Precise localization and nucleotide sequence of the two mouse mitochondrial rRNA genes and three immediately adjacent novel tRNA genes

Cell, Vol. 22, 157-l

70, November

1980

(Part

l), Copyright

0 1980

by MIT

Precise Localization and Nucleotide Sequence of the Two Mouse Mitochondrial rRNA Genes and Three Immediately Adjacent Novel tRNA Genes Richard A. Van Etten, Mark W. Walberg David A. Clayton Department of Pathology Stanford University School of Medicine Stanford, California 94305

and

Summary The complete DNA sequence of the ribosomal RNA region of mouse L cell mitochondrial DNA has been determined. Genes for the small (12s) and large (16s) rRNAs have been precisely located by direct sequencing of the termini of the two mature rRNAs. A comparison of the lengths (956 and 1582 nucleotides) and terminal sequences of the mature rRNAs with the DNA coding sequences indicates that mouse mt rRNAs are not spliced. Computer analysis of the complete DNA sequence has identified three potential transfer RNA genes. A gene for phenylalanine tRNA is located immediately adjacent to the 5’ end of the 12s rRNA gene, a valine tRNA gene occupies the entire region between the 12s and 16s rRNA genes and a leucine tRNA gene is located immediately adjacent to the 3’ end of the 16s gene. Hybridization of 32P-labeled, tRNA-sized mtRNA to selected DNA restriction endonuclease fragments from the rRNA region confirms the existence of small, abundant mtRNAs transcribed from these DNA sequences. All three tRNA genes and both rRNA genes are transcribed from the heavy strand of mtDNA. The mt rRNA sequences exhibit notable homologies to other rRNAs and, in particular, to those of E. coli. Within the 3’ terminal 50 nucleotides, the mouse mt 12s rRNA contains a potential 10 bp hairpin structure and a sequence of 15 consecutive nucleotides common to the RNA of the small ribosomal subunit in all systems, but does not contain the mRNA binding site (ACCUCC) found in E. coli and corn chloroplast rRNAs. The mt tRNA genes do not have the 3’ terminal CCA sequence encoded in the DNA, nor do they contain any intervening sequences. Two of the three tRNAs would lack many features which are known to be strictly conserved in all other nonorganelle tRNAs which have been sequenced. The fact that all the genes in this region are directly contiguous with at most one intervening nucleotide suggests that the entire region is transcribed into a polycistronic precursor RNA which is processed by endonucleolytic cleavages. The organization of the genes of the rRNA operon of mouse mtDNA, when compared to the organization of rRNA and tRNA genes in bacterial or eucaryotic nuclear genomes, provides evidence for the endosymbiotic hypothesis of the biogenesis of mammalian mitochondria.

Introduction The most aggressively expressed region of the mtDNA genome is the r-RNA region. rRNA genes have been mapped in close proximity to each other near the heavy strand (H strand) origin of DNA replication (Figure 1) in mouse (Battey and Clayton, 1978; Nagley and Clayton, 1980), human (Robberson et al., 1972; Wu et al., 1972) Xenopus laevis (Rastl and Dawid, 1979) rat (Parker and Watson, 1977) and possibly D. melanogaster (Klukas and Dawid, 1976). tRNA genes have been mapped adjacent to and between the two mt rRNA genes in human (Wu et al., 1972; Crews and Attardi, 1980) and Xenopus (Ohi et al., 1978). No 5S, 5.8S, other small rRNAs or genes for such rRNAs have been found in animal mitochondria. The order of transcription of the two mt rRNAs, small followed by large, is the same as in the nucleus of eucaryotes (Dawid and Wellauer, 1976; Reeder, Higashinakagawa and Miller, 1976) and in E. coli (Dunn and Studier, 1973). The organization of the rRNA region of mouse mtDNA has been determined at the nucleotide level. We have used the direct approach of combining DNA and RNA sequence information to precisely position and identify the two rRNA genes and three tRNA genes encoded in this region. The resultant map shows the same extreme economy of DNA sequence usage as is present outside the rRNA region in human mtDNA (Barrell, Bankier and Drouin, 1979; Battey and Clayton, 1980). Results DNA Sequence Analysis Sequencing of cloned mouse L cell mtDNA used the chemical method of Maxam and Gilbert (1980). In most cases DNA was 5’ end-labeled, and doublestranded DNA labeled at a unique 5’ end was subsequently generated by a secondary restriction enzyme cleavage. Figure 2 shows a fine-structure map of the rRNA region of mouse L cell mtDNA. The strategy used for sequencing the region is illustrated by the set of directed lines at the bottom of the figure. Typically about 230 bases from a labeled end could be read with confidence from a single gel. We estimate the accuracy of sequence obtained by this method to be -99%. A higher frequency of errors in reading sequence from gels occurs in the vicinity of a labeled end (Konkel, Maize1 and Leder, 1979). Whenever possible these regions were sequenced a second time, using a different restriction site for end labeling. Another occasional occurrence was the appearance of “gaps” in the sequence ladder at points corresponding to Eco RII restriction enzyme recognition sites. The cloned plasmid DNA is modified at these

158

sites, and the 5-methylcytosine residues are inert in the chemical cleavage reaction, leaving a space in the sequence (Ohmori, Tomizawa and Maxam, 1979). The presence of an Eco RII site was confirmed by sequencing the opposite strand. Identification of tRNA Genes The complete DNA sequence for the L strand of the rRNA region of mouse L cell mtDNA is shown in Figure 3. The DNA sequence was extensively analyzed by a modification of the program of Korn, Queen and Wegman (1977) to search for DNA sequences which permit the cloverleaf secondary structure characteristic of a tRNA molecule. Three such sequences were found by this method and their positions are indicated in Figure 3. The 5’ and 3’ borders of the tRNA genes were assigned by the condition that the 5’ border of the gene begins with the first base in the 7 bp long amino acid stem, while the 3’ end of the gene is one base after the 7 bp stem. No other such sequences were found in the region. The DNA sequence alone gives no information on whether the tRNAs are transcribed from the H or L strand, but evidence from Sl nuclease protection experiments (Nagley and Clayton, 1980) shows that these tRNAs are transcribed from the H strand (see Discussion). Based on their antico-

mt DNA

Figure

1. Location

and Orientation

of the Mouse

mt rRNA

Genes

The genes for 12s and 16s mt rRNAs are located close to each other near the origin of replication of heavy-strand DNA, OH. mt rRNAs are complementary to the heavy strand. The order of transcription is 125 followed by 16s and the shaded region represents the mtDNA sequence reported here.

dons they are genes for tRNAP”‘, tRNAVa’and tRNALe”. Figure 4 shows the hypothetical secondary structures of the tRNA sequences. The 3’ terminal sequence CCA, known to be a common feature of all sequenced tRNAs, is not encoded in the DNA sequence and must be added post-transcriptionally. Filter Hybridization of Mitochondrial tRNAs To provide evidence that these putative tRNA gene sequences are transcribed in vivo, a hybridization probe of tRNA-sized mtRNA was prepared by purifying LiCI-soluble total mtRNA on a 5% polyacrylamide, 50% urea gel. The region of the gel resolving the tRNAs was cut out and the RNA was eluted from the gel and labeled with Y-~‘P-ATP and polynucleotide kinase. This 3’P-labeled RNA was then hybridized to various restriction digests of mtDNA from the rRNA region of the genome which were blotted to nitrocellulose filters by the Southern method (1975). Figures 5A and 5B show the ethidium bromide staining pattern of the gels, along with the corresponding autoradiogram of the filter after hybridization. By referring to the accompanying restriction map (Figure 5C) it can be seen that tRNA-sized mtRNA transcripts have hybridized both to the 405 bp fragment containing the tRNAPhe gene and to the 415 bp fragment containing the tRNAVa’gene. In addition the 530 bp fragment from Xba I-B, which contains only sequences from the 12s rRNA, gene hybridizes to the probe. This hybridization signal is probably not due to contamination of the 32Plabeled RNA with degraded rRNA because neither the 340 bp Xba I-D fragment, which contains 12s rRNA gene sequences, nor the Xba I-C 380 bp fragment, which has 16s rRNA sequences, hybridizes with the probe. Both the 380 and 340 bp fragments were retained on the filter, so that the lack of hybridization is not due to inefficient transfer of smaller DNA fragments (data not shown). This hybridization could be due to a chance homology of DNA sequences in the 530 bp fragment to one or more RNAs in the probe, or it could represent one or more small specific transcripts from the H or L strand in this region. These possibilities are currently being investigated. The Hae Ill digest of HR-D shows that the 640 bp fragment containing the tRNALe” gene has strong homology to the probe. The 400 bp fragment, downstream from the tRNALe” gene, also shows some homology. This

Figure 2. Fine Structure Map of the rRNA gion of Mouse L Cell mtDNA b+ydmpen;pn ,, ,,,

H

we-

T’x$ Al”, ,j

EC0RI

tmf ,

Xb,

3j

cc3e/

DNA sequencmg end. 0, indicates

XbOl

cc-

>---

are shown. wth each directed lme at the bottom mdicatmg the strandedness the orwn of H strand DNA replication and the displacement loop (D loop).

