Proc. Nati. Acad. Sci. USA Vol. 77, No. 7, pp. 4196-4200, July 1980 Analysis of the nucleotide sequence of an invertible controlling element (flagellar phase variation/recombinational switch/ sequences/illegitimate recombination) JANINE ZIEG AND MELVIN SIMON Department of Biology, University of California, San Diego, La Jolla, California 92093 Communicated by Dan L. Lindsley, April 14,1980

ABSTRACr The nucleotide sequence of the inversion re- Mannheim. Hap II and Hae III were gifts from M. Hayashi and gion responsible for flagellar phase variation in Salmonela was R. Kolter, respectively. Pst I was prepared by the method of determined. Ihe inversion region is 995 base pairs (bp) in length P. Greene et al. (5). Enzymes used for labeling DNA fragments and is bounded by a 14-bp inverted repeat sequence. A homol- DNA large fragment (New England ogous recombination event between the 14-h inverted repeat included polymerase sequences would result in the inversion of the DNA segment BioLabs), bacterial alkaline phosphatase (Worthington), and between them. Sequence homologies with other systems suggest polynucleotide kinase (P-L Biochemicals). a-32P-Labeled de- that the 14-hp inverted repeat sequences may have some general oxynucleotide triphosphates were obtained from Amersham. significance as sites for specific recombinational events, The [,y-32P]ATP was either purchased from Amersham or prepared gene which specifies H2 ein synthesis begins 16 bp outside by modification of the Johnson and Walseth method (6). For the inversion region. Within the inversion region, an open the preparation of [ky-2P]ATP, 3sP-labeled phosphate was translational frame exists which could encode a low molecular obtained from ICN. weight polypeptide (190 amino acids). Plasmid Purification and Isolation of DNA Fragments. Hybrid phasmids were purified by the Sarkosyl lysis method of Phase transition in Salmonella is controlled by a novel kind of Bazaral and Helinski (7). Endonuclease digestion of plasmids regulatory unit in which a site-specific recombinational event was carried out in the buffer recommended by New England regulates gene expression (1, 2). An approximately 900-base- BioLabs for each enzyme. Slab gels containing 0.7-1.0% agarose pair (bp) DNA fragment adjacent to the H2 gene, which (SeaKem) or 9% acrylamide (BDH) were prepared in Tris/ specifies the synthesis of one of the flagellar antigens, can exist borate/EDTA buffer (TBE): 89 mM Tris, 89 mM boric acid, in either orientation with respect to the H2 structural gene. The and 2.5 mM EDTA. Gels were electrophoresed and treated as orientation of the inversion region controls expression of the H2 described by Bolivar et al. (8). gene-i.e., in one orientation the adjacent H2 gene is expressed DNA fragments were isolated from agarose or acrylamide and in the opposite orientation the H2 gene is not expressed. gels by the following procedures. Slices of agarose containing Genetic analysis of the invertible region has led to its subdi- DNA fragments were dissolved with 5 ml of 5 M Na perchlorate vision into five functional segments (3, 4). The deletion of two at 65°C for 40 min. DNA was bound to hydroxylapatite (Ha- small segments at opposite ends of the region completely patite C, Clarkson, Williamsport, PA), washed three times with eliminates inversion. Another segment which occupies about 5 mM NaPO4 (pH 7.4), and then eluted with 0.5 M NaPO4 (pH two-thirds of the inversion region controls the production of a 7.4). The eluent was dialyzed extensively against 10 mM Tris, factor which acts in trans to promote inversion. This has been pH 7.8/0.1 mM EDTA; then the DNA was precipitated with called the hin gene because its product is required for the in- ethanol. Alternatively, slices of acrylamide containing DNA version of the H2 controlling region. Finally, two other regions fragments were finely ground between layers of Parafilm. The map within the invertible segment. One is characterized by the pieces were then added to 1 ml of elution buffer (0.5 M NH4 