Gene. 121 (1992) 103-110 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

103

0378-l 119/92iSO5.00

06750

Protein-coding introns from the 23s rRNA-encoding circles in the hyperthermophilic archaeon Pyrobaculum (Recombinant

DNA;

RNA splicing;

Jacob Z. Dalgaard Institute

Received

of Biological

bulge-helix-bulge

motif; intron-encoded

gene

form

stable

organotrophum

proteins)

and Roger A. Garrett

Chemistry, B, Copenhagen

University,

by M. Belfort: 4 May 1992; Revised/Accepted:

Sslvgade 83, DK 1307 Copenhagen

6 July/l3

July 1992; Received

K, Denmark

at publishers:

27 July 1992

SUMMARY

Two archaeal

have been discovered in the single-copy 23s rRNA-encoding gene of the hyperthermophile, After excision from rRNA transcripts, both introns circularize and are stably retained in the cell. Putative proteins encoded by the introns and covering most of the intron sequence share a decapeptide motif with proteins encoded by another archaeal intron and by group I introns.

Pyrobaculum

introns

orgunotrophum.

INTRODUCTION

Archaeal introns located in tRNA- and 23s rRNAencoding genes have been detected in hyperthermophiles and extreme halophiles (Brown et al., 1989). They are excised from rRNA transcripts by a protein enzyme which recognizes a ‘bulge-helix-bulge’ motif at the exon-intron junction and cuts in the bulges (Thompson and Daniels, 1988; 1990; Kjems and Garrett, 1988; 1991); a similar motif, which may also be cleaved by this enzyme, occurs in the processing stems of the large rRNAs (reviewed by Garrett et al., 1991). The 23s rRNA intron of the archaeon Desulfurococcus mobilis circularizes after excision and forms a normal 5 ’ ,3’ phosphodiester bond at the ligation junction. This circular

Correspondence

Copenhagen

to: Dr.

R. A. Garrett,

University,

Institute

Serlvgade 83, DK

mark. Tel. (45-33)156338;

for Biologisk

1307 Copenhagen

Kemi B, K, Den-

Fax (45-33)145058.

Abbreviations: aa, amino acid(s); bp, base pair(s); DC., Desuijurococcus; EtdBr, ethidium bromide; kb, kilobase or 1000 base pairs; nt, nucleotides; oligo, oligodeoxyribonucleotide; ORF, open reading frame; Ph., Pw2xzulum:

PCR, polymerasc

rDNA. gene encoding dodecyl sulfate.

rRNA;

chain reaction; RT, reverse

rRNA,

transcriptase;

ribosomal

RNA;

SDS,

sodium

intron (622 nt) contains a putative ORF which crosses the ligation junction and covers most of the intron (Kjems and Garrett, 1985; 1988); the other characterized archaeal introns from tRNA-encoding genes (reviewed by Brown et al., 1989) and other rRNA-encoding genes (Kjems and Garrett, 1991) are small (< 110 nt). In the present experiments, we used PCR to screen hyperthermophiles (Stetter et al., 1990; Kjems et al., 1992) for the presence of larger introns in the DNA region encoding the functionally important domains IV and V of 23s rRNA where most rRNA introns are located in archaea and in eukaryotic nuclei (reviewed by Garrett et al., 1991).

RESULTS

AND DISCUSSION

(a) The single 23s rRNA-encoding gene of Pyrobaculum organotvophum contains two introns PCR products were obtained from DNA samples of the hyperthermophiles Archaeoglobus fulgidus, Thermoplasma volcanium and a Pyrodictium-like isolate (AV2) (Stetter el al., 1990) using oligo primers bordering the domain IV-V region (Leffers et al., 1987) of the 23s rRNA gene; they were all the same size as the control product from the hyperthermophile DC. mucosus (Fig. 1) which contains no

