Medical Genetics

Transcription

1 Medical Genetics

2 Cytogenetics Human chromosome Chromosomal aberration Chromosome disease

3 History of Ct Cytogenetics ti Dark Ages Painter TS. Studies in mammalian spermatogenesis. II. The spermatogenesis of man. JExp Zool. 1923; 37:

4 Dark Ages Hypotonic Period Hsu TC. Mammalian chromosomes in vitro. I. The karyotype of man. J Hered. 1952; 43: Tjio JH, Levan A. The chromosome number of man. Am JObstet Gynecol. 1956; 130:

5 T. C. Hsu ( 徐道觉,April 17,1917-July,9,2003)

6 Tjio & Levan (1956) Showed d the normal human chromosome number to be 46.

7 Dark Ages Hypotonic Period Trisomy Period Lejeune J, et al. Etude des chromosomes somatiques de neuf enfants mongoliens. G. R. Acad. Sciences. 1959; 248: Ford CE, et al. Asex chromosomal anomaly in a case of gonadal dysgenesis (Turner's syndrome). Lancet. 1959; 1: JACOB PA, et al. A case of human intersexuality having a possible XXY sex determining mechanism. Nature. 1959; 183:

8 Jérôme Lejeune (1959)

9 Dark Ages Hypotonic Period Trisomy Period Banding Era Caspersson T, et al. Differential banding of alkylating fluorochromes in human chromosomes. Exp Cell Res. 1970; 60:

10 Dark Ages Hypotonic Period Trisomy Period Banding Era Molecular l Era Pardue ML, et al. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl. Acad. Sci. USA. 1969; 64: Pinkel D, et al. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA. 1986; 83:

11 History of Cytogenetics Morgan, The Theory of the Gene, --beginning i of cytogenetics 1923 Pit Painter, 2n= n=48 48, XX or XY 1952 Hsu TC, hypo-osmoticosmotic preparing 1956 Tjio & Levan, 2n= n=46 46, beginning i of human cytogenetics 1959 Lejeune, Trisomy Denver system 1970 Caspersson &Q-banding FISH( FISH(1986), microdissection technique( ), connect to cytogenetics molecular cytogenetics

12 Human Chromatin Definition Components Structure Compaction

13 Human Chromatin -Definition Dfiiti Chromatin is the protein-dna complex in which h genetic material exist in interphase nucleus During metaphase, chromatin condenses into chromosomes

14 2.1.1 Human Chromatin -Definition Euchromatin Lightly stained Chromatin Loose structure Genetically active Heterochromatin Deeply stained Compact structure Genetically silent

15 2.1.1 Human Chromatin -Definition

16 2.1.1 Human Chromatin -Definition Constitutive Heterochromatin Always highly condensed Heterochromatin Enriched at centromeres and telomeres Y chromatin Facultative Heterochromatin Specifically inactivated- specific times Switch between two states X chromatin

17 2.1.1 Human Chromatin-Componentsp Chromatin is the protein-dna complex in which h genetic material exist in interphase nucleus Components-DNA, associated proteins, and small amount of RNA

18 Human Chromatin-ComponentsComponents DNA: 24 different kinds of molecules Two types of proteins Histones Small, well-defined, highly conserved basic Non Non-histone chromosomal proteins Large number Diverse Mostly not well characterized

19 Human Chromatin-ComponentsComponents Histones Small proteins: Arginine or lysine rich: positively charged Interact with ih negatively charged DNA Can be extensively modified(-make make them less positively charged Five types: H1, H2A, H2B, H3, H4

20 Human Chromatin-Structure Nucleosomes: fundamental subunits of chromatin Evidences Electronic Microscope Nuclease digestion

21 Human Chromatin-Structure EM of chromatin shows presence of nucleosomes as beads on a string

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23 Nucleosome Structure Contains ~ 200 bp of DNA DNA wrapped around a core of eight basic proteins called histones histone octamer Linker DNA- linking nucleosome together H1 binds to linker DNA

24 The Histone Octomer Four proteins: H2A, H2B, H3, and H4 H3 and H4 are arginine rich and highly conserved H2A and H2B are slightly enriched in lysine Both arginine and lysine are basic amino acids making the histone proteins both basic and positively charged The octomer is made of two copies of each protein poe

25 The Fifth Histone, H1 A A fifth protein, H1, is part of the nucleosome, but resides outside the octomer H1 varies between tissue and organisms and seems to stick to the 19 bases attached to the end of the core sequence

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27 Histone H1 plays an important t role in organizing the chromatin fiber

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29 A Portion of the Nucleosome Looking from the top and showing the DNA wrapped around the Histones

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32 Human Chromatin-CompactionCompaction Human DNA s total length is ~2 meters! This must be packaged into a nucleus that is about 5 micrometers in diameter This represents a compression of more than 100,000 times! It is made possible by wrapping the DNA around nucleosomes and then packing these in helical filaments

33 Human Chromatin-CompactionCompaction Packing ratio is the length of the DNA divided id d by the length into which it s packaged Smallest human chromosome (21) has 4x10 7 bp of DNA Equivalent to 14mm of extended DNA In most condensed state the chromosome is about 2mm long Packing ratio = 14000/2 = 7000

34 Human Chromatin-CompactionCompaction There are many levels of DNA compaction First level of DNA compaction is to form nucleosome Nucleosomes become helically arranged into a 30nM chromatin fiber (solenoid) The fiber is super coiled by attachment to a non-histone protein scaffold (not completely understood)

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36 30 nm fiber

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38 Packaging DNA Histone octomer Histone proteins DNA Helix 2 nm

