YEAST

VOL:~:

403410 (1990)

Nucleotide Sequence of the Cytochrome Oxidase Subunit 2 and val-tRNA Genes and Surrounding Sequences from Kluyveromyces lactis K8 Mitochondria1 DNA* CHRIS M. HARDY? AND G. D. CLARK-WALKERS Molecular and Population Genetics Group, Research School of Biological Sciences, Australian National University, Canberra. A.C. T.. 2601 Australia Received 18 December 1989; revised 24 February 1990

The nucleotide sequence of the cytochrome oxidase subunit 2 ( ~ 0 x 2 and ) Val-tRNA genes and surrounding regions from Kluyveromyceslactis mitochondrial DNA is reported. Analysis of the coding region shows that the codons CUN (Thr), CGN (Arg) and AUA (Met) are absent in this gene. A single sequence, ATATAAGTAA, identical to the baker's yeast mtRNA polymerase recognition site, was detected upstream of Val-tRNA. This sequence is absent from regions between val-tRNA-cox2 and ~ 0 x 2 - c o x lIn . addition a sequence AATAATATTCTT, identical to the mRNA processing site in other yeast mitochondrial genomes is present 32-43 bp downstream to the TAA stop codon for the cox2 gene. Another short conserved sequence of 5 bp, TCTAA, is present upstream of the coding regions of cox2 genes in several yeasts, including K . lactis, but is not present upstream of other genes. Comparison of cox2 sequences from other organisms indicates that the mitochondrial DNA of K . luctis is closely related to that of Saccharomyces cerevisiae. K E Y WORDS - Kluyveromyceslactis; budding

yeast; mitochondrial DNA.

INTRODUCTION understanding the molecular basis for the obserThe gene for cytochrome oxidase subunit 2 ( ~ 0 x 2 ) vation that some yeasts are unable to produce and surrounding regions has been well characterized respiratory-deficient mutants having large deletions in the yeast Saccharomyces cerevisiae (Coruzzi and in their mtDNAs. By this criterion, K. lactis is norTzagoloff, 1979; Coruzzi et al., 1981; Pratje et al., mally classified as a petite-negative yeast (Bulder, 1983; Sevarino and Poyton, 1980). Intervening 1964). However, in one case, after fusion of this sequences appear to be absent from this gene in yeast with S. cerevisiae, we have obtained a strain yeasts (Coruzzi and Tzagoloff, 1979; Lawson and that can form respiratory-deficient mutants with Deters, 1985), fungi (Macho and Morelli, 1983; large deletions in K. lactis mtDNA (Hardy et al., Dyson et al., 1989), mammals (Anderson et al., 1989). In the course of characterizing the mtDNA 1981; Bibb et al., 1981; Brown and Simpson, 1982; deletion sites, we have obtained the sequence of the Young and Anderson, 1980), insects (Clary and cox2 gene because one frequently occurring deletion Wolstenholme, 1983) and some plants (Hiesel and site is close to the 5' end of this gene (Hardy et al., Brennicke, 1983) but are present in several other 1989). In this report we present the sequence of the plants (Fox and Leaver, 1981; Bonen el al., 1984; cox2 gene and flanking regions from K. lactis Kao et al., 1984). In addition, the cox2 gene is of mtDNA. Subsequently, by sequence comparisons interest because in yeasts and many other organisms we have examined the phylogenetic relationship of this yeast to other yeasts and higher eukaryotes. i t appears to be the only mitochondrially encoded polypeptide synthesized as a larger precursor (Machleidt and Werner, 1979; Pratje et al., 1983; MATERIALS AND METHODS Sevarino and Poyton, 1980). Our interest in the mitochondrial genome of Strains and plasmids Kluyveromyces lactis stems from studies aimed at K . lactis K8 was derived from a cross between *EMBL Accession No. X15999. W599C and W23 1B (A. Herman, Northern Region +Corresponding author. :Reprint requests. Research Laboratories, Preoria, Ill., USA). S. 0749%503Xj90105040348 $05.00 0 1990 by John Wiley &Sons Ltd

404

-

0

H

S

LrRNA

C. M. HARDY AND G. D. CLARK-WALKER

in 5m B Sm ' B E

EY~P I

m -

EE

X EEEEEP

H

* I I

/

0

/'

cox I

-----

ATPase SU 6 4

I

/'

