Genetic switches by DNA inversions in prokaryotes

Biochimica et Biophysica Acta, 782 (1984) 111-119

111

Elsevier

REVIEW BBA 91366

GENETIC SWITCHES BY DNA INVERSIONS IN PROKARYOTES R O N A L D H.A. PLASTERK and PIETER VAN DE PUTTE

Laboratory of Molecular Genetics, State University of Leiden, Department of Biochemistry, Wassenaarseweg 64, 2333 AL Leiden (The Netherlands) (Received March 26th, 1984)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111

II.

lnvertible DNA determines the host range of phage Mu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112

III. The flagellar phase variation of Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

113

IV. The invertible P-segment in the el4 element in the E. coil chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

114

V.

Related DNA invertases in other organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

114

VI.

Homology between the DNA invertases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

115

VII. DNA inversion studied in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

115

VIII.Genetic switches by DNA recombinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

118

IX.

118

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

118

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction

The paradigm of the lactose operon for regulation of gene expression [1] has been shown to hold for the regulation of many operons in Escherichia coli, not only those involved in sugar metabolism, but also operons involved in amino acid synthesis, DNA repair, etc. In all cases repressor proteins regulate the transcription initiation by reversible binding to the DNA. In these systems the DNA plays a more or less passive role in the regulation, 0167-4781/84/$03.00 © 1984 Elsevier Science Publishers B.V.

and it remains unchanged throughout. It has become dear that genes can also be switched on and off by specific rearrangements of DNA, and that this phenomenon plays an important role in prokaryotes as well as in eukaryotes [4]. The first report of a 'structural anomaly' in the DNA of Enterobacteriaceae came in 1956. It had been found previously that the bacterium Salmonella typhimurium alternatively expressed two different flagellar genes [2]. The flagellar phase

112

was determined by the Phase Determinator [3]; the phase could be transduced from one bacterium to another by exchange of this region of the genome. This showed that an inheritable reversible state of expression was determined by the Salmonella DNA. The state of expression was found to reside in the orientation of an invertible DNA segment [19]. Recently several invertible DNA segments have been found in different organisms of which the function is not yet known: yeast [5], herpes simplex virus [6], a plasmid of Staphylococcus aureus [7] and a phage of the archaebacterium Halobium [53]. In the Enterobacteriaceae switches by DNA inversions have been studied of which the function is reasonably well understood. Surprisingly these switches turn out to be evolutionarily related, even to the point that the recombinases (DNA invertases) can complement each other. We will describe three well-analysed examples of three D N A inversions in more detail, and then discuss their evolutionary relationship. Experiments will be described which demonstrate the inversion reaction in vitro; these will probably provide the answer to the question of how the D N A invertase finds its target sequences, how it cuts and rejoins the D N A strands, and how the tertiary structure of the DNA affects the recombination reaction. Another review on the subject of gene regulation by DNA recombination has been published recently [28]. II. lnvertible D N A determines the host range of phage Mu

Mu is a temperate phage of E. coli. Electron microscopy studies revealed a 3000 bp invertible segment (the G-segment) in the phage D N A [47]. Mutants were isolated that were unable to invert the G-region (gin) [9]° The Gin protein was identified in minicells [48]. The function of the flip-flopping between the two forms remained long unclear, especially since the G ( - ) phages turned out to be noninfectious for E. coli [8]. However, the G ( - ) phages were not 'dead' phages, but were found to plate on hosts other than E. coli K12: Citrobacter freundii, E. coli C, Shigella sonneii [10], Enterobacter cloacae, and several Erwinia species (A. Toussaint, personal communication). From the mapping of mutants [11], and analysis of plasmid

