Construction of a mitochondrial consensus sequence for the Khoi-San population of Southern Africa

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Construction of a mitochondrial consensus

sequence for the Khoi-San population of

Southern Africa

BY

MICHELLE KOEKEMOER, B.Sc. (Agric.), M.Sc. (Agric.)

Thesis submitted for the degree Philosophiae Doctor (Ph.D.) in Biochemistry at the North-West University

PROMOTOR: Professor Antonel Olckers

Centre for Genome Research, North-West University (Potchefstroom Campus)

CO-PROMOTOR: Doctor Gordon Wayne Towers

Centre for Genome Research, North-West University (Potchefstroom Campus)

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Samestelling van ‘n mitokondriale

konsensusvolgorde vir die Khoi-San

bevolking van Suidelike Afrika

DEUR

MICHELLE KOEKEMOER, B.Sc. (Agric.), M.Sc. (Agric.)

Proefskrif voorgelê vir die graad Philosophiae Doctor (Ph.D.) in Biochemie aan die Noordwes-Universiteit

PROMOTOR: Professor Antonel Olckers

Sentrum vir Genomiese Navorsing, Noordwes-Universiteit (Potchefstroom Kampus) MEDEPROMOTOR: Doktor Gordon Wayne Towers

Sentrum vir Genomiese Navorsing, Noordwes-Universiteit (Potchefstroom Kampus)

(3)

This thesis is dedicated to my late grandmother, Hester Margaret Freeman, and to my husband, Theunis Johannes Koekemoer

(4)

To know where we are going, we have to know were we are; to know that we have to know where we came from

Filipino version of an Oceanic proverb Stephen Oppenheimer, 2003

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ABSTRACT

The revised Cambridge Reference Sequence (rCRS) is used as a standard for the human mitochondrial DNA (mtDNA) sequence in studies of human evolution and the identification of disease-causing mutations. Due to the large number of differences observed between the rCRS and mitochondrial sequences obtained from individuals of African descent, it is frequently difficult to differentiate between alterations that are population-specific or have possible pathological significance. To address this problem, two human consensus sequences consisting of mitochondrial sequences from different continents and different haplogroups have been constructed. However, combining data from different continents and haplogroups led to a loss of variation in the human mitochondrial consensus sequences. This can be countered by using an African reference sequence, of which two sequences are currently available, namely NC_001807 (L3a1) and D38112 (L0c2). However, these sequences are not representative of the most ancient African populations (i.e. hunter-gatherers) or haplogroups (i.e. L0).

In the current investigation, the complete mitochondrial genome sequences of 30 Khoi-San individuals from southern Africa were determined. Twenty-two of these Khoi-San sequences, which belong to the L0a and L0b sub-haplogroups, were combined with 13 L0 Khoi-San sequences generated previously to compile a consensus sequence for the Southern African Khoi-San population. This Khoi-San consensus sequence will represent the first example of an African population-specific consensus sequence.

The results presented in the current investigation provide support for previous findings regarding the existence of a high level of genetic variation in the mtDNA of the Khoi-San population, as well as the ancient character of the Khoi-San population. In addition, it offers novel insights into the complexity of the L0 haplogroup within the Khoi-San population and the African population as a whole. The high level of genetic variation and increased age of mtDNA lineages in the Southern African Khoi-San population compared to other populations, support the use of the Khoi-San consensus sequence, alone or in conjunction with the rCRS, as a standard in studies of human evolution and mitochondrial disease.

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OPSOMMING

Die hersiene Cambridge Verwysingsvolgorde (rCRS) word as ‘n standaard vir die menslike mitokondriale DNS volgorde aangewend in studies van menslike evolusie en in die identifikasie van siekteveroorsakende mutasies. As gevolg van die groot aantal verskille wat waargeneem word tussen die rCRS en mitokondriale volgordes van individue van Afrika-oorsprong, is dit moeilik om te onderskei tussen veranderinge wat bevolkingsspesifiek is, of moontlike patologiese betekenis het. Om die rede is twee menslike konsensusvolgordes bestaande uit mitokondriale volgordes van verskillende kontinente en verskeie haplogroepe saamgestel. Die samevoeging van data van verskillende kontinente en haplogroepe het egter gelei tot ‘n verlies in variasie in die menslike konsensusvolgordes. Dit kan teëgewerk word deur die gebuik van ‘n Afrika verwysingsvolgorde en daar is tans twee volgordes beskikbaar, naamlik NC_001807 (L3a1) en D38112 (L0c2). Hierdie twee volgordes is egter nie verteenwoordigend van die oudste Afrika bevolkings (d.i. jagter-versamelaars) of haplogroepe (d.i. L0) nie.

In die huidige ondersoek is die volledige mitokondriale genoomvolgordes van 30 Khoi-San individue bepaal. Twee-en-twintig van hierdie Khoi-San volgordes, wat aan die L0a en L0b sub-haplogroepe behoort het, is met 13 L0 volgordes wat voorheen bepaal is, gekombineer om ‘n konsensusvolgorde vir die Suidelike-Afrika Khoi-San bevolking saam te stel. Hierdie Khoi-San konsensusvolgorde verteenwoordig die eerste voorbeeld van ‘n Afrika bevolkingsspesifieke konsensusvolgorde.

Die resultate wat aangebied word in die huidige ondersoek, ondersteun vorige bevindinge rakende die bestaan van ‘n hoë vlak van genetiese variasie in die mitokondriale DNS van die Khoi-San bevolking, asook die antieke aard van die Khoi-San bevolking. Dit bied verder nuwe insigte in die kompleksiteit van die Khoi-San bevolking as ‘n geheel. Die hoë vlak van genetiese variasie en die verhoogde ouderdom van die mtDNS lyne in die Suidelike-Afrika Khoi-San bevolking in vergelyking met ander bevolkings, ondersteun die gebruik van die Khoi-San konsensusvolgorde, alleen of saam met die rCRS, as ‘n standaard in studies van menslike evolusie en mitokondriale afwykings.