observation that insertions and deletions generated by a 5-ki- acetate/0.01 M Mg acetate/0.1% NaDodSO4/0.1 mM EDTA) lobase within this region cause a 5-fold and incubated at 650C for at least 12 hr. The slurry was then decrease in the frequency of inversion. The second region filtered through glass wool and the DNA in the filtrate was corresponds to a promoter-like function; deletion of this region ethanol precipitated. Yields ranged from 75% to 95% for does not significantly affect inversion but eliminates formation fragments 700-10 bp long. of the H2 gene product. Labeling of DNA Fragments and Nucleotide Sequence In order to delineate precisely the genetic functions and to Determination. Labeling of 5' ends with polynucleotide kinase understand the molecular basis of the control of H2 gene ex- and [h-32P]ATP after bacterial alkaline phosphatase treatment pression and of the inversion process, it was necessary to de- was done as described by Maxam and Gilbert (9). 3'-End la- termine the nucleotide sequences that correspond to each of beling of DNA fragments with DNA polymerase large frag- these functions. In this paper, we present the nucleotide se- ment and [a-32P]dNTPs was also performed by the procedure quence of the inversion region and correlate parts of the se- of Maxam and Gilbert (9). Single end-labeled fragments were quence with these genetically defined functions. obtained by either secondary cleavage with a restriction enzyme or by strand separation. The strand separation procedure in- MATERIALS AND METHODS volved resuspending the double-strand DNA pellet in dimethyl Enzymes and Radioactive Isotopes. Alu I, Hae II, Hha I, sulfoxide/half-strength TBE, 60:40 (vol/vol). The solution was HinfI, Hpa II, and Taq I were purchased from New England boiled for 5 min and then added immediately to a 5% nonde- from naturing acrylamide gel. The gel and running buffer were BioLabs; Hpa II and Alu I were obtained Boehringer prepared in half-strength TBE. All sequence determination was done by the method of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Maxam and Gilbert (9, 10). vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: bp, base pair(s). 4196 Genetics: Zieg and Simon Proc. Nati. Acad. Sci. USA 77 (1980) 4197

Amino Acid Analysis of H2 Flagellin. H2-enx flagellin was Fig. 3 illustrates a possible mechanism for the inversion isolated from cells harboring pJZ1OO (11) as described (12). The process involving these repeated sequences. A homologous re- purified protein was subjected to solid-phase amino acid combination event between the inverted repeat sequences could analysis by the thioacetylation stepwise degradation procedure result in an inversion of the DNA segment. By using restriction (13) by R. Doolittle (Department of Chemistry, University of endonuclease analysis, we have found that pJZ143 and pJZ121 California at San Diego, La Jolla, CA). retain the ability to invert (unpublished observations). Thus, sequences on the arm segments are not required for the inver- RESULTS AND DISCUSSION sion event. It is possible that the 14-bp inverted repeats alone Strategy for Sequence Determination. The restriction en- are required as sites for the inversion event. donuclease cleavage maps of plasmids pJZ121 and pJZ143 that Analysis of the 14-bp inverted repeat sequence revealed that were used to determine the nucleotide sequence are shown in it shares homology with DNA regions in other systems that are Fig. 1. These plasmids are in vdvo recombinants derived from involved in recombination and transposition. For example, the populations of the plasmid pJZllO, and their construction has A att core sequence resembles this sequence, as do regions on been described (2, 11). pJZll0 carries a fragment derived from the A P and A P' arms (14, 15). Two regions within the TnlO the Salmonella which includes the inversion region inverted repeat are also strikingly similar to the 14-bp repeat and flanking sequences. It is capable of inverting, and it is (16). Also, we find homology between the 