0.5

1.0

.--._ 1.5 . I.

s

PSm'Sm

I

fox2

ATPase cox3 su 9

--

30

71)

m

cytb

SrRNA

2.0

40kbo

S

I

LrRNA

2.5kbp

__-

Val-tRNA

cox I

cox 2 I

I

pCH 14

-

30bp (G+C1

: =+

___) -c_-

---t

__-

-

c

_

c -

f-

c _ _ -

-

Figure 1. Restriction map of the K. lactis K8 mtDNA region encoding the cox2 gene. Location of genes hybridizing to S. cerevisiae probes are shown beneath the map. A 952 bp FnuDII cloned segment of K . lactis K8 mtDNA containing the cox2 gene (pCH14) is also shown beneath the map. Arrows indicate the extent and direction of sequence determinations. Restriction sites: S = SdI;H =HindIII; Sm = ma^; B = B~WIHI; E = ECORI;P = PVUII;x = X ~ O I .

cerevisiae strain DS302 is a petite strain containing the gene for cox2 (Coruzzi et al., 1981).

and Sriprakash, 1981). Agarose gels were washed in 0.5N-NaOH for 30 min. and transferred to Pal Biodyne nylon filters under alkaline conditions (Reed and Mann, 1985). Hybridization conditions were as described (Clark-Walker and Sriprakash, 1981) and filters were washed in 2 x SSC (0.3 MNaC1,0.03 M-Na citrate) at 56°C. Autoradiography took 12-24 h at -70°C.

Preparation of mitochondria1 D N A Yeasts were grown in GlyYP medium (2% glycerol, 1 YOBactopeptone, 0.2% yeast extract) to late exponential phase, harvested by centrifugation and treated with Zymolyase (Seikagaku Kogyo Co. Ltd) in osmotically stabilized buffer (0.5 M-sorbitol, 0.05 M-EDTA, pH 8.5) to form protoplasts. D N A sequencing Cells were then lysed by passage through a Mitochondria1 DNA fragments were cloned into French Press and the mtDNA was isolated from pTZ18 and pTZ19 (Bio-Rad) in the Escherichia coEi cesium chloride, bisbenzimide H33258 gradients as strain XLl-Blue (Blue Scribe) and sequenced described (Clark-Walker et al., 1981). according to the dideoxy chain-termination method (Sanger et al., 1977) with dGTP replaced by deazaRestriction endonuclease analysis GTP to resolve compressions. Large fragments were All digests wereconducted in TA buffer (O'Farrell sequenced using nested sets of deleted plasmids proet al., 1980). Fragments were electrophoresed in 1YO agarose slab gels, stained with ethidium bromide duced by digestion with Ex0111 nuclease (Henikoff, and DNA was collected using Geneclean (Bio 101). 1984). Restriction enzymes were obtained from Pharmacia or Biolabs. RESULTS AND DISCUSSION D N A hybridization [32P]-labelledprobes were prepared by the random priming method as described (Clark-Walker

Cloning and sequencing of the cox2 region The gene for cox2 and part of the cytochrome oxidase subunit 1 ( c o d ) gene have been shown by

405

NUCLEOTIDE SEQUENCE OF THE CYTOCHROME SUBUNIT 2

5'-GMTTCCAGGGMTMGCCtCCGGTGTATAMT 35

MTTTTATATACTCTTCTCCCGGGGACTCCTTCCCATMCGTGTATACCTGGGMGAGTCCCCGGAGTTMTATMTTMT~CCCCACCCCCTTTMG

0

135

GGGGGTGCGGAGGMTACGCMTTGGGGGMTTTATMTMTTATTATATMTATTATTATATAG A T A T M G T M GTATMTATTATTATMTMTM

233

TMTAGAGATTAGCTTMTTGGCAGAGCATTCGTTTTAGACGC~GGACATGAGTT~TCTCGTATCTCTMTATATATT~TATAT~TT~

333

TAT~TTTMTMTMTATAT~TATACTTATTTMTATTATTATMTTATTTATMTMTTTATTATTMTMGTTTTTTTCCTMTTGCGMC

433

T

533

ATG TTT TAT TTA TTA M T TCA ATT ATT ATG M T GAT GTA CCT ACA CCT TAT GGT ATG TAT TTC C M GAT TCA GCT M e t P h e T y r L e u L e u A s n Ser I l e I l e M e t A n n A s p V a l P r o T h r P r o T y r G l y Met T y r P h e G l n A s p Ser A l a

