Variability of antigen genes in African trypanosomes

TIG --January 1986

reviews Variability of antigen genes in African trypanosomes

Trypanosomiasis affects central and east Africa in a belt south of the Sahara desert. In addition to causing human sleeping sickness, trypanosomes can infect cattle, preventing livestock proEtienne Pays duction on a quarter of the In African to~panosomes, the expression of surface antigen genes is controlled by several African continent. There is no m~.~nisms which lead to their mutually exclg¢iveactivation, along with ral~d evolution need to stress the considerable of their fOeflo/fes. Tl2is allows the parasite to escape the immune response of its impact of these diseases on mammalian host, and to det~lop waves of chronic infedion. Analysis of these public health and economic medumisms has uncwJered several int~res6ng features for molecular ~,netieis~, concerning DNA recombination, transc~tion and rOlication in tdomeres. Although development in some of the this potemtialfi~, M ~ mu,ia6onprech~lesshod-term vaccim~lion,somepossibil'~iesfor most fertile areas of Africa. prop~la~ against human sle#n'ngs i c k s can ~ ¢nvisag~ Vaccination against these parasites has met with little success. Although the life cycle of the parasite has been known since the beginningof the Although many antigen genes are located within century, and trypanosomes have been thoroughly chromosomes, transcription of these sequences has studied after isolation and cultivation both in mtro and in never been detected in situ. The reasons for this are vivo, all efforts to eradicate the disease have failed. obscure. They could be related to specific characterisTrypanosomes are transmitted by G/oss/na flies to tics of the RNA polymerase transcribing antigen genes. mammals, where they can survive for long periods by It exhibits a sensitivity to ~-amanitin distinct from that maintaining chronic infection characterized by succes- observed for transcription of other geness. Moreover, sive waves of parasitaemi~ The number of parasites in contrary to what occurs for other genes, that RNA the blood undergoes successive increases and sharp polymerase is rapidly released from its template when reductions, correlated with fluctuations in acuteness of the trypanosomes are isolated from the bloods. It is the disease. There is thus a mechanism keeping the possible that the RNA polymerase devoted to antigen trypanosome population more or less controlled during gene transc~otion only recognizes telomeric DNA, and infection, and preventing premature death of the host by needs the presence of a blood factor to be active. However, the telomeric position of an antigen gone is avoiding overgrowth of the parasites. This mechanism involves antigenic variation. In not sufficient for expression. Many antigen genes are mammals, the parasitic trypanosomes are completely located at chromosome ends6'7, and the mechanisms surrounded by a thick coat, consisting of a tight network involvedin the selective transcriptional activation of only of a single glycoprotein species, of about 65 kD& The one of these telomeres ~are as yet unknown. Two protein moiety is the major antigenic determinant uf the hypotheses can account for this. Either a unique factor parasite. The immune system of the host reacts against determines the activation of a single telomere, or strict this surface antigen, thereby destroying the trypano- selection allows only andgenically homogeneous parasome population. However, at any given time, a few sites to survive. Some observations support the second individual trypanosomes (less than 0.1~) exhibit a hypothesis. Firstly, although only one antigen gene can surface protein o f a different antigenic specitidty, and be transcnl~ in a clone, the sequences upstream from therefore escape this antibody response. This allows different telomeric antigen genes can be simultaneously some of them to proliferate into new populations, against transcribed in distinct chromosomess. This suggests which the immune system willagain rea~ The interplay that several active expression sites may coexist arguing between antigenic variation of the trypanosome and the against the existence of a unique activation factor. immune response of the host therefore shapes the Secondly, formation of the surface coat requires chronic infection characteristic of African trypano- cooperation between antigen oligomers, and does not take place properly when different antigens are mixed. somiases (for a review, see Ref. 1). Since trypanosomes are destroyed by non-immune serum if their coat is incomplete1, the expression of a The expression of antigen genes is selective single antigen type may be the only way for the parasite In the blood, each trypanosome appears to express to survive in the blood. only one antigen type at a time. To date there are no reports of parasites with stably heterogeneous coats. In any trypanosome clone, there is only one type of antigen-specific mRNA. However, in each cell, the How switching of antigen gene expression repertoire of antigen genes is extensive. It accommo- occurs The telomeric ~sition of the expressed antigen gone dates information for up to 1000 different antigen types, provided all antigen-specific sequences can encode renders it parti¢,~y prone to recombination. Telofunctional proteins. It is therefore likely that specific meres are prefe'~ential targets for several mechanisms control mechanisms ensm'e the selective transcription of of DNA reanar~:~!;ement, probably because they contain a single antigen gene from a large collection of different repeated sequer c ~ interrupted by single°~trand r~cks, and because they are closely associated in mitosis9. sequences 1. A str~dnf characterstic of the transcription of antigen Evolution may have favoured the location of the antigen genes is that it occurs onlyat the end of a chromosomeTM- gene expression site in such a recombination hot-spot to 21 © ~ F~v~ ~ ~ B.v., ~ 0xes-ssz~s~so.z0o

