Download A pseudogene cluster in the leader region of the Euglena

volume 10 Number 51982
Nucleic Acids Research
A psendogene dusteT in the leader region of the Euglena chloroplast 16S - 23S rRNA genes
Takashi Miyata, Reiko Kikuno and Yasumi Ohshima
Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, and Institute of
Biological Sciences, University of Tsukuba, Sakura-mura, Ibaraki-ken 305, Japan
Received 10 December 1981; Revised and Accepted 4 February 1982
ABSTRAa
The nucleotide sequence of a region( leader region ) preceding the 5'end of 16S-23S rRNA gene region of Euglena gracilis chloroplast DNA was
compared with the homologous sequences that code for the 16S-23S rRNA operons
of Euglena and E.coli. The leader region shows close homology in sequence to
the 16S-23S rRNA gene region of Euglena( Orozco et al.(1980) J.Biol.Chem. 255,
10997-11003) as well as to the rrnD operon of E.coli, suggesting that it was
derived from the 16S-23S rRNA gene region by gene duplication. It was shown
that the leader region had accumulated nucleotide substitutions at an extremely
rapid rate in its entirety, similar to the rate of tPNA Ile pseudogene identified in the leader region. In addition, the leader region shows an unique
base content which is quite distinct from those of 16S-23S rRNA gene regions
of Englena and E.coli, but again is similar to that of the t a w 1 * 6 pseudogene.
The above two results strongly suggest that the leader region contains a
pseudogene cluster which was derived from a gene cluster coding for the
functional 16S-23S rRNA operon possibly by imperfect duplication during
evolution of Euglena chloroplast DNA.
INTRODUCTION
Euglena gracilis chloroplast genome contains three tandemly repeated DNA
regions of 5.6 Kbp each that codes for rRNAs and tRNAs (1). The arrangement
of the genes within each repeat unit was shown to be 5' - 16S rRNA - tRNAs 23S rRNA - 5S rRNA - 3' (refs.2,3), which is identical to that of the rrnA,
D and X operons of E.coli (4,5). Recently the DNA sequence of the 16S-23S
rRNA spacer region, together with the flanking sequences that code for the 3'terminal region of 16S rRNA and for the 5'-terminal region of 23S rRNA, was
determined by two groups (2,3). Furthermore, Orozco, Jr. et al. (2) determined the DNA sequence of a region preceding the 5'-end of the 16S rRNA gene,
and showed that this 16S rRNA leader region is 68% homologous 1n sequence to
the 16S-23S rRNA spacer region, suggesting that they shared the same ancestral
DNA sequence at one time (2). Interestingly, the leader region was shown to
contains a tRNA I1e pseudogene (2).
Pseudogene is considered to be a region of DNA that shows marked homology
© IRL Prow Limited, 1 Falconberg Court, London W1V 5FG, U.K.
0305-1048/82/1005-17718 ZOO/0
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t o a f u n c t i o n a l gene i n sequence b u t has mutations t h a t prevent i t to f u n c t i o n
normally ( 6 ) .
Since the f i r s t r e p o r t o f Xenopus 5S RNA pseudogene ( 7 ) ,
several pseudogenes i n d i f f e r e n t multigene f a m i l i e s have been I d e n t i f i e d and
sequenced, i n c l u d i n g a - and 6-glob1n pseudogenes from mouse ( 8 - 1 0 ) , r a b b i t
(11),
goat (12) and man ( 6 ) , inmunoglobulin v a r i a b l e domain pseudogenes from
man (13) and mouse ( 1 4 ) , leukocyte i n t e r f e r o n pseudogene from man (15) and
small nuclear RNA pseudogenes from mouse (16) and man ( 1 7 ) .
Nishioka and
Leder (8) and Maniatis and others (6,11) pointed out t h a t pseudogenes could
accumulate mutational changes at a r a p i d r a t e .
We (18-20) and others (21-23)
showed t h a t pseudogenes evolve at an e x t r a o r d i n a r y r a t e t h a t i s higher than
the rate o f changes between synonymous codons, one of the most r a p i d l y e v o l v i n g components of f u n c t i o n a l genes ( f o r r e f e r e n c e s , see r e f . 2 4
).
