Vol. 168, No. 2JOURNAL OF BACTERIOLOGY, Nov. 1986, P. 471-4780021-9193/86/110471-08$02.00/0Copyright © 1986, American Society for Microbiology

MINIREVIEW

Molecular Biology of ArchaebacteriaPATRICK P. DENNIS

Department ofBiochemistry, The University of British Columbia, Vancouver, British Columbia V6T I W5, Canada

Why has there been so much recent interest in ar-chaebacteria? The answer to this question is related to ourchanging perception of early cellular evolution and thepotentially important information that archaebacteria canprovide to us about this process. Before 1977, most biolo-gists visualized evolution as a linear progression from simpleprocaryotic eubacteria to more complex, multicellular eu-caryotic organisms. Implicit in this scheme were the con-cepts (i) that the primary genomic lineage of the eucaryoticcell, the nuclear genome, evolved in some complex andunknown manner from a eubacterial-like nucleoid and (ii)that the molecular features of genome organization, regula-tion, and expression in eucaryotes were more sophisticatedand advanced than in procaryotes.Using 16S (small ribosome subunit) RNA sequences as a

phylogenetic index, Woese and his collaborators have pro-vided convincing evidence suggesting that evolution has notbeen a progression from simple procaryotes to the morecomplex eucaryotes (19, 36, 46, 47). Instead, they haveargued that organisms can be grouped into three distinctgenomic lineages or kingdoms: eubacteria, eucaryotes, andthe new group, archaebacteria. No one of these threeappears to be more ancient from an evolutionary point ofview than either of the other two. That is, each of the threekingdoms appears to have arisen independently some 3.5 x109 years ago from a common but ill-defined ancestral formdesignated the progenote.

Eucaryotic cells contain a primary nuclear genome as wellas mitochondrial and (in plants) chloroplast genomes. Thenuclear genome is believed to have evolved from theprogenote giving rise to an evolutionary intermediate, theurkaryotes. Endosymbiotic relationships between theurkaryote and purple sulfur bacteria and between theurkaryote and cyanobacteria account for the introduction ofmitochondrial and chloroplast genomes (organelles), respec-tively, and the gradual evolution towards the modem eu-caryotic cell. These proposed nuclear, mitochondrial andchloroplast genome lineages are supported by 16S rRNAsequence data.What was the state ofgenomic organization and regulation

in this progenote, and how exactly was this primitive state ofbiological organization achieved? It has been pointed outthat many of the molecular features of the eucaryoticgenome previously imagined to be advanced, such as multi-ple chromosomes with reiterated replication origins, inter-ruptions in coding sequences, monocistronic mRNA, etc.,may well have been characteristic features of the progenotestate (for recent reviews, see references 10 and 15). Incontrast, certain eubacterial features, such as a unitarygenome with a unique replication origin, uninterrupted cod-ing sequences, polycistronic mRNAs, etc., probably repre-

sent substantial refinements from the progenote state thatwere required to achieve rapid and efficient growth in highlycompetitive environments. The present states of genomeorganization and regulation in eubacteria, archaebacteria,and eucaryotes are thus independent adaptations to theproblems of efficiency and accuracy of information transfer.The intellectual challenge of studying archaebacteria is thatthey provide us with a vitally needed but almost totallyunexplored third perspective from which to view the pro-cesses of early cellular evolution.What is the current state of our knowledge relating to the

molecular biology of archaebacteria? Although archae-bacteria exhibit a procaryotic cell structure and organiza-tion, they are quite distinct from eubacteria and in factpossess some features reminiscent of eucaryotes. Some ofthe major distinguishing and unifying characteristics include(i) the sequences and structures of rRNAs, ribosomal pro-teins, and tRNAs (18, 21, 22, 33, 48); (ii) the presence ofintervening sequences in genes encoding rRNA, tRNA, andpossibly also proteins (7, 8, 25, 28, 31); (iii) an RNApolymerase subunit complexity reminiscent of and immuno-logically related to eucaryotic RNA polymerase (20, 50); (iv)a translation apparatus that utilizes 16S, 23S, and 5S rRNAs,that is sensitive to some eucaryotic ribosome-targeted anti-biotics and to ADP ribosylation by diphtheria toxin, and thatinitiates protein synthesis with methionine rather thanformyl-methionine; and (v) cell envelopes that lackpeptidylglycan and contain ether- rather than ester-linkedlipids (14, 26, 30). (Readers wishing more details on thesetopics are referred to recently published collections of re-view articles on archaebacteria [27, 32, 49]. One of thesevolumes [27] is a collection of papers given at an archaebac-teria workshop in Munich in June 1985.)

