臺灣博碩士論文加值系統

本研究有兩個重點：（1）人類與三種現存巨猿（黑猩猩、大猩猩、紅毛猩猩）之間的遺傳距離以及（2）人類與黑猩猩之間遺傳距離的深入研究。
過去對物種間遺傳距離的研究均過於狹隘，以少數幾個基因推演概括物種間之基因組整體距離。但若欲完成整個基因組定序後再加以比對，又遠非個別實驗室財力與人力所能及。本研究示範了利用隨機取樣定序的方式，以最小的物力、人力與時間成本，得到正確的基因組遺傳距離，並且首度展示如何對包含重複序列在內的大片段序列進行排序。對於爾後分子演化相關領域的研究，為一先驅性的研究示範。
我們選取了53段基因間片段，分別測定其在人類、黑猩猩、大猩猩與紅毛猩猩之中的去氧核糖核酸序列並計算其遺傳距離。人類─黑猩猩、人類─大猩猩、黑猩猩─大猩猩之間的平均遺傳距離分別是1.24% ± 0.07%、1.62% ± 0.08%，以及1.63% ± 0.08%，此三組遺傳距離均顯著小於之前以偽基因為基準的估計。紅毛猩猩與人類、黑猩猩、大猩猩之間的遺傳距離分別是3.08% ± 0.11%,，3.12% ± 0.11% 與 3.09% ± 0.11%。其他不同區域的遺傳距離可由基因庫中的序列求得。若依照遺傳距離的大小順序排列，阿魯序列的遺傳距離最大，其餘依序為Y染色體非編碼基因序列、假基因、同義突變、體染色體基因間片段、X染色體非編碼基因序列、內含子、與非同義突變。
我們依據這53個片段相接而成的去氧核糖核酸序列（24,234 鹼基對）畫成鄰接演化樹，該演化樹以100％的重複取樣統計值支持人類和黑猩猩是最相近物種。然而，若我們將各個片段分別畫成鄰接演化樹，則有22個片段（約42％）的基因樹與物種樹不同，此一現象暗示人類與黑猩猩共同祖先的有效族群相當龐大。若以最大簡約法分析，則由53個基因間片段與37個蛋白質基因可推得人類─黑猩猩共祖的有效族群約為55,000-100,000個個體。此估計量是其他資料組演算出之人類長期有效族群（約10,000個個體）的5-10倍，故人類這一支系必然在與黑猩猩支系分離後經歷族群量遽減的過程。此一族群量估算的前提是分子鐘必須成立，而此一前提經由對53段基因間片段之相對速率測試而獲得驗證。若取紅毛猩猩種化的時間12-16百萬年前為參考基準，則人類─黑猩猩之分隔時間約在4.8-6.4百萬年前，而大猩猩種化約在6.5-8.7百萬年前。大猩猩種化與人類─黑猩猩分離之間的時間間隔約為1.7-2.3百萬年。
因人類與黑猩猩的基因序列數量較大猩猩與紅毛猩猩為多，此二者之遺傳距離可以更深入探討。我們從基因庫取得二者之基因序列，並進行大規模基因排序（這些基因先以RepeatMasker處理，除去重複片段後，再進行排序）。非重複序列排序完成之後，再將重複片段插入其中，重新進行排序。如此排序完成的序列共計2.3百萬鹼基對，其朱克斯─坎特遺傳距離為1.22%。將非重複序列（共1.44百萬鹼基對）與重複序列(共0.86百萬鹼基對)分開計算，其遺傳距離分別為1.14%與1.34%，重複序列之遺傳距離略高於非重複序列。
註解過之編碼基因與非編碼基因亦從基因庫中取得並排序比較。88個編碼基因之同義突變與非同義突變分別為1.48%與0.55%。而5端外襯序列，3端外襯序列，啟動子以及假基因之遺傳距離分別是1.47%，1.41%，1.68%，0.75%以及1.39%。這些區域的遺傳距離比2.3百萬鹼基對基因組序列的遺傳距離更大的原因並不清楚，很可能是因為這些片段恰好落在突變速率較高的區段之中。
本研究的主要貢獻可歸結如下：
（1）本研究展示了一個有效而迅速獲取整個基因組遺傳距離的方法，研究者得以極短的時間、少量的費用獲致相當可靠的物種間遺傳距離，而不必取得整套基因組序列再加以比較。
（2）本研究提供了人類與巨猿間基因組遺傳距離的重要參考值，可為未來相關研究的比較基礎。
（3）本研究提供了大規模基因排序的範例與障礙排除的經驗，對未來類似研究有重要參考價值。

This study focuses on two subjects: the divergences between human and the great apes, and an in-depth research of the divergence between human and chimpanzee using a relatively large data set.