From the observations of rapid r a t e o f e v o l u t i o n and unusual base contents
i n the 16S rRNA leader region of Euglena c h i o r o p l a s t DNA, both o f which are
s i m i l a r to those of the tRNA
e
pseudogene but are q u i t e d i s t i n c t from those
o f f u n c t i o n a l 16S-23S rRNA gene regions of Euglena and E . c o l i rrnD operon,
we r e p o r t here t h a t the leader region contains a c l u s t e r of pseudogenes.
This may be a f i r s t reported example f o r a pseudogene c l u s t e r derived from an
operon.
Hereafter
we w i l l call the 16S-23S rRNA region i n c l u d i n g the non-
coding spacers as the f u n c t i o n a l
tional
region t o d i s t i n g u i s h i t from the non-func-
leader r e g i o n .
METHODS
F1g.l schematically shows the l o c a t i o n of the f u n c t i o n a l 16S-23S rRNA
region and the n o n - f u n c t i o n a l 16S rRNA leader region i n the Euqlena c h l o r o p l a s t
, the DNA sequences o f which were compared.
The sequences of the two d i f f e r e n t
DNA regions were aligned w i t h t h a t o f the f u n c t i o n a l 16S-23S rRNA region of
E . c o l i rrnD operon (4,5) ( F i g . l b ) .
Based on the alignment, we have c a r r i e d
out a pairwise comparison among the three homologous DNA sequences, and the
extent of sequence d i f f e r e n c e per s i t e ( K ) was c a l c u l a t e d f o r each o f the
f o u r d i f f e r e n t f u n c t i o n a l b l o c k s , I n c l u d i n g the 3 ' - t e r m i n a l
region o f 16S rRNA
lie
gene, the adjacent noncoding spacer, the tRNA
gene and about first twothird of the tRNA
gene. Since, 1n Euqlena chloroplast DNA, the noncoding
spacer between the tRNA e and the tRNA a genes 1s short in size, we excluded
this segment from further analysis. In calculating the K, a gap was considered
as a base change. When a gap consisting of more than one base sites was found
in any one of sequences compared, it was regarded as one base change in one
site. When gaps of the same length were found in the same position of both
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a)
16S
23S 5S 16S 23S 5S 16S
23S 5S
5'HZH
23S
b)
16S rRNA
TGACTGGGGTGAAGTCGTA
ACAAGOTAGCCGTACTGGAAGGTGTGGCTGGAACAACT
A
TA C T T C GTGTA TT TT
TCTT C
T
A
GG
CC C T
T C
Spacer
CTTTAGTTTTTTAACTAAAAAAA
TGTATAGATTGAAAACAATGAAAAA-TA
-- TT A
T
T C
C
A
T
A
TT*|
C T
G GCGTAC T GC G CTC
C» TTGTC G
tRNA-Ile
AAAAAAATAAGTAGGGAAACCTCTTATTTTTCCAAGA3GGCTATTAGCTCAGTTGGTTAG
TC AA
TT T
T TTTA TA T
TG
G C TA
ACt
TG
AGCATACCCTTGATAAGGGTAAGGTCGCTAGTTCAAGTCTAGCATGGCCCTA
T
G
TGGA G TC T T TAG A
GC
C
G
G G
C CTCA
TACCAHATTTG
tRNA-Ala
CTTGGTTTT 5GGGGTATAGCTCAGTTGGTAGAG
AA AT
T CTT A AA CC AG
CACGGCAAATTTGAAGAGGTTTTAACTACAT
C
C G
CGCTGCCTTTGCAAGGCAGATGTCA
TA GTC CCA AC T
A
CTG
C CAG G T
Fig.l. Alignment of sequences of the functional 16S-23S rRNA region and the
non-functional 16S rRNA leader region of Euglena chloroplast with that of the
functional 16S-23S rPNA region of E.coli rrnD operon. a) The arrangement of
the rRNA gene clusters on Euglena chloroplast DNA is schematically shown.
Hatched bars: regions where DNA sequences have already been determined (2,3).
Arrows indicate regions whose sequences were compared with that of the homologous region of E.coli rrnD operon. b) Alignment of the sequences among the
functional 16S-23S rRNA region (top) and the non-functional leader
region
(middle) of Euglena chloroplast and the functional 16S-23S rRNA region of E_.
coll rrnD operon (bottom). Only the sites where bases differ from the top~are
shown for the middle and the bottom. RNA coding sequences are boxed. Dashes
indicate gaps. Sequence data were taken from ref.2 for the two homologous
regions of Euglena and from refs.4 and 5 for E.coli.