14-bp sequence and shown in Fig. 1 in the orientation that leads to the expression a DNA region just outside the inverted repeat within the Tn3 of H2 gene function (i.e., the H2 "on" configuration). Plasmid transposon (17). Table 1 shows the extent of these sequence pJZ121 contains the entire inversion region with the H2 en- homologies. It is possible that the phase-variation inverted re- coding arm on both sides. Plasmid pJZ143 also contains the peat sequences may have evolved from some general site which entire inversion region flanked by the opposite or non-H2 en- takes part in various specific recombinational events. A simple coding arm. Fig. 1 also illustrates the strategy for determining recombination event within the inverted repeats can account the nucleotide sequence from the endonuclease cleavage map. for inversion in the phase variation system, as illustrated in Fig. The nucleotide sequence of the inversion region and proximal 3. arm regions is shown in Fig. 2. The G-loop region of bacteriophage Mu and the C loop of Determination of the Ends of the Inversion Region. It was bacteriophage P1 are genetic elements capable of inversion (18). possible to determine the extent of the inversion region by In addition, it is known that the orientation of the G loop reg- comparing sequences of fragments that contained the ends of ulates specific gene expression in Mu. It is possible that the in- the inversion region from both pJZ121 and pJZ143. For ex- version events in Mu and P1 are similar to the inversion event ample, by comparing the sequences of fragments a and b shown in phase variation. Thus, there may be some sequence homology in Fig. 1, we were able to locate the left end of the inversion between the inverted repeat regions in the three systems. It will region precisely at the point of diversion between the two se- be possible to compare them when the nucleotide sequence of quences. Likewise, comparison of the sequences of fragments the G-loop or C-loop region is determined. c and d allowed us to locate the right end of the inversion region. 2 Gene Promoter. Genetic evidence has suggested that Furthermore, when we compared these sequences with each transcription of the H2 gene is in the left-to-right direction as other, we discovered that the ends of the inversion region were drawn in the figures and that the promoter region regulating bounded by a 14-bp inverted , T-T-A-T- the expression of the gene is located within the inversion region. C-A-A-A-A-A-C-C-T-T. The inverted repeats occurred at po- Inspection of the nucleotide sequence for possible ATG se- sitions 1-14 and 982-995, defining a 995-bp invertible region. quences on the 5'-to-3' strand corresponding to the translational The position of these repeated sequences suggests that they start signal on the mRNA reveals that only one ATG was not correspond to the cis-acting genetic elements that map at the followed by an in-phase termination codon. This ATG codon ends of the inversion region and are required for the inversion is 16 bp outside the inversion region, at position 1012. It is event. preceded, at position 1002, by the sequence G-G-A-A which

H2 GENE pJZI1O -NC pJZ121 FIG. 1. Strategy for nucleotide sequence de- Hoe m termination. Plasmid pJZ110 is shown in the ori- Hpa U - -~ D TT~ entation that corresponds to H2 gene expres- sion-i.e., the H2 "on" orientation. The same ori- AluI entation is shown for the other plasmids. Plasmids pJZ121 and pJZ143 are derivatives of pJZ110. En- Toq I donuclease cleavage maps ofpJZ121 and pJZ143 are Hoe given. Arrowheads pointing downward reflect cleavage sites on pJZ121, and those pointing upward Hinf I * indicate sites on pJZ143. Solid, horizontal arrows above and below the plasmids indicate 32P-5'-end- HhaI labeling at the corresponding restriction site and t_ 5'-to-3' sequence determination. The dashed arrows indicate 32P-3'-end-labeling and 3'-to-5' sequence determination. All sequence information was con- ~~~~~-. pJZI43 firmed on both strands. Fragments a, b, c, and d were < ~ ~ 4--_.+4- > < < used to locate the ends of the inversion region (see text). 4198 Genetics: Zieg and Simon Proc. Natl. Acad. Sci. USA 77 (1980)