Within the archaebacterial kingdom there is a clear anddeep evolutionary division between the sulfur-dependentthermoacidophiles and the methanogens and halophiles (36,48). This division is exemplified by many differing charac-teristics, including the morphological structures on the largeribosome subunits as visualized in the electron microscope(although there is disagreement on the extent and signifi-cance of these structural differences; see references 29 and43) and by the protein content of ribosomal particles.Ribosomes from the halophilic branch in general have aeubacterialike protein content with few or no proteinsgreater than 30,000 daltons in mass, whereas ribosomes fromthe sulfur-dependent thermoacidophilic branch have a muchhigher eucaryotelike protein content with many proteinsgreater than 30,000 daltons in mass (3). The amino acidsequences of at least some archaebacterial ribosomal pro-teins exhibit more amino acid sequence homology with theireucaryotic equivalents than with their eubacterial equiva-

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lents (33). However, amino acid sequence homology, like16S rRNA homology, is greater for member species withinthe archaebacterial kingdom than for member species be-tween kingdoms.

Organization, transcription, and processing of stable RNAgenes. The ribosomal RNA genes from more than 10 differentarchaebacterial species have now been cloned and eitherpartially or completely sequenced (13, 48). The primarysequence data are being used to support the evolutionaryuniqueness of archaebacteria and to establish phylogeneticrelationships within the kingdom. In general, the copy num-ber for rRNA genes in archaebacterial species ranges fromone to four copies per genome. The gene order is 5'-16S-23S-5S-3' except in the sulfur-dependent thermoacidophiles,in which the 5S gene is not linked to the 16S and 23S genes.An alanine tRNA gene is present within the 16S-23Sintergenic space in several halophilic and methanogenicspecies, and in halophilic species a cysteine tRNA gene isfound distal to the 5S gene. When the rRNA genes aremulticopy, the tRNA gene sequences are not always presentin all copies. In the sulfur-dependent thermoacidophilicspecies Desulfurococcus mobilis, the single-copy 23S genecontains a 622-base-pair (bp) intron in domain IV; this intronhas features of both class I (rRNA) and III (tRNA) introns ofeucaryotes (28, 31).

It has been shown that the single-copy rRNA gene clusterin Halobacterium cutirubrum is contained in a 6-kilobase(kb) region of genomic DNA; this entire region has now beensequenced (24; A. Mankin and V. Kagramanova, Mol. Gen.Genet., in press; K. Roy, personal communication; H.cutirubrum, H. halobium, and H. Salanarium are closelyrelated organisms and probably represent a single species).The nucleotide sequence analysis revealed several interest-ing features including (i) the presence of long, nearly perfectinverted-repeat sequences surrounding the 16S and 23SrRNA genes; (ii) the presence of an alanine tRNA gene in the16S-23S intergenic space and a cysteine tRNA gene distal tothe 5S gene; (iii) a series of three highly conserved and twoless highly conserved bipartite direct-repeat sequences in the900-bp 5' flanking region; (iv) a short inverted repeat fol-lowed by a T5GCAGT4 sequence between the 5S and cyste-ine tRNA genes; and (v) an A+T-rich sequence preceded bya G+C-rich sequence located distal to the cysteine tRNAgene (24).