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the sequences compared, the corresponding sites were excluded from the calculation.
RESULTS AND DISCUSSION
Table 1 shows sequence differences among the functional 16S-23S rRNA region of Euglena ( Gf ) and E.coli ( Cf ) and the 16S rRNA leader region of
Eugl.ena ( Gl ). Appreciable homology between Gf and Gl for overall the regions
suggests that they were derived from a conmon ancestor by gene duplication (
2 ). A plausible phylogenetic relationships among the Gf, Gl and Cf based on
the K values of Table 1 would be that, after the separation of chloropiast and
prokaryotic genomes, the DNA sequences of Gl were diverged by gene duplication
and accumulated mutations at a higher rate than did the Gf and Cf ( Fig.2 ):
The greater homology between Gf and Cf than between Gf and Gl for the rRNA and
tRNAs regions 1s presumably due to strong functional constraints on the functional Gf and Cf sequences. This 1s reverse of the corresponding comparisons
for the noncoding spacer, the sequence of which 1s constraint much more weakly
than the RNA coding sequences. The difference 1n K value between Gf/Cf and
Gf/Gl for the noncoding spacer might be useful to fix the relative point 1n
time at which the duplication took place relative to the Gf and Cf split. The
smaller K value between Gf and Gl than between Gf and Cf for this region
supports the phylogeny in Fig.2. In addition, difference in K values between
Gf/Gl and Cf/Gl could be understood on the basis of phylogeny in Fig.2: Writing "0" for a common ancestor of Gf and Gl, we have for sequence difference
between Gf and Gl ( designated as K(Gf-Gl) ), K(Gf-Gl) = K(O-Gf) + K(O-G1),
here K(O-Gf), for example, means difference between 0 and Gf. Similarly we
Table 1. Pairwise comparison among the sequences of the functional 16S-23S
rENA region ( Gf ) and the non-functional leader region ( Gl ) of Euglena
chloroplast and the functional 16S-23S rHNA region ( Cf ) of E.coli for each
of the 3'-terminal 16S rRNA gene, 16S-tRNAIle spacer, tRNA Ile and tRNAAla genes.
K:difference per site
Gf/Cf
Gf/Gl
Cf/Gl
,
16S rRNA
noncoding spacer
0.167
0.463
0.240
0.245
0.393
0.263
0.365
0.533
0.446
0.607
0.480
0.622
,r/o-m+<j
K(O-Gf)±O
v(n-rn+o
K{O-G1)±O
0.057±0.032
0.06O+.0.029
0.063*0.029
0.078*0.041
0.336+0.077
O.2O4±O.O54
0.303*0.064
0.455*0.099
Sequence differences ( K ) per site were calculated on the basis of the alignment shown in Fig.l. K(O-Gf), for example, means difference between O and Gf.
Assuming that the total number of base changes be distributed in a poisson
fashion, standard error a was estimated as 0 - /K/N , where N is the number of
sites in the sequence compared.
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Fig.2. A schematic picture representing
the phylogenetic relationships among
three homologous rRNA gene clusters of
Euglena chloroplast and E.coli. Gf and
Gl: functional 16S-23S rRHA region and
non-functional leader region of Euglena
, respectively. Cf: rrnD operon of E_.
coli. O: common ancestor of Gf and Gl.
Gl
Cf
have K(Cf-Gl) = K(O-Cf) + K(O-G1). Since K(Gf-Gl) and K(Cf-Gl) contain
K(O-G1) in common, the different values between the two could be attributable
to the different time Intervals between functional lines O-Gf and O-Cf. Thus
the smaller values of K(Gf-Gl) than those of K(Cf-Gl) in Table 1 support the
argument that Gf and Gl diverged after Gf and Cf split.
Orozco, Jr. et al. (2) described a tRNA Ile pseudogene 1n the 16S rRNA
leader region of Euglena chloroplast. This gene shows close homology in sequence to a functional tRNA e gene and the sequence could be folded into a tRNA
-like cloverleaf structure but has changed considerably from the functional
counterpart, both in terms of alternations in Invariant sites and 1n loss of
base pairing 1n stem regions. These alternations rendered 1t unable to function normally, and thus this gene was identified as a pseudogene. Similar
consideration might also be possible for the DNA segment corresponding to the
tRNA Ala gene region, although, in this case, the tRNA A1a anticodon 1s altered
into tRNA P anticodon due to mutational change(s). The loss of function frees
the gene from several functional constraints, thereby allowing accumulation of
mutations at an elevated rate. Recent Investigations (18-23) on pseudogene
evolution showed the extraordinarily rapid rate of evolution. In contrast,
functional tRNAs are thought to be the most slowly evolving molecules (25).