Heavy and light strands of nated H and L. respectively. H strands 1s 5’ to 3’ from that of the L strand is right endonuclease sates used in and extent of sequence obtained

Re-

mtDNA are deslgOrientation of the left to right, while to left. Restriction 5’ end labeling for from each labeled

Mouse 159

MitochondrialrRNAandtRNA

Sequences

5’

TTAGTACTTG

60 TAAAAATTTT ACAAAATC:::

140 GTAGCTTAAT

150 160 170 AACAAAGCAA AGCACTGAAA ATGCTTAGAT

180 GGATAATTGT

230 ATTAGAGGTA

240 25Q AAATTACACA TGCAAACCTC CATAGAC;;:

TGTAAAA:;;

50

CCTGATCA:!

TCTAGTAG:!

100

TAATCATACT

110

CTATTACGCA

12s rRNA

CCCAAAAT:!

GACTTATA::

120 ATAAACATTA

tRNAPh’ ACA&TTAAT

-

190 210 220 ATCCCATAAA CA1cyIAAGGTT TGGTCCr$tc;cTTATAATTA 280 CTTAAACATT

290 TACTTAAAAT

370 380 CAGCAGTGAT AAATATTAAG

TGTTCCGT::

ACCAAAAC;;

300 310 320 TTAAGGAGAG GGTATCAAGC ACATTAAAAT

330 340 AGCTTAAGAC ACCTTGCCTT

350 360 AGCCACACCC CCACGGGACT

390 400 CAATAAACGA AAGTTTGACT

420 TCTTAGGGTT

440 GTGCCAGCCA

410

AAGTTATACC

HincII

480 "kEm3 ATCTTCGGCG TAAAACGT

550 560 CATTGTTAGG ACCTAAACTC

570 580 AATAACGAAA GTAATTCTAG

600 610 620 TACACGACAG CTAAGACCCA AACTGGGATT

630 AGATACCCCA

640 650 CTATGCTTAG CCATAAACCT

660 670 680 690 700 710 AAATAATTAA ATTTAACAAA ACTATTTGCC AGAGAACTAC TAGCCATAGC TTAAAACTCA

720 AAGGACTTGG

730 CGGTACTTTA

750 760 GAGGAGCCTG TTCTATAATC

590 TCATTTATAA

GAATTAAAAT

530 CCAACTTATA

540 TGTGAAAATT

790 ACCATCTCTT

800 810 GCTAATTCAG CCTATATACC

820 830 840 GCCATCTTCA GCAAACCCTA AAAAGGTATT

850 860 870 880 AAAGTAAGCA AAAGAATCAA ACATAAAAAC GTTAGGTCAA

890 900 GGTGTAGCCA ATGAAATGGG

910 920 AAGAAATGGG CTACATTTTC

930 TTATAAAAGA

940 ACATTACTAT

1000 TAGAGAGCTT

1020 1030 1040 1050 1060 AGCAATGAAG TACGCACACA CCGCCCGTCA CCCTCCTCAA ATTAAATTAA

1010 AATTGAATTG

770 780 GATAAACCCC GCTCTACCTC

520

450 CCGCGGTCAT

460 470 ACGATTAACC CAAACTAATT

740 TATCCATCTA

5QQ 510 ATAAA TAAATAAATA

430 GGTAAATTTC

950 ACCCTTTATG

960 970 980 AAACTAAAGG ACTAAGGAGG ATTTAGTAGT

990 AAATTAAGAA

1070 1080 ACT AACATA ATTAATTTCT

tRNA 61

1090 1100 1110 1120 1130~~~~~ 1140 AGACATCCGT TTATGAGAGG AGATAAGTCG TAACAAGGTA AGCATACTGG-4AAGTGTGCT

60 1170 TGGAATAAT,C ATAGTGTAGC TTAATAHTAA

1180 1190 1200 AGCATCTGGC CTACACCCAG AAGATTTCAT

1240 CCTAG~~~~,(

1270 TATATAAATC

1280 AAAACATTTA

1290

1300

TCCTACTAAA

1310

1330

1410 TTTGCATAAT

1420 1430 GAACTAACTA GAAAACTTCT

1460 1470 1480 1490 GAAACCCCGA AACCAAACGA GCTACCTAAA AACAATTTTA

1500 TGAATCAACT

1510 CGTCTATGTG

1560

1570

1580

TAACGAGCTT

GGTGATAGCT

1630 AACAAAATCA

1640 AAAAGTAAGT

1650 TTAGATTATA

GCCAAAAGAG

GGACAGCTCT

TCTGGAACGG

AAAAAACCTT

1720 AACAGCTTTT

AACCATTGTA

1750 CAGCCACCAA

1760 TAAAGAAAGC

1770 GTTCAAGCTC

AACATAAAAT

1820

TATAACAACT

1990 CTGTTAACCC

AACACCGGAA

2080 2170 TAATCACTTG

TTCCTTAATT

2180

2260

2530

ATGAACGGCT

2280

.2620

AGAGAAGGTTIATTAGGGTGG

2640 2i&m

2980

2920 CATTCTAATC

3000 TTGTTGGTCC

2930 GCCATAGCCT

3010 ATACGGCATT

3090 CCTTATTTAT

AGACTTAAAA

3020 TTACAACCAT

3100 TATTGCACCT

3110 ACCCTATCAC

2680 TATTTACGAT

2760 ATAAAAICTA

2840

2590 GTGTAGAAGC

2670 GGTTTCTATC

2750 TAATTTATGA

2830

2910

3080 ACCTCTATAT

2740 TCAACT

2500 TACCCTAGGG

2580 CATCCCAATG

2660 AATCCAGGTC

AAATTGCGTA

TCCTCGTCCC

2990 GGCCCTAACA

3070 CCCTTTAACA

G

2820

2900

2570

2650

ACAAATA

2490 CGGACCAAGT

TGGATCAGGA

AGACCGGAGC

aeII

CAGAGCCAGG

ATCCTAACAC

ACTACGAAAA

2560 ACCTCGATGT

ATCTGAGTTC

28QQ tRNALa;810

2480 TTTTGATCAA

2410

2770

2850 CCTTGTTCCC

2940 TCCTAACATT

3030 TTGCAGACGC

3120 TCACACTAGC

2420 GATTATAACC

AGAGGTTCAA

2950 AGTAGAACGC

3Q4Q CATAAAATTA

3130 ATTAAGTCTA

2430 TAGACTTACA

2510 ATAACAGCGC

2520 AATCCTATTT

2600 TATTAATGGT

2610 TCGTTTGTTC

2690 2780 ATACGTACAC

2860

2340 AAACCTAATG

TTCTCCCAGT

AATAAAATAT

2250 TTGACCTTTC

2330 TTAATTTATT

TCCTCCGAAT

2160 AAAGGTAGCA

2240

2320

2400

2070 TACCAAAAAC

2150 CTGACCGTGC

ATCAGTGAAA

ACTTATCTAT

GAATAAAAAA

2060

2230

2310 AAATTATATA

2390 TGACCTCGGA

2470 CCCAGATATA

2550

2720 GAGCCANCTT

2890

2380

2460

2630

2300 TATGGAGCTT

TCGGTTGGGG

AGGGTTTACG

GTCCTACGTG

CTTTATTAAT

2290

1980 TATAACTTCT

CCCGCCTGTT

2140

CTTATCTTTA

CAATTCTCCA

1970 ATAATCCACC

CCGCGGTATC

2220 CCAACTGTCT

1890

ACA 2% AATTC

2050

2130 AGTTTAACGG

2210 AAACGAGGGT

GAGAAGACCC

2370

2540

2120

1800 NCCATAATTT

E

1960

AAACAAGAAC

1710 TAATTAACAA

1790 TTCAATTAAT

CACACTATAA

2040 GGAACTCGGC

CAGTGACTAA

2200

ATCTTATTGA

ATCGACAATT

2710 AAGAGAAATA

2190 AGGGACTAGC

2450

2030 AAAGATAAAA

2110 ACTGCCTGCC

GTTTGAAATT

AAATCAACAT

AACGATTAAA

2100 TATTAGAGGC

2360

2440

2020

1700

1780

1950 AGGCAATAAT

1620 CTAAAAAAAC

TAATAGTGAA

1870

1440 AACTAAAAGA

1610 TTTAAACTTG

AGTATGAGTA

1350 CAAGGGAAAG

1520 1530 GCAAAATAGT GAGAAGATTT

1690

1860 CAACACTGTT

1940 ATCAGACTAT

AAAGATCCAA

ATAATAAGAC

TATAGTATAA

AAGAGTTCAT

2010

2270 CTGAAATATA

2350

1930

TAAGTTCAAT

1680

1850 TTTATAGATG

TTGTTAGTTA

TGCCTAAAGG

2090 GCATTACAAG

AGTCAAAGTA

1920

2QQQ

1670

1840 AATCTATAAC

CGGATAACCA

ATCACCTCTA

GCCCAAAAAC

1830 AAATTGGGTT

1910

GACATACGCG

AGTGAAGAGG

1740 GGCCTAAAAG

CCTAAACTTA

1900

1660

AAAATGAATT

ACTAGTACCG

1600

GTGAAAAGCC

1730

GGTTACCCAA

1590

TTAGGTAGAG

1810 ACACCAACTT

1340

1380 1390 1400 AAGAACAAGC AAAGATTAAA CCTTGTACCT

1550

AAAGAAATTC

1320

GAGCTATAGA

1540

AGTATTGGAG

1250 CACAAATATA ATTATA;::;

GTACATCTAG

1360 1370 ATGAAAGACT AATTAAAAGT 1450 ATTACAGCTA

1210 12FrRNA-iX GACCAATGAA CAC1CTGAP.C TAATCCTAGC

7870 ATCCTCTCCC

2960 AAAATCTTAG

3050 TTTATAAAAG

3140 TGAGTTCCCC

2700 ACGAAAGGAC

----2790 LCJCTAACCT

2880 TAATdGTGTT

2970 GGTACATACA

3060 AACCAATACG

3150 TACCAATACC

3’ Flgure

3.

Complete

DNA Sequence

of the rRNA

Region

of Mouse

L Cell mtDNA

All sequence shown is of the L strand of mtDNA, which is the sense strand with respect to transcripts from th!s regton. Genes for the tRNAs and rRNAs are indicated by boxed regions. Recogmtlon s&s for the restrlction enzymes Hint II, Eco Rland Hae Ilare shown. Nucleohdes designated Nat numbers 1167 and 2717were not determined by DNAsequencmg.