clones [12] a picture emerged of the molecular basis of this genetic switch. This is given in Fig. 1. Two sets of genes are alternatively expressed; these are involved in the assembly of the tail fibers (F. Grundy, M. Howe, personal communication). The orientation of the G-region determines which type of tail fiber is synthesized by the phage, and consequently to which host cell wall the mature phage will be able to absorb. Note that the recombination sites are located within the reading frame of one of the genes involved; as a consequence the inversion reaction in fact 'splices' a different Cterminal part of the S or S' gene to the common Sc domain. Possibly the common part of the S and S' proteins are attached to the phage, whereas the specific recognition of the different host cell wall structures resides in the variable parts. The frequency of the inversion reaction is low under normal circumstances, mainly as a result of the low level of expression of gin which is responsible for the inversion reaction [13,49]. This low inversion reaction is understandable: whatever the advantage of being able to be in two distinct states, it would be lost if the two states were mixed continuously [14]. Phage P1 also contains in its DNA an invertible segment, the C-segment. This was shown to be highly homologous to the G-region of Mu [15], and mutants in the Mu genes S or U can be rescued by P1 [16]. The interesting feature of the C-region is that the orientation of the cin gene relative to the invertible segment is opposite to the situation for gin [17,18]; see Fig. 2. The two Cterminal codons of cin overlap the inverted repeat of the C-region. The promoter of cin shows homology to part of the sequence of the inverted repeats, and therefore it has been speculated that this sequence may have diverged from an original recombination site [18]. It has previously been found that the fraction of G ( - ) phages in an induction lysate of Mu is 50%, and less than 1% when the phages were grown lytically [8,9]. gin is expressed in a lysogen under phage immunity [49,13]. Therefore in a cell culture the fraction of G ( - ) lysogens reaches 50% in a few generations; these will produce G ( - ) phages upon induction. When Mu is grown lytically on E. coli K12 only G( + ) phages can absorb; the G( - ) phages found in the lysate will virtually all result

113

. . . . .

I

"

o

I

IR

Sc

~ ,v'E'

IR

Sv

U

U'

Sv'

gl

morn

Fig. 1. The invertible G region of Mu. The G region is here shown in the (+) orientation. Transcription reads from a promoter in the alpha region of Mu; the S and U genes are transcribed. These are essential for growth of Mu on a G( + ) host. Inversion of the G region is catalysed by Gin; the gin gene is located adjacent to the G region. The recombination sites which flank the invertible segment are 34 bp inverted repeats (IR). After inversion of G the genes S' and U' are expressed which are essential for growth on G ( - ) bacterial hosts. Note that S and S' share a common region of approximately 500 bp in the non-inverting DNA. This region is indicated as Sc (S-common), as opposed to Sv and Sv' (S-variable and S'-variable). The sequence of the G region has been determined by Dr. R. Kahmann (unpublished data); the sequence of the beta region (containing gin and morn) has been published [24,50].

S typh,mur,um

II"~-~J

ATT el/,

/ ~ l

•

i

ATT e1~ pseudo IR

Fig. 2. The invertible G, H, C, and P segments compared. The genetic organization of the Mu G-region is discussed in the legend to Fig. 1. The promoter of gin overlaps the inverted repeat sequence at the right side of the G region [24]. The invertible H segment S. typhimurium is presented in detail in Fig. 3. Note that the hin gene is located within the invertible segment. The relative orientation of hin towards its promoter/recombination site is the same as for gin; therefore the inverted repeats flanking the invertible segment are 'insideout' as compared to the G region. The third line shows the P region which was mapped within the element el4 in the chromosome of E. coll. The location of pin towards the invertible segment is as for gin; the size of the invertible segment is 1800 bp. The P region shows no homology to the Mu G-region tested by Southern hybridization under stringent conditions (Plasterk, unpublished information). The bottom line shows the C segment of phage P1; although the genetic organization of this region has not been determined it was proposed to be similar to that of the Mu G-region [18]. Note that the cin gene is the only of the four DNA-invertase genes for which the promoter does not overlap the recombination site. There is homology between the cin promoter and the recombination site, which led to the hypothesis that the organization of the C/cin region is the product of a genetic rearrangement [18].