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LIST OF ABBREVIATIONS AND SYMBOLS

Symbols and abbreviations are listed in alphabetical order:

LIST OF SYMBOLS

α alpha: denoting gamma shape parameter

∆ delta: denoting change

°C degrees centigrade

η eta: denoting total number of mutations

γ gamma

µ micro:10-6

m milli: 10-3

µ mu: denoting mutation rate

n nano: 10-9

p pico: 10-12

% percent

π pi: denoting nucleotide diversity or the average number of pairwise differences

between nucleotide sequences

∏_n average number of nucleotide differences between two sequences

Ψ psi: denoting pseudouridylate

ρ rho: denoting average number of sites differing between a set of sequences and a

specified common ancestor

Σ sigma: denoting summation

σ2 _{sigma squared: variance}

√ square root

θ theta: denoting the expected pairwise nucleotide site differences or nucleotide

diversity parameter

θπ the mean number of nucleotide differences between two sequences

 _{registered trademark}

 trademark

= equal to

≥ greater than or equal to

≡ identical to

≤ less than or equal to

+ plus

± plus-minus

$ dollar: denoting incompletely classified sequences

- minus or gap (in a nucleotide sequence alignment)

? missing character (in a nucleotide sequence alignment)

green square: outgroup (in phylogenetic trees)

red star: Khoi-San sequences generated in the current investigation (in phylogenetic trees)

turquoise octagon: rCRS (in phylogenetic trees)

violet triangle: African reference sequence (in phylogenetic trees) LIST OF ABBREVIATIONS

3’ 3 prime

5’ 5 prime

5S 5S ribosomal RNA

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LIST OF ABBREVIATIONS AND SYMBOLS

ii

12S 12 Svedberg units

12S rRNA 12S ribosomal RNA

16 16S rRNA (in L0-specific haplogroup networks)

16S 16 Svedberg units

16S rRNA 16S ribosomal RNA

A alanine (in amino acid sequence)

A, A or a adenine (in DNA sequence)

A.D. Anno Domini: of the Christian era

A260 absorbance of samples at 260 nm

A280 absorbance of samples at 280 nm

A260/A280 ratio of absorbency measured at 260 nm and 280 nm

ADP adenosine diphosphate

AF African mtDNA haplogroups

.aln CLUSTAL format file extension

Ala alanine

Alu I restriction endonuclease isolated from Arthrobacter luteus with recognition site 5’- AGCT -3’

AMP adenosine monophosphate

Arg arginine

AS Asian mtDNA haplogroups

Asn asparagine

Asp aspartic acid

ATP adenosine-5-triphosphate

ATP6 ATP synthase F0 subunit 6 (in L0-specific haplogroup networks)

ATP8 ATP synthase F0 subunit 8 (in L0-specific haplogroup networks)

atp6 gene encoding ATPase6

atp8 gene encoding ATPase8

ATPase6 adenosine triphosphatase subunit 6 or ATP synthase F0 subunit 6 ATPase8 adenosine triphosphatase subunit 8 or ATP synthase F0 subunit 8

ATT membrane attachment site

Ava II restriction endonuclease isolated from Anabaena variabilis with recognition site 5’- GG(A/T)CC -3’

b branch length

Bam HI restriction endonuclease isolated from Bacillus amyloliquefaciens H with recognition site 5’- GGATCC -3’

boric acid boracic acid: H3BO3

bp base pair(s)

BstN I restriction endonuclease isolated from Bacillus stearothermophilus N with recognition site 5’- CC(A/T)GG -3’

C cysteine (in amino acid sequence)

C, C or c cytosine (in DNA sequence)

CA California

(CA)n cytosine and adenine nucleotide repeat stretch

ca. circa: approximately

Ca2+ calcium (II) ion

CAP CONTIG ASSEMBLY PROGRAM (included in BioEdit version 5.09)

CAR Central African Republic

CBI codon bias index

CGR Centre for Genome Research

CI conservation index

CM cardiomyopathy

CNS central nervous system

CO cytochrome c oxidase subunit

CO2 carbon dioxide

COI cytochrome c oxidase subunit I

COII cytochrome c oxidase subunit II

COIII cytochrome c oxidase subunit III

CoQ coenzyme Q or ubiquinone

CoQH2 reduced coenzyme Q

Cov covariance

CPEO chronic progressive external ophthalmoplegia

CR control region

CRS Cambridge Reference Sequence

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CSB2 conserved sequence block 2

CSB3 conserved sequence block 3

CT Connecticut

CuA copper atom

CuB copper atom

Cys cysteine (in amino acid sequence)

Cytb cytochrome b

cytb cytochrome b (in L0-specific haplogroup network)

Cytc cytochrome c

∆-mtDNA deleted mitochondrial DNA

d maximum number of differences

D Tajima’s D test statistic

D adenine, guanine or thymine (in DNA sequence)

D aspartic acid

d deletion

D* Fu and Li’s D* test statistic

dATP 2’-deoxyadenosine-5’-triphosphate

DC District of Columbia

dCTP 2’-deoxycytidine-5’-triphosphate

ddATP 2’,3’-dideoxyadenosine-5’-triphosphate

Dde I restriction endonuclease isolated from Desulfovibrio desulfuricans with recognition site 5’- CTNAG -3’

ddH2O double distilled water

ddNTP(s) 2’,3’-dideoxynucleotide triphosphate(s)

DEL or del deletion

DGGE denaturing gradient-gel electrophoresis

dGTP 2’-deoxyguanosine-5’-triphosphate

dij and dkl estimate of the number of nucleotide substitutions per site between DNA sequences i (k) and j (l)