608

ACA CCT M T C M G M GGT ATT TTA G M TTA CAT GAT M T ATT ATG TTC TAT TTA TTT ATT ATT TTA GGA TTA GTA T h r P r o A s n G l n G l u G l y I l e L e u G l u L e u His A s p A s n I l e M e t P h e T y r L e u P h e Ile I l e L e u G l y Leu V a l

683

TCA TGA TTA TTA TTT ACA ATT GTA AGA ACT TAT AGT AAA M T CCT ATT GCT TAT MA TAT ATT MA CAT GGT CAA S e r T r p L e u L e u P h e T h r I l e V a l A r g T h r T y r Ser L y s A s n P r o I l e A l a T y r L y s T y r I l e L y s His G l y G l n

758

ACT ATT G M ATT ATT TGT TCA ATT TTC CCT GAT GTA ATT TTA TTA ATT ATT GCT TTC CCT TCA TTC ATT TTA TTA T h r I l e G l u I l e I l e C y s Ser I l e P h e P r o A s p V a l I l e L e u L e u I l e I l e A l a P h e P r o Ser P h e I l e L e u L e u

833

TAT TTA TGT GAT G M GTT ATT TCT CCA GCA ATG ACT ATT AAA GCT ATT GGT TTA C M TGA TAT TGA AAA TAT GAA T y r L e u C y s A s p G l u V a l I l e S e r P r o A l a Met T h r I l e L y s A l a I l e G l y L e u G l n T r p T y r T r p L y s T y r G l u

908

TAC TCA GAT TTC ATT M T GAT M T GGT G M ACA GTA G M TTT G M TCA TAT GTT ATT CCT GAA GAT TTA TTA G M T y r Ser A s p P h e I l e A s n A s p A s n G l y G l u T h r V a l G l u P h e G l u S e r T y r V a l I l e P r o G l u A s p L e u L e u Glu

983

GAT GGT C M TTA AGA TTA TTA GAT ACT GAT ACT TCA GTA GTA GTA CCT GTT GAT ACA CAT ATT AGA TTT GTT GTT A s p G l y G l n L e u A r g L e u L e u A s p T h r A s p T h r S e r V a l V a l V a l P r o V a l A s p T h r His I l a A r g P h e V a l V a l

1058

ACA GCA GCT GAT GTT ATT CAT GAT TTT GCT GTA CCT AGT TTA GGT ATT AAA ATT GAT GCA GCT CCT GGT AGA TTA T h r A l a A l a A s p V a l I l e His A s p P h e A l a V a l P r o S e r L e u G l y I l e L y s I l e A s p A l a A l a Pro Gly A r g L e u

1133

M T C M GTA TCA GCT TTA ATT C M AGA GAA GGT GTA TTC TAT GGA C M TGT TCA G M ATT TGT GGA C M TCA CAT A s n G l n V a l Ser A l a Leu I l e G l n A r g G l u G l y V a l P h e T y r G l y G l n C y s S e r G l u I l e C y s G l y G l n Ser His

1208

TCA GCT ATG CCT ATT AAA ATT G M GCA GTT TCA TTA CCA GCA TTC TTA G M TGA TTA M T G M C M TAA Ser A l a Met P r o I l e L y s I l e G l u A l a V a l Ser L e u P r o A l a P h e L e u G l u T r p L e u A s n G l u G l n O c h

