Cell, Vol. 61, 885-894,

June

1, 1990, Copyright

0 1990 by Cell Press

An Extensively Edited Mitochondrial Transcript in Kinetoplastids Encodes a Protein Homologous to ATPa& Subunit 6 G. Jayarama Bhat, Donna J. Koslowsky, Jean E. Feagin, Bob L. Smiley, and Kenneth Seattle Biomedical Research Institute 4 Nickerson Street Seattle, Washington 98109-1651

Stuart

The mitochondrial MURF4 gene of T. brucei has pronounced G versus C strand bias and heterogeneously sized transcripts, characteristic of genes encoding extensively edited transcripts. We find that MURH transcripts of T. brucei are extensively edited throughout by the addition and deletion of numerous uridines, creating potential initiation and termination codons and a continuous open reading frame. A potential guide RNA sequence occurs in a minicircle between inverted repeats. The 5’region of L. tarentolae MURF4 transcripts is also extensively edited, with a created initiation codon. The predicted MURF4 amino acid sequences have homology to those of mitochondrial ATPase subunit 6 genes from a variety of organisms. In addition, their hydropathic profiles are quite similar to those of other species. We therefore conclude that MURF4 encodes ATPase subunit 6 genes.

Genetic information is stored in an unusually compact form in the mitochondrial DNA of kinetoplastid flagellates as a result of the remarkable process of RNA editing (for reviews see Benne, 1989; Simpson and Shaw, 1989; Stuart, 1989). This process alters mitochondrial transcripts by the insertion of uridines not encoded in their genomic sequence and by the deletion of uridines encoded in the DNA. Editing produces apparently functional transcripts with homology to genes in numerous other organisms whose transcripts are not edited. Although the overall role of RNA editing is unclear, it creates initiation codons and extends open reading frames (Feagin et al., 1987, 1988a; Feagin and Stuart, 1988; Shaw et al., 1988) creates termination codons (Feagin et al., 1988b), eliminates internal frameshifts (Benne et al., 1986; van der Spek et al., 1988; Shaw et al., 1989), and modifies the nucleotide sequence within the 3’ untranslated region and the poly(A) tail of transcripts (Benne et al., 1986; Feagin et al., 1987, 1988b; van der Spek et al., 1988, 1990; Campbell et al., 1989). Thus, editing appears to regulate gene expression at the RNA level. The mitochondrial DNA of kinetoplastids is composed of two unrelated types of circular DNA molecules. Maxicircles encode mitochondrial ribosomal RNAs and components of the mitochondrial respiratory system (for reviews see Benne, 1985; Simpson, 1987) and minicircles encode small transcripts (Rohrer et al., 1987) but have no known protein coding function. No genes for tRNAs or subunits

of the ATPase complex, which are present in nonkinetoplastid mitochondrial genomes, have been identified, but several small regions of the maxicircle that have no identified function are candidate sites for these genes (Feagin et al., 1985; Jasmer et al., 1985, 1987; Simpson et al., 1987). The regions with no known function have pronounced G versus C strand bias and encode transcripts that are larger than the regions and are G biased. This resembles the cytochrome oxidase Ill (COIII) gene of Trypanosoma brucei, which, as recent studies from this laboratory have shown, is extensively edited (Feagin et al., 1985, 1988b). The COIII transcript sequence aligns perfectly, except for the uridine additions and deletions, with the strand-biased region upstream of the cytochrome b (CYb) gene. Consequently, the COIII gene of T. brucei exists in an abbreviated form. This obscures its homology to the COIII gene from other organisms, even in the closely related organisms Crithidia fasciculata (Sloof et al., 1987) and Leishmania tarentolae (de la Cruz et al., 1984) where the COIII gene has the same position, upstream of the CYb gene. In this study we have analyzed the region between the CYb and MURFl genes in T. brucei and L. tarentolae that was previously called ORFGDE in T. brucei (Simpson et al., 1987) (and ORFIIDE in T brucei [Feagin et al., 19851 and ORF12 in L. tarentolae [Simpson et al., 1987). We previously noted that this region had G versus C strand bias and encoded transcripts larger than the DNA sequence (Feagin et al., 1985). We report here that transcripts from this region are extensively edited throughout in T brucei and the 5’region is extensively edited in L. tarentolae. The edited transcripts have substantial nucleotide and predicted amino acid sequence homology between the two species, and thus the genes are temporarily termed MURF4 (for maxicircle unidentified reading frame 4) according to our previous convention (Simpson et al., 1987). However, editing creates a continuous open reading frame in both cases that has a predicted translation product homologous to ATPase subunit 6 (ATPase 6) of other organisms. Thus, MURF4 appears to be an ATPase 6 gene. Results The T. brucei MURF4 Transcript Is Extensively Edited The sequence of edited RNA cannot as yet be predicted from the DNA sequence. However, since editing occurs in the 3’to 5’direction (Abraham et al., 1988) partially edited T. brucei MURF4 cDNA clones were isolated using a probe for the unedited 5’ sequence. Analysis of four clones that were isolated from a hgtll library in this fashion revealed that they are not edited at their 5’ ends but are edited to different extents at their 3’ ends. A series of oligonucleotide probes was prepared based on the edited sequences and used for RNA sequencing and to isolate and sequence additional cDNA clones containing edited sequences. Comparison of cDNA sequences with

