*Laboratory of Molecular Evolution and Genome Diversity, and Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Yunnan, People's Republic of China;
Yunnan University, Yunnan, People's Republic of China;
Human Genetic Center, University of Texas at Houston
It has gradually become clear that the transfer of genetic material between mitochondrial and nuclear genomes may still be occurring in a diverse array of eukaryotic lineages (Nomiyama et al. 1985
; Zullo et al. 1991
; Smith, Thomas, and Patton 1992
; Lopez et al. 1994
; Arctander 1995
; Collura and Stewart 1995
; Blanchard and Schmidt 1996
; Moreiro and Seuanez 1999
). Most of these mitochondrial DNA (mtDNA)-like sequences in nuclear genomes are believed to be nonfunctional and thus pseudogenes. Although nuclear pseudogenes may mislead some studies of molecular phylogeny based on mtDNA, they are very useful for inferring genetic and evolutionary processes (Zhang and Hewitt 1996
; Bensasson, Zhang, and Hewitt 2000
; Mirol, Mascheretti, and Searle 2000
). Because of a higher evolutionary rate in the mtDNA than in the mtDNA paralogs in the nuclear genome, mtDNA-like pseudogenes may preserve more ancestral characteristics than do their homologous mtDNA.
Based on this assumption, the pseudogenes can be regarded as molecular fossils that can be used both to reconstruct more accurately the common ancestral state in phylogenic studies and to address issues about selective constraints on cytoplasmic mitochondrial genes (Zischler et al. 1995
; Perna and Kocher 1996
; Lopez et al. 1997
; DeWoody, Chesser, and Baker 1999
). However, the evolutionary process governing the transfer from mitochondrion to nucleus is incompletely understood. It is still an open question whether the pseudogenes evolve with a constant rate after transfer, and it is often unclear as to how and when the nuclear copies became nonfunctional. More analyses of the tempo and mode of base substitutions of mtDNA-like pseudogenes will shed light on the evolutionary process of transfer and pseudogenization.
In our previous study (Lü, Wang, and Zhang 2001
), mitochondrial cytochrome b (cytb) sequences of Nycticebus coucang and N. pygmeause were obtained with the universal primers L14724-H15149 and L15136-H15915 (Irwin, Kocher, and Wilson 1991
; Yu et al. 2000
). In this study, we used the cytochrome b universal primers L15408 and H15915 to amplify and directly sequence a PCR product from the N. coucang individual. This sequence differed by 16.2% from the cytb gene of the same N. coucang individual. The unexpected sequence contained one (TAG, at site 250252) or numerous stop codons in the three different reading frames based on the mammalian mitochondrial or nuclear universal code, respectively. Therefore, this fragment is presumably nonfunctional and likely a pseudogene of the mitochondrial cytochrome b (
cytb).
A pair of primers (cytbL 5'-TAGTACTATTCTCCCCTGACCTTC-3' and
cytbH 5'-GGCTTGTTAGTGGTATGAGGA TT-3') was designed to specifically bind to the pseudogene. PCR conditions were as followed: 94°C for 2 min, then 35 cycles of 94°C for 30 s, 65°C for 60 s, and 72°C for 60 s, followed by 72°C for 10 min. The cytb pseudogene fragment (390 bp) was successfully amplified from seven individuals of N. pygmaeus and three of N. coucang. To identify where the pseudogene copy was located, PCR amplification with purified mtDNA (Zhang and Shi 1989
) as the template was performed using primers
cytbL and
cytbH. No amplified product was detected, which confirmed that the fragments were in the nuclear genome of N. pygmaeus and N. coucang.