This study differs from previous ones in that it demonstrates an approach to efficiently obtain authentic genomic divergences without sacrificing accuracy or reliability. Most of the previous studies are flawed in that they include only small numbers of loci. Therefore, the results of the previous studies are susceptible to strong sampling bias. In contrast, the method employed in this study not only eliminates sampling bias but also proves to be highly efficient. Moreover, this study demonstrates an algorithm for large-scale sequence alignments that involve repetitive sequences. This pioneer research has significant impacts on future studies in molecular evolution.
To study the genomic divergences among hominoids and to estimate the effective population size of the common ancestor of human and chimpanzee we selected 53 autosomal intergenic noncoding DNA segments from the human genome and sequenced them in a human, chimpanzee, gorilla, and orangutan. The average sequence divergence was only 1.24% ± 0.07% for the human-chimpanzee pair, 1.62% ± 0.08% for the human-gorilla pair, and 1.63% ± 0.08% for the chimpanzee-gorilla pair. These estimates, which were confirmed by additional data from GenBank, are substantially lower than previous ones, which included repetitive sequences and might have been based on less accurate sequence data. The average sequence divergences between orangutan and human, chimpanzee, and gorilla were 3.08% ± 0.11%, 3.12% ± 0.11% and 3.09% ± 0.11%, respectively, which are also substantially lower than previous estimates. The sequence divergences in other regions between hominoids were estimated from extensive data in GenBank and the literature, and Alus showed the highest divergence, followed in order by Y-linked noncoding regions, pseudogenes, synonymous sites, autosomal intergenic regions, X-linked noncoding regions, introns, and nonsynonymous sites.
The neighbor-joining tree derived from the concatenated sequence of the 53 segments, 24,234 bp in length, supports the Homo-Pan clade with a 100% bootstrap value. However, when each segment is analyzed separately 22 of the 53 segments (~42%) give a tree that is incongruent with the species tree, suggesting a large effective population size (Ne) of the common ancestor of Homo and Pan. Indeed, a parsimony analysis of the 53 segments and 37 protein coding genes leads to an estimate of Ne = 55,000 — 100,000. As this estimate is 5 to 10 times larger than the long-term effective population size of humans (~10,000) estimated from various genetic polymorphism data, the human lineage apparently had experienced a large reduction in effective population size after its separation from the chimpanzee lineage. Our analysis assumes a molecular clock, which is in fact supported by the sequence data used. Taking the orangutan speciation date as 12 to 16 million years (Myr) ago, we obtain an estimate of 4.8 to 6.4 Myr for the Homo-Pan divergence and an estimate of 6.5 to 8.7 Myr for the gorilla speciation date, suggesting that the gorilla lineage branched off 1.7 to 2.3 Myr earlier than the human-chimpanzee divergence.
To study the genomic divergence between human and chimpanzee, large-scale genomic sequence alignments were performed. The genomic sequences of human and chimpanzee were first masked with the RepeatMasker and the repeats were excluded before alignments. The repeats were then reinserted into the alignments of non-repetitive segments and the entire sequences were aligned again. A total of 2.3 million base pairs (Mb) of genomic sequences, including repeats, were aligned and the average nucleotide divergence was estimated to be 1.22%. The Jukes-Cantor (JC) distances (nucleotide divergences) in non-repetitive (1.44 Mb) and repetitive sequences (0.86 Mb) are 1.14% and 1.34%, respectively, suggesting a slightly higher average rate in repetitive sequences.