f r o m t h e last i n f e c t i o n cycles, a n d t h e s e are l i m i t e d in t i m e (the M u life cycle takes a b o u t 3 0 - 4 0 min). T h e f r a c t i o n o f G ( - ) p h a g e s in the r e s u l t i n g lysate will t h e r e f o r e b e low. III. T h e flageilar p h a s e variation of Salmonella typhimurium L i k e m a n y b a c t e r i a l species, Salmonella typhim u r i u m was f o u n d to e s c a p e t h e a c t i o n of a n t i s e r a r a i s e d a g a i n s t a s e e m i n g l y m o n o c l o n a l cell c u l t u r e . T h e m a j o r a n t i g e n i c d e t e r m i n a n t o f the cell surface, t h e flagellum, was f o u n d to v a r y w i t h i n a cell c u l t u r e d e r i v e d f r o m o n e p a r e n t cell (the t w o diff e r e n t t y p e s o f flagella w e r e n a m e d H 1 a n d H2). T h e flageUar state was d e t e r m i n e d b y an e l e m e n t in the c h r o m o s o m e ( w h i c h was t r a n s d u c i b l e to d i f f e r e n t g e n e t i c b a c k g r o u n d s [3]). U s i n g r e c o m b i n a n t D N A t e c h n i q u e s the n a t u r e of this e l e m e n t was f o u n d [19,20]. Fig. 3 s h o w s a d i a g r a m o f the s w i t c h s y s t e m : the o r i e n t a t i o n o f t h e H - s e g m e n t d e t e r m i n e s in cis w h e t h e r the H 2 g e n e a n d the r i l l g e n e are t r a n s c r i b e d , the R h l p r o t e i n d e t e r m i n e s in trans w h e t h e r t h e H I g e n e is t r a n s c r i b e d . T w o d i f f e r e n c e s w i t h the G - s w i t c h are clear: t h e H segment contains a promoter which alternatively p o i n t s to t h e H 2 - r h l o p e r o n o r the o p p o s i t e direction, w h e r e a s the G - s e g m e n t ( p a r t i a l l y ) c o n t a i n s t w o sets o f g e n e s w h i c h are a l t e r n a t i v e l y l o c a t e d d o w n s t r e a m o f a p r o m o t e r in t h e n o n - i n v e r t i n g D N A . T h e H - s w i t c h has a n effect in trans o n the e x p r e s s i o n o f H1, w h e r e a s the G - s w i t c h is t h o u g h t

po

H1

hit,

~

P

H2 • r . , ~ "~ ]

Rhl I

E~ Fig. 3. Genetic organization of the S. typhimurium invertible DNA segment. The genetic organization has been described in Refs. 19 and 20. The invertible segment contains the bin gene, and a promoter which reads out of the segment. The top line shows the situation in which the promoter initiates transcription of the 1t2-rhl operon. The Rhl protein then represses the transcription of the HI gene, which is located elsewhere in the Salmonella chromosome. The bottom line shows the HI-state: the 1-12 flagellar gene and the gene coding for the HI repressor are not transcribed; the HI gene is transcribed.

114

to act only in cis. A third difference was predicted and first demonstrated by Kahmann and Kamp, 1981 [21]: the recombination sites, inverted repeats flanking the invertible segment, are located insideout in the H-segment relative to the situation in the G-segment (see Fig. 2). This is a more or less logical consequence of the fourth difference: the hin gene is located wi'thin the invertible segment, gin is outside it. IV. The invertible P-segment in the e l 4 element in the E. coli chromosome

Whereas Gin and Hin are known to be responsible for a genetic switch, the phenotypic effect of inversion of the P segment is not known. Mugin phages were found to be phenotypically Gin + when grown on certain strains of E. coli [23]. Similarly, hin mutations were complemented by a function in certain strains of E. coli [25]. The gin complementing function was mapped within the element e14 in the chromosome of E. coli. e14 is an element of 14 kb, which can be excised from the chromosome after ultraviolet irradiation of the cell, and cannot replicate autonomously [22]. It has the properties of a defective prophage. After