Nuclease Si protection of end-labeled DNA fragments bytotal in vivo RNA has been used to demonstrate cotranscrip-tion of this rRNA gene cluster and to examine excision andprocessing of the primary transcription product (4, 12; Fig.1). The long, nearly perfect inverted-repeat sequences sur-rounding the 16S and 23S rRNA genes are used by anRNaseIII-like activity to excise precursor 16S and 23SRNAs from the primary transcript. Similar inverted repeatssurrounding the 16S and 23S genes have been observed inother archaebacterial species including methanogens andsulfur-dependent thermoacidophiles (31, 45; J. Kjelms,R. A. Garett, and W. Ansorge, Syst. Appl. Microbiol., inpress) (Fig. 2). The precursor rRNAs from the primarytranscript are excised by endonuclease cutting on oppositesides of the helical structure at the bulge (unpaired bases)sequences.

Additional conserved sequences in the regions flanking themature 16S, 23S, and 5S genes may also be used forsubsequent rRNA maturation and assembly (31; Mankin andKagramanova, in press). The alanine and cysteine tRNAgene sequences are cotranscribed as part of the primarytranscript but are processed with reduced efficiency relative

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MINIREVIEW 473

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FIG. 2. Complementary processing sequences used to excise precursor 16S and 23S rRNAs from a long primary transcript. Inverted-repeat sequences surrounding the 16S (A) and 23S (B) rRNA genes are a conserved feature in archaebacteria (24, 31, 45; Kjelms et al., inpress). These sequences presumably form stable duplex structures; an endonuclease activity introduces a break at each side of the helix withinor adjacent to the unpaired bases (bulges) to liberate precursor 16S and 23S sequences. For each duplex illustrated, the top strand 5' -* 3'

(left to right) precedes the coding sequence, and the bottom strand follows the coding sequence. Only the part of the duplex in the vicinityof the processing sites is illustrated.

to the rRNA sequences (4, 5). Endonuclease incisions intothe middle of the tRNA sequences on the primary transcriptshave been detected; these incisions possibly represent earlysteps in an alternate processing pathway in which the tRNAsequences are not preserved.

Characteristic features of archaebacterial tRNAs based onthe sequences of 41 separate tRNAs from Halobacteriumvolcanii have been described (21, 22). Genes encodingtRNAs have been characterized from a number ofarchaebacterial species (8, 25, 44). None of the genes exam-ined encodes the CCA 3' termini found on all mature tRNAs,and the genes encoding tRNAs can occur as singlets, inclusters, or associated with rRNA genes. The serine andleucine tRNA genes from the sulfur-dependent thermo-acidophile Sulfolobus solfataricus and the tryptophan tRNAgenes from H. volcanii and other halophilic species containintervening sequences at the standard eucaryotic positionadjacent to the tRNA anticodon (8, 25). The serine tRNAand leucine tRNA introns are relatively short (25 and 15nucleotides, respectively), whereas the tryptophan tRNAintron is much longer (108 nucleotides). Northern hybridiza-tion with total H. volcanii in vivo RNA resulted in theidentification of the following tryptophan tRNA containingsequences: (i) a 184-nucleotide-long precursor transcriptcontaining the tryptophan tRNA and the intron sequence, (ii)a 108-nucleotide-long intron, and (iii) a 76-nucleotide-longmature tRNA. The tryptophan tRNA gene is unique single-copy DNA and corresponds in sequence to the trp tRNA.The mechanisms for tRNA processing and intron excisionare currently under investigation.

Organization and mRNA translation of protein-encoding

genes. Genomic DNA from Methanococcus voltae,Methanococcus vannielii, and Methanobrevibacter smithiicontains open reading frames that are separated by regionsof very high A+T content (2, 6). These spacers appear to beunique single-copy DNA and are typically about 200 bp inlength with an A+T content of about 80%. Genomic frag-ments from these organisms have been cloned into Esche-richia coli and are capable of complementing hisA, argG,purE, and proC mutations (6, 23). The expression of thesemethanogenic sequences in E. coli apparently results fromtranscription initiation events at sequehces within the A+T-rich spacers that fortuitously resemble eubacterial promoterconsensus sequences. Both mono- and polycistronic genearrangements appear possible. The ATG translation itlitia-tion codons are preceded by well-defined purine-rich se-quences that are complementary to the 3' end of the 16SrRNA; these same translation initiation signals are probablyalso recognized with reasonable efficiency by E. coliribosomes (41; Fig. 3).The hisA gene of M. voltae has been shown to be under