Annotated coding and noncoding regions of homologous and chimpanzee genes were also retrieved from GenBank and compared. The average synonymous and nonsynonymous divergences in 88 coding genes are 1.48% and 0.55%, respectively. The JC distances in intron, 5’ flanking, 3’ flanking, promoter, and pseudogene regions are 1.47%, 1.41%, 1.68%, 0.75% and 1.39%, respectively. It is not clear why the genetic distances in most of these regions are somewhat higher than those in genomic sequences. One possible explanation is that some of the genes may be located in regions with higher mutation rates.
The major contributions of this study can be summarized as follows:
(1)It demonstrates an effective and efficient method of fathoming genomic divergences between species without having to compare entire genomes.
(2)It offers an important reference that can be applied to future studies of hominoid evolution.

Content
ACKNOWLEDGEMENTI
中文摘要II
ABSTRACTIV
TABLEIII
FIGUREIV
CHAPTER ONE: GENERAL INTRODUCTION1
CHAPTER TWO: GENOMIC DIVERGENCES BETWEEN HUMAN AND OTHER HOMINOIDS AND THE EFFECTIVE POPULATION SIZE OF THE COMMON ANCESTOR OF HUMAN AND CHIMPANZEE5
INTRODUCTION5
MATERIAL AND METHOD6
Selection of Regions6
DNA Samples7
PCR Amplification and Sequencing7
Data Retrieval from GenBank7
Data analysis7
RESULTS8
Genomic divergence8
Phylogeny11
Molecular clock12
Divergence times13
Effective size of the ancestral population14
DISCUSSION16
Genomic Divergences16
Molecular Clocks and Divergence Dates18
Population size estimation19
CHAPTER THREE: LARGE-SCALE SEQUENCE ALIGNMENT OF GENOMIC SEQUENCES AND GENETIC DISTANCES BETWEEN HUMAN AND CHIMPANZEE21
INTRODUCTION21
MATERIAL AND METHOD22
Data retrieval22
Sequence alignment and distance calculation23
RESULTS23
Alignment of genomic sequences23
Genomic sequence distance24
Coding region distance25
Divergences in other noncoding regions26
DISCUSSION26
Implications for large-scale genomic studies26
Genetic distance between human and chimpanzee28
CONCLUSION32
REFERENCES33
Tables
TABLE 1. PCR AND SEQUENCING PRIMERS39
TABLE 2. AUTOSOMAL DNA SEGMENTS SEQUENCED AND PAIRWISE DIVERGENCES AMONG HUMAN (H), CHIMPANZEE (C), GORILLA (G) AND ORANGUTAN (O).41
TABLE 3. JUKES-CANTOR DISTANCES (%) AMONG HUMAN (H), CHIMPANZEE (C), GORILLA (G) AND ORANGUTAN (O) NON-CODING SEQUENCESA.43
TABLE 4. DISTANCES BETWEEN HUMAN (H), CHIMPANZEE (C) AND GORILLA (G) INTRONS.44
TABLE 5. DISTANCES BETWEEN HUMAN (H), CHIMPANZEE (C) AND GORILLA (G) PSEUDOGENES.45
TABLE 6. CODING REGION DISTANCES BETWEEN HUMAN (H), CHIMPANZEE (C), GORILLA (G) AND ORANGUTAN (O).46
TABLE 7. JUKES-CANTOR (JC) DISTANCES BETWEEN GENOMIC SEQUENCES OF HUMAN AND CHIMPANZEE.48
TABLE 8. KS AND KA VALUES IN CODING GENES BETWEEN HUMAN AND CHIMPANZEE49
TABLE 9. JUKES-CANTOR DISTANCES IN INTRON, FLANKING REGION, PROMOTER AND PSEUDOGENE SEQUENCES BETWEEN HUMAN AND CHIMPANZEE.51
Figures
FIGURE 1. THE JUKES-CANTOR DISTANCE DISTRIBUTIONS OF INTERGENIC REGIONS (A), INTRONS (B) AND ALUS (C) BETWEEN HUMAN AND CHIMPANZEE.53