Cin

ML

Hin

MAT

g

T

SAG

L

KAE

SAN

R

IA

Cin

HLSR

VWKLDRLGR

R

V

Hin

YVNK

VWKLDRLGR

K

A

V. Related D N A invertases in other organisms

As will be described below, Gin, Cin, Hin and Pin are closely related. This was a reason to expect that possibly other phase variation phenomena in bacteria could be catalysed by proteins similarly related to the known DNA invertases. Indeed such functions have been found, e.g., in Citrobacter [28]. In this case the inversion (catalysed by Vin) is responsible for a switch in flagellum expression, and it may therefore be expected that the system is closely related to the Hin system.

I

-10

Gin IRR CTTTACCGTriCTGtCC AATGGT TTi

Pin

Cin

cloning of pin, it was found that it was responsible for the inversion of a 1800 bp segment within el4 [23]. Analysis of proteins coded by both P ( - ) and P ( + ) clones indicates that indeed the inversion determines the differential expression of one or two proteins (Plasterk, unpublished observations), but it is not yet clear where they are located with regard to the P-region. Although variation in pili, in flagellar phase and colony morphology have been described for E. coli [26,27,31], these have not been correlated to the orientation of the P-segment (Van de Putte, unpublished results).

Pin

IRR(-)

A G T T T T G C T C C r T TCCC AACC

TTT T

Hin

IRL

CCATTTATTCC ITCTTG

AACC

TTT

AA.TGAA AAAGCA

Pin

IRR(*)

AATGCAGAGCC

AACC

TT

AATGAA

Bin

IRR

C T GC C AT A A A ~ r T[TT C C TT T T G G ~ A ~ G T T T ~ T [ G A ~ A A A G CA

Cin

IRL

CTGGTACCGA6

PKLTPEQW

M

E

Q

DVA

K

SFQS

TCC

CTCT

AACC

> "~

TGAAAT

(b)

Fig. 4. Comparison of the amino acid sequences of Gin, Pin, Cin and Hin, and the D N A sequences of their recombination sites. (a) The D N A invertases aligned. The sequences of Gin and Pin were derived from the D N A sequence by us [24], hin was sequenced by Zieg and Simon [51] and cin by Hiestand-Nauer and Iida [18]. The four proteins are largely homologous; most of the variability is in the C-terminal part. As can be seen, the Hin protein is least related to the other three proteins in amino acid composition. (b) The recombination sites aligned. The target sequences for Gin and Pin were from Ref. 24, for Hin from Ref. 51 and for Cin from Ref. 18. The putative promoter of gin, hin, and pin is indicated. The homologous sequences are boxed. Within most recombination sites a palindromic sequence is found: A A A C C - - G O Y I ' i . The I R R of Hin misses the sequence AAACC.

115 Vl. Homology between the DNA invertases Gin (Mu), Hin (Salmonella typhimurium), Cin (P1) and Pin (Escherichia coli) complement each other [21,23,25,29]. Therefore it is not surprising to find that both the proteins and their recognition sites are largely homologous [24]; see Fig. 4. The comparison is expected to define the essential amino acids in the DNA invertases, and essential bases in the recognition sites; these are shown in Fig. 4. It was previously shown that there is considerable homology between the DNA invertases and the resolvases of the Tn3 class transposons [19,24]. The resolvase TnpP, resolves transposition cointegrates by a site-specific deletion reaction. It has been proposed that TnpR binds covalently to the phosphoester in the DNA backbone by a tyrosine residue in the N-terminal region [35]. This tyrosine residue is also found at the corresponding site in the DNA invertases, and so is another tyrosine at the C-terminus of the proteins [24]; see Fig. 4. A model for the evolutionary development of the related systems has been proposed recently [41]. It is proposed that the site-specific resolution system of a transposon has, by transposition, inserted into completely different operons. In the case of the flagellar phase variation system the original situation might have been the presence of one flagellar gene. The flagellar gene has duplicated, and the original promoter of one of the flageUar genes has been replaced by the promoter which inverts as a consequence of the site-specific recombination system. This model is based on the observation that the DNA invertases are partially homologous to TnpR of the transposon Tn3, and on the comparison of the sequences surrounding gin, hin and pin. The two flagellar genes of S. typhimurium (H1 and H2, located in different parts of the chromosome) are largely homologous, and H1 is homologous to the flagellar gene of E.