-20 -10 -1+1 10 20 30 40 50 60 CGATTTATTGGTTCTTGAAAACCpG-GTTGATlU CATCCTCCATGAGAAAAGCGACTAAATTCTTCCTTATCTGATGTA

70 80 90 100 110 120 130 AGGAGAAAATC ATG GCT ACT ATT GGG TAT ATT CGG GTG TCA ACA AUT GAC CM MT ATC GAT TTA CAG Met Ala Thr Ile Gly Tyr Ile Arg Val Ser Thr Ile Asp Gin Asn Ile Asp Leu Gln 140 150 160 170 180 190

CGT MT GCG CTT ACT AGT GCA MT TGT GAC CGC ATT T GM GAC CGT ATC AGT GGC MG ATT GCA Arg Asn Ala Leu Thr Ser Ala Asn Cys Asp Arg Ile Phe Glu Asp Arg Ile Ser Gly Lys Ile Ala 200 210 220 230 240 250 260

MC CGC CCC GGC CTG AAA CGG GCG TTA AAG TAT GTA MT AM GGC GAT ACT CTT GTC GTC TSG AAA Asn Arg Pro Gly Leu Lys Arg Ala Leu Lys Tyr Val Asn Lys Gly Asp Thr Leu Val Val Trp Lys 270 280 290 300 310 320 330 TTA GAC AGA CTG GGC CGT AGC GTG AMA MT CTG GTG GCG TTA ATA TCA GAA TTA CAT GM CGT GSA Leu Asp Arg Leu Gly Arg Ser Val Lys Asn Leu Val Ala Leu Ile Ser Giu Leu His Giu Arg Gly 340 350 360 370 380 390 GCT CAC TTC CAT TCT TTA ACC SIAT AGT ATT GAT ACC AGT AGC GCG ATG GGG CGA TTC T m CAT Ala His Phe His Ser Leu Thr Asp Ser Ile Asp Thr Ser Ser Ala Met Gly Arg Phe Phe Phe His 400 410 420 430 440 450 460 GTA ATG TCA GCA CTG GCC GAG ATG GAG CGA GM TTA ATC GTC GAG CGA ACC CTU GCC GGA CTG GCT Val Met Ser Ala Leu Ala Giu Met Glu Arg Glu Leu Ile Val Glu Arg Thr Leu Ala Gly Leu Ala 470 480 490 500 510 520 GCC GCC AGA GCG CM GMA CGA CTG SGA GGG CGC CCT CGG GCG ATC MC AM CAT GAA GAG GM CAG Ala Ala Arg Ala Gin Gly Arg Leu Gly Gly Arg Pro Arg Ala Ile Asn Lys His Glu Gln Glu Gln 530 540 550 560 570 580 590 ATT AGT CGG CTA TTA GAG AM GGC CAT CCT CGG CAG CAA TTA GCT ATT ATT UTT GGT ATT GGC GTA Ile Ser Arg Leu Leu Glu Lys Gly His Pro Arg Gln Gln Leu Ala Ile Ile Phe Gly Ile Gly Val 600 610 620 630 640 650 660 TCC ACC TTA TAC AGA TAC TTT CCG GCA AGC AGT ATA AAA AM CGA ATG MT TM ATAAATCACMC FIG. 2. Nucleotidesequenceofthe Ser Thr Leu Tyr Arg Tyr Phe Pro Ala Ser Ser Ile Lys Lys Arg Met Asn END inversion region. The 5'-to-3' strand of 670 680 690 700 710 720 730 740 750 the nucleotide sequence of the inversion AGGATGGATATMCATTTTTGTTMTACAGGCGTATGGCATAAATAMACCGAAAGGGTATACAAAAAAGACAGCATCTMTTAMAAG region and proximal arm sequences are shown. The inversion region which ex- +1 760 770 780 790 800 810 820 830 tends from to +995 includes flanking inverted repeat sequences (shown AGAAAAAATTCMCGTATTAACATATATAGTGTMCGCGCTCACGATMGGCCTATGTTACATCCAGCTATAGACGACATCGCTCM within boxes). The underlined se- quences between nucleotides 40 and 53 840 850 860 870 880 890 900 910 920 and between 1000 and 1012 constitute AACACTACCAGACACAGTATTCACCTGGAMGGCTTTTTMTCMAAATGTTAGATGTMGCAATTACGGACAGAMAAATAGTAMG entation and direct repeatsHH2in the "off" orientation. Amino acid sequences 930 940 950 960 970 980 990 1000 1010 predicted by the nucleotide sequence UTTATGCCTCMGTGTCGATMCCTGGATGACACAGGTMGCCTGGCATMACATTW*ITAWXUTK!I'tAMA/4GGAAAATTTT are shown for the hin and H2 structural genes. The hin gene is located between nucleotides 76 and 648. The H2 gene 1020 1030 1040 begins at position 1012 and continues ATG GCA CM GTA ATC MC ACT MC AGT CTG rightward beyond the end of the nucle- Met Ala Gin Val Ile Asn Thr Asn Ser Leu otide sequence shown. resembles the sequence proposed for ribosome binding (19). not appear to be any further NH2-terminal processing required The phase of the ATG sequence remains open for the following for the formation of the flagellar filament. 