Cell 160

5’ A

3’

%

2 ?A TAA~;$~

A

ACA

A

GGATA AT ATA nn; ^^

G

TAAAI@ A NTA

A *

Figure 4. Structures

5’A f

g

18

GC

GC EC”

T’ dAGTAACC

T” ‘CCTATGT

A

3’

3’

5’ A CG

TTCATG

“:ccGA::c&G A

GAAA

TA

T C-k%TT)

Proposed Cloverleaf Secondary for the Three RNA Genes

Anticodons and unorthodox base pairs are indicated by boldface type. The N in tRNA”“‘is a nucleotide which was not determined by DNA sequencing.

r Tr UTG’T-i

AAT E

nnf

G

E A

Tc

A

TAA tRNALeU

homology is due to the presence of a gene for tRNA”’ which has been identified by examining the DNA sequence (data not shown). No other sequences show homology to the probe, including the 250 and 210 bp fragments, which contain 16s rRNA gene sequences. Sequencing of mt rRNA End-labeled mt rRNAs were isolated by electroelution from 4% polyacrylamide, 50% urea gels, and their termini were sequenced enzymatically according to the method of Donis-Keller, Maxam and Gilbert (1977). 65-120 bases of nucleotide sequence could be read from the autoradiograms for each mt r-RNA end, except for the 3’ end of the 16s mt rRNA. On some of the autoradiograms the relative positions of G and A can be read up to 200 bases from the end of an RNA, although the number of pyrimidine bases between G and A is difficult to determine accurately for the internal 100 nucleotides. All the RNA sequences can be matched with mtDNA sequence without interruptions. For RNAs labeled at the 5’ end the first few bases are difficult to determine with confidence from sequencing gels alone due to the presence of artifactual bands on the autoradiograms of the 20% acrylamide sequencing gels. Therefore the identities of the terminal bases were determined using thin layer chromatography on PEI-cellulose after digestion of 5’ labeled RNA with nuclease Pl and of 3’ labeled RNA with RNAase T2 (Figure 6). The end analyses demonstrate unambiguously that the 5’ terminal base of both mt rRNAs is A. This helps identify the first few 5’ terminal bases on the 20% sequencing gels and allows precise placement of the 5’ ends of both mt rRNAs on their DNA coding sequences (Figure 3). The 5’ end of the 12s rRNA gene is nucleotide 194, one nucleotide away from the 3’ end of the tRNAPhe gene. The 5’ end of the 16s rRNA gene is nucleotide 1219, immediately adjacent to the 3’ end of the tRNAVa’gene. The 3’ terminal nucleotide of the 12s mt rRNA migrates with or near 3’ AMP on the PEI-cellulose thin layer (Figure 6). However, this base is identified as a

pyrimidine on the autoradiogram of a sequencing gel (Figure 7) because it is not recognized as G by RNAase Tl or as A by RNAase U2. Lack of cleavage by RNAase U2 cannot be explained by the terminal position of this nucleotide since the enzyme is able to cleave dinucleotides (Uchida and Machida, 1978). The band labeled 1 Y on the sequencing gel shown in Figure 7 cannot be an artifact caused by unincorporated 5’ 32P-pCp because the labeled RNA was electrophoretically purified before it was sequenced. By matching the direct RNA sequence of the 3’ end of the 12s mt rRNA with its DNA coding sequence (Figure 3), the band labeled 1 Y (Figure 7) and representing the 3’ terminal nucleotide of 12s mt rRNA is clearly identified as U. Anomalous migration on PEIcellulose (Figure 6) suggests that this terminal U is post-transcriptionally modified. The nature of this modification has not been determined. The 3’ end of the 12s mt rRNA gene is therefore nucleotide 1149, immediately adjacent to the 5’ end of the tRNA”“‘gene (Figure 3). Ribonucleotides 18 and 19 of the 12s mt rRNA (Figure 7) are not recognized as A by RNAase U2; however, the sequence of the 12s rRNA gene indicates that these nucleotides are transcribed as As (nucleotides 1131 and 1132 in Figure 3). Presumably these A nucleotides are post-transcriptionally modified, but the nature of these modifications has not been identified. Attempts to determine the sequence of the 3’ end of the 16s mt rRNA have so far been unsuccessful. The 3’ terminal base of the 16s rRNA as determined by PEI-cellulose chromatography is U (Figure 6). This, combined with other available information (see Discussion), allows a tentative assignment of the 3’ end of the 16s mt rRNA gene at nucleotide 2799 or 2800, zero or one nucleotides away from the 5’ end of the tRNAL”” gene (Figure 3). Discussion The first evidence that the rRNA coding region of mouse mtDNA might be entirely represented by tran-

Mouse 161

Mitochondrial

rRNA

and tRNA Sequences

HR-B Xba I B DC HaelII Hinf I

HR-D

Hae III

HR-B Xba I B D C HaeIlI Hinf I

640530480-

A

B

tRNALeU 3’

~

16s

rRNA

tRNnv”’

125 rRNA

\::

t RNAPhe :a

io

5’

A HR-D

1 EcoR I

HR.8

7 XbaI

t Hae Jll

k Hinf I

scription products was provided by experiments in which DNA-RNA hybrids were formed between total mtRNA and restriction fragments of mtDNA which spanned the rRNA region (Nagley and Clayton, 1980). Mild digestion of these hybrids with Sl nuclease generated several discrete RNA-protected DNA species which were greater in length than the sum of the lengths of the 12s and 16s rRNAs. This result is best explained by the presence of several short transcripts interspersed among the rRNAs in the hybrids. If the distances between the ends of the various transcripts in the hybrid were small, then Sl might not cut the resulting short single-stranded DNA regions under these less stringent conditions. The data indicated that four small RNAs mapped in the rRNA region. One small RNA about 60 nucleotides in length mapped at the 5’ end of the 12s rRNA gene, a transcript of about 80 nucleotides in length mapped in the region between the 12s and 16s genes and two transcripts of about 80 nucleotides each were mapped at the 3’ end of the 16s rRNA gene. In addition, to account for the Sl protection results, these short RNAs had to map very closely to the ends of the rRNAs, within several nucleotides at most. This remarkably economical usage of DNA coding sequence has been detailed here at the nucleotide