transcriptional regulation (42). Addition of the histidineanalog aminotriazole results in at least a twofold increase inhisA (but not argG) mRNA. In the absence of the inhibitor,the transcript size is about 1.5 kb, and the transcript iscapable of encoding the hisA protein and possibly a secondsmall protein encoded by a downstream open reading frame.In the presence of inhibitor, the major hisA transcript is 9 to10 kb in size. Whether this represents a dual system fortranscription of the hisA gene or mRNA processing has notbeen determined.

Perhaps the best-studied example of an archaebacterial

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474 MINIREVIEW

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5' ...TT T R R 6 G T G R T T R T R T R T 6 6 R 6 ...

FIG. 3. Comparison of translation initiation site sequences with the sequence at the 3' end of 16S rRNA. The DNA sequences precedingthe ATG translation initiation codon of several archaebacterial genes are aligned with the sequences at the 3' ends of the corresponding 16SrRNAs. Complementary bases are indicated (0). The rpo sequence is one of three possible initiation codons for an RNA polymerase subunitgene. The hisA and purE sequences are from the archaebacterial genes that complement the corresponding mutants in E. coli. The H.cutirubrum (halobium) L12e translation initiation site is at least 1,500 nucleotides from the transcription start site. Three other noninitiationATG codons from within the LlOe open reading frame (non init. A, B, and C) exhibit better complementarity to the 3' end of 16S rRNA thanthe bona fide L12e initiation site. The sequences in front of brp and bop genes of H. halobium also exhibit complementarity to the 3' end of16 rRNA, but these sequences are not part of the primary rRNA transcripts since initiation of transcription occurs immediately in front ofthe ATG translation initiation codons. The L12e gene from S. sulfataricus also exhibits poor complementarity with its 16S rRNA.

gene is the bop gene from H. halobium (1, 11, 39). This geneencodes bacterio-opsin, a transmembrane protein of 246amino acids, which carries out a light-dependent vectoralproton translocation, thus generating a transmembrane elec-trochemical gradient that can be used to generate ATP.Purple membranes (membranes containing bacterio-opsin)and the ability to produce bacterio-opsin are features ofsome, but not all, halophilic species (F. Pfeifer, in F.Rodriguez-Valera, ed., Halophilic Bacteria, in press). Thesequence AGGT is present 8 bp upstream from the ATGtranslation initiation codon for the bop gene and is comple-mentary to the pyrimidine-rich sequence at the 3' end of 16SrRNA. However, transcript analysis with nuclease Si indi-cates that the in vivo mRNA start site (defined as a triphos-phate end) is only two nucleotides upstream from the ATGtranslation initiation codon (11). Because of the short leaderregion, no Shine-Dalgarno analog can be present upstream ofthe initiator codon; possibly, ribosomes initiate at the firstAUG as in most eucaryotic mRNAs. The sequence GAGG,also complementary to the 3' end of the 16S rRNA, ispresent immediately downstream from the initiator codon,but its role in translation initiation has not been established.A hairpin structure at the 5' end of the transcript might alsoplay a role in translation or mRNA stability. It is possible

that, in the evolutionary past, the bop gene mRNA mighthave contained a longer 5' leader sequence and that aShine-Dalgarno-type interaction might have been used toidentify the protein synthesis initiation site. It is interestingthat the bop gene, in contrast to many biosynthetic genesfrom methanogenic organisms, is not easily expressed in E.coli even when cloned into efficient expression vectors.Mutants ofH. halobium which fail to express the bop gene