D-loop displacement loop

DNA deoxyribonucleic acid

DnaSP DNA Sequence Polymorphism

dNTP(s) deoxynucleotide triphosphate(s)

dsDNA double stranded DNA

DTT dithiothreithol-1,4-dimercapto-2,3-butanediol: C4H10O2S2

dTTP 2’-deoxythymidine-5’-triphosphate

E glutamic acid

e- electron

e.g. exempli gratia: for example

ECM encephalomyopathy

EDTA ethylenediamine tetra-acetic acid: C10H16N2O8

.emf extended (enhanced) Windows metafile format file extension (to produce

phylogenetic tree images)

emPCR emulsion-based clonal amplification

ENT ear-nose-throat

et al. et alia: and other people/things

EtBr ethidium bromide: 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide:

C21H20BrN3

EtOH ethanol: CH3CH2OH

EU European mtDNA haplogroups

F forward

F phenylalanine

F* Fu and Li’s F* test statistic

F0 component of ATP synthase, oligomeric enzyme complex located in integral

membrane

F1 component of ATP synthase, water-soluble oligomeric enzyme complex bound to F0

.fas FASTA format file extension

FAD flavin adenine dinucleotide (oxidised form)

FADH2 flavin adenine dinucleotide (reduced form)

FBSN familial bilateral striatal necrosis

Fe-S iron-sulphur protein

Fi(t) the probability that two random neutral genes will differ at exactly i nucleotides in generation t

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iv

FMN reduced flavin mononucleotide

formamide carbamaldehyde: CH3NO

FST fixation index (a measure of population subdivision)

G glycine

g gravitational force

G + C G + C content: refers to the composition of nucleotide sequences, specifically to the number of cytosine and guanine nucleotides

G + C2 G + C content at second codon positions

G + C3s G + C content at third (synonymous) codon positions

G + Cc G + C content at coding positions

G + Cn G + C content at non-coding positions

G, G or g guanine (in DNA sequence)

g.cm-3 _{gram per cubic centimetre}

gDNA genomic DNA

GenBank®1 NIH genetic sequence database, an annotated collection of all publicly available DNA sequences

GI gastrointestinal system

GI number GenInfo Identifier sequence identification number

Gln glutamine

Glu glutamate or glutamic acid

Gly glycine

GTP guanosine-5'-triphosphate

GTPases a large family of hydrolase enzymes that can bind and hydrolyze GTP

H histidine

h hour(s)

H+ _proton

H2O water

Hae II restriction endonuclease isolated from a recombinant E. coli strain with recognition site 5’- (A/G)GCGC(T/C) -3’

Hae III restriction endonuclease isolated from a recombinant Haemophilus aegyptius with recognition site 5’- GGCC -3’

Haeme a a-type haeme

Haeme a3 a3-type haeme

Haeme bH high potential b-type haeme

Haeme bL low potential b-type haeme

Haeme c1 c-type haeme

HeLa human cervical carcinoma

Hha I restriction endonuclease isolated from Haemophilus haemolyticus with recognition site 5’- GCGC -3’

Hin fI restriction endonuclease isolated from Haemophilus influenzae Rf with recognition site 5’- GANTC -3’

Hinc II restriction endonuclease isolated from Haemophilus influenzae Rc with recognition site 5’- GT(T/C)(A/G)AC -3’

His histidine

HMG high mobility group

h-mtTF-1 mitochondrial transcription factor, involved in binding to enhancer elements h-mtTFA mitochondrial transcription factor, involved in binding to enhancer elements h-mtTFB mitochondrial transcription factor, involved in binding to enhancer elements, also

referred to as TFB2M

Ho null hypothesis

Hpa I restriction endonuclease isolated from Haemophilus parainfluenzae with recognition site 5’- GTTAAC -3’

HR high-resolution

HSP H-strand promoter

HSP1 major H-strand promoter

HSP2 minor H-strand promoter

H-strand heavy strand of the mitochondrial DNA molecule

HVS1 hypervariable segment 1

i insertion

1_GenBank®_{is a registered trademark of the U.S. Department of Health and Human Services, Independence Avenue, S.W.,}

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I internal branch (in L0-specific haplogroup network)

I isoleucine

i.e. id est: that is to say

I/T RFI/RFT

IgE immunoglobulin E

Ile isoleucine

IMM inner mitochondrial membrane

IN Indiana

INS insertion

ITH1 major initiation site for H-strand transcription

ITH2 minor initiation site for H-strand transcription

ITL initiation site for L-strand transcription

k average number of pairwise nucleotide differences or number of alleles

K lysine (in amino acid sequence)

K, K or k guanine or thymine (in DNA sequence)

K, K or k guanine or uracil (in RNA sequence)

kb kilobase pair

kDa kilodalton

kij number of nucleotide differences between the i th and j th sequences KS_CGR_### three digit sample number given to each Khoi-San individual

KSC Khoi-San consensus sequence

KSS Kearns-Sayre syndrome

L leucine

L0aA L0a (branch A)

L0aA1 L0a (branch A, group 1)

L0aA2 L0a (branch A, group 2)

L0aB L0a (branch B)

L0aB1 L0a (branch B, group 1)

L0aB2 L0a (branch B, group 2)

L0aC L0a (branch C)

L0bA L0b (branch A)

L0bA1 L0b (branch A, group 1)

L0bA2 L0b (branch A, group 2)

L0bB L0b (branch B)

L0bB1 L0b (branch B, group 1)

L0bB1a L0b (branch B, group 1, part a)

L0bB1b L0b (branch B, group 1, part b)

L0bB1b1 L0b (branch B, group 1, part b, section 1)

L0bB2a1 L0b (branch B, group 2, part a, section 1)

L0c1A L0c1 (branch A)

L0c1B L0c1 (branch B)