Cell 888

C

A CATG

Figure

1. Nucleotide

TGX

Sequence

Analysis

of Edited

MURF4

M

E PF CATG

CATGX

F

PF CATGX

L, CATGX

Transcripts

(A) The sequence of cDNA clone M4-251 using MURF4-9 as primer. Dots indicate uridines (lane A) in the cDNA but not the gene; the dots are discontinued in the upper portion due to space limitations, but numerous uridines are added in that region. The arrowhead indicates a site of uridine deletion. (8) RNA sequence of T. brucei bloodstream (BF), procyclic (PF), and dyskinetoplastic (Dk) mutant RNA using MURF4-2 as primer. (C and D) cDNA sequences of clones M4-251 and M4-5Pl1, respectively. (E) MURF4 procyclic RNA sequence using MURF4-14 as primer. (F) T. brucei procyclic and L. tarentolae MURF4 RNA sequences using MURM-11 and LtORF12-2 primers, respectrvely. Note the greater sequence heterogeneity near the 5’ end of T. brucei MURM RNA in (E) and (F). Putative initiation and termination codons are indicated with arrows marked I and T, respectively. Minus dideoxy nucleotides (X) and minus primer(M) controls for RNA sequences are indicated.

the corresponding DNA sequences reveals numerous uridines in the cDNA that are not in the gene and the absence of fewer encoded uridines (Figure 1A). The cDNA and RNA sequences match the MURF4 DNA sequence exactly, except for the presence and absence of numerous uridines (Figure 2). Analyses of total DNA with oligonucleotide probes did not detect a DNA copy of the edited version of the MURM transcript (data not shown). Editing of the MURF4 transcript produces a continuous open reading frame bounded by putative initiation and termination codons that are created by editing (Figure 2A). The termination codon, UAA, that is created by editing is evident in the sequence from total RNA (Figure 16) and more clearly in cDNA clones (Figure 1C). Two cDNA clones have the same S’terminus and a third has a poly(A) tail at the same 3’ terminal nucleotide, thus defining the 3’ boundary of the MURF4 transcript. The few 5’-most nucleotides could not be determined by sequencing the 5’ terminus (Figure 1E); however, it corresponds to a run of seven adenines in the DNA sequence. The poly(A) addition site of the CYb transcript, encoded immediately upstream of the MURF4 gene, is in the same run of adenines (Feagin and Stuart, 1989). Editing creates an in-frame AUG codon that is evident in a cDNA sequence (Figure 1D). RNA sequence analysis (Figure 1E) reveals that the sequence from the most intense bands matches that of the cDNA shown in Figure lD, including the presence of the AUG codon. However, the RNA appears heterogeneous, especially near the 5’ terminus (compare Figures

1E and lF), based on ambiguity in the RNA sequence. This is to be expected from the presence of partially edited RNAs. Analysis of cDNA S’sequences shows that they differ slightly 5’to the AUG (Figure 26). It is uncertain if these differences reflect microheterogeneity in fully edited and presumably functional RNAs or if these are partially edited RNAs. Figure ll3 also reveals that the MURF4 transcript is edited in both bloodstream and procyclic forms. Bloodstream forms lack the mitochondrial respiratory system that is present in procyclic (insect) forms (Englund et al., 1982; Stuart, 1983) and editing of COII and CYb transcripts is developmentally regulated, occurring primarily, if not solely, in procyclic forms (Feagin et al., 1987; Feagin and Stuart, 1988). The nucleotide sequence of the fully edited 7: brucei MURF4 transcript (Figure 2A) contains 821 nucleotides, excluding the poly(A) tail, of which 448 are uridines that are added at 173 sites. In addition, 28 encoded uridines are removed from 12 sites by RNA editing. This RNA sequence matches the maxicircle gene sequence, except for the uridines that are added or removed, as is the case for all edited transcripts examined to date, including the extensively edited T brucei COIII transcript (Feagin et al., 1988b). There are 143 nucleotides of edited sequence 3’ to the apparent termination codon, giving a much larger 3’ untranslated region than that of other maxicircle transcripts (Feagin et al., 1988b; Feagin and Stuart, 1989; Campbell et al., 1989). The first 32 nucelotides at the 5’ end and the last 37 nucleotides at the 3’ end match the

Editing

of ATPase

087

6 mRNA

Tb Tb

DNA AAAAAZATAAGTATTTTGATATTATTAAAGTAAA A G A RNA AAAAAUAAGUAWWGAUAWAWMAGUAAAc&&uuuuAuuuuuuuuuuGuGAuuuAWWGGu

Lt Lt

DNA RNA

A A

G GA

ATTTTGG

G CG

G

A

AGAG

u G TTG

GA

AG AA

ATTGCG

A

~~GUUUUUUUUGUWGUGAUUUAGUAAU~A*UGCGUAUUU~A~UA~GUUUU

GGA

A

GGA

A

GUUUUAUUGUGUAUUUUA

G AAGG GG A ~G~~A~AG~G~~~GAUCCAGAA~AGAA~~A~~G~G~~AUUU~AUA**A~

A

A TG

A UG

AA G GA GA A A G uAAuGuuGAUUUUUGAUUUUUUAUUAUUUUGUUUU~T;SI;