104

M 1 2

1

EXON 1 -

CGGGGGTGGC

TAGGTCCCCG AATTGTATAG

TGGCTATGTT GATTACAGAG D G Y V D Y R GGAATTCCTC GAG-TAT KEFL EEI CGCTTGCGCA AGAGACAGAG Y AC A R D R AATCTTGAAC TCCAGCCTTG

bp 2323 1929

R I L N

s s I,

GAATGTGAAG AAATACAGAA G N V K K Y R GGCTAGACGA ATTGAAGATG MARR IED GAAAGGAGGT AGATACACTG K K G G RYT TCGGGTAGTT AAAATACGCC Y R"" KIR CTAGGCCGAA CAAGTTGGCT *

1371 1264 702

CGAGCCCGTA~AGGGCGGTA GGGAGCCAAG CTTAAAAATA TGGATATATT "D I TCGGCAAAAA "G K GCAACGAACT C N E CCTTTAGGCT A F R CCCCCGAGGT A P E ATCAGCCTTT N Q P CGCTCACTGC AL T AAATCGTCGT EI" ACCCCCAAAA HPQ CCCTTCCGCC EXQN

I CGTTTATTTT GGGACGCTGT G T L GTGGAGTACG

D SD

P

c

c

CTCTAGCCCC

GCGCTTTTGA S A F ATATGTCTAA D M S GCGGAGTCTA C G" CTGTCGATAG S "D ATGCCGAGGG D AE AGAGTGACAT K s D AGATCACTAG KIT -GCTGTA

30

90 150 210 270

390 450 510

Q K L ATCTTGACCT

570

1722 ntl

I AGGAGGGGGT

TTTTTATACC

30

TCTCCATGGG M ACCACAACAA Y H N TGGCGATTCT

CTGTGATTTG GAGTATCTAG G C D L E Y L GAAGGCTAGG GAGTATGTGG KKAR EYV GGTTGATATG TTGAAGAGTT L ” D M L K S CAGGGTTAGG GTGAACAGTA Y R VR VN s TCTAGTATCT CCGACGGTGC LLVS PT" CTTTGACAGG AGGAAGAGGC Y F D R R K R AGTTGTAAAC GCCGCTGCTG R v v N A A A CTATGGGGGG AGGTTCTTCA s Y G G RF F GTCAAGCCTC TCCACCCGGT LSSL STR CCCCCCA& CC’XCCATTA

CTGGCGTGGT A G V TTGAGATCTA "EI GCGGTTTAAA C G L GGGAGTTTTA R E F CGTTTGTGCG P F v GTCTCTATCC R L Y TGGTGCTTTC V" L AGTTGGTGGT K L v GAAGTTCTCC *

TAAGGGTGAT v K G D TGATAGGGAT YDRD TCCCCATGTG N P H" CGAATCTGTC YE s v TGGGTTGTTT R G L F CGTGGTGGAG P V" E GTCGTTTGGC s s F G TAGGGGGACG "R G T CCTCTTTCTT

90

GGAACTATTA G N Y TTGAGCGGCT IER GCACTCTCTA GTL CGGATTGGAG S D w GCGTTAAGAG S"K TGCCAGAGTT LPE CTCCAGCCCG

lntron I (Domain IV)

330

GGGTGGWCT~AGGCGGGTGC

“AI

TTGGGGAATT L G N GTTAAGTTTA "K F CCGTGTTGTT

2

AAAGCTCTCA K A L GTAATCGCCG V I A GGCGTGAGTT G V S GGCAAGGAGT G K E GCTGCAATAG AA I ATTGTCGTAA I vv AGATACGTTA RI'" GAAGAGAACC E E N GTGTGCCATT

CAGCCGCCM

VEY

AGGTCGTATG R S Y AGAGGCGCCA HGA GATAGCGACG

Fig. 1.

CCAGTATGTC F Q Y V CTACGAAATC NY E I AAGAAAATAC L R K Y TAGAATATAC L R I Y ACTATTAGCG V L L A TAGAACCAGA F R T R ACTATCGATA AL S I ATCTGGTAAG "SGK ATTACAGGTG KL CCGGGA

AAGGGGAA

-

EXON 3

150 210 210

Intron 2 (Domain V)

330 390 450 510 570

I598

Fig. 2. Fig.