These lines of evidence support the phylogeny in F1g.2.
On the basis of the phylogeny, we have carried out a further analysis for
the different rates of evolution between the functional 16S-23S rRNA region
and 16S rRNA leader region of Euglena on statistically solid background (20).
Knowing the sequence differences between temporal sequences, the K(O-Gf) and
K(O-G1) can be estimated for each of the four different functional blocks from
the equations:
K(O-Gf) = [ K(Gf-Gl) + K(Cf-Gf) - X(Cf-Gl) ] / 2
K(O-G1) = [ K(Gf-Gl) + K(Cf-Gl) - K(Cf-Gf) ] / 2
,
.
(1)
(2)
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The estimated values are shown in the last two columns of Table 1. Since time
interval between 0 and Gf is the same as that between 0 and Gl, the values of
K(O-G1) could be compared directly with that of K(O-Gf), and thus they could
be considered as relative evolutionary rates. It is apparent that the K(O-G1)
is significantly larger in value than the K(O-Gf) for all the gene regions
lie
compared without exception. For the tRNA
gene region, this result is well
understandable on the viewpoint that pseudogenes evolve much faster than functional genes. The result that all the gene regions of the 16S rRNA leader
lie
region evolve at rates being similar to that of the tRNA
pseudogene, but
much higher than those of the functional region genes strongly suggests that
the leader region examined consists of a cluster of pseudogenes.
Table 2 shows the base contents of the functional 16S-23S rRNA region from
Euglena and E.coli and of 16S rRNA leader region from Euglena for each of the
three RNA coding regions and the noncoding spacer region. In the functional
16S-23S rRNA region, base contents are similar between Euglena and E.coli and
also between different RNA coding sequences except for the noncoding spacer.
In contrast, Euqiena 16S leader region is quite different in base contents
from the functional 16S-23S rRNA region. Indeed, goodness of fit test (26)
shows that the base contents of the former deviate significantly from those of
the latter for all the RNA coding regions. In Euglena, the noncoding spacer
of the functional 16S-23S rRNA region strongly avoids using C and G and 1s
rather similar 1n base contents to the 16S rRNA leader region, which may contribute to reduce the K(O-G1) to some extent for this block, compared with
other RNA coding blocks.
The tRNA
pseudogene 1s rich in T and A, relative to the functional
tRNA
genes. It is remarkable that T content increases significantly 1n the
former relative to the latter, while G appears to be in much lesser extent.
lie
In comparison between the functional tRNA
gene and its pseudogene counterpart of Euglena, base changes from G and C in the functional gene to T and A in
the pseudogene are four times more frequent than changes from T and A in the
functional gene to G and C 1n the pseudogene. Similar situation 1s also found
for other different blocks. This may implies that the loss of function allows
to accumulate base changes from G and C to T and A more preferentially than
vice versa, which leads to higher T+A contents than G+C in the pseudogene. In
this regard, the following may be worth comment. Functional globin genes
prefer to use G- and C-ending codons and strongly avoid using A-( and T- )
ending codons. Whereas 1n the pseudogene counterparts, significant increase
of A-ending codons were observed (19), which is analogous to the present case.
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Table 2. Comparison of base contents among the three homologous 16S-23S rRNA
gene clusters.