The cytb PCR products were determined by cycle sequencing on an ABI 377 sequencer. The ends were trimmed to create a 390-bp alignment (DNASTAR package, Dnastar Inc., 1996) for further analysis. Using MEGA Ver.1.02 (Kumar, Tamura, and Nei 1994
), Li93 (Li 1993
), and DAMBE (Xia 1998
), we compared the characteristics of nucleotide substitutions of cytb and
cytb sequences (table 1 ).
|
We divided nucleotide substitutions into transitions and transversions, and the two genes revealed marked differences. A bias toward transitional substitutions has been observed for mitochondrial genes in closely related species (Yoder, Vilgalys, and Ruvolo 1996
; Yang and Yoder 1999
). Between N. coucang and N. pygmaeus, transitions in cytb accumulated 13.3 times faster than those in the corresponding
cytb sequences. Cytb transitions appear to have a nonrandom distribution among the three codon positions: most transitions (31 of 40) occur at the third coding position. However, there is only one transition at the third coding position in the
cytb. The difference in substitution rate is less distinct when considering only transversions, of which there are five and four for the cytb and
cytb copies, respectively. Four of five cytb transversions occur at third positions, whereas the distribution is more even along the sequence of
cytb copy. The pattern of nucleotide substitution in pseudogenes reflects the trend of spontaneous point mutation. The transition-transversion ratio within mitochondrial cytb (8.0) is much higher than that within
cytb (0.75).
In general, the selective pressure acting on protein-coding genes is greater in nonsynonymous substitutions than in synonymous substitutions. Pseudogenes are free from the selective constraints of coding sequences as reflected by the lack of a bias in the ratio of synonymous substitution per site (Ks) to nonsynonymous substitution per site (Ka) (Li 1997
, pp. 177213). The equal values of Ks and Ka between the two pseudogenes (Ks/Ka = 1.00) in comparison with the large difference between the two mitochondrial cytochrome b's (Ks/Ka = 25.99) is strong evidence that the
cytb gene has been evolving devoid of selective pressure since the divergence of the two species.
The high proportion of nonsynonymous base changes in cytb relative to cytb, the presence of premature termination codons, as well as the lack of bias toward transitions and synonymous substitutions strongly suggest that
cytb sequences are nuclear cytochrome b pseudogenes.
Based on the molecular phylogeny of Strepsirrhine primates (Yoder et al. 1996
), we chose multiple cytochrome b sequences from African species to use in phylogenetic analyses. These analyses helped reveal the origin of
cytb sequences. All phylogenetic trees obtained by different methods unambiguously support two monophyletic clades: one consisting of the cytb copies from each species and another consisting of the
cytb copies. The phylogenies also clearly showed the out-group status of Loris tardigradus for the Nycticebus sequences (fig. 1
). The
cytb clade originated after the divergence between Nycticebus and L. tardigradus but before the split between N. coucang and N. pygmaeus.
|
Divergence () between a pair of sequences is commonly expressed as a linear function of time (T),
= 2
T, where
is the absolute rate of substitution expressed as percent base pair divergence per million years (%bp/Myr). Assume that the mt-cytb sequences of the two species evolve at a uniform rate (
m) during both time periods T1 and T2, whereas the two
sequences had different rates,
n1 and
n2, respectively. We reconstructed the ancestral states of DNA sequences and estimated the number of substitutions between neighboring nodes on the phylogenetic tree with MacClade (Maddison L. H. and Maddison D. R. 1992
). There were 24 substitutions on the branch from A to AN and 20 substitutions on the branch from A to AM. Using these numbers of substitutions and the method for
(table 1
), we estimated 0.0642 (
2) and 0.0531 (
1) for sequence divergence between A and AN, and A and AM, respectively. Let
1,
2,
3, and
4 (
3 and
4 showed in table 1
) be the values of pairwise divergence between the most recently common ancestor of two cytb copies (A) and the common ancestor connecting the mitochondrial sequences (AM), between A and the common ancestor connecting the pseudogene sequences (AN), between the functional cytochrome b sequence in N. coucang (cytbNcou) and that in N. pygmaeus (cytbNpyg), as well as between the sequence of cytochrome b psuedogene in N. coucang (
cytbNcou) and that in N. pygmaeus (
cytbNpyg), respectively.