Figure 5. Hybridizahon to Restriction Fragments

of Labeled mt WINAs of L Cell mtDNA

(A) Hae Ill fragments of HR-D. Ethidium bromide staining pattern of the gel (1.8% agarose) is at left, with the accompanying autoradiogram of the hybridized filter on the right. Sizes of restriction fragments are indicated. The 70 bp fragment has run off the gel and was not transferred to the filter. (B) Xba I fragments of HR-B. Xba I-B. -C and -D were isolated from an Xba I digest of HR-B by gel electrophoresis and electroelution. Xba I-B was cut a second time with Hae III and Xba I-C was cut with Hinf I while Xba I-D was not restricted further. Ethidium bromide staining of a 1.8% agarose gel of the fragments is at left, with an autoradiogram of the filter at the right. 0 Map of restriction sites of the rRNA regron of mtDNA used in generating Southern filters. Sizes m base pairs of restriction fragments are indicated. Only a porhon of HR-B is shown.

level. The 5’ boundaries genes and the 3’ boundary identified precisely by DNA sequence. There is ing between the 3’ end first 5’ nucleotide of the

of the 12s and 16s mt rRNA of the 12s gene have been matching RNA sequence to a single nucleotide intervenof the tRNAPh” gene and the 12s rRNA gene. There are

no intervening nucleotides between the 3’ terminal nucleotide of the 12s gene and the 5’ end of the tRNAVa’gene, and there is none between the 3’ end of the tRNAVa’ gene and the initial 5’ nucleotide of the 16s rRNA gene. The 3’ end of the 16s rRNA gene was not determined directly but has been tentatively assigned as being immediately adjacent to the gene for tRNALeU. The gene for tRNALe” is located 1582 nucleotides from the 5’ end of the 16s rRNA gene, while the length of the 16s rRNA, as determined by electrophoresis of glyoxalated RNAs (McMaster and Carmichael, 1977), is approximately 1650 nucleotides (data not shown). This places the 3’ terminus of the 16s rRNA gene very close to the 5’ end of the tRNAL”” gene. The two nucleotides immediately upstream of the tRNAL”” gene are both T whereas the 3’ terminal nucleotide of the 16s rRNA has been shown to be U. Therefore a tentative assignment of the 3’ end of the 16s rRNA gene to one of these two T residues is consistent with both the experimental determination of the 3’ terminus of the 16s rRNA and the requirement that the end of the 16s gene be very close to a gene for a small RNA. No second tRNA gene can be identified on either side of the tRNALe” gene and it has now become clear that this second small RNA mapped at the 3’ end of the 16s rRNA was an artifact of the Sl digestion. The cloned mtDNA used in forming the rRNA-DNA hybrids had been cleaved at the unique Hae II site where it was inserted into the vector. However, the 3’ end of the 16s rRNA, as placed above, overlaps the Hae II site by about 70 nucleotides, creating a nick in the DNA strand of the

Cdl 162

hybrids 70 nucleotides internal to the 3’ end of the 16s rRNA. Sl cleavage of the intact 16s rRNA across from the nick would therefore appear to demonstrate a second small RNA adjacent to the 16s rRNA. We conclude that there is only one small RNA encoded at the 3’ end of the 16s rRNA gene, tRNAL”“. Consistent with this is the fact that an open reading frame for mitochondrial translation begins just downstream from the tRNAL”“gene and extends for 1054 nucleotides to the gene for tRNA”‘, indicating that the next transcript 3’ to tRNALe” may be an mRNA for an unidentified mitochondrial protein. interesting Properties of the Two mt rRNAs Absence of Splicing Three lines of evidence indicate that splicing of mouse mt rRNAs does not occur. First, direct comparison of the RNA and DNA sequences yields no discontinuities. Therefore there can be no splices within 100-200 nucleotides from either end of the 12s mt rRNA or the 5’ end of the 16s mt rRNA. Second, the lengths of the rRNAs determined by denaturing gel electrophoresis are 950 and 1650 nucleotides (data not shown).

F+

C A U G 0-b

-II

16s 12s 16s 12s 5’ 3’ Figure 125

6. and

5’ and 3’ End Analyses 16s

mt rRNAs

were

of 12s digested

and 16s

mt rRNAs

to 5’ mononucleotides

by

nuclease Pi for 5’ labeled RNA or to 3’ mononucleotides by RNAase T2 for 3’ labeled RNA. The PEI-cellulose thin layers were developed 4 cm in 1 N acetic acid followed by 0.3 M LiCl to 15 cm. The positions of unlabeled marker mononucleotides, circled with dashed lines, were determined using short-wave ultraviolet light. The positions of labeled terminal mononucleotides were determined as shown by autoradiography. The 3’ terminal nucleotide of the 12s mt rRNA appears to be A; however, the RNA sequence shown in Figure 7 suggests that this nucleotide is a pyrimidine and the DNA sequence suggests that it is U. (0) the origin. (F) the solvent front.

Within the limit of resolution of electrophoretic determination of the length of RNAs, these lengths are the same as the genomic distances between the regions coding for the 5’ and 3’ ends of the mt rRNAs, 956 and 1582 bases, respectively. Therefore the mt rRNA genes contain no long intervening sequences. Finally, Battey and Clayton (1978) have shown that digesting mouse mt rRNA-DNA hybrids with nuclease Sl reduces the size of hybridized DNA to roughly the same length as that of the mt rRNAs. Therefore there are no intervening sequences in the midportions (more than 200 bases from the ends) of the mt rRNA genes. Although the possibility of a very short intervening sequence within the 3’ terminal 200 nucleotides of the 16s mt rRNA gene has not been ruled out, it is probable that mt rRNAs are not spliced. Attardi et al. (1979) have also shown data from Sl nuclease and Exo VII protection experiments which suggest that HeLa cell mt rRNAs are not spliced. Since these RNAs are not spliced, the entire sequence of mouse 12s and 16s mt rRNA is determined from direct RNA sequence at the 5’ and 3’ ends and the DNA coding sequence between those ends. Comparison to Other rRNAs The mt rRNA sequences were searched for significant dyad symmetries using a computer program developed by Korn et al. (1977). A large number of long dyads with stems greater than 8 bp and as long as 20 bp could be formed. However, there are no data to support the existence of these hairpin structures. Many of the possible hairpin structures are mutually exclusive. Dyad symmetries therefore are not reported here, with the exception of one near the 3‘ end of the 12s mt rRNA which is discussed later. Both mt rRNAs contain sequences and structures which are homologous to other rRNAs. Except for 5s and 5.85 RNAs the only complete rRNA sequences available are those of E. coli 16s rRNA (Brosius et al., 1978; Carbon et al., 1978), E. coli 23s rRNA (Branlant et al., 1979; Brosius, Dull and Noller, 1980) and Zea mays chloroplast 16s rRNA (Schwartz and Kiissel, 1980). The 16s rRNAs of E. coli and corn chloroplast are very similar, showing nearly 75% homology. The major comparisons of mouse mt rRNAs will be made with E. coli rRNAs. Using the computer program developed by Korn et al. (1977) the rRNAs can be compared to each other for homologous sequences. In addition to identifying homologous sequences the program calculates the probability of finding such a homology in two random sequences. Initial comparisons of mouse mt rRNAs to E. coli rRNAs were made by considering only strong homologies, defined as 75% or greater homology and having less than a 1 Oe5 chance of occurring between random sequences. These homologies are shown in Tables 1 and 2. Most of these are in the same order relative to one another in the mouse mt and E. coli rRNAs. The relative locations of most of the strong homol-