FIGURE 2. THE PHYLOGENY OF HOMINOIDS. BRANCH LENGTHS ARE COMPUTED UNDER THE ASSUMPTION OF RATE CONSTANCY AND ARE SHOWN IN JUKES-CANTOR DISTANCES IN PERCENT.54
FIGURE 3. AN EXAMPLE OF NORMAL ALIGNMENT (A) AND EXCEPTIONAL ALIGNMENT (B, C).55
FIGURE 4. DISTRIBUTION OF KS (A) AND KA (B) VALUES BETWEEN HUMAN AND CHIMPANZEE.56

Arcot SS, DeAngelis MM, Sherry ST, Adamson AW, Lamerdin JE, Deininger PL, Carrano AV, Batzer MA (1997) Identification and characterization of two polymorphic Ya5 Alu repeats. Mutat Res 382: 5-11
Bailey WJ, Fitch DH, Tangle DA, Czelusniak J, Slightom JL, Goodman M (1991) Molecular evolution of the ψη-globin gene locus: gibbon phylogeny and the hominoid slowdown. Mol Biol EvoL 8: 155-184
Batzer MA, Stoneking M, Alegria-Hartman M, Bazan H, Kass DH, Shaikh TH, Novick GE, Ioannou PA, Scheer WD, Herrera RJ, Deininger PL (1994) African origin of human-specific polymorphic Alu insertions. Proc Natl Acad Sci 91: 12288-12292
Blumenbach JF (1791) Handbuch der Naturgeschichte, Vierte Auflage. Johann Christian Dieterich, Gottingen, Germany
Bohossian HB, Skaletsky H, Page DC (2000) Unexpectedly similar rates of nucleotide substitution found in male and female hominids. Nature 406: 622-625
Breguet G, Butler R, Butler-Brunner E, Sanchez-Mazas A (1990) A worldwide population study of the Ag-system haplotypes, a genetic polymorphism of human low-density lipoprotein. Am J Hum Genet 46:502-517
Cann RL, Stoneking M, Wilson AC (1987) Mitochondria DNA and human evolution. Nature 325: 31-36
Costache M, Apoil PA, Cailleau A, Elmgren A, Larson G, Henry S, Blancher A, Iordachescu D, Oriol R, Mollicone R (1997) Evolution of fucosyltransferase genes in vertebrates. J Bio Chem 272:29721-29728
Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991) “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucl Acids Res19: 4008
Dorit RL, Akashi H, Gilbert W1(995) Absence of polymorphism at the ZFY locus on the human Y chromosome. Science 268:1183-1185
Duret L, Mouchiroud D, Gouy M (1994) HOVERGEN: a database of homologous vertebrate genes. Nucl Acids Res 22: 2360-2365
Fay J, Wu C-I (2000) Hitchhiking under positive selection. Genetics 155: 1405-1413
Glusman G, Sosinsky A, Ben-Asher E, Avidan N, Sonkin D, Bahar A, Rosenthal A, Clifton S, Roe B, Ferraz C, Demaille J, Lancet D (2000) Sequence, structure, and evolution of a complete human olfactory receptor gene cluster. Genomics 63: 227-245
Goodman M (1963) Serological Analysis of the Systematics of Recent Hominoids. Hum Biol 35: 377-424
Goodman M, Tagle DA, Fitch DH, Bailey W, Czelusniak J, Koop BF, Benson P, Slightom JL (1990) Primate evolution at the DNA level and a classification of hominoids. J Mol Evol 30: 260-266
Goodman M, Porter CA, Czelusniak J, Page SL, Schneider H, Shoshani J, Gunnell G, Groves CP (1998) Toward a phylogenetic classification of Primates based on DNA evidence complemented by fossil evidence. Mol Phylogenet Evol 9: 585-598
Graur D, Li WH (2000) Fundamentals of Molecular Evolution. 2nd ed. Sinauer Associates, Sunderland, Massachusetts.