reviews

TIG--]anuary1986 tolomero

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Fig. 1. The different mechanisms alloun'ngantigen ~ s~tching in bloodstream tr~nosomes. Before antigenic variation, the two antigen genes involved, X and Y, are located at the end of chromosomes C1 and C2, respectively. The telomere of chromosome C.! is activated, as represented by the star, leading to transcription ofgene X (wavy anow). Thefactors conditionfng the selective telomere activation are still conjectural. Antigenic vaTiation can be achieved in at least three ways, which can alternatively applyfor the same telomericgene. In (A), a variably sizedgene conversion replaces thepredously expressedgene (X) by thecopyoftheensuinggene, which may be telomericas represented here (Y), or chromosome-internal.In some instances,when the conversion involves extensivestretchesof DNA encompassing elements allowing autonomous replication, the target (sequenceX) couldpossibly be conseroed in theform of a minichromosome (mc). In (B), exchange of two tdomeric genes occurs, leading to the expression o/gone Y in the active tdomere of chromosome C1, along with the inactivation of gene X. In (C), the te:omere of chromosome C I is de.activatsd and the resident antigen gene (X) silenced, while the telomere of chromosom~ C2 becomes the e~ession site of gene ]I. The mechanism involved is totally unknown. VSA = vaviant-epeci.fic antigen.

provoke extensive antigenic variation. Indeed, antigen switching often resultsfrom DNA rearrangements. At least two recombination mechanisms allow antigen genes to replace each other in a given expression site: gene conversion and reciprocal recombination (see Fig. 1). Gene conversion is relatively frequent, and can lead to total or partial replacement of the gene, probably depending on the extent and location of homology between the donor and target sequences (for review, see Ref. 10). It sometimes extends for considerable distances, and can replace whole telomeres ,~itn copies of others u. This may explain the extensive homology observed between different chromosome ends6. Reciprocal recombination results in telomere exchange. This mechanism has only been described oncelz, although in theory, the high homology between telomeres should favour homologous recombination. Why gene conversion predominates over reciprocal recombination is unclear. 22

It may be that the chromatin structure of the transcribed antigen gene is responsible for the preferential succession ofdifferent DNArearrangements in the same site. Being'open' for transcription, it is indeed more susceptible to cutting by endonucleases, such as DNAase I. This may explain the highrate ofdeletionin the terminal portion of the expressed telomeres, and may also make this telomere a preferential target for any recombinase. However, there is a mechanism which can achieve antigen switching without detectable DNA rearrangement 13'14. It involves inactivation of the expressing telomere and activation of another tek,.nere resulting in a new expression site (Fig. 1). What governs this differential telomere activation is unknown. It could result from differential binding to a unique activating structure (such as the nuclear matrix) or the presence of a unique transposable activator, but there is no experimental evidence for either hypothesis. The only characteristic that could be specifically related to the telomere activation is differential telomeric DNA modification. Two obse/'vations indicate that telomeric sequences are modified in deoxycytidines in the inactive state and that this modification is removed when they are transcribed. First, the overall base composition of a DNA fraction enriched in telomeric sequences reveals a strong depletion in deoxycytidine, due to extensive modification of this nucleoside3. The nature of this modification is unknown. Secondly, some restriction endonucleases do not cleave their target site completely in inactive telomeric sequer.ces, while digestion is complete when the DNA is transcribedls'le. This is probably a result of differential activity of a special modification system which is able to work only on telomeric DNA. There is a strict correlation between antigen gene activity and telomeric de-modification, suggesting that the telomere activation prevents the modification, or vice versa. Telomeres of 'procyclic' trypanosomes (the uncoated, insect-specific form) grown in vi~.o are not modified, even though they are not transcribed "°. Thus, it seems ~b~t the absence of modification is not sufficient to trigger transcription in telomeres.