Mouse 163

Mitochondrial

rRNA

and tRNA

Sequences

12s -3’ T, OH-U,

ogies between mt rRNAs and E. coli rRNAs is shown in Figure 8. The strong homologies, indicated by bold lines, vary in length from 10 to 40 nucleotides. The regions of lesser homology, indicated by thin lines, are either longer or of equal length in the E. coli rRNAs relative to the mt rRNAs. Due to the constraints imposed by a two-dimensional figure only homologies which are in the same relative order in the two rRNAs were used in constructing the diagrams shown in Figure 8. The criteria used for constructing these diagrams included the length and relative positions of homologies such that the greatest number of homologous sequences could be shown. Although not shown in Figure 8, strong homologies which are not in the same relative positions in the rRNAs are included in Tables 1 and 2. Regions of strong homology between E. coli rRNAs and mouse mtrRNAs are limited to stretches of 40 nucleotides or less. Regions of a lower degree of homology do not fall into an ordered pattern in a manner similar to the regions of strong homology and, therefore, the significance of the lesser homologies is difficult to assess. It is clear, however, that the degree of homology between mouse mt rRNAs and E. coli rRNAs does not approach the degree of homology between Zea mays chloroplast and E. coli 16s rRNAs (Schwartz and Kossel, 1980). The presence of strong homologies shown in Tables 1 and 2 does, however, suggest that the rRNA genes of animal mitochondria, chloroplasts and E. coli have evolved from common

-

40

30

Table 1. Regions of Strong rRNA and E. coli 16s rRNA Homology

Mouse

Homology

mt 12s

237-246

IV

Y = PYRIMIDINE Figure 7. Determination mmw of 125 mt rRNA

of the Nucleotide

Sequence

of the 3’ Ter-

The 3’ termlnal sequence of the 12s mt rRNA is shown as displayed on a 20% acrylamide. 50% urea gel. The OH- lane, wth cleavages at all bases, serves as a size standard. RNAase Tl cuts primarily after G and RNAase U2 cuts primarily after A. Material run in the (-) lane was incubated under conditions Identical to those for Tl and U2 lanes except that no enzyme was added. The 3’ terminal base of the 125 mt rRNA IS not recognized as A or G by the enzymes and is therefore assigned as a pyrimidine (Y). According to DNA sequence in this region of the genome this nucleotide IS U. C and U cannot be distinguished by the enzymes used in these experiments. The bases marked with asterisks are As according to the DNA sequence but are not recognized as As by RNAase U2.

between

Mouse

mt 12s

E. coli 16s 51-60

2’

365-374

1161-1170

3

402-415

459-472

4’

413-427

418-431

5

422-454

504-538

6’

472-496

803-826

7’

556-570

906-918

8

61 O-629

778-796

9

699-724

900-925

10’

706-718

1144-1158

11’

987-l

003

1429-1446

12

1027-l

044

1393-1411

13

1105-1145

1491-1530

Homologies were located using a computer program developed by Korn et al. (1977). Insertions or deletions of up to three nucleotides were allowed. The criteria for including a homology in this table were 75% or greater homology and less than a 1 Om5 probability of occurrmg by chance between random sequences. Sequences are numbered according to Ftgure 3 for mouse 125 mt rRNA and according to Brosius et al. (1978) for E. coli 16s rRNA. Asterisks indicate that a homology was not used in constructing Flgure 8.

Cdl 164

protoribosomal RNA genes of a procaryotic or preprocaryotic organism. mt rRNA genes appear to have diverged to a greater degree than the chloroplast or E. coli rRNA genes. The presence of a number of strong homologies between the mt rRNAs and E. coli rRNAs which do not fall into the same relative order as those shown in Figure 8 suggests the possibility of rearrangement of blocks of sequence within the mt rRNA genes during the course of evolution. Crews and Attardi (1980) have reported the sequence of 285-286 nucleotides at the 5’ end of the 12s mt rRNA gene of HeLa cells. This sequence is 70% homologous to the corresponding region of mouse 12s mt rRNA. It is interesting that this degree of homology is less than that between corn chloroplast and E. coli 16s rRNAs, about 74% (Schwartz and

Table 2. Regions of Strong rRNA and E. coli 23s rRNA Homology

Mouse

Homology

mt 16s

between

Mouse

Mt 16s

E. co11 23s

1235-i

253

332-349

2’

1262-l

301

2259-2279

3’

131 O-l 324

2305-2318

4

1327-1357

5

1396-1412

6’

1538-l

551

1276-l

290

7’

1704-l

717

1780-l

793

8

1728-l

763

1058-l

094

9’

2025-2038

2429-2442

10

2030-2044

1664-l

11*

2039-2055

12

2057-2072

1774-l

789

13

2102-2116

1829-l

842

14

2153-2172

1927-l

946

447-479 561-577

678

Kossel, 1980). Crews and Attardi (1980) point out three regions of homology between HeLa 12s mt rRNA and E. coli 16s rRNA. The three analogous regions of mouse 12s mt rRNA are 91-97% homologous to HeLa 12s mt rRNA. Nisen and Shapiro (1979) have shown that E. coli rRNAs contain regions of strong homology to the sequences of insertion elements IS1 (Ohtsubo and Ohtsubo, 1978) and IS2 (Ghosal, Sommer and Saedler, 1979). Both mouse mt rRNAs contain regions strongly homologous to IS1 and IS2. Using the criteria for comparing mt rRNA to E. coli rRNA sequences, six strong homologies were found between mouse 12s mt rRNA and ISl, seven were found between 12s mt rRNA and IS2, six were found between mouse 16s mt rRNA and IS1 and fifteen were found between 16s mt rRNA and IS2. 40% of the regions of mt rRNAs homologous to insertion elements are also strongly homologous to E. coli rRNAs. The homology between IS1 and E. coli 16s rRNA reported by Nisen and Shapiro (1979) is strongly conserved in mouse 12s mt rRNA, as shown by homology number 6 in Table 1. One of the most interesting regions of homology is contained within the terminal 50 nucleotides at the 3’ end of all small ribosomal subunit rRNAs for which the sequence is available (Figure 9). This region of the E. coli 16s rRNA is known to contain an mRNA binding site (Shine and Dalgarno, 1974; Steitz and Jakes, 1975), a 10 bp hairpin structure with two dimethyladenines in a 4 nucleotide loop (Yuan et al., 1979). as well as the colicin E3 cleavage site (Nomura et al., 1974). A 10 bp hairpin structure containing two As in a 3 or 4 nucleotide loop can be drawn for all small subunit rRNAs for which sequences are available (Fig-

473-488

15’

2189-2207

1127-1147

16

2233-2248

2007-2022

17’

2234-2249

2089-2106

18’

2250-2267

967-981

19

2277-2301

2051-2069

20’

2396-2415

217-235

21

2499-2508

2445-2454

22

2522-2537

2468-2483

23

2552-2576

2497-2521

24

2616-2647

2560-2592

25’

2650-2663

2294-2308

26

2684-2706

2649-2671

27’

2793-2813

924-945

Homologies were located as described In Table 1. Sequences are numbered according to Figure 3 for mouse 16s mt rRNA and according to Brosius et al. (1980) for E. coli 23s rRNA. Asterisks indicate that a homology was not used In constructing Figure 8.

MITOCHONDRIAL 12s E COL, 16s 5’W

MITOCHONDRIAL

E.CoLI

16s

23s

Figure 8. Relative and E. coli rRNAs

0

3’

2 3 4 5 6 7 8 9 10 II I2 13 14 15

5w3’ Locations

of Homologous

Sequences

rn Mouse

mt

mt rRNAs are represented as bold straight lines divided into units of 100 nucleotides. Strong homologies between mt rRNAs and E. coli rRNAs are indicated by short bold segments of the thin lines representing E. coli rRNAs. Strong homologies are defined as in Table 1. Only those homologies in the same relative order in mt rRNAs and E. coli rRNAs were used in constructing thus figure. The locations of all strong homologres between mouse mt rRNAs and E. coli rRNAs are shown in Tables 1 and 2. All rRNAs are drawn to the same scale in a 5’ + 3’ orientation. The 16 nucleotide homology shown nearest the 5’ end of the smaller rRNAs is 82% homologous and has a 2 X 1 Om5 probability of occurring by chance wrthin the two sequences. It is included here because of its similar location near the 5’ end of both rRNAs (nucleotides 2-l 7 from the 5’ end of mt 12s rRNA and nucleotides 7-23 rn E. coli 16s rRNA).