occur spontaneously at a high frequency (-10-4; 1, 39; F.Pfeifer, in press). Virtually all of these mutants are the resultof transposition of insertion elements either into the bopgene or into the 1.4 kb of 5' flanking sequence. Datailedanalysis of the upstream insertions has revealed the exist-ence of a second gene, brp (for bacterio-opsin-related pro-tein), which is oriented in the divergent direction; theintergenic space between the two genes is 526 bp (1). Likethe bop transcript, the brp mRNA is initiated very near theATG translation initiation codon and contains a hairpin at its5' end. In addition, the two transcripts are complementaryfor 13 residues near their 5' ends, and there are a number ofidentical sequences in the 120 nucleotides of 5' flankingsequence of the two genes that might represent conservedregulatory elements. The integrity of the brp gene andpresumably its expression are essential for the expression of

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MINIREVIEW 475

the bop gene. Only one strain exhibiting reversion from theBop- back to the Bop+ phenotype has been isolated (38).This was clearly not a simple excision event of the primaryinsertion into the brp gene but a rather more-complexsecondary insertion event into brp; this somehow altered brpexpression thereby restoring the Bop+ phenotype.The genes encoding the L12 equivalent ribosomal protein

(L12e) from S. solfataricus and H. cutirubrum have beencloned and partially characterized (L. Shimmin, C. Ramirez,A. McKee, A. Matheson, and P. Dennis, unpublished re-sults). In contrast to the brp and bop genes, the Halo-bacterium L12e gene is transcribed on an mRNA that beginsmore than 1,500 nucleotides upstream from the ATG startand is almost certainly polycistronic. The sequence up-stream from the L12e initiation codon exhibits only limitedcomplementarity to the 3' end of 16S rRNA; it is unclearhow this limited interaction could be sufficient to identifyunambiguously the translation initiation site on the longmRNA transcript (Fig. 3). Three other ATG sequences thatappear in the LlOe open reading frame preceding the L12egene exhibit better complementarity to the 3' end of 16SrRNA than the authentic L12e initiation region. Sites A andC (Fig. 3, non init. A and C) appear to be read as methioninecodons within the LlOe gene, whereas site B (Fig. 3, noninit. B) is apparently out of frame. The authentic LlOeinitiation site has not yet been identified with certainty. Thesequence preceding the L12e ATG initiation codon in S.solfataricus also exhibits poor complementarity to the 3' endof 16S rRNA; the in vivo transcript of this gene has not yetbeen examined.

In summary, it seems likely that methanogens utilize aShine-Dalgarno type interaction to identify translation initi-ation sites on their mRNAs. In halophiles (and possibly alsoin the sulfur-dependent thermoacidophiles), such a mecha-nism seems less likely since some mRNAs (those of the brpand bop genes) lack a 5' leader region and at least one othermRNA that has a leader region (L12e mRNA) exhibits onlymarginal complementarity to the 3' end of 16S rRNA whencompared with noninitiation ATG sequences. Clearly moreexamples need to be examined.Enzyme structure of RNA polymerase and nucleotide rec-

ognition signals. Archaebacteria appear to contain a singleDNA-dependent RNA polymerase. The enzyme is amultisubunit complex that is somewhat simpler than eucary-otic RNA polymerase I, II, or III but more complex than theeubacteria a2P,B'o enzyme (20, 50). The functional relation-ships among the larger subunits of the eucaryotic,archaebacterial, and eubacterial enzymes have been estab-lished by using immunological cross-reactivity. The largestsubunit in the thermophilic enzymes (subunit B, equivalentto the 13 subunit of E. coli polymerase and the second largestsubunit of eucaryotic polymerase) is replaced by two inter-mediate-sized subunits (B' and B") in the halophilic andmethanogenic enzymes. Another large subunit (subunit A) isrelated to the 13' subunit of E. coli polymerase and the largestsubunit of eucaryotic polymerase. Halophilic and methano-genic enzymes are somewhat simpler, containing fewerlow-molecular-weight subunits than the sulfur-dependentthermophilic enzymes.Are there defined promoter and terminator sequences that

are recognized by archaebacterial RNA polymerases andthat contribute to the regulation of gene expression? If suchsequences exist, are they related to eubacterial or eucaryoticconsensus sequences? In searching for such sequences, it isimportant to recognize that rRNA, tRNA, and mRNA geneswithin a species might utilize totally different recognition

signals and that the recognition signals themselves are likelyto be different among the halophilic, methanogenic, andsulfur-dependent thermoacidophilic organisms.The 5' flanking regions of a number of tRNA genes from