L0c2A L0c2 (branch A)

L0c2ABCE L0c2 (branch ABCE)

L0c2ACE L0c2 (branch ACE)

L0c2B L0c2 (branch B) L0c2B1 L0c2 (branch B, group 1) L0c2B2 L0c2 (branch B, group 2) L0c2C L0c2 (branch C) L0c2D L0c2 (branch D) L0c2D1 L0c2 (branch D, group 1) L0c2D2 L0c2 (branch D, group 2) L0c2E L0c2 (branch E) Leu leucine

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vi

LR low-resolution

LS Leigh syndrome

LSP L-strand promoter

L-strand light strand of the mitochondrial DNA molecule

Ltd. limited

Lys lysine

M methionine

M molar: moles per litre

M myopathy

m number of nucleotides examined per sequence or rate of migration

M, M or m adenine or cytosine (in DNA sequence)

µg microgram

µL microlitre

µm micrometre

µM micromolar

MA Massachusetts

MALDI-TOF MS matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Max. maximum

Mbo I restriction endonuclease isolated from Moraxella bovis ATCC 10900 with recognition site 5’- GATC -3’

MD Maryland

ME Multiregional Evolution

.meg MEGA alignment file

MEGA Molecular Evolutionary Genetics Analysis

MELAS mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes

MERRF myoclonic epilepsy with ragged-red fibres

Met methionine

Mfold multiple fold program

mg milligram

MgCl2 magnesium chloride

[MgCl2] magnesium chloride concentration

MILS maternally inherited Leigh syndrome

min minute(s)

Min. minimum

mL millilitre

mm millimetre

mM millimolar

MM molecular marker: FastRuler™ DNA Ladder, High Range, ready to use

MN Minnesota

Mnl I restriction endonuclease isolated from an E. coli strain that carries the Mnl I gene from Moraxella nonliquefaciens (ATCC 17953) with recognition site

5’- CCTC(N)7 -3’

MO Missouri

mol mole: unit describing the amount of a particular chemical species; the amount being equal to one Avogadro’s number (6.02 x 1023_{) of atoms, ions, molecules, or electrons}

MP maximum parsimony

MRCA most recent common ancestor

mRNA messenger RNA

Msp I restriction endonuclease isolated from a Moraxella species with recognition site 5’- CCGG -3’

mt3H mt3 H-strand control element

mt3L L-strand control element

mt4H mt4 H-strand control element

Mt5 control element

MT-CO1 cytochrome c oxidase subunit I

MT-CO2 cytochrome c oxidase subunit II

MT-CO3 cytochrome c oxidase subunit III

MT-CYTB cytochrome b

MT-DLOOP control region, including displacement loop

mtDNA mitochondrial deoxyribonucleic acid

mtEF-G mitochondrial elongation factor

mtEF-Ts mitochondrial elongation factor

mtEF-Tu mitochondrial elongation factor

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mtETC mitochondrial electron transport chain

mtIF-2 mitochondrial initiation factor

mt-MRCA mitochondrial most recent common ancestor

MT-ND1 NADH dehydrogenase subunit 1

MT-ND4L NADH dehydrogenase subunit 4L

mtRNase P mitochondrial RNase P

MT-RNR1 12S ribosomal RNA

MT-RNR2 16S ribosomal RNA

mtSSB mitochondrial single-stranded binding proteins

MT-TC tRNA cysteine

MT-TE tRNA glutamic acid

mtTERM mitochondrial transcription terminator

MT-TF tRNA phenylalanine

mtTF1 mitochondrial transcription factor

MT-TG tRNA glycine

MT-TH tRNA histidine

MT-TI tRNA isoleucine

MT-TL1 tRNA leucine (UUR)

MT-TL2 tRNA leucine (CUN)

MT-TQ tRNA glutamine

MT-TS1 tRNA serine (UCN)

MT-TS2 tRNA serine (AGY)

MT-TT tRNA threonine

MT-TV tRNA valine

MT-TW tRNA tryptophan

MT-TY tRNA tyrosine

Myr million years

N asparagine

η_s number of singletons (mutations appearing only once among the nucleotide

sequences)

N, N or n adenine, cytosine, guanine or thymine (in DNA sequence)

N, N or n adenine, cytosine, guanine or uracil (in RNA sequence)

N, N, n or n sample size

N/A not available

NA Native American mtDNA haplogroups

Na2EDTA di-sodium ethylenediamine tetra-acetic acid: C10H14N2Na2O8.2H2O

NaCl sodium chloride

NAD+ _{oxidised nicotinamide adenine dinucleotide}

NADH reduced nicotinamide adenine dinucleotide

NaOAc sodium acetate

NARP neuropathy, ataxia, and retinitis pigmentosa

NC effective number of codons

NC non-coding NC North Carolina NC1 non-coding nucleotide 1 NC2 non-coding nucleotide 2 NC3 non-coding nucleotide 3 NC4 non-coding nucleotide 4 NC5 non-coding nucleotide 5 NC6 non-coding nucleotide 6 NC7 non-coding nucleotide 7 NC8 non-coding nucleotide 8 NC9 non-coding nucleotide 9 NC10 non-coding nucleotide 10

NCBI National Center for Biotechnology Information

ND NADH dehydrogenase subunit

ND1 NADH dehydrogenase subunit 1

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viii

ND4L NADH dehydrogenase subunit 4L

ndh4 gene encoding ND4

ndh4L gene encoding ND4L

nDNA nuclear DNA

Ne effective population size

NEG negative control

ng nanogram

NIH National Institutes of Health, USA

NJ New Jersey

N-J neighbour-joining

Nla III restriction endonuclease isolated from an E. coli strain that carries the Nla III gene from Neisseria lactamica (NRCC 2118) with recognition site 5’ -GATG -3’

nm nanometre

nM nanomolar

No. number

NON-SYN non-synonymous (describing a nucleotide substitution)