1381

GAGACGGCGATTCTTACTTATTTTATATCCCGC~TTCATGTTAT~TCCGCGGATTATTATATTATATTATATTATATTATATTATATTTTTAAATAT

1481

MTMTMTTATATATAAATMGAGTAT~TMTTTATTGATCTCATGCTATACTCCCCCCCCTTCCTGTCGCACACGATTGGTGCGCAGATATTGA

C

A

T

C

~

C

A

T

T

A

G

T

T

G

G

A

T

G

A

G

T

T

C

G

C

G

G

C

A

W

U

T

A

A

T

T

TTATAT

1581

AGGGGGGGAGTAGGGAGTMTAAATTTAGGATMC~~ATTATMTMTTMTTATTATMTMTATTTTTCCCCCtCGGTCTCTCACCCTACGMTGT

1681

AGGGTGATGAGCCGCGGGGTMTTCTTATTTATTATATATTATMTATTMTMT~CCCCGCGGACTCCTTTAAAGGAGTCCGCG~,TMMGTATTAT

1781

CCGCGCCTCATCCATTATAAAGGATGAGGCGCGGATTMTATACATATATATATTTTMTATMTTATATATTATATTTATTTATMTATTATTATTTAT

1881

TATAT,TMMTMTACTMTGAGGGGGGTTATTTCCACCATATGGTGG~TAAGCCTACCTAATATATTTATTTATTTTTTATTATTMTATMTTA~

1981

T M T A T T T T T C T T T M G A G G C T T A T T A T A T A T T M T A G M T A A M C C C C C G C G - - - - -

2081

-----30

2181

GAT,TMMCTTAGTCNNNNNNNNN~~NCCC~TAAAGGGATGGG~CGAGCGCGGGGGTMCGMTTTATCTATMTTATATMTTATMTMTMTT

2281

ATATATMCTMTTMTTMTTMTMT~CATMTGTATATTACATACTMTCCCCGCGGCTCCCCCCCTTCAGGGGGGGAGCC~GGGT~TA

2381

TMCTMTMTMTAGTTTATTATATTATATATTTTAMTATTTA

bp (GC-rich)-----GGGGMTGTATATTTTACCGATCTGCATAGT~TTGGT~TTTATATATTTTATATATAT~TTATATATM

ATG Met

- -(cytochrome

oxidam)( s u b u n i t 1)

-

Figure 2. DNA sequence of the K. /actis K8 cox2 gene. The sequence shown is that ofthe non-transcribed strand. Numbering of the nucleotides commences with the first base of the EcoRIsite upstream of the cox2 gene. The sequence has been translated according to the yeast mitochondria1 genetic code. TGA codons are translated as tryptophan and ATA codons as methionine as in S. cerevisiae (Bonitz efa/., 1980b; Hudspeth et a/., 1982).The gene for Val-tRNA is underlined. Putative RNA transcript start and stop signals are boxed.

406

C. M. HARDY AND G. D. CLARK-WALKER

Table 1. Codon usages in the cox2 gene of K . lactis. Ala

Arg Arg

Gln

GCA 5 GCU 9 GCC 0 GCG 0 AGA 5 AGU 0 CGA 0 CGU 0 CGC 0 CGG 0 CAA 10 CAG 0

Asn AAU 9 AAC 0 Asp GAU 15 GAC 0 Cys UGU 4 UGC 0 Gly GGA 3 GGU 9 GGC 0 GGG 0

Glu GAA 15 GAG 0 His CAU 5 CAC 0 Ile AUU 30 AUC 0 Leu UUA 26 UUG 0 Lys AAA I AAG 0

DNA hybridization studies to be located on a 3.6 kb EcoR1 fragment (Figure 1).This fragment was purified and digested with further enzymes to identify a smaller fragment for cloning (Figure 1). One clone (pCH14), containing a 952 bp FnuDII fragment that hybridized to a probe from S. cerevisiae DS302 mtDNA (Coruzzi et al., 1981), was found by sequence analysis to encompass the entire cox2 coding region (Figure 1). This gene, which is uninterrupted by introns, encodes a protein of 247 amino acids (Figure 2). Flanking regions were determined from other overlapping clones prepared from K. lactis mtDNA digested with various restriction enzymes (Figure 1). Sequences with homology to the S. cerevisiae genes for val-tRNA and coxl were subsequently obtained (Figure 2). The illustrated sequence extends from the EcoR1 site 5' to the valtRNA gene to the first coding triplet of the coxl gene. The FnuDII sites used to clone the FnuDII fragment into pCH14 occur at positions 459 and 1311 in the illustrated sequence (Figure 2). A gap of 30 nucleotides is present 300 nucleotides from the start of the coxl gene and corresponds to an intergenic GC-rich cluster that was not sequenced. Codon usage in the cox2 gene Examination of codon usages in the cox2 gene, translated according to the yeast mitochondria1 system, indicates that codons AUA, CUN and CGN are absent (Table 1). TGA is assumed to encode tryptophan since four codons are found in identical locations to those in S. cerevisiae (Coruzzi and Tzagoloff, 1979). AGU is used twice for serine in the cox2 gene, once in the same location as in S. cerevisiae. Also UAC is used once in K. lactis as opposed to four times in Hansenula saturnus (Lawson and Deters, 1985) and not at all in S. cerevisiae. How-