M. vannielii and H. volcanii have been sequenced, and Siprecursor transcript mapping has been carried out on severalof the methanogen genes (8, 45). For the methanogen tRNAgenes a conserved bipartite sequence is present in the 5'flanking region. The transcription start site is at the first Gresidue in the consensus sequence TGCAAGT (Fig. 4, BoxB); this sequence is located between 10 and 29 bp in front ofthe tRNA coding sequence. Centered about 30 nucleotidesupstream from the start point is a 20-nucleotide-long, con-served A+T-rich sequence (Fig. 4, Box A). The 5' flankingregions of two rRNA operons from M. vannielii have alsobeen shown to contain the Box A and B sequences; initiationof transcription occurs 158 nucleotide bp in front of the 16Sgene at the first G of the Box B sequence. Transcriptiontermination signals at the ends of the rRNA and tRNAtranscription units consist of A+T-rich sequences about 15nucleotides in length. These are not preceded by the in-verted-repeat symmetry found in eubacterial rho-indepen-dent terminators, although the terminating nucleotide isusually located in a run of T residues on the DNA plusstrand.

In H. volcanii, tRNA genes are not preceded by theA+T-rich Box A-like sequences, although sequences resem-bling Box B are sometimes present (7). What appears to beconserved is the sequence GAGAGA that occurs at avariable position upstream from the coding sequences. Re-gions that are A+T rich are generally less frequent inHalobacterium spp. because of the unusually high G+Ccontent (68%) of single-copy DNA. Again, the genes arefollowed by T-rich sequences that probably function astranscription termination signals.The rRNA gene cluster in H. cutirubrum is unique single-

copy DNA and contains five highly conserved direct-repeatsequences in the 900 bp of 5' flanking sequence (24; Mankinand Kagramanova, in press). The 5' ends of the primaryrRNA transcript have been mapped to a highly conservedoctanucleotide element of each repeat (see Fig. 1 and 4). Theoctanucleotide resembles the Box B sequence observed infront of the M. vannielii rRNA and tRNA genes (45). Thefive different start sites in H. cutirubrum are used withdifferent efficiencies (over a 50-fold range), and their relativestrengths are subject to growth rate variation (12). Thisimplies that the conserved bipartite sequences around thesetranscription start sites might be important in specifying thetranscription start point but do not determine promoterstrength or growth rate regulation. For example, the secondelement of the conserved bipartite repeat is 27 bp in length,centered about 30 nucleotides in front of the transcriptionstart site and perfectly conserved in one of the two strongestpromoters, P4 (Fig. 1, P4), and in the two weakest promot-ers, p3 and p5 (Fig. 1, P3 and P5); the other strong promoter(Fig. 1, P1) retains only a 5-bp section of this second element(-35 sequence element; see Fig. 4). By comparing thenucleotide sequences upstream from the two strongest pro-moters, a third region of sequence homology centered about60 nucleotides (-60 sequence element) in front of the startpoint was identified (5, 12). As would be expected, theintermediate and weak promoters show less homology (45 to70%) to this third sequence; however, its role in promoteractivity remains to be demonstrated.The 5' flanking region in front of the Halococcus

morrhuae rRNA genes exhibits three repeat sequences that

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M. vannielii (rRNA and tRNA)

5'...R R R NH T T T R T R T R It T R..(18-20 n) ..T 6 C R R 6 T...

H. vo1caniu (tRNA)

5'..6 R 6 R 6 R...

H. cutirubrum (rRNA)

5'.. .T C C(C/R)T 6 6 T G T(C/6)6 G. .(21-25 n). T I C 6 R. (33 n). .T G C 6 R R C G ...