protein. No E. coli equivalent of Rhl is known, however. The obvious question is then why the hag gene should be repressible by Rhl. Possibly the one-flageUar situation of E. coli does not represent the evolutionarily older situation, but, on the contrary, has developed from the phase variation system in S. typhimurium. In that case one could assume that the equivalent in E. coli of the Salmonella hin-H2-rhl region has been lost. 2. Is it merely coincidental that both the Mu G-inversion and the S. typhimurium H-inversion involve the variation of genes coding for proteins which form part of a multimeric structure that can bind to the cell wall (phage tail fiber and flagellum)? It might be argued that there is a continuous line from flagellar genes, through F factors coding for their own conjugation pili, through filamentous phages, to the more complicated phages with their own infection apparatus. It would be interesting to compare the sequences of the the Mu S and S', U and U' proteins, the H2 and H1 proteins, the subunit of F pili, and possibly the proteins coded by the E. coli P-region. The alternative for evolutionary divergence is that the analogy of variation in both flagella and phage tail fibers is based on the usefulness of variation in proteins which contact an organism with its (always varying) environment. From the complementation of Gin by Pin one might conclude that the recombination sites of the two systems are interchangeable. This turns out not to be completely true: it was observed that whereas a gin gene cloned on the vector pBR322 was responsible for a high inversion rate of a G-segment on a compatible plasmid, this rate was considerably lower when a pin gene was used instead. Similarly, the P-region is inverted at a higher rate by a cloned pin gene than a gin gene (Plasterk, unpublished observations). This leads to the conclusion that the two recombination systems have indeed diverged somewhat.

coli (hag). Some questions still have to be answered: 1. It has been demonstrated that when the invertible segment and adjacent sequences of S. typhimurium are transduced to E. coli the bacterium starts to show phase variation between H2 and hag expression [52]. This implies that hag is not only homologous to H1, but also repressible by the same Rhl

Vll. DNA inversion studied in vitro

Initial attempts to demonstrate DNA inversion in vitro by Gin were unsuccessful, probably due to the low levels of Gin protein synthesized in the cell under normal conditions, and to the absence of a sensitive assay for the detection of an inversion.

116

41- .

<----~--(LacALacY LacZ . . . .

%•,,

LacZ

Sv, I

LacY LacA

Fig. 5. The plasmid to assay G-inversion. The plasmid pGP231 contains a G region, with the lac operon of E. coli fused to S ' of Mu. The plasmid is gin - . The G( + ) form of the plasmid is lac - , the G( - ) form lac + .

The sensitive assay could be obtained by the fusion of an easily selectable and detectable gene to the S ' gene of Mu on a cloned plasmid. In this plasmid (see Fig. 5) a functional fusion gene S ' lacZ is present in a G( - ) clone; the G( + ) clone is lac [13]. As the plasmid does not code for a functional gin gene the G region is frozen. This plasmid (pGP231) can be used as a substrate for Gin, both in vivo and in vitro: Gin activity can