NRY non-recombining portion of the Y chromosome

NS non-synonymous (describing a nucleotide substitution)

Nucl # nucleotide number

O.D. optical density

O2 molecular oxygen

OC Oceanic mtDNA haplogroups

OH H-strand origin of replication

OL L-strand origin of replication

OMM outer mitochondrial membrane

Orange G dye used in preparation of loading dye, 7-hydroxy-8-phenylazo-1,3-naphthalenedisulfonic acid: C16H10N2O7S2Na2

OTUs operational taxonomic units

OXPHOS oxidative phosphorylation

P proline

P P-value

p(kθ) probability of having k alleles in a sample of n sequences

PA Pennsylvania

PCR polymerase chain reaction

PDF portable document format

PEO progressive external ophthalmoplegia

Pfu DNA polymerase deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase from Pyrococcus furiosus: EC 2.7.7.7

pH a measure of acidity: numerically equal to the negative logarithm of H+ concentration expressed in molarity

.phy PHYLIP format file extension

PH1 major H-strand promoter

PH2 minor H-strand promoter

Phe phenylalanine

PHYLIP Phylogeny Inference Package

Pi inorganic phosphate.

.pir NBRF/PIR format file extension

PL L-strand promoter

pmol picomole

PNS peripheral nervous system

POS positive control

PPK palmoplantar keratoderma

Pro proline

PS Pearson syndrome

Pty. Proprietary

P-value probability value, indicates statistical significance

Q glutamine

R arginine

r average radius of the rotor in millimetre

R reverse

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R2 Ramos-Onsins and Rozas statistic

RAO Recent African Origin

rCRS revised Cambridge Reference Sequence

rDNA ribosomal DNA

RE restriction enzyme

RefSeq NCBI reference sequence

RF replacement mutation frequency

RFI frequency of replacement mutations in internal branches

RFLP(s) restriction fragment length polymorphism(s)

RFT frequency of replacement mutations in terminal branches

rg raggedness statistic

RNA ribonucleic acid

RNase ribonuclease

RNase MRP mitochondrial RNA processing endonuclease

ROS reactive oxygen species

rpm revolutions per minute

rRNA(s) ribosomal RNA(s)

RSCU relative synonymous codon usage

S number of segregating (polymorphic) sites

S serine

S Svedberg units

S synonymous (describing nucleotide substitution)

S, S or s cytosine or guanine (in DNA sequence)

S.W. South West

S’ probability of having no fewer than k0 alleles in a sample provided that θ = π

SAK Southern African Khoi-San

SChi2 _{scaled chi square}

SD standard deviation

SDS sodium dodecyl sulphate: C12H25NaSO4

sec second(s)

Ser serine

SIDS sudden infant death syndrome

Sk coefficient of θkπin Sn

SNP(s) single nucleotide polymorphism(s)

SSCP single stranded conformational polymorphism

ssDNA single stranded DNA

STR short, tandemly repeated

∑(y - y)2 _{sum of squared deviations}

SYN synonymous (describing a nucleotide substitution)

t time in generations

T terminal branch (in L0-specific haplogroup network)

T threonine

TΨC loop the loop region of the tRNA molecule containing pseudouridine (a modified uracil nucleotide in a UUCG sequence)

T, T or t thymine (in DNA sequence)

Ta annealing temperature

Taq polymerase deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase from Thermus aquaticus: EC 2.7.7.7

TAS termination-associated sequence

TBE Tris®_{borate-EDTA buffer}

Ter termination

Tfam mitochondrial transcription factor, involved in binding to enhancer elements (previously known as h-mtTF-1 or h-mtTFA)

TFB1M mitochondrialtranscription specificity factor

TFB2M mitochondrialtranscription specificity factor, also referred to as h-mtTFB

TFX mtTF1 binding site

TFY mtTF1 binding site

Thr threonine

Tm melting temperature

TMRCA(s) time(s) to most recent common ancestor

Tris®1 _{tris(hydroxymethyl)aminomethan: 2-amino-2-(hydroxymethyl)-1,3-propanediol:}

C4H11NO3

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x

Tris®-HCl 2-amino-2(hydroxymethyl)-1,3-propanediol hydrochloride: C4H11NO3.H2O

tRNA(s) transfer RNA(s)

tRNAAla _{tRNA alanine}

tRNAArg tRNA arginine

tRNAAsn _{tRNA asparagine}

tRNAAsp _{tRNA aspartic acid}

tRNACys _{tRNA cysteine}

tRNAGln tRNA glutamine

tRNAGlu _{tRNA glutamic acid}

tRNAGly _{tRNA glycine}

tRNAHis _{tRNA histidine}

tRNAIle tRNA isoleucine

tRNALeu tRNA leucine

tRNALeu(CUN) _{tRNA leucine (CUN)}

tRNALeu(UUR) _{tRNA leucine (UUR)}

tRNALys tRNA lysine

tRNAMet _{tRNA methionine}

tRNAPhe _{tRNA phenylalanine}

tRNAPro _{tRNA proline}

tRNASer tRNA serine

tRNASer(AGY) _{tRNA serine (AGY)}

tRNASer(UCN) _{tRNA serine (UCN)}

tRNAThr _{tRNA threonine}

tRNATrp tRNA tryptophan

tRNATyr tRNA tyrosine

tRNAVal _{tRNA valine}

Trp tryptophan

TS transition

TV transversion

Tyr tyrosine

U unit(s)

U, U or u uracil (in RNA sequence)