G

M&4-Y ATTA

A GATTCGTGTTATTTAATTTTTATGGATTGATT u~SUUU~UUULXIAUGAWCGUGWAWUZMJUUWA

LtORFlZ-2 A A G GA GTTA TG G GA G A GAA GCA G G UUG *UA~~UGUUGUUUUG~AUUGUUUUUU~UUG~~UUGCAUUUU~UUUUUGUUUUGUUUUU~UGUGAUUUUUUUUUGUU~~UU AATGTT TATATTTTATmGTATAGTTTTTATGTATGTATGGCATTTGCC

G

‘f?$&

G TAG GG GA ATTTTG A GGA G ATTCTTG G GG AGAG G ~G~UAG~~GG~GA~A****GU~~~AUGGAUGU~U~~~U~AUUC**G~~~~~~G~~G~G~UUUU~AGAG~G~G~UUUU~~~G~~GUG~CG~~G~~~G~C TTTTAGTCGGAGATG GACG

CATTTATGGATGTATTTTTTTTAC GTTTTG

GCG

AA

A

C

G

AAAA

GGCG

G

GC

GATATTTATTATGTGTTTTAGAATGTTTTTCTTTATTATGTAGATGTATATC

A CA CCCA

TTTTTA

G

GA G

GACGUUUUUGCGUUUGUUUUGUAAUUUAUUAUCC~U~AUUGUUGAUGUUUUUU~~UUUUUUU~~UUU~UUUUU~UUUUUUUUUUUU~

TACATTTTTACGAATGTTTTGTAAmATTATCTTCACATTTTTT~TGTT~TGTT~GTGAC~~TATA GAGA

GG G

A

A

A

GG

TTTTTTTATAATATTTTTTTTATTJF G

G

A

A

ATG

G

A A TTG

GGA

ATT

GCCTTT

uGGuGuuuuuuGuuA~~GA~~uuAuuuuAuuuAuuuuuG~G~~uuGuuuuuGuu~u~u~~~UG~Guuuu~~~WGuu~A~~~W~GCC***

GCCA

A

ACTTTTAG

A

A

GCCAUAUUAC****AGUUAUUUAUUUUUU

TTTGTGCATT

~u%uuuG

GAAG

A

AA

G

G

Lt DNA 5P18LT 5P2LT Figure

GAAA

GTTG TG ATTTTGGAGTT uuG**GuuU~uuA**WGGAGW

AAAAATAAGTATTTTGATATATTAAAGTAA AAAAATAAGTATTTTGATATTATTAAAGTAA AAAATAAGTATTTTGATATTATTAAAGTAA AAGTATTTTGATATTATTAAAG*AAtA AAAAATAAGTATTTTGATATTATTAAAGTAAtA

TATATAAAAAATTATATCAGATTAAGATAAATAA TATATXAAAAATTATATCAGATTAAGATAAAT TATATAAAAAATTATATCAGATTAAGA*AAA*

2. Comparison

of MURF4

G

RNA and DNA Sequences

G

GG

G

AG

GA

48

ATTACAAATATTTATATTTTGTAATATGATAATGCAGTTAAT

ELUUUUGAUAGUUAUUAUAUUGUUGUUGM~AU

Tb DNA 5P15 5P6 5P2 5Pl7

GCAG GA AA GGTTA CAGuuGAuA?dGG**

A A A G A ttttAttttttttttGtGAtttA A ttttAttttttttttGtGAtttA ttt.tAttttttttttGtGAtttA ttttAttttttttttGtGAtttA

G t 33 t

CAAATAAGTTAATAATA @&AUAAGWAAUAAUAAMAAAA h

GGA

G TTG G ttGttttttttGtTTGtGAtttA ttGttttttttGtTTGtGAtttA

from T. brucei

GA

A

A

and L. tarentolae

(A) The consensus T. brucei and L. tarentolae MURF4 RNA sequences, as determined by RNA and cDNA sequencing, are aligned below their corresponding DNA sequences (Simpson et al., 1967). Added uridines are indicated in lower case, and gaps are left at the corresponding positions in the DNA sequence. The positions of genomically encoded uridines deleted from the transcript are marked with asterisks, Where additions or deletions occur next to genomically encoded uridines, the 3’-most uridines are arbitrarily indicated as encoded. Gaps occurring in both the DNA and RNA sequences of one species relative to the other were placed to maximize amino acid sequence homology. The putative initiation and termination codons are boxed, and the caret indicates the polyadenylation site in T. brucei. The locations of oligonucleotides used in this study (see Experimental Procedures) are underlined. (6 and C) Sequence microheterogeneity at the 5’ ends of T. brucei and L. tarentolae MURF4 cDNAs, respectively. X indicates an ambiguous nucleotide.

maxicircle DNA sequence, and therefore these sequences are not edited in the final transcript. However, some cDNAs have sites that are edited 5’ to the most 5’ site of editing in the final transcript (Figure 28). Since editing can remove uridines, even those added by editing (Abraham et al., 1988) these regions that match the DNA sequence may have undergone editing.