I, Comparison

of PCR products

run on a 0.75”,, agarose tracted

gel alongside

from the domain

IV-V region of the 23s rRNA-encoding

(lane M) a BstEII

from frozen cell mass of the hyperthermophiles

(Sambrook sequence

restriction (Kjems

and Garrett,

1987), and the selcctcd

et al., 1989) using two oligo primers; one (5’-CCTGACTGTTTAATAAAA) near the 5’-end of domain

of 23s rRNA.

Primers were annealed

in bp). Methods:

DNA

was complementary

IV and the other (5’-CCCGTTCCTCTCGTACT) at 47°C and extended

genes of (lane 1) DC. mucos~~s and (lane 2) Ph. ~~rgmorrophwn

digest of phage 1. DNA (left margin,

to the strand encoding

was complementary

at 72°C. liry DNA polymerase

(Stratagene.

chromosomal

region was amplified to a conserved

DNA was cx-

by the PCR method rRNA at a conscrvcd

scqucncc

within domain

La Jolla, CA) and a thermocqclcr

(Hybaid)

VI were

used. Fig. 2. The nt sequences

of the two inserts (introns)

(exons) are boxed. The identities putative

ORFs

in a low-melting-point

agarose

gel. Possible recessed

with restriction

using the dideoxynucleotide

entered in the GenBank/EMBL

database

enzymes. procedure

in the 23s rRNA-encoding

where the RNA introns

are given below the nt sequences.

et al., 1985) and mapped sequenced

of the domains

Asterisk

indicates

gene of Ph. orgunotrophum.

arc located

a stop codon.

are indicated Sequencing

Short sequences

procedure

(Sanger

with the accession

fragments

corresponding

was as follows: PCR products

ends were filled in by the Klenow enzyme, and the product Overlapping

of the flanking 23s rDNA

on the right. The aa sequences

to the

wcrc purified

was cloned into pUC19 (Yanisch-Perron

were inserted

into M13mp18

and M13mp19

vectors.

et al., 1977). To avoid errors,

three different

PCR products

were sequenced.

and both strands Sequences

were arc

No. M86622

introns (Kjems and Garrett, 1985). None of these products was subcloned and sequenced. In contrast, the DNA sample of Pyrobaculum organotrophum (Huber et al., 1987) yielded a larger PCR product after an increased extension time compatible with the presence of one or more inserts (Fig. 1). The sequence of this DNA product was determined and aligned with 23s rRNA-encoding gene sequences from other hyperthermophiles. This revealed that it contained two additional sequences which could constitute introns (Fig. 2); both exhibited ORFs which, in contrast to that of DC. mobilis 23s rRNA (Kjems and Garrett, 1985), do not cross the insert junctions (Fig. 2). A Southern blotting analysis was made to determine the number of 23s rRNA-encoding genes present in the organism. Five restriction enzymes BarnHI, HindIII, Pstl,

Sac1 and Sac11 were used in the analysis, one of which, SUCH, cut twice within the amplified rDNA fragment (Fig. 3). Size markers prepared from a digest of phage ;1 DNA with BstEII were co-electrophoresed on an agarose gel with the restriction digests. After blotting onto a Hybond N filter, 32P-5’-end-labelled restriction fragments were used to probe inserts 1 and 2 and the 23s rRNAencoding gene + insert 2 (Fig. 3). The hybridization results (Fig. 3) show that for the four restriction enzymes which did not cut within the PCR product (lanes 2-5), single bands were observed in each lane when the probe for the 23s RNA-encoding gene + insert 2 was hybridized (Fig. 3B). This suggests that the genome of Pb. orgunotrophum exhibits a single copy of the 23s rRNA-encoding gene which always contains insert 2.