The succession of antigen switches may be interdependent Although no specific inducer for antigen gene expression has been found in the blood, the appearance of antigen types seems more or less programmed during chronic infection. The expression of some antigens occurs only late, while others are frequently found early. In addition, preferential switching has been reported. Finally, some rules seem to govern very early antigen expression, the antigen type present before transmission through the fly being transiently re-expressed on parasite invasion in the mammalian bloodstream (see Ref. 1). The molecular explanation of the last phenomenon is still lacking, but it is known to involve reactivation of the gene repressed when the trypanosomes were taken up by the fly17. This gene is inactive throughout the development of the parasite in the fly, including the metacyclic stage, where expression of some antigen types is induced (see below). When the trypanosomes are in blood again, the gene is reactivated. This suggests that some blood factor may condition the activity of the

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TIG--]anuary1986

'bloodstream' antigen gene expression site, and this site may r e ' ~ for long ~fieds in the fly, the memory of its former activation state. S~il~rly, when the expressed copy of a gene is silenced along with activation of an alternate telomere (mechanism C in Fig. 1), it seems subsequently to be preferentially reactivable, by either of the two major gene activation mechanisms d e s ~ to datell, lsao. This is analogous to the previous example in that the former expression site 'retains a form of memory of its previous activation state. However, in 'natural' chronic infection, this preferential reactivation cannot occur, since the immune response does not allow reappearance of antigen types. Pertaining more to normal infection is the observation that expression of some antigen types is preferred over others. Apparently telomeric antigen genes are more likely to ~ ' expressed first1~4"~°. However, some telomeric genes are only transcribed late (IV[. LaurenL unpublished). A working hypothesis could be that the probability of gene expression depends on its degree of homology with the actual expression site. Since the latter is telomeric, telomeric genes may have greater access to it. Alternatively, if telomeric genes originate from inactivated expression sites (see below), their reexpression frequency is likely to be high. Usually, antigen genes are flanked by sequences sharing homology with the expression site~ These two blocks differ, and consist in arrays of 70 bp repestS extending from about 1.5 kb upstream of the genes'9l-m, and less structured conserved elements around the gene stop codon (see Ref. 1). These sequences flank numerous genes, allowing them to insert into the expression site. Their difference would ensure the correct orientation of the gene on insertion. However, the homology between genes may vary. Some genes may even lack one of the homology blocksz°'2a. The only chance for these genes to be expressed would be the presence, in the expression site, of a gene sharing with them unusual homology. Recombination at these sequences may allow the gene to be inserted in the expression site, although in the form of a cb~naeric gene with the previously expressed one. Obviously, such gene expression would depend on the previous expression of other genes, and would thus only occur late. Such a situation has indeed been observed in several instances TM. Such cases of partial gene conversions have been considered so far as oddities, but they may actually be relatively frequent during 'normal' chronic infection. Indeed, in the T. brucei EATRO 1125 repertoire, the antigen gene expressed in different cloned populations taken up directly from the original stock turned out to have undergone partial gene conversions~7a9. Moreover, in the T. equiperdum BoTAR I repertoire, where the orderly succession of the antigen types has been thoroughly described, late antigen types seem encoded by chimaeric genes (H. Eisen, pers. commun.). Antigen expression in the fly In the fly, the trypanosomes only synthesize variantspecific surface antigens in the salivary glands ('metacyclic' stage). Repression of the antigen gene might simply be due to release of the RNA polymerase from its template, since this enzyme is no longer associated with the DNA when trypanosomes are separated from the blood (see above). The gene activation at the metacyclic stage is clearly

Table 1. Evolufon of the antigengone reperto/reby the alternate use of differentgene adiva6on mechanisms(A, B orC, seeFig. 1) 1st switch 2nd switch Result Examplea A