Gu Z, Wang H, Nekrutenko A, Li WH (2000) Densities, length proportions, and other distributional features of repetitive sequences in the human genome estimated from 430 megabases of genomic sequence. Gene 259:81-88
Harding RM, Fullerton SM, Griffiths RC, Bond J, Cox MJ, Schneider JA, Moulin DS, Clegg JB (1997) Archaic African and Asian lineages in the genetic ancestry of modern humans. Am J Hum Genet 60: 772-789
Horai S, Satta Y, Hayasaka K, Kondo R, Inoue T, Ishida T, Hayashi S, Takahaka N (1992) Man’s place in Hominoidea revealed by mitochondrial DNA genealogy. J Mol Evol 35: 32-43
Hudson RR (1983) Testing the constant-rate neutral allele model with protein sequence data. Evolution 37: 203-217
Huxley TH (1863) Evidence as to Man’s Place in Nature. Appleton, New York.
JaruzelskaJ, Zietkiewicz E, Labuda D (1999) Is selection responsible for the low level of variation in the last intron of the ZFY locus? Mol Biol Evol 16: 1633-1640
Jukes TH, Cantor CR (1969) Evolution of protein molecules. Pp. 21-123 in Munro HN eds. Mammalian protein metabolism. New York
Li WH (1993) Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J Mol Evol 36: 96-99
Li WH, Gu Z, Wang H, Nekrutenko A (2001) Evolutionary analyses of the human genome. Nature 409:847-849
Kaessmann HF, Heissig F, von Haeseler A, Pääbo S (1999) DNA sequence variation in a non-coding region of low recombination on the human X chromosome. Nat Genet 22: 78-81
Keith A (1920) The Antiquity of Man. Williams and Norgate, London
King MC, Wilson AC (1975) Evolution at two levels in humans and chimpanzees. Science 188: 107-116
Labuda D, Striker G (1989) Sequence conservation in Alu evolution. Nucl Acid Res 17: 2477-2491
Li WH (1993) Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J Mol Evol 36: 96-99
Li WH (1997) Molecular Evolution. Sinauer Associates, Sunderland, Massachusetts
Mann A, Weiss M (1996) Hominoid phylogeny and taxonomy: a consideration of the molecular and fossil evidence in a historical perspective. Mol Phylogenet Evol 5: 169-181
Maruyama T, Kimura M (1980) Genetic variability and effective population size when local extinction and recolonization of subpopulations are frequent. Proc Natl Acad Sci USA 77: 6710-6714
Nachman MW, Crowell SL (2000a) Estimate of the mutation rate per nucleotide in humans. Genetics 156: 297-304
Nachman MW, Crowell SL (2000b) Contrasting evolutionary histories of two introns of the Ducheme muscular dystrophy gene, Dmd, in humans. Genetics 156: 297-304.
Nei M (1986) Stochastic errors in DNA evolution and molecular phylogeny. Pp. 133-147 in Gershowitz HD, Rucknagel L, Tashian RE eds. Evolutionary perspectives and the new genetics. Alan R Liss, New York
Nei M, Graur D (1984) Extent of protein polymorphism and the neutral mutation theory. Evol Biol17: 73-118
Nowak RM, Paradiso JL (1983) Walker’s mammals of the world Vol 1. The John Hopkins University Press, Baltimore and London
Nei M (1995) Genetic support for the out-of Africa theory of human evolution. Proc Natl Acad Sci USA 92: 6720-6722
Osborn HF (1927) Recent discoveries relating to the origin and antiquity of man. Science 65: 481-488
Reynolds V, Reynolds F (1965) Chimpanzees of the Budongo forest. Pp. 368-424.in DeVore I eds. Primate Behavior — field studies of monkeys and apes. Holt, Rinehart and Winston Inc New York
Rogers AR, Harpending H (1992) Population growth makes waves in the distribution of pairwise genetic differences. Mol Biol Evol 9: 552-569
Rowold DJ, Herrera RJ (2000) Alu elements and the human genome. Genetica108: 57-72
Rozas J, Rozas R (1999) DnaSP version 3.0: An integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15: 174-175