Fig. 6. G-inversion in vitro, pGP231 plasmid DNA was incubated with a partially purified extract from a Gin overproducing strain. After the indicated times of incubation the DNA was phenol-extracted, ethanol-precipitated and digested with E c o R l and H i n d l l I . These restriction enzymes cut asymmetrically in both the vector and the invertible segment, which results in a different restriction pattern from the G ( - ) and G( + ) forms of the plasmid. The inversion leads to a shift in the restriction pattern of two bands; the most easily recognized shift is that of the smaller band. A detailed restriction map of pGP231 has been published [13]. Lanes: 1, G ( + ) DNA; 2, G ( - ) DNA; 3-6, G ( + ) DNA incubated with Gin during 0 h, 0.5 h, 1 h and 16 h; 7, G ( + ) DNA incubated with Gin during 1 h in the absence of Mg 1+ . The figure was reprinted from Ref. 33.

switch the plasmid from Lac to Lac +. A Gin overproducing strain was constructed by insertion of the strong lambda pL promoter just in front of the gin gene, and cloning of this construct in a multicopy plasmid with a so-called runaway-replication origin [32,33]. Incubation of the plasmid pGP231 with an extract from a Gin overproducing strain results in inversion of the G-segment on the plasmid. This inversion can be detected by transformation of the plasmid D N A to a Lac strain, and scoring of Lac + colonies on plates containing the/3-galactosidase indicator Xgal. When the fraction of plasmids containing an inverted G-segment is high enough, the inversion can also be monitored by digestion of the plasmid D N A with restriction enzymes which cut asymmetrically in both the G-region and the vector D N A and analysed by agarose gel electrophoresis [33]. A time series of the inversion reaction is shown in Fig. 6. As can be seen after prolonged incubation with the Gin extract, equilibrium is almost reached between the two orientations G ( + ) and G ( - ) . The reaction requirements are simple: no exogenous ATP is needed; besides buffer and NaC1 only the addition of Mg 2+ is essential. The substrate D N A molecule has to be supercoiled to get an efficient inversion reaction [33]. The inversion activity is also found in partially purified preparations of a Gin-extract, and in all probability no host factors are required. These modest requirements of Gin contrast with, for example, the phage lambda integration/excision system, but resemble the requirements of the T n p R protein (the resolvase of Tn3 type transposons) [34,54]. Now that the complete recombination reaction can be carried out in vitro the mechanism of the reaction can be studied in detail. Some of the important questions to be answered are: which phosphodiester bonds in the recombination site are dissociated and religated during the recombination? How does the inversion reaction affect the --topological structure of the substrate DNA: is there a change in the degree of linking or knotting? For deletion-catalysing recombinases the topological changes have been studied [37,38]; in this case, however, the end-product is evidently no longer one circular D N A molecule (but two circular molecules). The Gin-catalysed inversion reaction is, however, reversible, and the end-product

117 of the reaction is again one circular D N A molecule. This makes it possible to check whether the inversion reaction is accompanied by a simple topological change. It has been observed that Gin, Hin and Cin preferentially catalyse inversions [13,36,49], whereas T n p R shows a preference for deletions [34]. This difference could be explained by the p a t h w a y leading to the recognition of the two recombination sites by the recombinase. Supposing the recombinase does find its target sequences by tracking along the D N A (as has been proposed for m a n y D N A - b i n d i n g proteins [35,39]), the decisive step is the initiation of the tracking after the recognition of the first recombination site (see Fig. 7). Two possibilities are shown which lead to a succesful reaction: in (A) the direction of tracking is opposite to the direction in (B). Therefore (A) can only lead to an inversion reaction and (B) to a deletion. N o t e that after the two recombination sites have met in the reaction complex the relative orientation of the sites is identical within the boxed area. Although in the figure two Gin molecules are shown, nothing is known about the n u m b e r of molecules which are involved in the reaction. A1-

Recognition

of

sites

Intliation

of

Irclcking

Formation

!

a

,/~"x?