MURF4 Transcripts Are Heterogeneous in Size Numerous partially edited MURF4 transcripts are present in both bloodstream and procyclic forms. The MURF4-1 oligonucleotide, which is complementary to the unedited 5’ MURF4 sequence, hybridizes in Northern blots to a 450 nucleotide RNA, about the size predicted for unedited MURF4 transcripts (Figure 3A). It also hybridizes to heter-

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0.780.53-

C

BPD

PD

Lt

-0.89

-0.78

z::$:

-0.45

0.400.280.16-

Frgure 3. Northern Transcripts

Blot Analysis

of T. brucei

and L. tarentolae

MURM

Ten microgramsof total RNA from bloodstream (lane B), procyclic (lane P), and dyskinetoplastic (lane D) mutant T. brucei were electrophoresed in formaldehyde-agarose gels, transferred to nylon membranes, and hybridized to end-labeled oligonucleotides MURH-1 (A) or MURF4-14 (6) complementary to unedited or edited sequences, respectively, near the 5’end of the RNA. In (C). a similar blot of L. tarentolae total RNA hybridized to the LtORF12-3 oligonucleotide, complementary to edited sequences, is shown. Nonspecific hybridization to one cytoplasmic rRNA subunit (R) is evident in (B) and (C), and such hybridization is more extensive in (A).

ogeneous sizes of RNA of up to 780 nucleotides, as would be predicted for partially edited RNAs where the region complementary to the oligonucleotide is not edited. The MURF4-14 oligonucleotide, which is complementary to edited sequence in the same region as MURF4-1, hybridizes primarily to a 890 nucleotide RNA, about the size predicted for fully edited MURF4 transcripts (Figure 38). It also hybridizes to heterogeneous sizes of slightly smaller RNAs, again as expected for partially edited RNA. None of these transcripts are detected in RNA extracted from dyskinetoplastic mutants devoid of mitochondrial DNA (Figures 3A and 36). To examine MURF4 transcripts further, MURF4 reverse transcriptase products were amplified by the polymerase chain reaction (PCR). Nucleotide sequence analysis of clones of these products, to be presented in detail elsewhere, revealed a diverse set of MURF4 cDNAs that are edited to various extents in the 3 region and unedited in the 5’ region, as described previously for partially edited COIII transcripts (Abraham et al., 1988). The 5’ End of the L. tarentolae MURF4 Transcript Is Extensively Edited The extensive editing of the T. brucei MURF4 transcript produces a potentially functional transcript with initiation and termination codons (boxed in Figure 2A) and a con-

tinuous open reading frame that predicts an amino acid sequence (Figure 4) with homology to the amino acid sequence predicted from the L. tarentolae MURM DNA sequence (Simpson et al., 1987). The two amino acid sequences have substantial homology in the internal region, but they abruptly diverge in the N-terminal and C-terminal regions. This suggested that the L. tarentolae MURM transcript may be edited near the S’end, although this was not detected previously by RNA sequencing (Shaw et al., 1988) since the oligonucleotide primer used was complementary to unedited sequences in the divergent N-terminal sequence. RNA sequencing using an oligonucleotide primer for the 5’ end of the region of conserved amino acid sequence revealed that the L. tarentolae MURF4 transcript is edited in the 5’ region (Figure 1F). As shown in Figure 2A, there are 106 uridines added at 46 sites and 5 encoded uridines removed from 4 sites in a 112 nucleotide region of editing. An initiation codon is created by editing in the L. tarentolae MURF4 transcript at the same position as in T brucei. Editing 5’ of the created AUG differs slightly in different cDNAs (Figure 2C), as was seen for T brucei. The N-terminal amino acid sequence predicted from the edited L. tarentolae transcript shows 79% homology to the T. brucei sequence (Figure 4). The sequence of the 3’ region of the L. tarentolae MURF4 transcript has not yet been determined, but the divergence of the predicted amino acid sequence from that of T. brucei suggests that it may also be edited. Northern blot analysis of L. tarentolae RNA reveals two sizes of transcript with both edited and unedited probes (Figure 3C). The basis for the size difference is not known. Sequence Characteristics of Edited Regions The T. brucei MURF4 gene, which encodes an extensively edited transcript, is GC rich, relative to other maxicircle sequences, and has a pronounced G versus C strand bias (Figure 5) such that the transcript is G biased. The L. tarentolae MURF4 gene is larger than the T. brucei gene, presumably reflecting less total editing, and encodes a transcript that is extensively edited in the 5’ region. The region that encodes the edited portion of the transcript is also GC rich with G versus C strand bias (Figure 5). The T. brucei COIII gene encodes an extensively edited transcript (Feagin et al., 198813) and exhibits similar G versus C strand bias, as do other regions of the maxicircle (Feagin et al., 1985; Jasmer et al., 1985, 1987; Simpson et al., 1987). Thus, this strand bias may help identify gene regions encoding extensively edited transcripts. Guide RNA A sequence that could encode an RNA that is complementary to 35 nucleotides of edited RNA and 13 nucleotides of adjacent unedited RNA, allowing G-U base pairing, at the 3’ end of the MURF4 mRNA has been identified (Figure 6). This sequence could encode a guide RNA (gRNA) that has been proposed to have a role in RNA editing (Blum et al., 1990). The gRNA sequence is in a minicircle (Jasmer and Stuart, 1986a) and, intriguingly, is located between 18 nucleotide inverted repeats that we previously identified (Jasmer and Stuart, 1986b).