114 bp whose sequence hasbeen determined. All other reading There are only 16 bp between the right-hand inverted repeat frames in this region contain several termination signals. sequence and the beginning of the H2 gene. Part of this region In addition, direct determination of the NH2-terminal amino encodes the putative ribosome binding site and none of the acid sequence of H2-enx flagellin agrees with the amino acids sequences in this region corresponds to known sequences that predicted by the nucleotide sequence. The results of the analysis are involved in transcription initiation. were that the NH2-terminal amino acids are Ala-Glx-Val-Ile(or It seems reasonable to conclude, therefore, that initiation of Tyr)-Asx-Thr(or Ser)-Asx. The amino acid sequence predicted transcription occurs within the inversion region. This is also from the nucleotide sequence agrees with this order found by consistent with all of the previous genetic and physiological amino acid analysis. These data are consistent with the con- evidence. Sequences resembling prototypic sequences for clusion that the coding region of the H2 gene begins at nucle- promoter regions can be found within the inversion region. otide 1012. It is interesting that although the flagellin molecule Sequence T-A-A-C-A-T-T at positions 973-979 resembles the is transported to the tip of the growing flagellar filament during consensus sequence that precedes transcription initiation-i.e., the assembly of bacterial flagella (20, 21) there is no apparent the Pribnow sequence (22, 23). It is accompanied by a region signal-like sequence at the beginning of the gene. Furthermore, resembling the prototypic RNA polymerase recognition site at aside from the removal of NH2-terminal methionine, there does positions 950-961 (24). Other sequences that show homology Genetics: Zieg and Simon Proc. Natl. Acad. Sci. USA 77 (1980) 4199 51 TTGAAUAC >AGTTTTTGAT..AGCAATCCT TAKAT TG QiTA A fACCTT CA AA AG GA * AACTTTTGrT1rCCAAAAAATCTATTTCGTTAGGA AT TGTA AC qAMATAS TT TG GA GTTT TC CT , H2 'or

TA AT TA £4 TA AT c&6 C AT TA ATtl

SCTA TA AT A AT TTGAAA AACTTTTACrC6S 6 tTTTCCT

H2 'off' TTGAAAAC cGSGTTTTTGATA CAATGTTA.ATGATTGCATfTATCAAiCCJC CAAAMSA6* AACT TT TSG TC CAAAA CT AT GTTACAAT TCCTAACSAAATAGTTTT TO TTTTCCT FIG. 3. Model for inversion. A homologous recombination event anywhere within the 14-bp inverted repeat sequences would result in the inversion of the region between the repeats. Thus, the H2 "on" orientation could be converted to the H2 "off' orientation and vice versa. The 14-bp inverted repeat sequences are shown within the boxes. with the Pribnow sequence appear at positions 944-950 (T- suggestion has been made by Ikeda and coworkers (25, 26) and A-A-C-C-T-G) and 776-782 (T-A-T-A-G-T-G). However, a by Ohtsubo and Ohtsubo (27) that transcription may play a role corresponding region for RNA polymerase recognition for ei- in enhancing specific recombinational events. ther of these Pribnow sequences is not apparent. There are no it is known that transcription of the H2 gene is regulated by apparent promoter-like sequences on the 3'-to-5' strand that various trans-acting elements (28). It is not apparent which could initiate transcription in the H2 "off" orientation. parts of the sequence preceding the H2 gene, if any, might act It is clear from the position of the translational start codon as a site for binding a regulatory protein. There is an open phase that transcription begins within the inversion region and that that would allow for the synthesis of a polypeptide of 56 amino at least a part of the inverted repeat is transcribed. Furthermore, acids encoded by the nucleotides in positions 805-975. There transcription should proceed independently of the orientation is no evidence, however, to indicate that this sequence is tran-. of the inversion region. In fact, we have presented evidence that scribed or translated. A striking feature of the sequence between suggests that transcription operates in the opposite direction 648 and 981 is the presence of regions of high A+T content, a when the inversion region is in the "off" configuration (4). The common characteristic of sequences upstream of promoters (29, 30). Fig. 2 shows the distribution of these A+T-rich regions with Table 1. DNA sequence homology between the 14-bp inverted respect to the position of the translational start of the H2 gene. repeat and regions of genetic elements involved in Regions between nucleotides 648 and 783 and between 863 and transposition or recombination 928 have A+T contents of approximately 72%. They