U.S. United States

Ui number of singleton mutations in sequence i

UK United Kingdom

UPGMA unweighted pair group method with arithmetic mean

UQ coenzyme Q or ubiquinone

USA United States of America

UV ultraviolet

V valine

V volt

V(π) sample variance of nucleotide diversity

V.cm-1 _{volts per centimetre}

v/v volume per volume

Val valine

vs. versus: against

W tryptophan

W, W or w adenine or thymine (in DNA sequence)

W, W or w adenine or uracil (in RNA sequence)

w/v weight per volume

WI Wisconsin

.wmf Windows metafile format file extension (to produce phylogenetic tree images)

x mismatch distribution

y value of observation

Y tyrosine

Y, Y or y cytosine or thymine (in DNA sequence)

Y, Y or y cytosine or uracil (in RNA sequence)

YBP years before present

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LIST OF EQUATIONS

Equation

No. Title of Equation

Page

4.1 Conversion of gravitational force to revolutions per minute……… 123 4.2 Calculation of DNA concentration from the absorbance at 260 nm……….. 130 4.3 Calculation of arithmetic mean and standard deviation……….. 156 4.4 Average number of nucleotide differences……… 157 4.5 Calculation of nucleotide diversity, sampling variance and standard

deviation……….. 158

4.6 Tajima’s D statistic……… 159

4.7 Fu and Li’s D* and F* test statistics……… 161 4.8 Calculation of the pairwise number of sequence differences under the

assumption of constant population size………. 162

4.9 Raggedness statistic………. 163

4.10 The Ramos-Onsins and Rozas statistic………. 163 4.11 Fu’s FS statistic……….. 164

4.12 Calculation of the replacement mutation frequency………. 166 4.13 Calculation of the average number of sites differing between a set of

sequences and a specified common ancestor………. 168

4.14 Calculation of the TMRCA……… 169

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LIST OF FIGURES

Figure

No. Title of Figure Page

2.1 The structure of the mitochondrion………... 8 2.2 Schematic representation of the mitochondrial electron transport chain…….. 12 2.3 Diagrammatic representation of the mitochondrial genome……… 16 2.4 Diagrammatic representation of the replication cycle of mitochondrial DNA .. 19 2.5 Morbidity map of the human mitochondrial genome……… 47 3.1 Different models of human evolution……….. 53 3.2 Human migration pattern based on regional distribution of mitochondrial

haplogroups………..……….. 56

3.3 Spread of Bantu-speakers in Africa during the first movement of the Bantu

expansion during the Early Iron Age………..………. 61 3.4 Spread of Bantu-speakers in Africa during the second movement of the

Bantu expansion………..………….. 62

3.5 A schematic representation of a global phylogenetic tree of mtDNA

3.6 A schematic representation of the mtDNA phylogeny of African mitochondrial haplogroups………..………….………… 84 3.7 Classification network for African mtDNA haplogroups…………..………. 86 3.8 The Khoi-San language classification system………..………. 117 4.1 Schematic representation of the single nucleotide polymorphisms used to

characterise African mtDNA L haplogroups………...……… 139 5.1 Location of the collection site and the geographic origin of the Khoi-San

individuals studied in the current investigation………..……… 175 5.2 Photographic representation of the variation in amplification efficiency

observed between amplified PCR products for different samples………..…... 180 5.3 Photographic representation of the variation in amplification efficiency

observed between different amplified PCR regions………..……... 181 5.4 Photographic representation of the background smear observed for

amplified PCR products………..……….. 183 5.5 Photographic representation of secondary amplification observed for

amplified PCR products………..……….. 184 5.6 Photographic representation of primer-dimers observed for amplified PCR

products………..………. 185

5.7 Photographic representation of an artefact observed in the gel matrix…..….. 186 5.8 Photographic representation of the distortion of amplified fragments…..……. 187 5.9 Photographic representation of slanted fragments………..…. 188 5.10 Representative electropherogram indicating background noise……….... 194

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LIST OF FIGURES

xiv

5.11 Representative electropherogram depicting low signal strength………..……. 196 5.12 Representative electropherogram illustrating poor mobility correction……... 196 5.13 Representative electropherogram illustrating the insertion and deletion of

nucleotides………..………… 197

5.14 Representative electropherogram illustrating excess dye peaks…..…………. 198 5.15 Representative electropherogram depicting dye blobs………..…………. 199 5.16 Representative electropherogram depicting slippage in a homopolymer

region………..……. 200

5.17 Photographic representation of amplified products of PCR region 1…..…….. 205 5.18 Representative electropherograms of PCR region 1 generated using

forward primers F32 to F3………..…….. 206 5.19 Representative electropherograms of PCR region 1 generated using

reverse primers R32 to R3………..………. 207 5.20 Proposed secondary structure and sequence alignment of tRNAPhe

containing the A647G alteration………..……… 212 5.21 Photographic representation of amplified products of PCR region 2…..…….. 215 5.22 Representative electropherograms of PCR region 2 generated using

forward primers F4* to F7………..……... 216 5.23 A representative electropherogram of PCR region 2 generated using

reverse primer R4………..……… 217

5.24 Photographic representation of amplified products of PCR region 3..……….. 220 5.25 Representative electropherograms of PCR region 3 generated using

forward primers F8* to F11………...……… 221 5.26 A representative electropherogram of PCR region 3 generated using

reverse primer R8………..……… 222

5.27 Proposed secondary structure and sequence alignment of tRNATrp

containing the A5515G alteration………..……….. 223 5.28 Photographic representation of amplified products of PCR region 4…..…….. 227 5.29 Representative electropherograms of PCR region 4 generated using

forward primers F12* to F15………..……….. 228 5.30 Representative electropherogram of PCR region 4 generated using the

reverse primer R15………..……….. 230 5.31 Proposed secondary structures and sequence alignment of tRNATyr

containing the T5865A alteration………..……….. 230 5.32 Photographic representation of amplified products of PCR region 5……….... 235 5.33 Representative electropherograms of PCR region 5 generated using

forward primers F16* to F19………..….. 236 5.34 A representative electropherogram of PCR region 5 generated using

reverse primer R16………..….. 237 5.35 Photographic representation of amplified products of PCR region 6……..….. 244 5.36 Representative electropherograms of PCR region 6 generated using

forward primers F20 to F23………..……… 245 5.37 Photographic representation of amplified products of PCR region 7…..…….. 248 5.38 Representative electropherograms of PCR region 7 generated using