Editing 889

of ATPase

6 mRNA

HYQFNFILSPLDQFEIRDLFSLNANVLGNIHLSITNIGLY

An Tb Lt An Tb Lt An Tb Lt Figure

4. Comparison

of the Predicted

MURF4

Amino

Acid Sequences

of T. brucei

and L. tarentolae

to that of A. nidulans

ATPase

6

The T. brucei MURF4 amino acid sequence and the N-terminal 67 amino acids of L. tarentolae MURF4 were predicted from the edited RNA sequence presented in Figure 3. The remaining L. tarentolae amino acid sequence is predicted from the DNA sequence (Simpson et al., 1987) and is underlined. The A. nidulans sequence is from Netzker et al. (1982). The boxed areas indicate amino acid identity and the shaded areas show conservative replacements. An, A. nidulans; Tb, T brucei; Lt, L. tarentolae.

various organisms. This homology is illustrated (Figure 4) by the comparison of the T brucei MURF4 amino acid sequence with the ATPase 6 amino acid sequence of Aspergillus nidulans (Netzker et al., 1982). These amino acid sequences have 37% total homology, including 23% representing perfect matches. While ATPase 6 amino acid sequences are not highly conserved among species, the C-terminal region is more conserved than elsewhere (Schneider and Altendorf, 1987). The degree of conserva-

We have also found a gRNA sequence for T brucei MURFB RNA that is similarly located on a minicircle between 18 nucleotide inverted repeats. The MURH Protein Product Is Homologous to ATPase 6 The amino acid sequence predicted from the edited transcript sequence of T. brucei has 214 residues and has limited but significant homology to that of ATPase 6 from

Tb Y I s Lt

I

J

250

MURP4

500

750

1000

1250

5. G versus

C Strand

1750

Bias of the MURF4

bp

2000

1

--j t-

Figure

1500

and MURFl

MURFI

Genes

of T. brucei

and L. tarentolae

The values are %G minus %C over a window of 50 nucleotides in 5 nucleotide steps for the coding strand. The coding strand of MURF4 is on the strand opposite to that of MURFl. The shaded regions show substantially greater strand bias than MURFI, for which no editing has been detected, and match regions of extensive editing. A gap has been left between MURF4 and MURFl of T brucei to preserve the alignment with the larger L. tarentolae MURF4 gene.

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8. CUUUAUAAUAWAUUJAUCUAAUIV\GUUAUUGUAAUAUA *t*t*

l t*tttttttt

tt

tt

l

l **

AAAAUGAUAUAAUAGAUAUGUAAUGAUAGAAUUAAUGUA

Figure

6. Potential

gRNA

Sequence

in a Minicircle

(A) The H40 minicircle (Jasmer and Stuart, 1986a) gRNA coding sequence (lower sequence) is aligned with the 3’ end of edited MURF4 mRNA (upper sequence). Uridines added by editing are in lower case; matches, including G-U base pairs, are indicated with asterisks and the 18 bp inverted repeats are underlined. The 13 nucleotides at the 3’ end of the block of perfect homology are present in both edited and unedited RNA. (6) Alignment of the 18 bp repeats showing potential base pairing.

tion between the T brucei MURM C-terminal sequence and that of other species is similar to the conservation among these other species (Figure 7). Strikingly, a number of sites that are conserved or conservatively replaced among species are conserved in T. brucei, including an arginine (position 210 in the Escherichia coli sequence), which has been implicated in proton translocation (Cain and Simoni, 1989). The predicted L. tarentolae MURM amino acid sequence shows the same degree of homology to ATPase 6 as does T. brucei MURF4 (Figure 4) except at the extreme C-terminal region, which may be edited (see above). In addition, the hydropathic profile of the T. brucei MURF4 amino acid sequence is similar to that of ATPase 6 profiles of other organisms (Figure 8) showing the alternating hydrophobic and hydrophilic regions characteristic of many proteins of the respiratory complexes. We conclude on these bases that the MURF4 gene encodes the ATPase 6 polypeptide. HS Xl Dm An ZUI EC Tb Hs Xl Dul An ZRl EC Tb

138) 138) 139) 163) 182) 190) 126)

Discussion Editing of the MUFlF4 transcript is extensive and combines many features of the remarkable RNA editing process that have also been demonstrated for the CYb, COII, COIII, MURF2, and MURF3 transcripts (Benne et al., 1986; Feagin et al., 1987, 1988a, 1988b; Feagin and Stuart, 1988; Shaw et al., 1988, 1989; van der Spek et al., 1988). Editing creates initiation and termination codons and a single continuous open reading frame in the fully edited MURM transcript. This results in a potentially functional transcript that has a predicted amino acid sequence with homology to ATPase 6 from other organisms. Importantly, the amino acid sequence is more conserved at the C-terminal region, as it is among other organisms, and amino acid residues that are conserved among all species examined are also conserved in T. brucei. Furthermore, the hydropathic profile similarity to that of ATPase 6 of

SS'flK *

*

*

&IGSATLAnSTINLPSTL

HS Xl m An ZUI EC Tb Figure 7. Comparison ATPase 6 from Other

of the Amino Organisms

Acid Sequence

Predicted

from Edited T. brucei

MURF4

mRNA

with the C-Terminal

Amino Acid Sequences

of

The conserved amino acids are boxed, conservative replacements are shaded, and ammo acid residues that are conserved in all the species are indicated with asterisks. The position of the beginning of each sequence relative to the N-terminal end of each ATPase 6 is shown in parentheses. Hs, Homo sapiens (Anderson et al., 1981); XI, Xenopus laevis (Roe et al., 1985); Dm, Drosophila melanogaster (de Bruijn, 1983); An, A. nidulans (Netzker et al., 1982); Zm, Zea mays (Dewey et al., 1985); EC. E. coli (Gay and Walker, 1981); Tb, T. brucei.