A

Replacement of

1A--,10A~IB

additionalcopy from (Ref.27) 1st switch A C (or B) Conservationof 1A-,3A-,6A additionalcopy from (Ref. 14) 1st switch C (or B) A Loss of g e n e 3A--.6A-,?~C activated in 1st (Ref. 14) switch C (or B) C (or B) Reversionof gene 1B--,6C--,1G expression without apparent reazrm~ement a T. ~ EATRO 1125. induced. The expression of some antigen types is indeed specifically triggered in all metacyclic trypanosomes simultaneously. Moreover, this expression is unstable, but switches only to other metacyclic types. The analysis of this activation is still speculative, since, for technical reasons, it has been impossible to study the DNA of'true' metacyclics isolated from the fly salivary glands. To determine the mode of metacyclic gene expression, the best that can be done at present requires a few days of ~ypanosome growth in the bloo(L This form of analysis, although subject to certain limitations, has produced some interesting results. Metacyclic genes can be expressed via either of the two major activation mechanisms (Refs 14, 24 and M. F. Delauw and M. Laurent~ unpublished). An metacyclic genes analysed so far are telomeric. More specifically, they appear to be located on large chromosomes (D. Barry, pers. commun.; M. F. Delauw and M. Laurent, unpublished). This particular location could perhaps provide the specific metacyclic expression character. However, the metacyclic gene repertoire is not rigorously stable. New variants may become metacyclic, at the expense of others. It remains to be seen if this evolution is linked to translocation of the concerned sequences. Building and hyper-evolution of the antigen gene repertoire Comparison of the antigen repertoires of related T. bmcd stocks reveals their extensive variability. Antigen types shared by different stocks, even those originating from the same region, are only rarely found (these antigen types are called 'isotypes'). "lids high variation potential is reflected in the DNA. Many antigen genes, especially telomeric ones, are limitedto certain stocksz~. The fast evolution of the antigen gene repertoires can be tentatively related to the nature and alternate use of the different mechanisms used to develop antigenic variation, as discussed in the following paragraphs. Building of the antigen repertoire implies numerous duplications, and subsequent alterations, of ancestor genes. These may be found in the rare examples of genes, such as AnTat 1.8, which are extraordinarily well represented and conserved between stocks, even from different subspecies. The expansion of the number of antigen genes may have been achieved by occasional c~nservation of the target of some gene conversion 23

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T I G - - J a n u a r y 1986

several trypanosome stocks, from various geographical o~ins, have been hybridized with two antigen-s~cific ~robesfrom theT. bruceiEATRO 1125 repertoire, as well as wilh a probe probably unrelated to antigen genes (modif~d, from R ef. 25). The arrowpoint to the froonents containing the AnTat 1.13 and 1.8 genes, as analysed in the EATRO 1125 stock. Arrows i~licate the sole variations detected in T. gambiensehybrut"ization patterns, from a study involving ten different probes.

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events. Usually, this target (the replaced gene) is lost, but it is conceivable that when the conversion is extensive and involves a whole telomere, the converted sequence could be saved in the form of a minichromosome (see Fig. 1). This could occur if the target contains some element allowing for its autonomous replication and if it receives telomeric ends. There are several examples showing that DNA fragments can acquire & novo synthesized telomenc sequences, perhaps by the activity of a terminal transferase-like enzyme9. Moreover, telomeres can carry replication origins9. Occasional conservation of large conversion targets would account for the highnumber of minichromosomes, characteristic of the trypanosome genome7. Firstly, the size of these minichromosomesis compatible with that of the target sequence of some gene conversion events n. Secondly, the minichromosomes resemble inactivated antigen gene expression sites, as expected for intact conversion targets. :n particular, they carry haploid antigen genes, and have the potential to be used for antigen gene transcription~e. Of course, other interpretations, such as amplification ~ e that observed for drug-resistance genes, could also account for the generation of minichromosomes. Whatever their origin, these minichromosomes constitute a large reservoir of telomeric antigen genes. Telomeric genes evolve bypoint mutations scattered throughout the coding region~7. Only a few nucleotide ~ubstitutions are sufficient to change the antigenic specificity of the gene27. Therefore, related sequences, altered by mutations but still belonging to the same 24