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LIST OF FIGURES

5.39 Representative electropherograms of PCR region 7 generated using

reverse primers R24 to R27………..……... 250 5.40 Photographic representation of amplified products of PCR region 8……..….. 258 5.41 Representative electropherograms of PCR region 8 generated using

forward primers F28* to F31………..…….. 259 5.42 Global neighbour-joining tree constructed using the All L Sequences dataset

(dataset 1a)………. 279

5.43 Africa neighbour-joining tree constructed using the All Africa L Sequences

dataset (dataset 2a)………..………. 298 5.44 Khoi-San neighbour-joining tree constructed using the All Khoi-San

L Sequences dataset (dataset 3a)………..……… 307 5.45 Global maximum parsimony tree constructed using the All L Sequences

dataset (dataset 1a)………..………. 311 5.46 Africa maximum parsimony tree constructed using the All Africa

L Sequences dataset (dataset 2a)………..……… 317 5.47 Khoi-San maximum parsimony tree constructed using the All Khoi-San L

Sequences dataset (dataset 3a)………..……… 321 5.48 Global maximum parsimony tree adapted to indicate the geographic origin

of the mitochondrial genome sequences………..………. 326 5.49 L0-specific haplogroup network based on the 22 Khoi-San L0 sequences

generated in the current investigation………..……….. 333 5.50 L0a section of the global L0-specific haplogroup network after addition of 46

L0 sequences……….. 337

5.51 L0b section of the global L0-specific haplogroup network after addition of 46

L0 sequences……….. 340

5.52 L0c1 section of the global L0-specific haplogroup network after addition of

46 L0 sequences………..……….. 342

5.53 L0c2 section of the global L0-specific haplogroup network after addition of

46 L0 sequences………..……….. 344

5.54 Graphical representation of pairwise differences for datasets 1b, 2b and 5 which exhibited deviation from neutrality and displayed characteristics of

population expansion………..……….. ₃₇₆ 5.55 Graphical representation of pairwise differences for dataset 6 which

exhibited deviation from neutrality but displayed no evidence of population

expansion………..……….. ₃₈₀

5.56 Graphical representation of pairwise differences for dataset 7 which did not exhibit deviation from neutrality and displayed characteristics of constant

population size………...………. ₃₈₁

5.57 Graphical representation of pairwise differences for datasets 3a and 4b which did not exhibit deviation from neutrality and displayed characteristics

of constant population size………..………. ₃₈₂ 5.58 Graphical representation of pairwise differences for dataset 8a which did not

exhibit deviation from neutrality and displayed characteristics of constant

population size………...…. 383

5.59 Geographic distribution of L0 sub-haplogroups and lineages within sub-haplogroups in Africa………..………... 413

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LIST OF FIGURES

xvi

5.60 Reduced global maximum parsimony tree based on the MP tree constructed

using the All L Sequences dataset (dataset 1a)……… 416 5.61 Revised classification scheme for the L0 haplogroup……..……… 421 5.62 Consensus sequence generated for the Khoi-San of southern Africa

presented in the form of a list of SNPs compared to the rCRS…..……… 429 5.63 Schematic representation of the global L0-specific haplogroup network

indicating new branches after the addition of 46 L0 sequences... 454 6.1 Model indicating the different factors that influence the genetic variation

observed in human mitochondrial genome sequences…………..………. 478 6.2 Updated model indicating the contribution made by the unique population

characteristics of the Khoi-San from southern Africa………..………. 491 C.1 Schematic representation of a global phylogenetic tree of mtDNA

I.1 Geographic regions in Africa………..……….. 597 J.1 Global and Africa phylogenetic trees………..……… 600 K.1 L0 haplogroup network obtained from MITOMAP…..……….. 610 L.1 Global L0-specific haplogroup network………..……… 612 N.1 Mitochondrial consensus sequence for the Khoi-San population of southern

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LIST OF TABLES

Table

No. Title of Table Page

2.1 The multi-subunit complexes which play a role in the electron transport

chain………....….. 10

2.2 Subunits of the electron transport system encoded by mitochondrial

genes………...………....…. 11

2.3 Functional elements located in the mitochondrial DNA genome………... 14 2.4 The mitochondrial genetic code………...………. 17 2.5 Genes encoded by the H-strand and the L-strand of the mitochondrial DNA

genome………..…...………… 21

2.6 Differences between the genetic code of mammalian mitochondria and the

universal genetic code……….…………... 25 2.7 Evidence in support or rejection of recombination events within

mitochondria………...……... 33

2.8 Adaptive mitochondrial mutations associated with specific haplogroups……... 42 2.9 Clinical features in mitochondrial diseases associated with mtDNA

mutations……….…………... 44

2.10 The genetic classification of human mitochondrial disorders………... 46 2.11 A selection of previously identified pathogenic mtDNA mutations………...…... 48 3.1 Regions in Africa that contributed slaves to other parts of the world………….. 66 3.2 Strategies that can be employed to avoid the inclusion of errors in

mitochondrial sequence datasets………...……….. 72 3.3 Evolutionary models that describe the nucleotide substitution process……... 74 3.4 Estimates of the α parameter of the gamma distribution of rate variation for

mitochondrial regions or genes………. 75 3.5 Aspects to take into consideration when deciding on the most appropriate

method for use in constructing phylogenetic trees………. 78 3.6 Restriction enzyme sites defining continent-specific African mitochondrial

haplogroups……….. 85

3.7 Geographic distribution of major African mitochondrial haplogroups…...…….. 88 3.8 Population-specific mitochondrial macrohaplogroup L lineages……….. 89 3.9 Sequence divergence times for major African mitochondrial haplogroups…… 90 3.10 Restriction enzyme sites used to define continent-specific Asian mtDNA

haplogroups……….. 96

3.11 Distribution of haplogroups A, B, C, D, E, F and G……….…...………... 97 3.12 Divergence times of the major Asian mitochondrial haplogroups……… 97 3.13 Restriction enzyme sites used to define continent-specific Native American