Editing 691

Xl

of ATPase

6 mRNA

I

Dm

Y ”

\I

Tb

I

Figure 6. Comparison of the Hydropathic Profile Predicted from the Edited T. brucei MURF4 mRNA to Those of ATPase 6 of Several Species Hydropathy values were calculated according to the method of Hopp and Woods (1961) and are aligned from the C-terminal end. The abbreviations are the same as in Figure 7.

other organisms predicted from the edited MURF4 amino acid sequence implies a similar structure and hence function. Taken together, these data strongly suggest that the MURM gene encodes ATPase 6. This is not a surprising finding since the mitochondrial DNAs of numerous other organisms encode ATPase 6 (reviewed in Wallace, 1982). These mitochondrial DNAs also encode other ATPase subunits, implying that other maxicircle regions may contain these genes, perhaps also encoded by extensively edited transcripts. G versus C strand bias serves as a convenient marker for sequences encoding extensively edited RNAs. Several small regions of the maxicircle in all three kinetoplastids examined exhibit these characteristics and encode heterogeneous sizes of G-rich transcripts that are larger than the coding sequence (Feagin et al., 1985; Jasmer et al., 1985, 1987). These regions probably encode extensively edited RNAs, a proposal now being assessed, and perhaps are genes normally found in mitochondrial genomes that have not yet been identified in the kinetoplastids. The bias for guanosines in extensively edited RNA sequences may reflect the involvement of G-U base pairing in gRNA binding. Regions where editing is less extensive, such as the 5’ region of L. tarentolae MURF3 transcripts (Shaw et al., 1988) do not always exhibit G versus C strand bias. However, many of these regions have purine versus pyrimidine strand bias, which is not surprising considering the paucity of encoded thymidines. Together, these characteristics may indicate edited regions that are not evident as interruptions in open reading frames.

The T brucei ATPase 6 transcript contains a larger 3’untranslated region than has been seen for other maxicircle genes examined to date. It is 143 nucleotides long, excluding the poly(A) tail, and contains 63 added uridines with 6 uridines deleted. No editing was seen in the poly(A) tail, but since only one cDNA examined had a poly(A) tail and it was quite short, editing in the poly(A) tail of ATPase 6 transcripts cannot be ruled out. Editing in the 3’ untranslated region and poly(A) tail are both seen in other transcripts (Benne et al., 1986; Feagin et al., 1988b; van der Spek et al., 1988, 1990; Feagin and Stuart, 1989); in fact, editing of the poly(A) tail of some transcripts occurs in the apparent absence of editing of the body of the transcript (Campbell et al., 1989; van der Spek et al., 1990). Poly(A) tail editing also appears heterogeneous, as different cDNAs for the same gene are edited differently in that region (Feagin et al., 1988b; van der Spek et al., 1988,199O). The 5’ editing of T. brucei ATPase 6 transcripts also exhibits microheterogeneity outside the reading frame, although it is uncertain whether this represents incomplete editing or less stringent control. The significance of editing in these regions is unknown; it could be a byproduct of the editing activity. Alternatively, it may affect functions other than protein coding, such as stability, in which 3’untranslated sequences have been implicated (Shapiro et al., 1987) and hence the abundance of the transcript and consequently the protein product. Editing also occurs in the 5’ region of the L. tarentolae ATPase 6 transcript, producing a predicted amino acid sequence with 79% homology to that of T brucei ATPase 6. It appears unlikely that the middle region of the L. tarentolae ATPase 6 transcript is edited, based on its 75% conservation of amino acid sequence homology to T. brucei. This conservation extends into the C-terminal region that is the most strongly conserved between species. However, the furthest C-terminal region of the L. tarentolae sequence diverges from those of ATPase 6 of T. brucei and other organisms, suggesting that editing may occur in the corresponding region of the L. tarentolae ATPase 6 transcript. Editing of ATPase 6 transcripts differs in both detail and extent between L. tarentolae and T. brucei. There are several sites where the same short (3-7 nucleotides) DNA sequence is edited to rather different sequences in the two species. For example, AGGAAAT is edited to Auu GuG UAU uuu AAu U in T brucei and Auu uuu GuG uuA uuu UAU K in L. tarentolae, which translate to IVYFN and IFVLFY, respectively. These differences may reflect differences in gRNAs used by the two species. While the short sequence given above occurs within two nucleotides of the same relative position in the two DNA sequences, the amino acid sequences are two codons out of register in the edited RNA. The differences in editing contribute to the different predicted amino acid sequences in these species, although several result in conservative amino acid replacements. The register is restored downstream by other editing differences, so that overall homology is maintained. The amino acid sequence predicted from the edited ATPase 6 transcript of T brucei has less homology to that of L. tarentolae and other organisms than is the