11ethanogens and Helophiles (mRNA)

5'...G R R H T T T C R..(35-135 n)..R T 6...

FIG. 4. Conserved sequences from the 5' flanking regions of archaebacterial genes. The rRNA and tRNA genes from M. vannielii arepreceded by Box B and Box A sequences. Transcription initiation occurs at the first G in the conserved Box B. In addition to the GAGAGAsequence illustrated, H. volcanii tRNA genes sometimes also contain a sequence resembling the Box B sequence from M. vannielii. The H.cutirubrum rRNA genes are preceded by five direct-repeat sequences that correspond to alternative transcript start sites. Initiation occurswithin the conserved octanucleotide start site; this sequence is similar to the Box B sequence of M. vannielii in both sequence and function.The - 35 sequence element is part of a much longer sequence that is conserved at three of the five start sites (p3, p4, and ps). The strongeststart site (pl) retains only this short pentanucleotide component of the longer sequence. The -60 sequence element is present in front of thetwo most active transcription start sites (p1 and p4). For the three less active start sites (P2, p3, and ps), this -60 sequence element is only45 to 70% conserved. A conserved sequence found 35 to 135 nucleotides in front of some but not all mRNA genes is also illustrated.Nonspecific bases in these conserved sequences are indicated by N.

are highly homologous to the start site and -35 sequenceelement of the H. cutirubrum repeat sequences (31). Asimilar sequence has also been observed in front of a 7Sstable RNA gene in H. halobium (35).There is an inverted-repeat sequence followed by a T-rich

sequence located immediately downstream from the 5S genein the rRNA transcription units of H. cutirubrum, H.halobium, and H. volcanii; in Halococcus morrhuae theinverted repeat is conserved, but the T-rich sequence isabsent (9, 24, 31; Mankin and Kagramanova, in press).These sequences resemble eubacterial transcription termina-tion signals. Nuclease Si protection experiments havefailed, however, to detect any 3' transcript ends in thevicinity of this sequence; all transcripts through the 5S genein H. cutirubrum apparently continue through the intergenicspace and into the cysteine tRNA gene (4). It has beensuggested that the termination site for the long primarytranscript lies within an A+T-rich sequence beginning 120nucleotides beyond the cysteine tRNA gene, although, fortechnical reasons, it has not been possible to demonstratethis unambiguously (4).

Definitive information relating to transcription initiationsignals in front of protein-encoding genes remains scarce.The transcription start points of the related brp and bopgenes of H. halobium are located immediately in front of theATG translation initiation codons (1, 11). There are a num-ber of short homologous regions in the sequences upstreamfrom these two genes, but whether they are fortuitoushomologies, remnants of a gene duplication, common con-trol signals, or RNA polymerase promoter recognition sig-nals remains unclear. The start sites for three transcripts inM. voltae, one extending into an RNA polymerase subunit

gene and the other two extending into open reading frames,have been located by S1 nuclease transcript mapping (2). Noconserved sequences in the vicinity of the start points areobvious. Hamilton and Reeve (23) have compared 5' flankingregions in front of the 10 different methanogen and halophilegenes (including six insertion element open reading frames)and have identified the common conserved consensus se-quence GAANTTTCA, located 35 to 135 nucleotides up-stream from the translation initiation codon (Fig. 4; N is anynucleotide). This sequence is not present in other 5' flankingsequences, and even when present, its position relative toknown transcription start sites would appear to be variable,its significance, therefore, remains in doubt. Clearly what isrequired to define transcription initiation signals is a greatervariety of gene sequences and detailed mapping of the invivo 5' transcript ends. The 3' ends of the bop and L12e genetranscripts have been mapped. Like stable RNA transcripts,these ends fall within A+T-rich sequences which in generalare not preceded by inverted-repeat symmetry (5, 11).Genome structure in halobacteria. The genomes of H.