Met AUA AUG Phe UUU UUC Pro CCA

ccu

Ser

CCC CCG AGU AGC

0 6 6 8 2 11 0 0 2 0

Ser

UCA 13 ucu 1 ucc 0 UCG 0 Trp UGA 4 UGG 0 Thr ACA 6 ACU 5 ACC 0 ACG 0

Thr CUA 0

cuu

CUC CUG Tyr UAU UAC Val GUA GUU GUC GUG

0 0 0

12 1 11

7 0 0

ever, the codon usage of K. lactis is closest to S. cerevisiae since CUN and CGN are used in H. saturnus but not in the former yeasts. A strong bias in favour of A + T in the third position of the codons was observed, with only nine out of 247 having a C in the third position. Comparison of cox2 genes At present the cox2 nucleotide and amino acid sequences from only three yeasts and a few higher organisms have been described. Amongst the yeasts, nucleotide sequence homologies have been determined from the first nucleotide of the putative mature protein. The nucleotide sequence of the K. lactis cox2 gene (from the 34th nucleotide) shows 88% base matching to the gene from S. cerevisiae (from the 46th nucleotide) and 78% base matching to the corresponding H. saturnus (34th nucleotide) gene. By contrast the gene for H . saturnusshows 85% base matching to the S . cerevisiae gene (Lawson and Deters, 1985). Amino acid homologies are similar, with homologies of 88% between K. lactis and S . cerevisiae, 82% between K. lactis and H. saturnus and 85% between S . cerevisiae and H. saturnus. Interestingly, the cytochrome b genes of K. lactis and S . cerevisiae show only 83% base matching (Brunner and Coria, 1989). However, comparisons between the sequences of the cytochrome b genes may be influenced by the presence or absence of introns in these genes, since in some cases, this is associated with strain-dependent nucleotide differences in the vicinity of intron splice sites (Hensgens et al., 1983; Trinkl et al., 1985). Amino acid sequence comparisons of the available yeast cox2 sequences with sequences from other organisms are shown in Figure 3. A total of 54 amino acids were found to be conserved between all

**

J

Ref.

*

S.cerevisiae (1) MLDLLRLQLTTFIMN- DVPTPYACYFQDSATPNQEGILELHDNIWYLLVI MFYLLNSIIMN- DVPTPYGMYFQDSATPNQEGILELHDNIWYLFII K. lactis MLLLINNLILN- DVPTPWGLYFQDSSTPNQEPIIELHDNIWYLVLI H. s a t u r n u s (2) MGLLFNNLIMNF DAPSPWGIYFQDSATPQMEGLVELHDNIMYYLWI N.crassa (3) MAYPMQLGFQDATSPIMEELLHFHDHTLMIVFLI Beef (4,s) MAYPFQLGLQDATSPIMEELMNFHDHTLMIVFLI Mouse (6) MAHAAQVGLQDATSPIMEELITFHDHALMIIFLI Human (71 MSTWANLGLQDRASPLMEQLIFFHDHALLILVMI Drosophila (8) MILRSLECRFLTIALC DAAEPWQLGSQDAATPMMQGIIDLHHDIFFFLILI Maize (9) Oenothera (10) MIVN--ECLFLTIAPC DAAEPWQLGSQDAATPMMQGIIDLHHDIFFFLILI