Cell 892

case for other edited transcripts. This may reflect the relatively low conservation among species of ATPase 6 N-terminal sequences. The mechanism of RNA editing and how the final nucleotide sequence is specified are not clearly understood. Numerous sensitive studies have failed to detect a DNA template complementary to edited RNAs (Benne et al., 1986; Feagin et al., 1987, 1988b; van der Spek et al., 1988; Shaw et al., 1989) including ATPase 6. Several lines of indirect evidence support the hypothesis that edited molecules are not transcripts from an undetected template, but are posttranscriptionally altered by a novel RNA processing mechanism. The poly(A) tails are edited (Benne et al., 1986; Feagin et al., 1987, 1988b; van der Spek et al., 1988, 1990; Campbell et al., 1989; Feagin and Stuart, 1989), and editing occurs in the 3’ to 5’ direction (Feagin et al., 1988b; Abraham et al., 1988). Edited transcripts match the maxicircle gene sequence exactly, except for the addition and deletion of uridines, and unedited transcripts that match these genes are invariably present (Feagin et al., 1987, 1988a, 19886; Feagin and Stuart, 1988; Shaw et al., 1988). Finally, numerous partially edited molecules containing both edited and unedited sequences have been detected (Abraham et al., 1988; unpublished data). These have the characteristics expected for editing intermediates, which appears to eliminate a simple transcriptional mechanism. Short sequences that are complementary to adjacent edited and unedited sequences, allowing G-U base pairing, are likely to play a role in specifying the RNA sequence during RNA editing, as suggested by Blum et al. (1990). The presence of potential gRNA coding sequences in minicircles suggests that these molecules are a repository of gRNA sequences. The parallel between the greater complexity of minicircles in T brucei (300 times minicircle size) than in L. tarentolae (a few times minicircle size) and the much more extensive editing in T. brucei than in L. tarentolae support this notion. The inverted repeats may play a role in RNA editing or more likely in processing gRNAs since the gRNAs reported by Blum et al. (1990) are approximately the size of the sequence between the inverted repeats (Jasmer and Stuart, 1986b). The interaction of gRNAs with transcripts to be edited is presumed to be mediated by a macromolecular complex, termed the editosome, with endoribonuclease, uridine addition, and RNA ligase activities. Candidate activities have been detected in these organisms (White and Borst, 1987; Bakalara et al., 1989). The rationale for the existence of RNA editing is not inherently obvious. Its origin is a mystery; perhaps it arose in the era of the RNA genome as a regulatory mechanism. The ability to modify mRNA sequence and consequently affect initiation and termination codons, coding sequence, and untranslated regions, and thus mRNA function, has the potential to provide extensive flexibility in regulating gene expression at the RNA level. This may provide an important selective advantage to parasites with complex life cycles. Experimental

Procedures

Cell Culture and RNA Isolation T brucei clone IsTaR 1 from stock EATRO

184 was grown

and isolated

as described previously (Stuart et al., 1984). Bloodstream forms were harvested after 3 days of infection in rats and were virtually all long, slender forms. L. tarentolae (University of California strain) was grown in brain heart infusion medium supplemented with 10 nglml hemin as described (Simpson and Braly, 1970). Cells were frozen in liquid nitrogen and stored at -80% prior to RNA extraction. RNA was isolated as previously described (Feagin et al., 1985) and stored at -8OOC. Oligonucleotide Primers The MURF4-1 and LtORFlZ-2 oligonucleotides are complementary to the unedited MURF4 transcripts of T. brucei and L. tarentolae, respectively. The MURF4-2, MURF4-7, MURF4-8, MURF4-9, MURF4-10, MURF4-11. and MURF4-14 oligonucleotides are complementary to the edited MURF4 transcript of T. brucei, while the MURF4-5 oligonucelotide matches this transcript. MURF4-6 matches the unedited MURF4 transcripts. MURF4-2 has one difference from the edited RNA sequence since it was based on a cDNA that was later found to be partially edited. The LtORFlZ-3 oligonucleotide sequence is complementary to the edited MURF4 transcript of L. tarentolae. The Notl-polyC oligonucleotide was used for anchor PCR (Loh et al., 1989). The sequences of oligonucleotides used in this study are shown below, and their locations (except for Notl-polyC) are shown in Figure 2A. MURM-1: MURF4-2: MURF4-5: MURF4-6: MURF4-7: MURF4-8: MURF4-9: MURF4-10: MURF4-11: MURF4-14: LtORFlZ-2: LtORFlZ-3: Notl-polyC:

S-CCTTTCTCCTTCATTTCCTCTCCTGTCTCCTTCTCTTCCGCCC-3’ 5’CAACCAAATTTCAACAACAATATAATAACTATC-3’ 5’GGATTTTTTGTTGTTTTTGTTGTTTGTTTAG-3 5’GGGCGGAAGAGAAGGAGACAGG-3’ 5’-GATCTTATTCTATAACTCC-3’ 5’TCCATTATCAACTGCAAAATC-3’ 5’-ATGGGATGATAATAAATTAC-3’ 5’CAAATTCAAATAAGTAATAC3’ 5’sCACAAACCAACAAACAAATACAAATC 5’CACAATAATACATACATAATAACAAACGCAACC-3’ 5’GTAATACAATAATATACAATCAATCC-3’ 5’CATCAAAAATACAAAACATTAACTCGGTAC-3’ 5’-GTGGCGGCCGCCCCCCCCCCCCCCCCCC-3’