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family, can participate in the divers~cat=on of the antigen repertoire. This process is so last that a specific mechanism for hypermut~genesls has been invoked. However, as yet the nature of this mechanism is totally unexplained. That error-prone gene conversion may be involved was proposed, but detailed studies ruled this out 2s. Several successive gene conversion events can occur without the appearance of a single mutation. It is clear, instead, that gene conversion contributes to the evolution of antigen genes by creating chinmeric sequences, made up of fragments from different templates, and that these fragments can be rather smallzs. There are certainly ways for telomeric genes to switch to a chromosome-internal position. Antigen genes located within chromosomes are indeed flanked with sequences normally found in telomeres2o. An obvious mech~sm for this 'internalization' would be the translocation of a telomere to another chromosome; this has been observed3°. If the recipient chromosome carries a telomeric antigen gene, that gene would be internalized. Repetition of this process would progressively shift antigen genes towards the chromosome centre. This hypothesis provides satisfactory explanations for two puzzling observations: most antigenspecific sequences are clustered in tandem arrays2O ' 2 1 , and they appear to be haploid in a diploid genome 1'13. That particular organization could indeed result from successive translocations of minichromosomes, carrying haploid genes, to the ends of large, diploid, chromosomes.

reviews

TlG--January1986 Modulation of the size of antigen gene families may depend on the alternative use of the different gene activation mechanisms. As illustrated in Table 1, a succession of duplicative and non-duplicative modes of gene activation (A and C mechanisms: see Fig. 1) leads to either loss or gain of genes. Since at least some telomeric genes can be activated by any one of these mechanismstl-ts, a gene family can be expanded in some repertoires, or be diminished and even lost in others, even if they are from closely related stocks: it all depends on the random choice of the different gene activation mechanisms during chronic infection. It may be stressed here that the location of a gene strongly influences its probability for expressionxg. As mentioned above, some telomeric genes seem to be preferentially activate& Translocation of gene can therefore alter the pattern of antigen repertoire expression, probably accounting for observations that late antigen types from a given repertoire can be expressed early in others (N. Van Meirvenne, personal commun). Finally, the nature and size of antigen repertoires could be extens'nvely modified by transfers of genetic information between trypanoscmes from different stocks. Such a transfer has been observed experimentally in a case of mixed cyclical transmission of different trypanosomes in the same tsetse fly (P. Paindavoine, E. Pays, M. Steinert and L. Jenni, unpublished). It suggests that sexual conjugation can take place dmingthe parasite developmentin the fly. At present, it is difficultto assess if this is a natural phenomenon, or ifit results from some aberrant event. In support of the first alternaiive, some 'hybrid' patterns generated by the mixed transmission experiment were also found in the DNA of stocks from the field. Stability of T. gambiense antigen repertoires Unlike other trypanosome subspecies, T. gambiense exhibits high conservation in its antigen repertoires. Many antigen types are present in all gambiemsestocks exmnined, regardless of their geographical origin (D. Le Ray, pers. commun.). Comparisons of serodemes or isoenzyme patterns have indicatedthat 7".g m ~ e is a homogeneous entity, i.e. a distinct species, unable to recombine with others. Results obtained at the DNA level completely agree with this vierS. The pattern of antigen-specific sequences is strikingly conserved between T. gambiense stocks, whatever the probe, as shown in Fig. 2. Even probes supposed to be unrelated to antigen genes show a ren-~kable constancy in T. g~e hybridization patterns. This suggests that the •genome of T. gambienseis relatively stable compared to other trypanosome species. In the analyses referred to in Fig. 2 (Ref. 25), the pattern of instability of nongambie~se subspecies was shown to be unrelated to the variability of the telomere sizes. Rather, it appeared to reset from a high rate of mutagenesis. Therefore, one may tentatively conclude that in T. gambimse, the antigen genes are less prone to mutations than in other trypanosomes. Since the hyper-evolution of antigen genes may depend on a telomeric location (see above), their relative stability in 7".gambienseis probably linked to the fact that only a few of them are telomeric in that species. Indeed the gambio~e genome contains relatively few small chromosomes and consequently less telomeric antigen

genes than T. brucd (13. Dero, M. Guyaux and E. Pays, unpublished), even though, as in other species, the expressed ge~e is telomericst. One can only speculate about the reasons for such a difference. We have shown previously that two factors determine fast changes in antigen gene repertoires: the alternate use of different activation mechanisms, and at least occasional genetic transfers during the development in the fly. Maybe T. gambiotse can only use a single gene-activation mechanism, namely gene conversion. This would !e~ad, during chronic infection, to replacements between copies made on different genes, and would not alter the antigen expression potentiaL As yet, the use of different gene actw"ation mechanisms has not been demonstrated