X~ / ~ ' x

1

ternative models are also possible: e.g., Gin does not at all bring together its recombination sites by D N A tracking, but by three-dimensional diffusion. In that case the superhelical strain in the molecule might play a role in the chance for the two sites to meet [13]: local unwinding of the supercoil is needed to bring together two direct repeats, whereas by 'slipping' of the supercoiled molecule two inverted repeats could more easily meet. This would explain the preference of Gin for inversions. The crucial experiment is to test whether tracking plays a role in the in vitro reaction. This can be tested by determining whether the topology of the reaction p r o d u c t is a simple function of the topological structure of the substrate, and independent of the distance between the two recombination sites (which is what is expected if the two sites are brought together by ordered one-dimensional tracking). W h e n these questions have been answered we will have a detailed picture of how a simple genetic switch takes place. It is the relative simplicity of this inversion reaction (one substrate, one enzyme, and - probably - one reaction product) which makes the reaction accessible to mechanistic studies.

reac'~ion

: ),a

complex

)

f

Fig. 7. Model explaining the difference between the preference of Gin and tnPR for inversions and deletions, respectively. Three stages are shown: recognition by the recombinase of the recombination sites, initiation of the tracking and the situation after the tracking. The determining step for inversions or deletions is the second step: when pathway A is used and the recombination sites are in direct repeat no recognition will result. Similarly pathway B will only lead to successful recognition of direct repeats. It should be noted that it is not essential for this model that site 2 is occupied by a second er~zymemolecule. In fact it is not known how many molecules are involved in one recombination reaction. In this figure the protein on site 1 recognizes the protein which occupies site 2, but it might equally well recognize the DNA of site 2, The central role in this model is for the enzyme which occupies site 1 and starts pulling through the DNA (in either of two directions), tracking for the second site.

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VIII. Genetic switches by DNA recombinations It was suggested as early as 1957 by McClintock [42] that D N A recombination might be at the basis of cellular differentiation. Some cases studied on the genetic level point to a central role for D N A recombination in cellular differentiation (e.g., assembly of immunoglobulin genes [43]), and in variation (e.g., yeast mating type switching [44], and Trypanosoma antigenic variation [45]). It is possible that gene rearrangements contribute more to cellular differentiation than the few examples found thus far. D N A recombinations have thus far been classified in terms of E. coli genetics: homologous recombination (RecA-dependent) and non-homologous (RecA-independent) recombination. The latter class has further been subclassified into, for example, site-specific recombination and transposition. It is also possible to classify recombinations on the basis of their function. Several sitespecific recombination reactions are known which do not have a known function in genetic switches but in phage lysogenization/excision or in replicon resolution (Cre of phage P1 and resolution of transposon cointegrates). Different examples have been found, however, where the function of the recombination reaction is to determine the expression of genes within a cell. These programmed recombinations may be accurately site-specific (like the inversions described in this article) or more region-specific (like V-J joining in immunoglobulin gene assembly) [46]. But they have all been selected during evolution to create specific variability in originally isogenic cells by switching on or off the expression of genes. Different somatic cells can be generated within an organism, and still the original genetic information can be passed through to new generations by the germ cells.

IX. Conclusion It becomes increasingly clear that specific rearrangements of D N A segments play an important role in the regulation of gene expressicn, and in differentiation processes. A class of well-defined genetic switches is formed by D N A inversions in Enterobacteriaceae. The first indication of such a phenomenon came from the observation of the

instability in the flagellar antigen of Salmonella typhimurium strains. Another well-described example is the variation in the host range of E. coli-bacteriophage Mu (and probably also the related phage P1). Recently an invertible element has been discovered in an ultraviolet-excisable segment of the E. coli chromosome (e14). Complementation analysis and D N A sequence analysis have revealed that all these variations are the result of D N A inversions, catalysed by enzymes which are closely interrelated, and in addition show homology to other site-specific recombinases (the resolvases of Tn3 type transposons). An overview has been given of the different DNA-inversion systems in prokaryotes, and their evolutionary relationship has been discussed. The reaction can be demonstrated in vitro, which will allow studies of the mechanism of the inversion reaction.

Acknowledgements We would like to thank Dr. S. Iida and Dr. M. Simon for communicating their results prior to publication.

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