*

*

* *

** **

** *

LGLVSWMLYTIVITY--SKNPIAYKYIKHGQTIEVIWTIFPAVILLII~PSFILLYLCDEVI-SPAITI LGLVSWLLFTIVRTY--SKNPIAYKYIKHGQTIEIICSIFPDVILLII~PSFILLYLCDEVI-SP~TI LCTVSWLLFSIVKDS--SKNPLPHKYLVHGQTIEIIWTILPAWLLII~PSFILLYLCDEVI-SP~TI LFWGWILLSIIRNYISTKSPISHKYLNHGTLIELIWTITPAVILILIAFPSFKLLY~EVS-DPSMSV ss----- LVLYIISLMLTTKLTHTSTMDA-QEVETIWTILPAIILILIALPSLRIL~EIN-NPSLTV ss----- LVLYIISLMLTTKLTHTSTMA-QEVETIWTILPAVILPAVILIMIALPSLRILY~EIN-NPVLTV CF-- - -LVLYALFLTLTTKLTNTNISDA-QEMETVWTILPAIILVLIALPSLRILYMTDEVN-DPSLTI TV-LVGYLMFMLFFNNYVNRFLLHG-QLIEMIWTILPAIILLFIALPSLRLLYL~EIN-EPSVTL LVFVSWMLVRALWHFNEQTNPIPQR-IVHGTTIEIIWTIFPSVIPLFIAIPSFALLYSMGVLVDPAITI LVFVSWILVRALWHFHYKKNPIPQR-IVHGTTIEILWTIFPSIIPWIAIPSFALLYSMG~P~TL

---

* ** *

* **** * *

* * *

*

*

** *

KAIGYQWYWKYEYSDFINDSGETVEFESWIPDELLEEGQLRLLDTDTSIWPVDTHIRFWTAADVIHD KAIGLQWYWKYEYSDFINDNGETVEFESWIPEDLLEDGELRLLDTDTS~~THIRFWT~VIHD KAIGLQWYWRYEYSDFINDSGETIEFESYVIPEDLLEDGQLRLLDTDTSWCPVNTHIRFIVSAVIHD LAEGHQWYWSYQYPDFLDSMEFIEFDSYIVPESDLEEGALRMLEVDNRVILPELTHVRFIITAGDVIHD KTMGHQWYWSYEYTDYED----- LSFDSYMIPTSELKPGELRLLEVDNRWLPMEMTIRMLVSSEDVLHS KTMGHQWYWSYEYTDYED----- LCFDSYMIPTNDLKEGQLRLLDTDTSIVWVDTHIRFWTAADVIHD KSIGHQWYWTYEYTDYGG----- LIFNSYMLPPLFLEPGDLRLLDVDNRWLPIEAPIRMMITSQDVLHS KSIGHQWYWSYEYSDFNN----- IEFDSYMIPTNELAIDGFRLLDVDNRVILPMNSQIRILVTAVIHS L A I G H Q W Y W S Y E Y S D Y N S S D E N S L T F D S Y T I P E D D P E L G Q V P H S

LAIGHQWYWSYEYSDYNSSDENSLTFDSYTIPEDDLELGQSRLLEVDNR~PVKTNLRLIVTP~VPHS

* * *

* * *

* *

** *** ** *

*

FAIPSLGIKVDATPGRLNQVSALIQREGVFYGACSELCGTSLPKFLEWLNEQ

FAVPSLGIKIDAAPGRLNQVSALIQ~GVFYGQCSEICGQSHS~IKIEAVSLP~LEWLNEQ FAIPSLGIKWASPGRLNQVSALIQREGVYYGMCSETCGVAIEWSTKEFLTWLNEQ FAVPSLGVKCDAYPRRLNQVSVFINREGVFYGQCSEICGIIVIESVSLEKFLTWLEEQ

WAVPSLGLKTDAIPGRLNQTTLMSSRPGLYYG~SEICGSNHSFMPIVLELVPLKYFEKWSASML FAIPSLGLKTDAIPGRLNQATVTSNReGLFYGQCSEICGSIVLEMVPLKYFENWSASMI WAVPTLGLKTDAIPGRLNQTTFTATRPGVYYGQCSEICGAIVLELIPLKIFEMGPVFTL WTVPALGVKVDGTPGRLNQTNFFINRPGLFYGQCSEICGANS

WAVPSSGVKCDAVPGRSNLTSISVQREGVYYGQCSEICGTNH~TPIWEAVTL~Y~~SNQLILQTN WAVPSSGVKCDAVPGRLNQISMSVQ~G~YG~SEICGTNH~MPIVIEAVSATDYT~SNLFIPPTS Figure 3. Comparison of predicted amino acid sequences for the cox2 genes from various organisms. Amino acids are written in single letter code (IUPAC-IUB Commission, 1969). Amino acids conserved in all species are indicated above the sequence by an asterisk. The known and putative cleavage sites of the precursor polypeptides are indicated by an arrow. The predicted sequence for maize has been adjusted to allow for CGG to encode tryptophan rather than arginine as proposed in plant mitochondria (Hiesel and Brennicke, 1983). References: (1) Coruzzi and Tzagoloff (1979); (2) Lawson and Deters (1985); (3) Macino and Morelli (1983); (4) Young and Anderson (1980); ( 5 ) Steffens and Buse (1979); ( 6 ) Bibb et al. (1981); (7) Anderson ei al. (1981); (8) Clary and Wolstenholme (1983); (9) Fox and Leaver (1981); (10) Hiesel and Brennicke (1983).