Gel Electrophoresis, Blotting, and Hybridization RNA gel electrophoresis and blotting were done as described previously (Feagin et al., 1987). Prehybridization was carried out in 5x SSPE (lx SSPE: 90 mM NaCI, IO mM NaHsP04, 1 mM EDTA), 1% SDS, 200 nglml salmon sperm DNA, and 0.02% each of polyvinylpyrrolidone, Ficoll, and bovine serum albumin at 60%. Hybridization with end-labeled oligonucleotides was carried out in 5x SSPE, 0.1% SDS at 65%. Blots were washed five times with 5x SSPE, 0.1% SDS for 5 min each at room temperature, followed by a final wash with lx SSPE, 0.1% SDS for 1 min at 60% and exposed to film overnight. PCR Amplification and cDNA Cloning Twenty micrograms of total procyclic form T. brucei RNA was hybridized with MURF4-7, and first-strand cDNA synthesis was performed as described (Maniatis et al., 1982). RNA was hydrolyzed in the presence of 0.2 N NaOH at 65% for 45 min. After ethanol precipitation, half the cDNAwasamplified by PCR using MURF4-6and MURF4=Ioligonucleotides as described (Saiki et al., 1988). A total of 25-50 cycles of 1 min at 9Z°C, 2 min at 37OC, and 3 min at 72% were conducted. Anchor PCR was performed as described (Loh et al., 1989; Belyavsky et al., 1989). In brief, first-strand cDNA synthesized from procyclic T. brucei RNA using MURF4-11 or from L. tarentolae RNA using LtORFlZ-2 was tailed with dGTP and amplified using Notl-polyC as the 5’ member of the primer pair. PCR products were cloned into Bluescript vector either by GC tailing or by the addition of BamHl linkers (Maniatis et al., 1982). Library Screening and DNA and RNA Sequencing A procyclic form cDNA library prepared in Igtll (Feagin et al., 1987) was screened with end-labeled MURF4-1 oligonucleotide. Inserts from the positive clones were subcloned into the EcoRl site of Ml3mp18. Single-stranded DNA was isolated from Ml3 clones or, for PCR clones, from Bluescript vector using helper phage KO7, and sequenced by dideoxy chain termination using Sequenase (United States Biochemical), according to the manufacturer’s instructions. RNA was sequenced as previously described (Feagin et al., 1987, 1988a) using M-MLV reverse transcriptase (Bethesda Research Laboratories).

Editing 893

of ATPase

6 mRNA

Computer Analysis DNA strand bias was analyzed by the program BIAS (written by B. L. S.) set at a window of 50 bases and a step of 5 bases. BIAS calculates the percentage of each of the four bases within a user chosen window, and then repeats the calculation for a new overlapping window that starts one step value over from the original. For each window, the difference between percentages of chosen bases is printed along with the position of the center of that window. For the %G minus %C analysis, the value is positive if there are more Gs in the window than Cs, negative if there are fewer Gs than Cs, and zero if they are equal.

We thank Drs. Peter J. Myler and Augustine E. Souza for helpful suggestions and Andrea Perrollaz for excellent technical assistance. This work wassupported by National Institutes of Health grants Al14102 and GM42188 to K. S., who is also a Burroughs-Wellcome Scholar in Molecular Parasitology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. January

26, 1990; revised

March

8, 1990

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brucei.

Mol.

Feagin, J. E., and Stuart, K. (1989). Transcript alteration by mRNAediting in kinetoplastid mitochondria. In Molecular Biology of RNA, T. Cech, ed. (New York: Alan R. Liss). pp. 187-197. Feagin, J. E., Jasmer, D. P.. and Stuart, K. (1985). Apocytochrome b and other mitochondrial DNA sequences are differentially expressed during the life cycle of Tiypanosoma brucei. Nucl. Acids Res. 13. 4577-4596.

Abraham, J. M.. Feagin, of cytochrome c oxidase gion. Cell 55, 267-272.

J. E., and Stuart, K. (1988). Characterization Ill transcripts that are edited only in the 3’re-

Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L.. Coulson, A. R., Drouin, J.. Eperon. I. C.. Nierlich, D. P., Roe, 8. A., Sanger, F,, Schreier, F! H., Smith, A. J. H., Staden, Ft., and Young, I. G. (1981). Sequence and organization of the human mitochondrial genome. Nature 290. 457-465. Bakalara, N., Simpson, A. M., and Simpson, L. (1989). The Leishrnania kinetoplast mitochondrion contains terminal uridylyltransferase and RNA ligase activities. J. Biol. Chem. 264, 18679-18686. Belyavsky, A., Vinogradova. T., and Rajewsky, K. (1989). PCR based cDNA library construction: general cDNA libraries at the level of a few cells. Nucl. Acids Res. 17, 2919-2932. Mitochondrial

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Feagin, J. E., Shaw, J. M., Simpson, L.. and Stuart, K. (1988a). Creation of AUG initiation codons by addition of uridines within cytochrome b transcripts of kinetoplastids. Proc. Natl. Acad. Sci. USA 85,539-543. Feagin, J. E., Abraham, J. M., and Stuart, of the cytochrome c oxidase Ill transcript 53. 413-422.

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