Mouse 165

Mitochondrial

rRNA and tRNA Sequences

ure 9). The two As in the loop in this structure are known to be modified to 2, 6-dimethyladenine in the E. coli 16s rRNA (Carbon et al., 1978) and the rat 18s rRNA (Alberty, Raba and Gross, 1978). It is probable that the analogous As are modified in the mouse 12s mt rRNA since they are not recognized as A by RNAase U2 (Figure 7). It is interesting that the degree of sequence homology within the stem of this hairpin between the 12s mt rRNA and the other small subunit rRNAs is not impressively high. In this stem structure there is a higher degree of homology between the E. coli 16s rRNA and chloroplast or eucaryotic rRNAs than there is between the mouse 12s mt rRNA and any other rRNA. The strict maintenance of a secondary structure in the face of changing primary structure during the course of evolution suggests an important functional role in protein synthesis for this region of small subunit rRNAs. Azad (1979) has suggested that this structure may be important in binding

Rot I85

G A G +6 ” AA G A GIA 2 G-C G-C U-t C-G G-C G-C A-” A-U A-U U-G U-G U-G G-C G-C G-C C-G C-G C-G C-G C-C C-G G-C “-A *-” “-A A-” A-” U-G U-G U-G G G G G G G G G A G A G A A A ” A ” A A ” A C A C U A C c A c n c A n A C A U n A C A c A U A c ‘J u A ‘OH u u u ” ‘OH G C G C G C c c C c u uu u u ” G ” A r; -: n\ A bH A A OH A G n G n ” ”

G A* G Ax u-n C-G A-” U-0 A-” C-G G-C A-” A-L U-G G F A A c A A U G E ” G A

G i’

G 4

8A $“~.“A

G.C’ G-C A-U ::t C-G C-G “-A u-n U-G G G G A A ” A C h

A” ” P bH

I! 5’

Figure 9. Structural and Sequence Homology at the 3’ Ends of Mouse mt 125 rRNA and Procaryotic, Eucaryohc and Chloroplast Small Ribosomal Subunit rRNAs The 3’ end of mouse 12s mt rRNA contains a hairpin structure. similar to that found in all other small ribosomal subunit rRNAs, which is made up of a 10 bp stem and a 4 base loop. This hairpin has been shown to exist as a stable structure in E. coli 16s rRNA (Yuan et al., 1979). The loops all contain two adjacent As at the 3’ end of the loop which are known to be modified to m:A m E. coli and rat rRNAs. The analogous As (asterisks) of 12s mt rRNA appear to be modified since they are not recognized as A by RNAase U2. as shown in Figure 7. Analogous modifications have not been demonstrated directly In B. mori i&S rRNA (Samols, Hagenbdchle and Gage, 1979) or corn chloroplast 16s rRNA. 3’ terminal nucleotides of the rRNAs are indicated by (-OH). Nucleotides common to all sequences are indicated by bold letters. There are 15 contiguous nucleotides common to all sequences 5’ to the hairpin structure. This regtion in the E. coli sequence contains the cleavage site for collcin E3 (arrow). All sequences diverge at the same point 5’ to this conserved sequence except the E. coli and corn chloroplast 16s rRNAs. Only the E. coli and corn chloroplast 16s rRNAs contain the mRNA binding site ACCUCC (Shine and Dalgarno, 1974). 3’ to the hairpin structure.

the two ribosomal subunits through interaction of the larger rRNA and the 5s rRNA with this region of the small subunit rRNA. No 55 rRNA has ever been isolated from mitochondria. If binding the two ribosomal subunits does involve this region of the 12s mt rRNA then the interaction may involve only the 16s mt rRNA. The mRNA binding site, ACCUCC, is found at the 3’ end of E. coli (Shine and Dalgarno, 1974) and corn chloroplast (Schwarz and Kossel, 1980) 16s rRNAs. Mouse 12s mt rRNA and eucaryotic 18s rRNAs do not contain this sequence at their 3’ ends. Hagenbuchle et al. (1978) have suggested that a different sequence at the 3’ ends of eucaryotic 18s rRNAs may perform a similar function. A similar situation may apply to mt rRNA-mRNA interactions. However, in the only published sequence of an animal mitochondrial gene, the human cytochrome oxidase subunit II gene (Barrel1 et al., 1979), the protein coding sequence is immediately adjacent to the tRNAASp gene. Thus if there is no RNA splicing it is doubtful that there is a nontranslated leader sequence on mt mRNAs which binds to the 3’ end of the 12s mt rRNA. Although splicing of animal mt mRNAs has not been totally ruled out it does not seem to occur. It is possible that there is a region within the protein coding sequence of animal mt mRNAs which is involved in mRNA recognition by ribosomes. Little is known about the nature of the 5’ end of animal mt mRNAs; however, Grohmann et al. (1978) have shown that at least the majority of HeLa mitochondrial polyadenylated RNAs are not capped. Therefore it is unlikely that initiation of mitochondrial translation involves a cap structure on mt mRNA, as has been suggested for eucaryotic translation (Patterson and Rosenberg, 1979). The interactions responsible for ribosomal recognition of the 5’ ends of mt mRNA coding sequence are not clear and speculation on possible interactions requires more information concerning the structure of the 5’ ends of mature animal mt mRNAs.

Unusual

Characteristics

of Mouse

mt tRNAs

The proposed structures of the three mouse mt tRNAs are highly unusual. Each of the three tRNA genes has at least one non-Watson-Crick base pair in the DNA cloverleaf secondary structure. It is possible that the cloned plasmid DNA used in sequencing has diverged from the original mouse sequences, leading to the generation of aberrant base pairs when a secondary structure is drawn. This is unlikely, in view of the fidelity of the sequence of the adjacent rRNA genes obtained from the same DNA, when compared to the rRNA sequence itself. The most common type of mismatch is a GT base pair which would correspond to a GU pair in the tRNA. This type of interaction is permitted under the rules of Crick’s “wobble hypothesis” (Crick, 1966) and GU base pairs are present with some frequency in published tRNA sequences (Sprinzl et al., 1980). These base pairs probably can exist in

Cell 166

a stem without gross distortion of the RNA helix. The other three kinds of mismatches, an AA pair in the amino acid stem of tRNAPhe, an AC pair in the anticodon stem of tRNALe” and a UC pair in the amino acid stem of tRNAVa’, occur much less frequently in published tRNA sequences. It is not known what effects these unorthodox base pairs have on the double helical structure of the various stem regions of the tRNAs, but it is noted that the AA and UC base pairs occur as solitary mismatches in the middle of relatively long base paired stems. It also is not possible to tell from the sequence of the gene which if any of the nucleotides in the mature RNA contain modified bases. Location of modified bases in mouse mt tRNAs will require direct sequence analysis of the RNA. Several other mouse mt tRNA genes have been identified (R. A. Van Etten, unpublished data) and there are several features common to all the tRNAs. Each has an amino acid stem of 7 bp, an anticodon stem of 5 bp and an anticodon loop of 7 nucleotides. The first arm on the 5’ half of the molecule, traditionally called the D arm, has a stem of either 3 or 4 bp and a loop of between 3 to 10 nucleotides. There are two nucleotides between the amino acid stem and the D stem and a single nucleotide, always a purine, between the D arm and the anticodon arm. There is a small variable arm on the 3’ side of the anticodon stem which contains either 4 or 5 nucleotides. The major arm on the 3’ side of the molecule, called the TX arm by convention, has a stem of 5 bp and a loop size of from 4 to 8 nucleotides and there are no nucleotides between the end of the TW stem and the beginning of the 3’ half of the amino acid stem. The first nucleotide in the D stem is always a G, and it is always paired to a pyrimidine. The two nucleotides in the anticodon loop on the 5’ side of the anticodon are pyrimidines and the nucleotide immediately adjacent to the anticodon on the 3’ side is always an A. The tRNA genes from mouse mitochondria are very similar to those in human mitochondria (Barrel1 et al., 1979, 1980) and the proposed tRNA structures from both species lack sequences in the D and TW loops and elsewhere which are known to be conserved in all other nonorganelle tRNAs which have been sequenced. Procaryotic and eucaryotic cytoplasmic tRNAs have exactly 7 nucleotides in the T\kC loop and the loop always has the sequence 5’TSCPuANPy-3’, where Pu and Py are purine and pyrimidine, respectively, and N may be any nucleotide. The D loop of these tRNAs has from 7 to 11 nucleotides, including several which are invariant or semi-invariant. The most significant of these is a pair of adjacent G nucleotides. In the tertiary structure of yeast tRNAPh”(Kim et al., 1974) these two G nucleotides are involved in hydrogen bonding with the ‘kc pair of the TWZ loop in an interaction which forms the basis of the three-dimensional L-shaped conformation of the molecule. Of the three mouse mt tRNAs re-