408

C. M. HARDY AND G. D. CLARK-WALKER

K1

G C G G G A G A A T A A T A T A T T A C A A T G T ~ T A ~ ~ ~ T A T A T A G ~ T A T A T A T A G A T T ~ - T T A T A A AATG T

Hs

CCTATATTAATAATTATATAGATATTATATA~~~------------TA -TA -AMGAA

SC

AGTATTAACATATTATAAATAGACAAAAGAG'ZCI..AGGG--------------------

........ ......

*

:::: :

Tg

::::

: ::::::::

AATATATAAGGACACGAAAG~~~GA------------------

......... ATG ... T T m T T T A T T A A A ATG ...... T T e A A G T A T A A A T T ATG

Figure 4. Comparison of upstream untranslated regions of yeast cox2 genes. Sources of the sequences: Kl ( K . tactis, Figure 2; - 73+ 3); Sc (S.cerevisiae, Coruzzi and Tzagoloff, 1979; - 54- 3); Hs ( H . saturnus, Lawson and Deters, 1985; - 61- + 3);Tg (T.glabrata, Clark-Walker et al., 1985; 477-528). Numbering of the nucleotides is according to the authors. An asterisk (*) above the sequences denotes known transcript starts. Colons denote conserved stretches of nucleotide sequence. Emboldened letters represent the conserved 5 bp sequence which is present in all untranslated leader sequences upstream of the ribosomal binding sites. Underlined nucleotides represent possible ribosome binding sites. Dashes indicate gaps introduced to align the sequences.

+

organisms representing approximately 23 % of the glabrata (Clark-Walker et al., 1985).This consensus gene. In particular, three His and two Cys residues sequence is present 27-36 nucleotides upstream of are conserved in all genes examined. These amino the gene encoding Val-tRNA. In addition the dodeacidsmay be required for bindingCu*+ ions (Steffens camer sequence AATAATATTCTT, identical to and Buse, 1979). Notably there is a considerable RNA processing sites in S . cerevisiae (Osinga et al., difference between amino acid sequences in the 5' 1984b) and T . glabrata (Clark-Walker et al., 1985), region. In Neurosporra crassa a leader sequence of is found 3 2 4 3 b p downstream from the TAA 12 amino acids has been reported (Machleidt and termination codon of the cox2 gene (Figure 2). Werner, 1979), while a similar leader sequence of 15 Similar, but non-identical sequences have also been amino acids has been found in S. cerevisiae (Pratje et reported in H . saturnus (Lawson and Deters, 1985). al., 1983; Sevarino and Poyton, 1980). On the basis The transcript start signal, ATATAAGTAA, is of these comparisons, it appears that the cox2 gene functional in K. lactis at least for the rRNA genes of K. lactis has a leader sequence of 11 amino acids. (Osinga et al., 1982). The absence of a detectable The presence of differing leader sequences in both transcription initiation site for the coxl gene (Figure yeasts and fungi may reflect either alterations in pro- 2) suggests that the termination signal downstream tein processing in these organisms or that the leader of cox2 in K. lactis may act as an RNA processing sequence does not need to be conserved for cleavage site similar to the common transcript for coxl, to occur. This leader sequence is similar to nuclear- ATPase subunits 8 and 6 in S. cerevisiae (Osinga et encoded precursor polypeptides, where insertion of a[., 1984b). In S . cerevisiae, H . saturnus and T . these proteins into the mitochondrial membrane glabrata the cox2 gene is transcribed as a monocisrequires arginine, asparagine or lysine residues sur- tronic messenger RNA (Coruzzi et al., 1981; Clarkrounded by hydrophobic amino acids (Allison and Walker et al., 1985; Lawson and Deters, 1985). The Schatz, 1986; Roise and Schatz, 1988). Examination absence of a detectable transcription initiation sigof the amino-termini of cox2 genes with leader nal between the cox2 and coxl genes in K. lactis sequences indicates that they are all characterized suggests that they could be transcribed with the valby the presence of an arginine or asparagine residue tRNA gene as a polycistronic transcript. In addition surrounded by two or more hydrophobic residues. the Val-tRNA is not upstream of cox2 in these other All putative leader sequences appear to be spliced at yeasts. Recent experiments have shown that nucleara conserved aspartate residue (Figure 3). encoded genes are required for the translation of the cox2 gene in S. cerevisiae, which in turn may be Flanking nucleotide sequences of the cox2 gene regulated by other factors (Fox, 1986;Costanzo and Examination of the nucleotide sequence shows Fox, 1988; Schulze and Rodel, 1988). These studies that there is only one potential mitochondrial RNA have suggested that a specific structural element polymerase recognition site ATATAAGTAA, that may be present in the leader region of the mRNA is identical to another K. lactis promoter (Osinga et that is required for translation. Therefore, comparial., 1982;Wilson et al., 1989)as well as to those from sons were made between the non-translated regions S . cerevisiae (Biswas et al., 1987; Christianson and of the cox2 gene of several yeasts (Figure 4). A Rabinowitz, 1983; Michel, 1984; Osinga et al., 5 bp sequence, TCTAA, was detected at variable 1984a,b; Schinkel et al., 1988) and Torulopsis distances upstream of all the coding regions (Figure