in T. g a ~ e . Vaccination prospects Two lines of evidence point to metacyclic antigen types as ideal targets for vaccine development. First, these antigens cover the trypanosomes which initiate invasion of the blood of a mammalbitten by an infected fly. Secondly, they only represent a minor subset of the total repertoire. Current estimates are about ten, out of a possible total of 1000o In T. b~cd or 7". rkodes/ense, the metacyclic repertoire, like the whole repertoire, varies. Nothing is known about the metacyclic repertoire of T. &ambiense, but, from the striking stability of the antigen repertoire expressed in the blood, we may expect it also to be ra~er conserved among stocks. If so, vaccination against T. gambiensemetacyclic antigens would probably be effective in preventing sleeping sickness in humans. Acknowledgements I would like to thank Prof. P. Borst, Prof. M. Steinert and Dr N. Murphy for useful discussions and comments on the manuscript. Work reported herein from my laboratory was supported by grants from the Commission of the European Communities (TSD-M0.23-13), from the FRSM (Belgium), from the ILRAD/ Belgian Research Centres Agreement for Collaborative Research (Nairohi), and from the Trypanosomiases component of the UNDP/World Bank/WHO Special programme for Research and Ti-aining in Tropical Diseases (Geneva). References 1 Borst, P. and Cross, G. A. M. (1982)Cell 29, 291-303 2 De Lange, T. and Borst, P. (1982)Nafure 299, 4514,53 3 Ra~mud,A.etaL (1983)Proc.NailAcad.ScL USAgO, 4306-4310 4 Lament,M. eta/. (1983) Nature 302, 263-266 5 Kooter, J.M. and Borst, P. (1984) Nude/c .~dds Res. 24, 9457-9472 6 Pays, E. et aL (1983)Nude/c Adds Res. 11, 8137-8147 ? Van der Ploeg, L. H. T. et aL (1984) Cell 37, 77-84 8 Come~ssen, iL W. C. A. et aL (1985) CeU 41, 825-832 9 Blackburn,E. H. andSzostak,J. W. (1984)Annu.£ev. Biochem. 53, 163--194 I0 Pays, E. (1985)Pvog Nuddc Acid Res. Mol. Biol. 32, 1-26 11 Pays, E. et ai. (1983) Cell 35, 721-731 /2 Pays,E, Gnyaux,M.,Aerts, D., VanMeirvenne,N.andSteinert, M. 0985) Nature 316, 562-564 13 Beroards, A. et el. (1984) Cell 36, 163-170 14 Lament,M. eta/. (1984) Natme 308, 370-373 15 Bemards,A., Van Harten-Loosbmek, N. and Borst, P. (1984) Nude/c Adds Res. 12, 4153-4170 16 Pays, E. et al. (1984)Nuc/e~ Adds Res. 12, 5235-5247 17 Delauw,M. F. et aL (1985) EMBO ]. 4, 989-993 18 M/chels,P. A. NL ~ a/. (1984)EMBO ]. 3, 1345-1351 19 Laurent, M. et al. (1984)Nuc/dc Adds Res. 12, 8319-8328

25

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TIC --January 1986

20 Van der Ploeg, L. H. T. et al. (1982) Nude/c Ac/ds Res. 10, 5905-5923 21 IAu, A. Y. C. et ai. (1983) ]. blol. Biol. 167, 57-75 22 Campbell, D. A., Van Bree, M. P. and Boothroyd, J. C. (1984) Nude/c Ac/ds Res. 12, 2759-2774 23 Mne, R. et ai. (1985) Nuc/e/c Ac/ds Res. 13, 3161-3177 24 Lenardo,M. J. et ai. (1984)Proc. NatlAcag Sci. USA81, 66426646 25 Paindavoine, P., Pays, E., Laurent, M., Geltmeyer, Y., Le Ray,

D., Mehlitz,D. and Steinert,M. (1986)Paras/to/oo 92 (in press)

26 Guvanx.M. et al. (1985)EMBO]. 4, 995-998

27 28 29 30 31 10,

Pays, E. et ai. (1983) Cell 34, 371-.381 Pays, E, et ai. (1985) Cell 42, 821-829 Blackburn, E. H. and Challoner, P. B. (1984) Cell 36, 447-.457 Van der Ploe& L. H, T, eta/, (1984) Cell 39, 213--221 Pays, E., Lheureux, M. andSteinert, M. (1982)NudeicAcidsRes. 3149-3163