105

A

C

B

123451234512345

bp -14140

II

-I

EcoR

on a 0.75% agarose

Cla 1

I

Sma

mCi/mmol)

of (A) intron

I

of the size markers

specific for each of the introns was performed

iDra

-

1264

-

702

1, (B) the 23s rRNA-encoding

derived

restriction

enzymes:

DNA (Boehringer

under conditions

I

gene + in&on 2 and (C) intron 2. Chromosomal

1, SacII;

to a Hybond

2, ScrcI; 3, PsfI; 4, BarnHI; N filter (Amersham)

from phage I DNA digested

and the 23s rRNA

primed

II

4

EcoR

I

B

with each of the following

and a labelling kit for randomly

onto a filter. Hybridization

1929

C

gel. The gel was stained with EtdBr and transferred

et al., 1989). The positions labelled probes

analysis

-

9

A

blotting

4324

4

I qn+

Sac

Sac II 4 lntron 2

lntron 1

Fig. 3. A Southern

6369

-

Ip'

im

organotrophum was digested

-

with BstEII

gene were prepared Mannheim,

of high stringency

(Maniatis

using the capillary

Probes

restriction

fragments

were denatured

ct al., 1982). A scheme

method

(Sambrook

(in bp). Radioactively

using [z-32P]dATP

(3000

at 95°C for 10 min and absorbed

of the PCR fragment

fragment

and electrophoresed

transfer

are shown on the right margin

from purified

Germany).

DNA (2 pg) from Pb.

and 5, HindHI,

probes for introns

is given below showing

the two introns and the locations of two internal Sac11 sites. The hybridization 23s rRNA-encoding gene + intron 2 (B) are also indicated

sites for the restriction

1 (A), and 2 (C), and

implying that there is not a mixed population of intron+ and intron strains. The probes for the individual inserts hybridized exclusively to the same bands (Fig. 3A,C) which renders it very likely that both inserts are confined to a single 23s rRNA-encoding gene. These two inferences were reinforced for the Sac11 digest (lane 1) where the DNA was cut between the inserts at the start of insert 2 (see scheme in Fig. 3). Probes specific for the two inserts hybridized to different single bands, while the probe for the 23s RNAencoding gene + insert 2 hybridized to both bands. We concluded, therefore, that both inserts are associated exclusively with a single 23s rRNA-encoding gene. Next, we investigated whether the inserts were excised during maturation of the rRNA and could, therefore, con-

stitute introns. Oligo primers were hybridized to mature 23s rRNA, 3’ to intron 1(5’-GAAUCCUGGCCACUGGCGGUACG) and intron 2 (5’-AGUACGAGAGGAACGGG). The sequences at the 3’ boundaries of the inserts were then examined by the dideoxynucleotide sequencing method using RT. The results are shown for both inserts in Fig. 4 alongside the gene sequence showing the corresponding boundaries at the DNA level. Clearly, both inserts are absent from the mature 23s rRNA. Furthermore, the lack of RT termination at the putative exon ligation sites indicates that a normal 5’,3’-phosphodiester bond formed during maturation of the 23s rRNA in an efficient ligation reaction. Therefore, we concluded that both inserts are introns.

106

TCGA

UCGA

TCGA

UCGA

T

C

G

A

Fig. 4.

A

B

C A UCGA 5’

- 720

hltmn 1 i

lntmn 2 -t

I -

Ei

t

&OR,

CII I

B Fig. 5.

tf

t KP” I

sma

Prlmcr c

A Fig. 6.

107 (b) Both introns are present in the cell in a circular form The fate of the excised RNA introns in the cell was examined by a Northern blotting analysis. Total RNA (5 pg) from Pb. organotrophum was denatured in 17.5% formaldehyde, electrophoresed in a 1 “;b agarose gel containing 2.2 M formamide together with size markers and then blotted onto a Hybond N filter. Radioactively end-labelled restriction fragments and an oligo (5’-CCAGCTAGATACTCCAAATC) were used to probe the 23s rRNA and introns (see scheme in Fig. 5). The results revealed that both free introns were present in the cellular extracts and that they co-migrated with a 720-nt size marker (Fig. 5). The extended band in Fig. 5A probably reflects degradation of the 23s rRNA sample, but we cannot eliminate the possibility that other introns are present. Further analysis of RNA extracts on composite polyacrylamide gels revealed weak bands relative to those of 7s and 5s RNAs, suggesting that, in vivo, both introns are of limited stability postsplicing (data not shown). The possibility that the RNA intron moieties were circular, like that of DC. mobilk, was investigated by sequencing the RNA introns using RT primed by oligos hybridized close to the 5’-ends of introns 1 (5’-GGAATATATCCACTATACAATTGC) and 2 (see section a above). For both introns the reverse transcript extended from the 5’-end into the 3’-end of the linear intron sequence, indicating that circularization had, indeed, occurred (Fig. 6).