ported here only tRNALe” has these features. The gene for this tRNA has a GG in the D loop at the correct position and has a precisely conserved TW loop. The TX loop has 7 nucleotides, the usual number, and has the sequence 5’-TTCAAAT-3’. Pseudouridine is produced in tRNA by an enzymatic modification of a uridine (Altman and Smith, 1971) so that the second T residue could correspond to a \k in the RNA. This would mean that the T\kC loop of the tRNAL”” exactly conforms to the pattern shown by procaryotic and eucaryotic cytoplasmic tRNAs. The other two tRNAs, Phe and Val, do not show this pattern. There are no G nucleotides in either of the D loops of these tRNAs, and there is no conserved pattern discernible in the TW loops which, at 4 and 5 nucleotides, respectively, are both smaller than the 7 nucleotides in an orthodox TW loop. It is not apparent from examining the sequences of these two tRNAs whether the D and T9C loops can still interact in a manner similar to those in yeast tRNAPhe or whether they assume some different tertiary structure. Although this is the major interaction in the traditional tRNA structure several other hydrogen bonding interactions occur and serve to stabilize the conformation (Rich and RajBhandary, 1976). Each interaction corresponds to one or more nucleotides which are conserved in the sequences of all conventional tRNAs. Examples of this are a conserved purine at the second position of the D loop which interacts with an invariant pyrimidine at the end of the variable loop, a conserved CA pair at the first and last positions of the anticodon loop, an invariant U immediately 5’ to the anticodon and a GC pair at the end of the T9C stem. All these conserved nucleotides are found in the mouse mt tRNALe” but are not present in one or both of the other two tRNAs. It is clear that tRNALe” is very much like a conventional tRNA in terms of its conserved bases and potential tertiary structure, .while tRNAPhe and tRNA”“’ are considerably different in both features. If one considers any theory in which all mt tRNAs arose from ancestral procaryote-like tRNA genes, then it is not clear why at least one of these genes has changed so little while others have diverged drastically. This unexpected conservation could be accounted for if tRNALe” had some other function in the mitochondrion or elsewhere in the cell, in addition to its role in mitochondrial protein synthesis, which required maintenance of the original tRNA structure. As might be expected the mouse mt tRNAs show only slight homology with their cytoplasmic counterparts. The sequences for tRNAs Val and Phe show only about 38% homology with published sequences of mammalian cytoplasmic tRNAs for Val and Phe. No sequence information is available on mammalian cytoplasmic leucine tRNAs, but the mduse mt tRNALe” shows somewhat better homology with E. coli K12 tRNALe” (43%) and yeast cytoplasmic tRNALe” (45%) (Sprinzl et al., 1980). It is interesting to compare the

Mouse 167

Mitochondrlal

rRNA and tRNA Sequences

gene for mouse mt tRNAPhe and the corresponding gene from human mtDNA (Crews and Attardi, 1980). The mouse gene is 69 nucleotides long while the human gene is 71. The proposed secondary structures of the two RNAs are slightly different. The D loop of the human tRNA has 9 nucleotides while the corresponding loop in the mouse has 8; the TW arm in the human tRNA has a loop of 9 and a stem of only 3, while the mouse tRNA has a loop of 4 and a stem of 5. Taking these differences into account and matching comparable regions of the molecules yields a homology between the two of about 50 out of 70 nucleotides, or about 70%. A striking fact is that 15 of the 20 nonhomologous bases between the two tRNAs occur in the D and TW loops, while the stem regions and anticodon arm are closely homologous. The amino acid stem, in particular the fourth nucleotide from the 3’ end, the D stem, the anticodon arm and the variable arm have all been implicated as regions of E. coli tRNA molecules involved in recognition by aminoacyl tRNA synthetase enzymes which charge tRNAs with the correct amino acid (Schimmel and WI, 1979). This implies that perhaps those features of the two mammalian mt tRNAs which are important in the synthetase reaction have been conserved while the two loop regions have drifted. Conservative Gene Organization of the rRNA Region The delineation of the organization of the rRNA genes and associated tRNA genes of mouse mtDNA allows several hypotheses about the origin, transcription and processing of these genes to be made. The structure of rRNA operons in E. coli has been extensively studled (Dahlberg et al., 1978; Young, Bram and Steitz, 1979). The order of genes is 5-l 6S-23S-G-3’. Usually there are one or two tRNA genes in the region between the 16s and 23s genes and at the 3’ end of the 5s gene, with spacer regions between all the various genes. The operon is transcribed polycistronically into a single large precursor RNA and subsequently processed by the action of several enzymes mto mature rRNAs, 5s RNA and tRNAs. In eucaryotes the nuclear 18S, 5.8s and 28s rRNA genes are similarly transcribed as a single large RNA which is then processed to mature rRNAs (Hadjiolov and Nikolaev, 19761, but no tRNAs are present in the precursor RNA. The majority of eucaryotic nuclear tRNA genes appear to be transcribed as single cistrons (DeRobertis and Olson, 1979) or, rarely, as dimeric precursors (Beckman et al., 1979). Further evidence that transcription of eucaryotic nuclear rRNAs and tRNAs is independent is the fact that these two classes of genes are transcribed by different polymerases. RNA polymerase I transcribes eucaryotic rRNA genes while RNA polymerase Ill transcribes tRNA genes (Roeder, 1976). The arrangement of mouse mt rRNA and tRNA genes is reminiscent of procaryotic rRNA

operons and suggests that animal mt rRNA operons did not evolve from a nuclear rRNA operon. It seems more probable that mouse mtDNA arose from a procaryote-like genome by systematic deletion of nonfunctional spacer regions as well as of genes whose function could be assumed by the nucleus. The rRNA region of the mouse mtDNA genome shows amazing economy of organization. The close proximity of the rRNA and tRNA genes suggests that they are transcribed by a single RNA polymerase into a polycistronic transcript from which mature rRNAs and tRNAs are generated by single endonucleolytic cleavages at the 5’ and 3’ boundaries of the tRNAs. A high molecular weight transcript which hybridizes to mtDNA from the rRNA region has been observed in low abundance in previous transcript mapping experiments (Battey and Clayton, 1978). but the map position of this transcript is too imprecise to determine whether this RNA might be a precursor to the mt rRNAs. It is interesting to note that the 5’ end of the tRNAPhe gene is located only 200 bp from the D loop region of mouse mtDNA. This region contains the origin of H strand DNA replication and is maintained as a triple-stranded structure by continual synthesis and degradation of a family of short, single-stranded DNAs complementary to the L strand (Bogenhagen and Clayton, 1978). It therefore is possible that the origin of mtDNA replication and the major promoter for transcription of mtDNA are closely contiguous to one another, and interactions between the two functions may prove to be important in the expression of the mtDNA genome. It is not known whether a single enzyme might be involved in the processing of a polycistronic precursor or whether several enzymes participate. In E. coli, rRNA precursor sequences which are destined to become the terminal regions of mature rRNAs form part of large hairpin structures. These doublestranded RNA regions are then cleaved by the processing enzyme RNAase Ill to generate free 16s and 12s rRNAs (Bram, Young and Steitz, 1980). The sequences at and around the ends of the mouse mt rRNAs cannot form analogous hairpin structures, which suggests that these rRNAs are not processed by any RNAase Ill-like enzyme. At the same time it implies that the hairpin interactions involving the ends of the E. coli rRNAs are important only in providing information for processing the precursor and are probably not required for protein synthesis. In E. coli the enzyme cleaving precursor that the cloverleaf molecule processing mitochondrial in E. coli

RNAase P appears to be responsible for at the 5’ borders of the tRNAs in the rRNA (Lund and Dahlberg, 1977). It is possible mouse mt tRNA sequences could assume a conformation while still in the precursor and that this structure acts as a guide to a endonuclease which might function as a analog of RNAase P. Indeed, it is known that RNAase P must recognize some aspect

Cell 168

of tertiary structure since primary sequences adjacent to the cleavage site are different in different tRNA precursor species (Altman, 1978). Regardless of the exact mechanism it is clear that recognition and precise excision of tRNA sequences is a central feature of mtRNA processing. Experimental