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LIST OF TABLES

xviii

3.14 Single nucleotide polymorphisms used to define continent-specific Native

American mtDNA haplogroups………..……… 99 3.15 Sequence divergence times for Native American mitochondrial

haplogroups………..…… 100

3.16 Single nucleotide polymorphisms used to define continent-specific Oceanic

mtDNA haplogroups……… 102

3.17 Divergence times of some of the major Oceanic mitochondrial

haplogroups………..……… 103

3.18 Restriction enzyme sites used to define continent-specific European mtDNA

haplogroups………..……….…….. 105

3.19 Divergence times of the major European mitochondrial haplogroups…..…….. 105 3.20 Classification of haplogroup H into sub-haplogroups ………..…. 106 3.21 Age estimates determined for sub-haplogroups of haplogroup H………... 107 3.22 Classification of haplogroup U into sub-haplogroups ………...………… 107 3.23 Age estimates determined for selected sub-haplogroups of haplogroup U...… 108 3.24 Classification of haplogroup J into two sub-haplogroups ………. 108 3.25 Classification of haplogroup T into two sub-haplogroups ………...………. 108 3.26 mtDNA haplogroups which are associated with specific phenotypes and

susceptibility to specific disorders………. 109 3.27 Characteristics of the Marzuki human mitochondrial consensus sequence….. 114 3.28 Characteristics of the Carter human mitochondrial consensus sequence…... 115 4.1 Primer pairs used for amplification of the complete mitochondrial genome….. 125 4.2 Polymerase chain reaction thermal cycling conditions………..…… 127 4.3 Estimation of the quantity of the purified PCR product to be used in a cycle

sequencing reaction based on the size of the PCR product……… 131 4.4 Thermal cycling conditions to be used for sequence determination of

segments of the mitochondrial genome………..………. 131 4.5 Primers used for sequencing of the complete mitochondrial genome……….... 132 4.6 Thermal cycling conditions used during SDS/heat treatment of cycle

sequencing reactions………..…… 134

4.7 Keywords and phrases used to recover published mitochondrial genome

sequences belonging to macrohaplogroup L………..………… 138 4.8 Assignment of haplogroups to incompletely classified sequences included in

the current investigation………..…………... 140 4.9 Sequences obtained from GenBank®_{that were excluded from phylogenetic}

analyses………..…….. 142

4.10 Description of all datasets used in the current investigation for analyses…..… 145 4.11 Species used to calculate the conservation index……….………… 167 5.1 Primer pairs and PCR conditions used for amplification of the complete

mitochondrial genome………..……….. 177 5.2 The likely causes for the distortion of sample fragments and ways in which it

can be prevented………. 187

5.3 The possible causes of slanted sample fragments and ways in which it can

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LIST OF TABLES

5.4 The amount of purified PCR product template used for sequencing per

region……….…………... 192

5.5 Factors affecting template quality………..…………... 208 5.6 Sequence alterations observed between the complete Khoi-San

mitochondrial DNA genome sequences and the rCRS in the D-loop……..…... 208 5.7 Sequence alterations observed between the complete Khoi-San

mitochondrial DNA genome sequences and the rCRS in the tRNAPhe

gene………..…………. ₂₁₁

5.8 Sequence alterations observed between the complete Khoi-San mitochondrial DNA genome sequences and the rCRS in the 12S rRNA

gene………..…………. ₂₁₃

5.9 Sequence alterations observed between the complete Khoi-San mitochondrial DNA genome sequences and the rCRS in the 16S rRNA

gene………..……. ₂₁₇

5.10 Sequence alterations observed between the complete Khoi-San

mitochondrial DNA genome sequences and the rCRS in the ND1 gene……... 219 5.11 Sequence alterations observed between the complete Khoi-San

mitochondrial DNA genome sequences and the rCRS in the tRNA genes…… 222 5.12 Sequence alterations observed between the complete Khoi-San

mitochondrial DNA genome sequences and the rCRS in the ND2 gene……... 224 5.13 Sequence alteration observed between the complete Khoi-San mitochondrial

DNA genome sequences and the rCRS in the tRNATyr

gene………... ₂₂₉

mitochondrial DNA genome sequences and the rCRS in the COI gene……… 232 5.15 Sequence alteration observed between the complete Khoi-San mitochondrial

DNA genome sequences and the rCRS in the tRNASer(UCN)

gene………... ₂₃₇

mitochondrial DNA genome sequences and the rCRS in the COII gene……... 237 5.17 Sequence alterations observed between the complete Khoi-San

mitochondrial DNA genome sequences and the rCRS in the ATPase8

gene………... ₂₃₉

5.18 Sequence alterations observed between the complete Khoi-San mitochondrial DNA genome sequences and the rCRS in the ATPase6

gene………... ₂₃₉

mitochondrial DNA genome sequences and the rCRS in the COIII gene…….. 242 5.20 Sequence alterations observed between the complete Khoi-San

mitochondrial DNA genome sequences and the rCRS in the ND4L gene... 246 5.22 Sequence alterations observed between the complete Khoi-San

Construction of a mitochondrial consensus sequence for the Khoi-San population of Southern Africa