tionally important

(c) The splicing sites The locations of introns 1 and 2 in the latest secondary structural model of the domain IV-V region of the 23s rRNA from Pb. orgunotrophum are shown in Fig. 7 together with the positions of the other known archaeal rRNA introns. Many of the eukaryotic group I and group II introns found in pre-23S-like rRNAs also occur within this func-

(d) Functions of the putative proteins encoded by the archaeal rRNA introns Both introns of Pb. organotrophum and that of Dc. mobilis (Kjems and Garrett, 1985) contain ORFs, and circumstantial evidence suggests that they are expressed. First, only one reading frame is possible for each intron, and second, dot matrix analyses of the aa sequences (not shown) reveal

Fig. 4. Autoradiograms show nt sequences the exon-exon

of sequencing

junctions.

junction

They were determined

et al., 1982) close to the 5’-termini ers were annealed

gels demonstrating

across the 5’-exon-intron

at least one stable helix (Thompson and Daniels, 1990; Kjems and Garrett, 1991). These motifs can be discerned for both introns in the pre-rRNA of Pb. orgunotrophum (Fig. 7B,C). The presence of the motif requires, as for the other archaeal rRNA introns (Kjems and Garrett, 1991) that a local rearrangement of the exon structures occurs post-splicing in order to generate the mature rRNA structure. The location of intron 1 is of particular interest because it lies in the boxed stem-loop structure in Fig. 7A which constitutes the binding site of the primary binding protein EL2 that has an important role in ribosomal assembly (Egebjerg et al., 1991). Formation of the ‘bulge-helix-bulge’ motif in the pre-23s rRNA (Fig. 7B) will preclude binding of the hyperthermophile L2 protein and, therefore, of 50s subunit assembly, until the splicing reaction is completed. Intron 2 is located at an RNA site that has been implicated in A-site binding of tRNA (Moazed and Noller, 1989); thus the 50s subunit can neither assemble completely nor function while the two introns are present.

I (A-C) and 2 (D-F). The flanking gels

that intron excision and cxon ligation occurs for introns (A, D) and the intron-3’.exon

by extension

of the linear introns

at 5O’C, and the RT reaction

region of the rRNA (reviewed by Garrett

et al., 1991). There is strong evidence that the archaeal cleavage enzyme recognises a ‘bulge-helix-bulge’ motif at the exonintron junctions (Thompson and Daniels, 1988; Kjems et al., 1989) which constitutes two three-base bulges on opposite DNA strands separated by 4 bp and bordered by

from “P-5’~end at 48°C.

(C, F). The central gels (B, E) show RNA sequences

labelled oligo primers hybridized

using RT (Life Sciences,

was performed

junction

Arrows

under high stringency

FL) and the dideoxynucleotide indicate

the intron-exon

procedure

junctions

conditions

(Sanger

across

(Maniatis

et al., 1977). Prim-

(DNA) and the exon-cxon

junctions

(RNA), and the bordering sequences arc given for each gel. Compressions, indicated by stars, occurred in one DNA (A) and in one RNA sequence probably due to the presence of a stable secondary structure; the nt sequence was verified by sequencing the complementary strand.

(E)

Fig. 5. Northern analysis of the total RNA extracted from Ph. orgumtrnphum cells to examine for the presence of 23s rRNA and RNA introns. The hybridization pattern obtained with “P-5’-end labelled probes specific for (A) 23s rRNA, (B) intron I and (C) intron 2 are shown. The 23s rRNA and intron

I were probed with restriction

The locations

of co-clcctrophorcsed

fragments,

while intron 2 was probed with an oligo; their respective

23s rRNA

(2900 nt) and a transcript

of domain

hybridization

II of E. coli 23s rRNA

sites are indicated

(720 nt) arc indicated.

in the scheme.