Procedures

Abbreviations Used for DNA Restriction Fragments The six fragments produced by a combined digest of mouse L cell mtDNA by Eco RI and Hind III are designated HR-B, HR-C, HR-D, HRE, HR-F and HR-G. m order of decreasing size. The subfragments of HR-B generated by Xba I digestion are designated Xba I A, Xba I B, Xba I C and Xba I D. in order of decreasing size. Isolation of Plasmid DNA E. coli SF8C600 r-m- transformed with pBR322 vector containing either HR-B or HR-D were grown under P2-EKl conditions in BactoPenassay broth (Difco) supplemented with 10 mg per L thiamine and 25 pg/ml ampicillin. Cultures were grown to stationary phase and plasmid DNA was isolated as described by Battey and Clayton (1978). 5’ End Labeling and DNA Sequencing DNA was dephosphorylated by treatment with bacterial alkaline phosphatase. The 20 ~1 reachon mixture, containing 1 O-20 pg of DNA at pH 8.0 and 1 ~1 of BAP (12 U/ml), was incubated at 37°C for 30 min for staggered 5’ ends and at 6O’C for 1 hr for blunt or recessed 5’ ends. The reaction was stopped by extracting three times with phenol followed by three extractions of the aqueous phase wrth diethyl ether. The aqueous phase was made 0.3 M in NaOAc and precipitated by 2 vol of cold ethanol. 5’ end labeling was performed as described by Maxam and Gilbert (1977) for either staggered 5’ ends or blunt or recessed 5’ ends. Nucleotide sequences were determined by the chemical method of Maxam and Gilbert (1980). RNA Isolation mtRNA was obtained from the C2-1 lme of mouse L cells essentially as described by Battey and Clayton (1978) except that the mitochondrial pellet from 1.5 x 10’ stationary phase cells was resuspended m 4 ml of 10 mM Tris-HCI (pH 7.2). 1 mM EDTA and then added directly to an equal volume of neutral phenol, 0.5% sodium dodecylsulfate at 70°C. After mixing for 3-4 min at 7O”C, 2-3 ml of chloroform were added and the phases were separated by centrifugation in a Beckman JA-20 rotor in a J-21 C centrifuge at 17.000 rpm at 0°C for 20 mm. The hot phenol extraction process was repeated l-3 times until there was no visible denatured protein at the interface. Phenol was removed by extractmg the aqueous phase with chloroform. Nucleic acids were ethanol precipitated from the remaining supernatant. To remove tRNAs, DNA and other small RNAs the nucleic acrd was resuspended in autoclaved. distilled water and an equal volume of 4 M LiCl was added. After storing at 4°C for 8-24 hrs the precipitated RNA was pelleted in an Eppendorf centrifuge for 10 min. washed with cold 2 M LiCI, washed with ethanol and dried. Dephosphorylation and Labeling of 5’ and 3’ RNA Ends 5’ and 3’ phosphates were removed by treating the RNA with bacterial alkaline phosphatase (BAP) for 30 mm at 37°C. Dephosphorylation reactions contained about 100 pg of LiCl precipitated RNA at a concentrahon of 1 mg/ml in 0.1 M Tris-HCI (pH 8). and 0.5 units per ml of purifred BAP A sigmficant reduction of contamination by nbonuclease of Worthington’s BAP F was obtained by DEAE cellulose chromatography according to the method of Weiss, Live and Rtchardson (1968). This BAP was contributed by P. Fisher. BAP was removed from dephosphorylation reachons by phenol extraction. Phenol was removed by extracting with chloroform. After ethanol precipitation the RNA was labeled at etther the 3’ end or the 5’ end with ‘*P. LiCI-precipitated dephosphorylated RNA was 3’ end-labeled at 0°C for 8 hr in 10 ~1 of 50 mM HEPES (pH 7.5). 3.3 mM dithlothreitol. 15 mM MgCI,. 10% dimethylsulfoxlde, 0.001% serum albumm as

described by Peattm (1979) and Bruce and Uhlenbeck (1978). RNA was approximately 100 PM, ATP was 50 PM. and 5’ 32P-pCp was 40-50 pM. 50- 100 pg of LiCI-preciprtated dephosphorylated RNA was 5’ endlabeled at 37°C for 30 min by 2 units of polynucleotide kinase (Bohringer-Mannheim) in a 20 PI volume of 60 mM Tris-HCI (pH 9.0). 10 mM MgCl*, 50 mM dithiothreitol, 1.4 mM spermidine, 70 PM EDTA. 3 pM y-=P-ATP. After labeling RNAs were resolved on 0.15 cm X 23 cm 4% polyacrylamide (19 acrylamide:l bisacrylamide) gels containing 50% urea, 89 mM Tris-borate (pH 8.3), and 1 mM EDTA. After the xylene cyanole marker had migrated 15 cm at 150-200 V the gels were stained under sterile conditions with 2 pg/ml ethidium bromide. Bands containing mt rRNAs were excised and electroeluted in dialysis bags under sterile conditions in the presence of 50 pg/ml carrier yeast 45s RNA. Up to 750.000 cpm of each end-labeled mt rRNA could be obtained in this way. RNA Sequencing Enzymatic RNA sequencing was performed as described by DonisKeller et al. (1977) except that all reactions were scaled down in volume so that 5 ~1 samples could be loaded onto 0.35 mm x 40 cm sequencmg gels. The bases nearest the ends of the RNAs were resolved by allowing the xylene cyanole marker to migrate 11 cm at 2200 V through 20% acrylamide. 0.67% bisacrylamrde gels containing 50% urea. 50 mM Tris-borate(pH 8.3)and 1 mM EDTA. Sequence between 15 and 200 bases was resolved by allowing the xylene cyanole marker to migrate 20 cm at 1500 V through 7.6% acrylamide, 0.4% bisacrylamide gels containing 50% urea. 100 mM Tris-borate (pH 8.3) and 2 mM EDTA. To reduce RNAase activity on the gels a solution of 15-50 mM iodoacetate was prerun through all gels used for resolving RNAs. Autoradiograms of sequencing gels were made using Kodak X-Omat R film without intensifying screens at -76°C for 2-14 days. 5’ and 3’ End Analyses 5’ labeled RNA was digested to 5’ mononucleohdes by 0.5 mg/ml nuclease Pl in 50 mM ammonium acetate (pH 5.3), at 37°C for 4 hr. 3’ labeled RNA was digested to 3’ mononucleotides by 2500 units per ml of RNAase T2 in 100 mM Tris-HCI (pH 8) at 37°C for 4 hr. Digested samples were then spotted, with appropriate unlabeled markers, onto wet PEI-cellulose plates (Macherey-Nagel CEL 300 PEI) which had been predeveloped with distilled water. Mononucleotrdes were resolved as described by Randerath and Randerath (1967) by allowing 1 N acetic acid to migrate 4 cm followed by 0.3 M LiCl to a total of 15 cm. The positions of marker mononucleotides were determined under short-wave ultraviolet light. The positions of labeled mononucleotides were determined by autoradiography on Kodak XOmat R film. Preparation of mt tRNA Probe Crude mtRNA was isolated and fractionated by LiCl precipttation as described above. The LiCI-soluble RNA was precipitated by ethanol and mt tRNAs were purified by electrophoresis on a 5% polyacrylamide, 50% urea gel. The gel was stained with ethidtum bromide and the region containing the tRNAs was cut out and electroeluted under sterile condttions. Dephosphorylation and 5’ end labeling was as described above, except that unincorporated Y-~*P-ATP was removed from the probe before hybridrzation by G-75 Sephadex gel filtration. Southern Filter Preparation and Hybridization Blotting restrictron endonuclease drgests of mtDNA from agarose gels to nitrocellulose paper was performed by a modification of the Southern method (1975). The bottom wicks were dipped into reservoirs contaming 20x SSC and the transfer was carried out for 24 hr at room temperature. Filters were pretreated according to the method of Denhardt (1966). Hybridization was performed in 50% formamide, 4x SSC, 0.1 M Tris-HCI (pH 7.4). 0.02% polyvinyl pyrrolidone. 0.02% Ficoll. and 0.02% BSA at 37°C overnight The filters were washed in 2x SSC. 0.1% SDS and 0.1% NaPPi for several hours at 37°C. Filters were rinsed with 2x SSC. blotted dry wtth paper towels and analyzed by autoradrography using Kodak X-Omat R film.

Mouse 169

Mitochondrial

rRNA

and tRNA

Sequences

of tRNA

Acknowledgments We thank C. T. Wright, M. J. Bibb and J. N. Doda for techmcal assistance in the sequencing of DNA. This Investigation was supported by grants from the NIH and American Cancer Society. R.A.V. and M.W.W. are Medical Scientist Traming Program Trainees (NIH) and D.A.C. IS a Faculty Research Awardee (ACS). The costs of publicahon of this article were defrayed in part by the payment of page charges. This arhcle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

May

14, 1980;

revised

July 21, 1980

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