Euglena
functional
16S-23S
f
16S rRNA
T
C
A
G
Euglena
non-functional
leader
n
0.20(12)
0.19(11)
0.25(15)
0.36(21)
X
noncodlng
spacer
T
C
A
G
0.31(27)
0.09( 8)
0.48(42)
0.12(10)
x2
tRNA 1 1 6
T
C
A
G
0.28(21)
0.20(15)
0.23(17)
0.28(21)
x2
tRNA
T
C
A
G
0.25(12)
0.18( 9)
0.20(10)
0.37(18)
x2
f
n
E.coli
functional
16S-23S
f
n
0.41(23)
0.16( 9)
0.29(16)
0.14( 8)
0.23(14)
0.21(13)
0.24(15)
0.32(20)
20.3
(P< 0.005)
0.58
(P> 0.1)
0.40(31)
0.05( 4)
0.49(38)
0.05( 4)
0.25(17)
0.22(15)
0.34(23)
0.19(13)
6.3
(P~ 0.1)
19.4
(P< 0.005)
0.41(30)
0.14(10)
0.27(20)
0.19(14)
0.23(17)
0.23(17)
0.20(15)
0.34(25)
8.4
(P< 0.05)
2.1
(P> 0.1)
0.32(14)
0.21( 9)
0.32(14)
0.16( 7)
0.20(10)
0.25(12)
0.16( 8)
0.39(19)
9.4
(P< 0.025)
2.0
(P> 0.1)
n and f: the number of a given base and its frequency appeared in each of the
four different functional blocks of Fig.lb/ respectively. Considering the
base frequency of the Euglena functional region as an expected frequency,
goodness of fit test (26) was made for the Euglena leader region and the E.coli
functional region in every block. The x 2 value and the probability P that
the x 2 value realizes were shown for the two regions.
It 1s remarkable that, in the leader region, the base contents of the RNA
He
pseudogene. From the two
coding blocks are similar to those of the tRNA
lines of observations that the leader region genes evolve much faster than the
functional region genes and have quite distinct base contents from the latter
region genes, while they are quite similar 1n terms of evolutionary rate and
base contents to the tRNA
e
pseudogene, we conclude that the sequence of the
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16S rRNA leader region 1s a r e l i c of 16S-23S rRNA gene cluster having no functions for the genes.
By what reasons the pseudogene cluster emerged? A large rRNA precursor
containing both the 16S and 23S rRNA genes was observed in spinach (27,28) and
Chlamidomonas (29) chloroplasts. A large rRNA of similar size has been observed also 1n the Euglena chloroplast (30). Although, at present, there 1s no
experimental evidence for transcription of the 16S-23S rRNA spacer region, i t
is highly l i k e l y that the 16S-23S rRNA gene cluster in the Euqlena chloroplast
1s transcribed Into a single precursor RNA analogous to bacterial operons.
Thus 1f some of the signals essential for the proper function of the common
precursor are lost by aquisition of mutational changes, due to imperfect duplication or by other reasons, a l l the genes within the same transcription unit
may be unable to function normally and subsequently they became pseudogenes,
even 1f some of the genes preserve their sequences necessary to function as a
single gene completely. According to the restriction map of the rRNA transcription units of Euglena chloroplast DNA (2), there 1s no space to Include a
complete set of 16S-23S rRNA gene cluster between the 5S and 16S rRNA genes.
This suggests that the genes in the 16S rRNA leader region had no potential
for producing functional precursor RNA from the begining due to imperfect
duplication. Alternatively, these genes originally functioned as a perfect
set of duplicate and then accidentally aquired deletion mutation(s) that
resulted in inactivation. Although at present there 1s no additional evidence
to decide which of the two interpretations is more plausible, we rather prefer
the former.
Each of the three Identical repeat segments of Euqlena chloroplast DNA
contains 2.6 Kbp Ecp_ RI fragment Ec£ P which includes both the functional 16S23S rRNA region and 16S rRNA leader region (1,2). Orozco, Jr. et a l . (2)
showed that there is no large differences 1n sequence, nor significant Insertions and/or deletions among the three Ecp_ P DNAs. This observation suggests
that Euqlena chloroplast DNA contains three identical pseudogene clusters, each
of which flanks d i r e c t l y to the rRNA transcription unit analogous to Xenopus
5S rRNA gene system (31). Thus the evolutionary history of Euqlena chloroplast
rRNA gene clusters may be: The functional 16S-23S rRNA region and the 16S rRNA
leader region was f i r s t diverged from a conmon ancestor by gene duplication,
but presumably one of the duplicants had no functions from the begining due to
imperfect duplication. Subsequent tandem duplication of a unit DNA segment
including the above two regions had taken place to form the present-day rRNA
gene organization of Euglena chloroplast. The lack of difference between the
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three sets of pseudogene cluster would suggests that either some "homogenisation" process ( 32 for review ) 1s occurring all the time since the successive duplication events took place, or that the last triplication occurred
very recently. It would be interesting to know if there 1s also a corresponding leader region 1n E.coli.
ACKNOWLEDGEMENTS
We thank Prof. H. Matsuda, Dr. M. Hasegawa and Dr. T. Yasunaga for discussions and conments. This work was supported in part by grants from the
Ministry of Education, Science and Culture of Japan.
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