Zero denotes

the

origin of electrophoresis. Total RNA was extracted from 0.3 g frozen cell mass by a single-step procedure using acidified guanidinium thiocyanate, phenol and chloroform (Chomczynski and Sacchi, 1987) and stored in 0.5”, SDS (5 mg/ml) at -80°C. Total RNA (5 pg) from Pb. organotrophum was fractionated on a I ‘lo agarosc gel containing 2.2 M formamide (Sambrook et al., 1989). The gel was stained with EtdBr, and the RNA was transferred to a Hyhond N filter (Amcrsham). Fig. 6. Autoradiograms of RNA sequencing gels demonstrating the circularity of the introns. scribed in the legend to Fig. 4. The ligated junctions are indicated by arrows, and the bordering No termination

of reverse transcription

was observed

at the junctions.

RNA was isolated

Sequences sequences

as described

were generated using the RT procedure as dccorresponding to the rRNA template are given. in the legend to Fig. 5.

108

a decapeptide

sequence occurring twice in the ORF of the and inn-on 2 of Pb. organotrophum and once in intron 1 of Pb. organotrophum (Fig. 8). Moreover, despite little additional common sequence, the putative proteins resemble each other in their aa composition, net charge and predicted secondary structures. Strikingly similar are the predicted P-sheet distributions in the three puDC. mobilis intron

tative proteins (Fig. 9). These results (Fig. 9) also provide evidence for a repeated secondary structure within the two halves of each putative protein when they are aligned at the sequence motifs.

lntron 2

C

3’ 5 AG-C ~~ U

:

\GC_G AC

’

Ac”ZZ C-G C-GC C-GA\ C-G G-C C-G C-G C-G A-UG

A UG-C

A

C

E-0”” U-A C-G ;I: 6-G C-G A

G

u ‘550

u

_%J

Fig. 7. Secondary structure of the domain IV-V region of the 23s rRNA of Pb. organotrophum and sequences of exon-intron junctions. (A) The schematic structure of the IV-V domains is derived from that of the 23s rRNA of other hyperthermophiles (Leffers et al., 1987). The cleavage sites of the two newly discovered

introns

and those previously reported for DC. by arrows. (B and C). and putative secondary structures surrounding

mobilis (D.m.) and St. marinus (S.m.) are indicated

The nucleotide

sequence

the exon-intron

junctions

trons

1 and 2, respectively.

[including Arrows

the two boxed areas in (A)] for indenoting

the cleavage

sites and the

A comparative sequence search in the EMBL/GenBank databases revealed no genes or proteins with any overall similarity. However, a search made with the repeated decapeptide sequence (Fig. 8) revealed a similar motif characteristic of proteins encoded by self-splicing group-I introns (Michel et al., 1982; reviewed by Burke, 1988; Dujon, 1989) constituting either RNA maturases that effect intron splicing (Lambowitz and Perlman, 1990) or DNA endonucleases which promote intron mobility (Dujon, 1989; Perlman and Butow, 1989); these two enzyme activities may have a common evolutionary origin (Goguel et al., 1992). The sequence similarities between the putative proteins encoded by the archaeal introns and proteins encoded by group-I introns lend support to the hypothesis (BellPedersen et al., 1990) that the ORF of the group-I intron td4 of T4 phage and its catalytic RNA core have different evolutionary origins. The hypothesis derives from the inference that intron mobility, catalysed by the encoded protein, and self-splicing, catalysed by the catalytic RNA core, are functionally unrelated. The present data suggest further that the putative proteins encoded by archaeal introns could have a common evolutionary origin with those encoded by group I introns despite the apparently unrelated splicing mechanisms of the two intron classes. Circumstantial evidence suggests that the archaeal introns are mobile. Thus, they are located at different positions in the rRNA genes (Kjems and Garrett, 1991). Moreover, they are absent from DC. mucosus, which is closely related to DC. mobilis (Kjems and Garrett, 1985) and from Pb. islandicum, which is closely related to Pb. organotrophum (Kjems et al., 1992). Furthermore, the presence of common sequences at the exon-exon (GTA*AG) and intron-intron (GAG*AGGGC) junctions (where asterisks denote the ligation points) shared by the splicing sites of the DC. mobilis intron and intron 1 of Pb. organotrophum could also reflect common recognition sites. Nevertheless, the true function(s) of putative proteins encoded by archaeal rRNA introns remains to be established. sizes ofthe omitted intron sequences are given. RNA secondary structures were drawn using the EDSTRIJC program (N. Larsen. unpublished data).