Ruvolo M (1997) Molecular phylogeny of the hominoids: inferences from multiple independent DNA sequence data sets. Mol Biol Evol 14: 248-265
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425
Sarich VM, Wilson AC (1967) Immunological time scale for hominid evolution. Science 158: 1200-1203
Satta Y, Klein J, Takahata N (2000) DNA archives and our nearest relative: the trichotomy problem revisited. Mol Phlogenet Evol 14: 259-275
Shen P, Wang F, Underhill PA, Franco C, Yang WH, Roxas A, Sung R, Lin AA, Hyman RW, Vollrath D, Davis RW, Cavalli-Sforza LL, Oefner PJ (2000) Population genetic implications from sequence variation in four Y chromosome genes. Proc Natl Acad Sci USA 97: 7354-7359
Sibley CG, Ahlquist JE (1987) DNA hybridization evidence of hominoid phylogeny: results from an expanded data set. J Mol Evol 26: 99-121
Sigurdardóttir S, Helgason A, Gulcher JR, Stefansson K, Donnell P (2000) The mutation rate in the human mtDNA control region. Am J Hum Genet 66: 1599-1609
Schultz AH (1963) Classification and Human Evolution. Aldine, Chicago
Simpson GG (1961) Principles of Animal Taxonomy. Columbia University Press, New York
Smit AF (1999) Interspersed repeats and other mementos of transposable elements in mammalian genomes. Curr Opin Genet Dev 9: 657-663
Smith GE (1924) The Evolution of Man. Oxford University Press, New York.
Stringer CB, Andrews P (1988) Genetic and fossil evidence for the origin of modern humans. Science 239: 1263-1268
Szmulewicz MN, Novick GE, Herrera RJ (1998) Effects of Alu insertions on gene function. Electrophoresis 19: 1260-1264
Takahata N (1990) A simple genealogical structure of strongly balanced allelic lines and trans- species evolution of polymorphism. Proc Natl Acad Sci USA 87: 2419-2423
Takahata N (1993) Allelic genealogy and human evolution. Mol Biol Evol 10: 2-22
Takahata N, Satta Y, Klein J (1995) Divergence time and population size in the lineage leading to modern humans. Theor Popul Biol 48: 198-221
Tang R, Wang PF, Wang HC, Wang JY, Hsieh LL (2001) Mutations of p53 gene in human colorectal cancer: distinct frameshifts among populations. Int J Cancer 91: 863- 868
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22: 4673-4680
Thomson R, Pritchard JK, Shen P, Oefner PJ, Feldman MW (2000) Recent common ancestry of human Y chromosomes: evidence from DNA sequence data. Proc Natl Acad Sci USA 97: 7360-7365
Tremblay M, Vézina H (2000) New estimates of intergeneration time intervals for the calculation of age and origins mutations. Am J Hum Genet 66: 651-658
Vallois HH (1955) Primates Pp. 1854-2206 in Grassé PP eds. Traité de Zoologie Vol 17. Masson et Cie., Paris.
Wolpoff MH, Wu XZ, Thorne AG (1984) Pp 411-483 in Smith FH, Spencer F eds. The Origins of Modern Humans, Liss, New York
Wu CI (1991) Inferences of species phylogeny in relation to segregation of ancient polymorphisms. Genetics 127: 429-435
Wu CI, Li WH (1985) Evidence for higher rates of nucleotide substitution in rodents than in man. Proc Natl Acad Sci USA 82:1741-1745
Wyckoff GJ, Wang W, Wu CI (2000) Rapid evolution of male reproductive genes in the descent of man. Nature 403: 304-309
Xia X (2000) Data analysis in molecular biology and evolution. Kluwer Academic Publishers, Boston
Yu N, Zhao Z, Fu YX, Ramsay M, Jenkins T, Leskinen E, Patty L, Jorde LB, Sambuughin N, Li WH (2001) Global patterns of human DNA sequence variation in a 10-Kb region on chromosome 1. Mol Biol Evol 18: 214-222
Zhao Z, Jin L, Fu YX, Ramsay M, Jenkins T, Leskinen E, Pamilo P, Trexler M, Patthy L, Jorde L, Sambuughin N, Yu N, Li WH (2000) Worldwide DNA sequence variation in a 10 kilobase noncoding region on chromosome 22. Proc Natl Acad Sci USA 97: 11354-11358