E. Pays is at theDepartmentofMolecularBiolo~, Universityof Brussels, Rhode St Genese, Belsium.

Bacteriophage X development: temporal switches and the

The growth of phage ~, is a developmental system in the sense that a single genome in a single infected cell exercises a choice of temporal pathway. Phage 7`may be instructive also because we can now define most of the molecular events Harrison Echols that specify the lytic and lysoBacteriophage ~ executes a regulated temlZcal program along two lmthways, lyric or genic pathways and the choice lysogenic. Each pathway delz,nds on controlled, sequential synthesis and subsequent between them. Here, I will outactivity of A.emoded proteins. The choice of pathway requires an initial pa~'tion line for 7`the answers to some specified by the relativea ~ ' t y of the Acll regulatoryprotein,followed bya stabilization basic developmental quesof this choice by other ~ proteins. The stabiFtzation events involve armpetition by the tions: (1) What regulatory repressors cI and Cro for control of develOmental transmption and by the events define the temporal recombination proteins Int and Xis for the direaion of site.speafic recombination. sequence of the individual Several host and phage proteins exertpost-tmnscffptional control on the amount of clL pathways? (2) What deterpresumably transducingphysiological signals for choiceof pathway. mines the choice of pathway? (3) What does the regulatory strategy accomplish in ability of the bacterial genome to replicate, 7,escapes terms of the life-cycle of the creature? How generally from the endangered host by using a host distress one believes the molecular strategy of 7`development system (SOS) to derepress its genes. The 7, DNA is to apply is, currently, a matter of faith. For believers, excised from the host genome and enters the lytic the life and times of 7`are described in greater depth in pathway. old and new testament biographies 1'2 and in some general reviewss 5. References below are mostly to The early phase and the lytic pathway: recent specialized review articles that cover ia depth rolling down the DNA the topics outlined. As indicated above, Adevelopment proceeds mainly through the regulated, sequential synthesis and subseGeneral f e a t u r e s of 7`d e v e l o p m e n t quent activity of ?,-specified proteins. Most of this The pathways of 7`development are shown in Fig. 1. regulation involves control over transcription of the In the virus particle, the 7,genome is a double-stranded appropriate genes. Because A genes with related linear DNA (50 000 bp) with short (12 bp), comple- function are clustered, the regulatory circuits are mentary single-stranded ends that allow intracellular quite simple. circularization of injected 7`DNA (cos site of Fig. 1). The pattern of transcription for early development Starting from intracellular 7`DNA, development pro- and the lytic pathway is shown in Fig. 2. Immediately ceeds through a replication-oriented early phase com- after infection, the host RNA polymerase begins tranmon to the lytic and lysogenic pathways. A choice of scription at the promoter sites PL and PR. Because pathway follows: the 7, mid-life crisis. In the lytic these transcripts are soon terminated, this earliest (or pathway to the production of more virus, the head and 'immediate-early') stage of 7, development provides tail components of the mature phage are produced in mainly for expression of the N and cro genes (Fig. 2). large quantities. Replication also switches to a rolling- The N protein converts RNA polymerase into a form circle mode which produces the specialized encapsu- that is resistant to nearly all known termination lation substrate, a multimeric DNA with multiple cos signals6. The conversion requires sites coded in 7,(the sites. After phage-specified proteins lyse the cell, the nut sites) and several host proteins, including a pathway terminates with new free virus particles. ribosomal protein (S10) (Ref. 6). Thus the N-modified In the lysogenic pathway to the prophage state, the complex is probably much larger than that typically protein production of the lytic pathway is repressed, 7` studied @ vitro (but not necessarily larger than that replication ceases, and 7`DNA is inserted into the host active on typical bacterial genes in vivo). The genome by phage-directed localized recombination N-mediated early pattern of 7` DNA transcription between specific sites on the phage (attP) and host provides for the high level of 7`replication proteins (O, (attB). Once achieved, the lysogenic state is normally P) and recombination proteins appropriate for the stable; there is continued repression of lytic genes, replication-oriented early phase of development. Also and the prophage DNA replicates as a harmonious included in the early transcripts are the genes for the component of the host genome. However, 7`retains an cII, cIII and Q regulatory proteins, which control the option f.t~r divorce. If damage to DNA impairs the next events in 7`development. 26 ~) 1986.EIse~L-r~.~ncePublishersB.V..Amsterdam 0168- 9525/88/$0.200

choice of iysls or lysogeny