109

Fig, 8. Alignment decapeptide (Madison,

of the aa sequences

motif. These sequences WI) (Devereux

of the putative

intron-encoded

are also similar to consensus

et al.. 1984) was used for analysing

proteins

sequences

from the two archaea

within proteins

the aa and nt sequences

encoded

showing

the similarities

by eukaryotic

and for comparing

between

group I introns.

them to sequences

the repeated

The GCG

package

in the EMBL/GenBank

databases.

D.m.

Brown, J.W., Daniels, C.J. and Reeve, J.N.: Gene structure,

r-4

P.O.

and expression

in archaebacteria.

protein

factors

Chomczynski,

genetics of group I introns:

required

Fig. 9. Comparison

of p-sheet distributions

by the introns

from DC. mobilis (D.m.)

1 and 2) using the structure

(1978). The repeated

of the three putative

decapeptides

prediction

proteins

and Pb. orgunotrophum

algorithm

of Garnier

et al.

are boxed.

Biochem. Devereux,

for splicing.

P. and Sacchi,

by acid guanidinium encoded

16 (1989)

287-338.

I

Burke, J.M.: Molecular

(P.o.

organization

CRC Crit. Rev. Microbial.

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and

Gene 73 (1988) 273-294.

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method

of RNA isolation

thiocyanate-phenol-choroform

extraction.

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162 (1987) 156-159.

H., Haeberli,

P. and Smithies 0.: A comprehensive

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set of se-

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387-395. Dujon,

B.: Group

1 introns

as mobile genetic elements:

anistic speculations-a Egebjerg, J., Christiansen,

(e) Conclusions (1) Two introns occur within the single 23s rRNAencoding gene of Pb. organotrophum. Both RNA products circularize after excision from the 23s rRNA and are stable in the cell. (2) The putative proteins encoded by the two introns exhibit repeated secondary structures in their two halves. Moreover, one half of intron 1 and both halves of intron 2 contain a common decapeptide sequence which is shared by the putative protein encoded by both the archaeal intron of DC. mobilis and many group-I introns. This raises the possibility that the ORFs of the two classes of introns have a common evolutionary origin.

mary binding

J. and Garrett,

proteins

facts and mech-

(1989)91-l 14.

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R.A.: Attachment

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Ll, L2 and L23 on 23s ribosomal

RNA of

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J., Osguthorpe,

implications globular Garrett,

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and

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of

J. Mol. Biol. 120 (1978) 97-120.

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switching

A.S.:

Sci. 16 (1991) 22-26.

experiments.

RNA

RNA mat-

Mol. Cell. Biol.

12 (1992) 696-705. Huber,

R., Kristjansson,

nental

solfataras

K.O.: Pyrobaculum gen.nov.,

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a new genus of neutrophilic growing

rod-shaped

optimally

archaebacteria

at 100°C.

from conti-

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R.A.: An intron in the 23s ribosomal

RNA gene

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3 18 (1985)

of the archaebactcrium 675-677. Kjems, J. and Garrett,

ACKNOWLEDGEMENTS

extreme

R.A.: Novel expression

thermophile

of the rRNA genes in the Desulfurococcus mobilis.

and archaebacterium

EMBO J. 6 (1987) 3521-3530.

The research was supported by grants from the Danish Science Research Council and the NOVO Research Foundation. J. Z. D. received a Scholar Stipend from the Carlsberg Foundation. We are grateful to K. Stetter for providing the archaeal cells and thank J. Kjems, A. Liljas and A. Mankin for helpful discussions and Vicka Nissen for help with the manuscript.

Kjems, J. and Garrett,

R.A.: Novel splicing mechanism

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