (Clewell and Flannagan 1993; Salyers and Shoemaker 1994; Clewell etal. 1995; Salyers et al. 1995a, b; Scott and Churchward 1995). With respect to conjugation five major mechanistically different systems have been identified in bacteria. One or more of these systems can act as vehicles for horizontal genetic transmission, and certainly there is very good evidence for such a role in the trans- mission of resistance genes since many of these are located within transpos- able elements (Bennett 1995; Wilkins 1995).

7. Are there barriers to horizontal genetic transmission?

Whether or not barriers to horizontal genetic transmission exist in eukaryotes is not yet known, although there is evidence in Neurospora from transformation experiments that there are barriers at the nuclear level (Miao etal. 1995). There are also several lines of evidence suggesting that there are limits to such transmission in prokaryotes. For example, restriction (or destruction) of foreign DNA is a maj or and effective mechanism that has evolved to exclude, or limit, introduction of foreign DNA into a species (Barcus etal. 1995; Naito etal. 1995). In enteric bacteria, particularly in E. coli where the most information is available, there are at least 11 naturally occurring restriction/modifica- tion systems which fall into three families, hsdR, M and S. Most of these genes are chromosomally located but a few are plasmid borne (Barcus and Murray 1995; Barcus e~ aI. 1995). The alleles of the hsdR family are responsible for destruction of foreign DNA while those of the M and S families are responsible for methylating (protecting) those DNA that escape restriction thus facilitating recombination into the resident genome, However, it appears that these genetic systems might themselves be recombinogenic at least insofar as small DNA fragments are concerned, and thus some 'leakage' of foreign DNA might contribute to horizontal genetic transmission (Barcus etaI. 1995). A second apparent barrier to genetic transmission is the mismatch DNA repair system encoded by the mutL, routs and mutH genes of E. coli and S. typhimurium (Fox 1995; Matic et al. 1995). This genetic system severely inhibits recombination of nonhomologous DNA in interspecies transfers between E. coil and Salmonella, but another genetic system, the error-prone SOS system, stimulates such recombination by triggering the RecBC-dependent SOS system which enhances recombination by 228 Viii Krishnapillai increasing the synthesis of the recombinogenic RecA protein (Matic et al. 1995). Thus these processes govern the relative rates of exclusion or recombination of extraneous DNA in a species, providing a vital source of fine-tuning in bacterial evolution. In addressing the relative roles of the DNA transfer mechanisms between bacteria (i.e. plasmid-mediated conjugation, phage-mediated transduction, and transformation by naked DNA) and the restriction of foreign DNA in interstrain transductional recom- bination in E. coli, it has been shown that there are limits to the length of DNA that can recombine and that mosaic patterns of recombination are found that reflect the evolutionary potential of the species (McKane and Milkman 1995). At the experimen- tal level it can be shown that horizontal transmission of genes can be reduced 10g-10S-fold by the artificial creation, by recombinant-DNA methods, of genes linked to the bacterial antibiotic colicin E3 gene whose product cleaves the 3' ends of 16S ribosomal RNA of a wide range of taxonomically unrelated species of bacteria (Diaz et al. 1995). The significance of the target site is its extreme conservation in nearly all prokaryotes and many eukaryotes. It is thus of interest to ask whether natural equivalents of this type exist that could modulate or influence the relative rates of horizontal genetic transmission.

8. Evolutionary significance of horizontal genetic transmission

Although it is currently difficult to assess the real and specific evolutionary significance of horizontal genetic transmission, it is possible to speculate on evolutionary roles for horizontal transfer. Probably the best understood case of horizontal transfer is the one that occurs between the soil bacterium and many dicotyledonous plants. Here a T (tumour) DNA segment within the Ti and Ri plasmids ofA. mmefaciens and A. rhizogenes, respectively, is excised from the plasmid, transferred into the plant celI and finally integrated into the plant genome. The molecular-genetic details of the transfer process itself are well understood but much remains to be learned about events within the plant cell that result in the formation oftumours (Hooykaas and Beijersber- gen 1994). Other examples of interkingdom transfer are those of horizontal trans- mission of genes from bacteria (Gram-negative) to plants (Buchanan-Wollaston et al. 1987) and yeast (Heinemann and Sprague 1989). An interesting example of horizontal transfer, in the yeast S. cerevisiae, is the transfer of a mitochondrial gene to the nucleus, facilitated by a number of nuclear gene mutations (Thorsness and Fox 1993 ). It is sobering to realize that the human and mouse genomes contain more than 500,000 reverse-transcribed DNA segments generated by reverse transcriptases copying the RNA genomes of retroviruses into cDNA and their integration into the host genome. Many of these include well-recognized transposable elements common to all mam- malian genomes, for example the SINES and LINES referred to earlier, which occur in tens to hundreds of thousands of copies, and the retrovirus-like elements that may be present in hundreds to thousands of copies (Temin 1985; Lee et al. 1996). These transposable elements are ubiquitous in eukaryotes (vertebrates, invertebrates and plants) and prokaryotes. Another possibility that one might speculate about is generation of pseudogenes and their potential for recruitment as new genes. Recom- bination between transposons may generate duplications that might then evolve to create new genes. In a search of the human genome DNA sequence database, it has been estimated that 196 transposon 'fossils', accounting for about 150,000 elements and Itorizontal gene transfer 229 representing 1% of the sequences analysed, occur in the human genome (Smith and Riggs 1996). In addition, it has become apparent that there is a striking similarity in the molecular mechanisms governing transposition, retroviral integration, and the recom- binational rearrangements leading to the generation of immunoglobulin specificity in vertebrates (Craig 1995, 1996; Polard and Chandler 1995; van Gent er al. 1996). This applies to prokaryotic transposable elements such as Tn7, Tnl 0 and phage Mu, and eukaryotic retroviral elements. Fundamental to these mechanisms is the role of reverse transcriptases or integrases for retroviral integration, and transposases for site-specific recombination of all transposable elements be they eukaryotic or prokaryotic (Polard and Chandler 1995). It is thus impossible to deny a major role for horizontal genetic transmission of transposable elements in an evolutionary context. A significant role may also exist for non-transposable-element-based transmission events, though the mechanism of movement of these elements is presently unknown. It may be that in the past they too relied on a transpositional mechanism but that the signature sequences that typically identify transposable elements (e.g. the terminal inverted repeat se- quences) have since been erased. Perhaps this is to be expected if relies on horizontal genetic transmission but avoids retransposition by erasure of key features thus providing selective advantages in an evolutionary sense.

9. Conclusion

Horizontal (or lateral) genetic transmission is increasingly being recognized as of more than passing interest because of its extensive occurrence in the genomes of both eukaryotes and prokaryotes. Much has been learnt about its molecular-genetic characteristics and the underlying principles have been defined, particularly in relation to how transposable elements move from genome to genome by site-specific or nonhomologous recombination. Moreover, increasing numbers of examples of non-transposable-element-based nonhomologous recombination are being uncovered in prokaryotic genomes. Although it is difficult to quantify the real evolutionary significance of horizontal genetic transmission, it is inconceivable that it reflects only the parasitic nature of transposable elements and has little significance to the host . Future research is likely to focus particularly on this important aspect.

Acknowledgements

I thank Dr Vilma Stanisich for stylistic comments on the manuscript and Dr Dena Lyras for providing references on bacterial transposons.

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