NUCLEOTIDE SEQUENCE OF THE CYTOCHROME SUBUNIT 2

4) but is absent from the non-translated regions upstream of the cox1 genes in both K. lactis and S. cerevisiae (Bonitz et al., 1980a). Whether this common sequence element has a role in the translation of cox2 mRNA has yet to be determined. The gene for val-tRNA shows considerable sequence variation to the equivalent gene from S . cerevisiae (Thalenfeld and Tzagoloff, 1980) with 13 substitutions, two additions and three deletions. This contrasts with the reported average variance of only five changes between other mitochondrial tRNAs from S. cerevisiae and another strain of K. lactis (Wilson et al., 1989). However, the deduced secondary structure of this tRNA shows that this sequence encodes a bonajde valine tRNA with the anticodon 5'-GAC-3' and is the 22nd tRNA gene described from K. lactis mtDNA. In conclusion, analysis of the cox2 gene of yeasts indicates that this gene may prove to be a useful tool for comparing the evolution of mtDNA coding regions and signal sequences. Since the cox2 gene is relatively short and does not appear to contain introns (except in plants), this gene should be ideal for determining the molecular evolutionary relationships between yeast mitochondrial DNAs as more sequences become available. ACKNOWLEDGEMENTS The support of a Commonwealth Postgraduate Research Scholarship for C.M.H. is acknowledged. REFERENCES Allison, D. S. and Schatz, G. (1986). Artificial mitochondrial presequences. Proc. Natl. Acad. Sci. ( U S A ) 83, 9011-9015. Anderson, S., Bankier, A. T., Barrel, B. G., de Bruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, J. C., Nierlich, D. P., Roe, B. A,, Sanger, F., Schreirer, P. H., Smith, A. J. H., Staden, R. and Young, I. G. (1981). Sequence and organisation of the human mi tochondrial genome. Nature 290,457-465. Bibb, M. J., Van Etten, R. A,, Wright, C. T., Walberg, M. W. and Clayton, D. A. (1981). Sequence and gene organisation of mouse mitochondrial DNA. Cell 26, 167-180. Biswas, T. K., Tich, B. and Getz, G. S. (1987). In vitro characterization of the yeast mitochondrial promoter using single-base substitution mutants. J . Biol. Chem. 262,1369G13696. Bonen, L., Boer, P. H. and Gray M. W. (1984). The wheat cytochrome oxidase subunit I1 gene has an intron insert and three radical amino acid changes relative to maize. Embo J . 3,2531-2536.

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Nucleotide sequence of the cytochrome oxidase subunit 2 and val-tRNA genes and surrounding sequences from Kluyveromyces lactis K8 mitochondrial DNA.

The nucleotide sequence of the cytochrome oxidase subunit 2 (cox2) and val-tRNA genes and surrounding regions from Kluyveromyces lactis mitochondrial ...
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