alternate Source Sequence with regions that have A+T compositions of approximately 14-bp inverted 50%. These regions may be important in enhancing promoter repeat 5'-T-T-A-T-C-A-A-A-A-A-C-C-T-T-3' activity and thus regulating H2 gene expression. Tn3 4889 T-T-A-T-C-A-A-A-A-A-G-G-A-T 4902 hin Gene. Genetic studies have indicated that a region within X att core 7 T-T-AjT-AjA-A-A-A-AjG-C-T-G -9 the inversion segment is required to encode a product necessary G T A for the inversion event. It was proposed that a gene, hin, was X P arm -64 T-T-A-TjC-A-A-A-A-T-C-T-A-A -50 located within the inversion region and occupied approximately G two-thirds of the sequences corresponding to the region. The X P'arm 15 T-T-A-T-A-A-A-A-A-A-G-C-A-T 28 nucleotide sequence provides direct evidence for the existence TnlO a T-T-A-T-C-A-A-A-A-T-C-A-T-T of such a gene. An open translational frame beginning with the TnlO b T-TjA-T-A-A-A-A-A-T-C-A-T-T ATG at position 76 proceeds on the 5'-to-3' strand and termi- G nates at position 648 with a TAA sequence. Examination of the Bases that differ from the 14-bp inverted repeat sequence are shown DNA sequences of both strands that correspond to the inversion boldface. Inserted bases are indicated by an arrow and the corre- region indicates that there are no other open frames that could sponding nucleotide is written beneath. References for each of the lead to the synthesis of a protein composed of more than 70 systems cited are given in the text. amino acids in the region defined genetically. This ATG is 4200 Genetics: Zieg and Simon Proc. Natl. Acad. Sci. USA 77 (1980) preceded by the sequence G-G-A-G which could function to phase variation system and the more general question of the evolution provide a ribosome binding site. Furthermore, the sequence of controlling elements. at positions 40-46, T-A-A-A-A-T-T, similar to the Pribnow sequence, may indicate that the initiation of transcription occurs The authors thank Dr. R. Doolittle for the amino acid sequence within the inversion region. At positions 6-17 there is the se- analysis and for helpful discussions. We thank Marcia Hilmen for ex- quence T-T-T-T-G-A-T-A-A-A-G-C which resembles the cellent technical assistance and P. Deininger, T. Friedman, and D. consensus sequence for the RNA Stalker for introducing us to the techniques of DNA sequencing. This polymerase recognition site. work was supported by grants from the National Science Foundation This site would overlap with the 14-bp inverted repeat and and the American Cancer Society. J.Z. is a U.S. Public Health Service would exist in identically the same position in either orientation trainee. of the inversion region. It is also possible that the promoter of transcription for the 1. Zieg, J., Silverman, M., Hilmen, M. & Simon, M. (1977) Science synthesis of the hin gene product could occur outside the in- 196, 170-172. version region. In this arrangement, 2. Zieg, J., Hilmen, M. & Simon, M. (1978) Cell 15,237-244. promoters would exist on 3. Silverman, M. & Simon, M. (1980) Cell 19,845-854. both the H2 and non-H2 encoding arms. The relative strengths 4. Silverman, M., Zieg, J., Hilmen, M. & Simon, M. (1979) Proc. of these promoters may direct different levels of hin product Natl. Acad. Sci. USA 76,391-395. in the H2 "on" and "off" situations. Such a system could ac- 5. Greene, P. J., Heyneker, H. L., Bolivar, F., Rodriguez, R. L., count for the bias previously observed in the inversion fre- Betlach, M. C., Corarrubias, A. A., Backman, K., Russel, D. J., quency in the two directions (1). Other possible regulatory se- Tait, R. & Boyer, H. W. (1968) Nucleic Acids Res. 5, 2373- quences occur between nucleotides 40 and 53 and between 1000 2380. and 1012. They constitute an inverted repeat in the H2 "on" 6. Johnson, R. A. & Walseth, T. F. (1979) Adv. Cyclic Nucleotide configuration and a direct repeat in the H2 "off" configuration. Res. 10, 135-167. These sequences 7. Bazaral, M. & Helinski, D. (1968) J. Mol. Biol. 36, 185-194. may play a role in regulating the expression 8. Bolivar, F., Rodriguez, R., Betlach, M. & Boyer, H. (1977) Gene of the adjacent hin and H2 genes. 