|
|
|
Zischler, Geisert, and Castresana (1998)
suggested that an increase in evolutionary rate of a mitochondrial pseudogene could be due to a shift from the mitochondrial polymerase-based replication machinery to the nuclear polymerase system. However, such an explanation does not fit our data. We compared the pseudogene sequences from N. coucang and N. pygmaeus with published sequences of the mitochondrial cytb gene in primates. Based on the principle of maximum parsimony and the method proposed by Zischler, Geisert, and Castresana (1998)
, we determined the mutations that unambiguously occurred on the Numt-ancestor lineage. Few substitutions occurred in the conserved regions of cytb or in CpG-dinuleotides. Most of the changes in the branch leading to the AN (fig. 1
) were transitions (20/25 = 80%), occurred at third codon positions (18/25 = 72%), and were synonymous (Ks/Ka = 4.9). This pattern appears to reflect selective constraints on a functional mitochondrial gene. The most likely interpretation of our results is that the cytb pseudogene originated from a duplication event in the mitochondrial genome of common ancestor A. The duplicated copy of the cytochrome b gene was functional in the mitochondrial genome of the common ancestor of N. coucang and N. pygmaeus, diverged from the original copy, inserted into the nuclear genome, and pseudogenized. Under this scenario, the long branch from A to AN reflects fast mutation rates in the mitochondrion.
To our knowledge, no duplication or deletion of any of the mitochondrial genome has been observed in complete mtDNA sequences from vertebrates. But a few reports have shown the possibility that duplicated copies of a gene could exist in the mitochondrial genome over a long time span. For example, Lopez et al. (1994)
reported that the Numt gene in cats could have existed in the mitochondrion and evolved as mitochondrial genome for a long time. Similarly, Southern hybridization experiments using mtDNAs purified from multiple akamata individuals showed that the duplicate state of the control region was not a transient or unstable feature found in a particular individual but that it stably occurred in mitochondrial genomes of the species, which existed at least 70 MYA, according to fossil records (Kumazawa et al. 1998
).
According to the branch length of the gene tree (fig. 1
), and assuming the uniform evolutionary rate (m) of mt-cytb sequences during both time periods T1 and T2, we estimated that T1 is nearly equal to T2. That is T1 = (20/22.5) x 3.0 = 2.7 Myr, where 22.5 is the mean number of substitutions from present-day cytochrome b in N. coucang and N. pygmaeus to their ancestor. The translocation of the duplication to the nuclear genome occurred between 3.0 to 5.7 MYA (T1 + T2). Although it is difficult to be precise, the translocation event more likely occurred at a time closer to the speciation date than to the duplication date for the following reasons: (1) the fact that
cytb differs by only seven changes between species, compared with 45 changes in cytb, strongly supports that the rate of change slowed after the gene was translocated into the nucleus; and (2) if translocation occurred soon after duplication, it would be difficult to explain why the gene was evolving relatively fast before species' divergence but then slowed roughly 10-fold after speciation. If we assume that translocation occurred right before speciation, then the observations
n1 =
2/T1 = 0.0642/(2.7 x 106) = 2.38 x 10-8 and
n2 =
4/2T2 = 0.0182/(2 x 3.0 x 106) = 3.03 x 10-9 substitutions/site/year agree with the well-documented difference in rate between mitochondrial genomes (2.5 x 10-8 substitutions/site/year; Hasegawa, Kishino, and Yano 1985
) and nuclear pseudogenes (34 x 10-9 substitutions/site/year; Li 1997
).
Acknowledgements
We are grateful to the insightful advice of Dr. Arndt v. Haeseler and two reviewers. We thank Y.-W. Zhang, H.-P. Li, C.-H. Wu, and D. Pan for their constructive discussions and S.-K. Gou for technique assistance. This work is supported by Chinese Academy of Sciences (KSCX2-1-05) and National Natural Science Foundation of China.
Footnotes
Brandon Gaut, Reviewing Editor
Keywords: mitochondrial cytochrome b pseudogene
evolution
Nycticebus
Address for correspondence and reprints: Ya-Ping Zhang, Laboratory of Molecular Evolution and Genome Diversity, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, People's Republic of China. E-mail: zhangyp{at}public.km.yn.cn
.
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