halobium and related organisms (i.e., organisms capable ofproducing the bacterio-opsin-containing purple membranes)exhibit several interesting structural features. Their cellularDNAs can be separated into two fractions by either CsClbuoyant density gradient centrifugation or by malachitegreen bisacrylamide column chromatography (16, 17, 37; F.Pfeifer, in press). The major fraction representing 70 to 90%of the cellular DNA has a G+C content of 68% and repre-sents almost exclusively chromosomal, single-copy DNAsequences. The minor fraction representing 10 to 30% of thecellular DNA is more heterogeneous, has a G+C content of60% or less, and includes a variety of different covalently

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closed, circular plasmid DNAs as well as A+T-rich islandsfrom the chromosomal DNA. Several years ago Sapienzaand Doolittle (40) demonstrated that repetitive sequencefamilies clustered in the A+T-rich regions are associatedwith high-frequency genomic rearrangements. One A+T-rich island 70 kb in size has been analyzed and shown tocontain a variety of different insertion elements and relatedrepetitive sequences that are also found on plasmid DNA butonly rarely, if at all, in G+C-rich single-copy genomic DNA(37).Based on insertional inactivation of the bacterio-opsin

gene and insertional rearrangements in plasmid or halobacte-rial phage + H DNA, a total of nine different insertionelements have been identified and characterized in H.halobium (reviewed elsewhere [F. Pfeifer, in press]). Al-though there are exceptions, these insertion elements ingeneral exhibit many classical characteristics, such as atleast one major open reading frame within the element,terminal inverted repeats, and duplication of the target DNAsequences. Most of these sequences are present at about fivelocations in A+T-rich genomic DNA or plasmid DNA.The gene encoding bacterio-opsin (bop) is located in

unique G+C-rich genomic DNA. The frequency of inser-tional inactivation of this gene is very high, approaching10-4. Insertions which inactivate the gene are stable; rever-tants resulting from precise excision have never been de-tected (38). These observations raise several provocativequestions. Are insertions into unique single-copy DNArandom, or are they highly targeted towards limited seg-ments such as the bop region? If they are random and occurat such a high frequency, why are there not more insertionsequences found in the G+C-rich regions of the genome? Ifthey are not random but targeted, what makes the bop regiona preferred target, and what protects essential regions suchas ribosome or RNA polymerase component genes frominsertion? Do these insertion sequences play any definedrole in the general process of genome organization andreorganization or the specific process of selective generegulation? Although other archaebacterial species includingmethanogens have insertion elements, the number of suchelements is more limited; the characteristic of high-frequency rearrangement seems to be confined to purple-membrane-containing halophilic species.

Genetic exchange has recently been demonstrated for thefirst time in archaebacteria by using auxotrophic mutants ofH. volcanii, a non-purple-membrane-producing species thatexhibits little if any genomic instability (34). Exchangerequires cell-to-cell contact and occurs at a low frequency ofabout 10-6. It is believed that exchange proceeds by way ofcell fusion to produce a transient or stable diploid cell.Prototrophic recombinants can segregate the parental auxo-trophic variants in the absence of selection, suggesting thatcells remain heterozygous at a given locus for some timeafter the fusion process.There is as yet no transformation system available for

introduction and propagation of recombinant DNA mole-cules in archaebacteria. This, along with the absence of awell-developed genetic system, has greatly hinderedprogress in analyzing gene structure, function, and regula-tion. A number of laboratories are currently working on thecloning problem; efforts are focusing on (i) identification of avector, (ii) localization and introduction into the vector ofsuitable restriction sites for cloning, (iii) characterization ofa suitable selection system and its integration into the vectorsystem, and (iv) development of techniques for permeabil-izing cells to the recombinant DNA molecules. To my

knowledge, no substantive progress has yet been made inany of these areas. Such transformation and genetic sys-tems, when available, will greatly facilitate the unraveling ofthe mysteries and complexities of the archaebacterial king-dom. This information will provide the important thirdperspective on the solution of the efficiency and accuracyproblem in early cellular evolution.

ACKNOWLEDGMENTS

I thank my many colleagues, both at the University of BritishColumbia and elsewhere, who have shared with me their thoughtfulinsights and unpublished observations.

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