2,75-93. Further confirmation of the assignment of the hin gene to 9. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, this region comes from preliminary results of studies using in 499-560. vitro transcription and translation (unpublished data). In both 10. Maxam, A. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, of these systems, the presence of the invertible region leads to 560-564. the synthesis of a 19,000 molecular weight polypeptide. In- 11. Zieg, J., Silverman, M., Hilmen, M. & Simon, M. (1977) in Mo- terruption of the sequences within the inversion region by re- lecular Approaches to Eukaryotic Genetic Systens, ICN-UCLA striction endonuclease Symposia 8, eds. Wilcox, J., Abelson, J. & Fox, C. (Academic, New digestion eliminates the synthesis of this York), pp. 25-35. polypeptide. Furthermore, both pJZ143 and pJZ121 were found 12. Silverman, M. & Simon, M. (1972) J. Bacteriol. 112,986-993. to stimulate the synthesis of this polypeptide, indicating that 13. Doolittle, L. R., Mross, G. A., Fothergill, L. A. & Doolittle, R. F. the entire coding region for hin is located within the inversion (1977) Anal. Biochem. 78,491-505. region. It is interesting to note that the gin function of Mu has 14. Landy, A. & Ross, W. (1977) Science 196, 1147-1160. been correlated with the appearance of a 19,000 molecular 15. Landy, A., Hoesr, R., Bidwell, K. & Ross, W. (1978) Cold Spring weight polypeptide (31). Harbor Symp. Quant. Biol. 43, 1089-1097. The results of the nucleic acid sequence in terms of the as- 16. Kleckner, N. (1979) Cell 16,711-720. signment of specific sequences to genetically defined functions, 17. Heffron, F., McCarthy, B. J., Ohtsubo, H. & Ohtsubo, E. (1979) can Cell 18, 1153-1163. be summarized as follows. 18. Chow, L. & Bukhari, A. (1976) Virology 74,242-248. (i) Sequences prior to nucleotide 76 appear to be responsible 19. Shine, J. & Dalgarno, L. (1974) Proc. Natl. Acad. Sci. USA 71, for transcriptional initiation of the hin gene. 1342-1346. (ii) Sequences between positions 76 and 648 code for the 20. Emerson, S., Tokuyasu, K. & Simon, M. (1970) Science 169, structural gene that specifies the hin protein. 190-192. (iii) Sequences located between 649 and 1011 correspond to 21. Iino, T. (1969) J. Gen. Microbiol. 56,227-239. the "silent" region, the site of transcriptional initiation, and 22. Pribnow, D. (1975) Proc. Natl. Acad. Sci. USA 72,784-789. perhaps the control region for H2 gene expression. 23. Pribnow, D. (1975) J. Mol. Biol. 99,419-443. 24. Seeburg, P. H., Nusslein, C. & Schaller, H. (1977) Eur. J. Bio- (iv) The sequence beyond nucleotide 1012 encodes the H2 chem. 74, 107-113. structural gene. 25. Ikeda, H. & Kobayashi, I. (1977) Proc. Natl. Acad. Sci. USA 74, (v) The 14-bp inverted repeat sequences lie between nucle- 3932-3936. otides 1 and 14 and between 982 and 995. Their sequence ho- 26. Ikeda, H. & Matsumoto, T. (1979) Proc. Natl. Acad. Sci. USA 76, mology with other systems suggests that they may be important 4571-4575. as general signals for site-specific recombination events. 27. Ohtsubo, H. & Ohtsubo, E. (1978) Proc. Natl. Acad. Sci. USA 75, 615-619. Note Added in Proof. Computergenerated comparison of the amino 28. Silverman, M. & Simon, M. (1974) J. Bacteriol. 120,1196-1203. acid sequences of the hin protein and the tnpR protein which is a 29. Rosenberg, M. & Court, D. (1979) Annu. Rev. Genet. 13, component of the Tn3 transposon (17) reveals 33% identity. These two 319-353. proteins therefore have a common ancestor. They probably also have 30. Nakamura, K. & Inouye, M. (1979) Cell 18, 1109-1117. similar functions-i.e., to act as repressors of the formation of specific 31. Kamp, D., Chow, L. T., Broker, T. R., Kwol, D., Zipser, D. & proteins and to mediate site-specific homologous recombination. This Kahmann, R. (1978) Cold Spring Harbor Symp. Quant. Biol. 43, similarity may provide a clue to understanding the evolution of the 1159-1167.