39 Packaging DNA Histone octomer Histone proteins DNA Helix 2 nm

40 Packaging DNA Histone proteins 11 nm Histone octomer DNA Helix 2 nm Nucleosome

41 Packaging DNA Histone H1

42 Packaging DNA Histone H1

43 Beads on a string Packaging DNA 11 nm 30 nm Looped Tight helical 200 nm Domains fiber Protein scaffold

44 Packaging DNA Nucleosomes 11 nm 30 nm Tight helical fiber Metaphase Chromosome 700 nm 200 nm Looped Domains DNA Helix 2 nm Protein scaffold

45 2 nm DNA double helix 10 nm Nucleosome (10 nm fiber 30 nm 30 nm Fiber 300 nm Loops I 700 nm Loops II 1400 nm chromosome

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47 2.1.2 Metaphase Chromosomes Interphase - replication Cll Cell cycle Prophase - condensed Dividing stages Metaphase Anaphase - seperated td Telophase

48 Metaphase chromosome Preparation Cultured cells are arrested at metaphase by adding colchicine This is when cells are most condensed and easiest to identify

49 Metaphase chromosome Preparation Arrested cells are broken open Metaphase chromosomes are fixed and stained Chromosomes are photographed p through microscope Photograph of chromosomes is cut up and arranged to form karyotype diagram

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51 Then you can examine the preparations under microscope.

52 Chromosome Morphology Each chromosome has a primary constriction called a centromere. The centromere can be found almost anywhere along the chromosome near the top, near the middle, or in-between, but not at the very ends of the chromosome. The centromere divides the chromosome into two "arms arms, " and unless the centromere is right in the middle, there will be a short arm, labeled p, and a long arm, labeled q.

53 Ideogram: 1960, Denver System Autosomes were named in order of their size (1~22) and centromere position, and were divided into 7 groups (A~G). X chromosome belongs to group C, and Y chromosome belongs to group G.

54 Ideogram: A:1~3. 1,3 metacentric;2 submetacentric; 1q secondary constriction; largest B:4,5. submetacentric; C:6~12 12,X. submetacentric; 9q secondary constriction; D:13~15. acrocentric; satellites E:16~ metacentric, 16q secondary constriction; 17,1818 submetacentric F:19,20 metacentric G:21,22,Y acrocentric; 21,2222 satellites;22>21; smallest

55 Ideogram of human chromosomes showing the Giemsa banding patterns, arranged and numbered according to the Paris classification of 1971

56 2.1.4 Chromosome banding Centromere Staining with a dye called Giemsa chromosomes will appear to have between 400 or more alternating light and dark bands. This is called Giemsa banding or G-banding. These bands, which can vary in intensity from very light to almost solid black, have been numbered by scientists and provide location markers with which breakpoints and small structural aberrations can be identified.

57 Banding band A pattern of light and dark bands along the arms of chromosomes showed by using special staining techniques Specific Stable landmark Adistinctive sign on chromosome centromeres telomeres some bands distinctive region g the region landmarks between two neighborhood

58 Naming of bands Region namednamed from centromere to telomere Bands in the same region-- named from centromere to telomere Landmark asas the first band of the further region landmark band region

59 The standard nomenclature for banding of human chromosomes 1. the chromosome number; 2. the arm symbol (p or q); 3. the "region" number along that arm; 4. the band number within that region.

60 Description of bands Chromosome number arm symbol region number band number 1p32 1q23 1p32 The band divided by centromere were respectively named as p11 and q11. 1p11 1q1111 1q23

61 Sometimes, sub-bands, separated by a decimal point from the band designation, are also noted. For example, the location of a protooncogene (ABL1) associated with chronic myeloid leukemia, sub-band 1 of band 4 in region 3 of the long arm of chromosome 9. Centromere Though microscopic in size, a sub- 9q34.1 band can contain tens of individual genes. Karyotyping using Giemsa Banding ame_setup/cytoimages.html

62 Banding techniques Q-banding G-banding R-banding C-banding T-banding N-banding SCE staining High resolution G banding Other staining and banding techniques

63 Quinacrine banding, Q-Banding: A technique that stains chromosome preparations with Quinacrine mustard or similar fluorescent dye. This results in a pattern of light and dark bands that is unique for each human chromosome and allows the unequivocal definition of each chromosome. Earliest used. traits 1. very clear 2. can t be kept for along time 3. needsfluorescent microscope 2012 年 4 月 6 日星期五 Medical Genetics

64 Giemsa-stained stained banding, G-Banding A technique involves gentle trypsin treatment t t of chromosome preparations followed by staining with Giemsa traits: 1. Very clear, too 2. Can bekept for along time 3. Needs light microscope only Most widely used 2012 年 4 月 6 日星期五 Medical Genetics

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66 G-banded Chromosome telomere centromere G-pale bands G-dark bands GC-rich AT-rich few genes many genes telomere

67 light band in Q-banding dark band in G-banding dark band in Q-banding Staining with Giemsa chromosomes will appear to have 350~ alternating light and dark bands. light band in G-banding

68 Reversed G-band, R-Banding Pretreated with salt solution then stained by Giemsa. This Thisresults ina pattern of reversed bands to G-bands bands. light band in R-banding dark band in G-banding dark band in R-banding light band in G-banding For examining the light bands in G-banding, also terminal of chromosomes 年 4 月 6 日星期五 Medical Genetics

69 G-banding R-banding

70 Constitutive heterochromatin banding, or Centromeric banding, C-Banding Denatured with saturated Ba(OH)2 then stained with Giemsa. Specially stains centromeres and constrictions. titi Notice the large heterochromatic regions on chromosomes 1 and 年 4 月 6 日星期五 Medical Genetics

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72 Telomere banding or Terminal banding, T-Banding Specially stains telomeres. Study of telomeres can help us to obtain the structure and dynamics of chromosomes 年 4 月 6 日星期五 Medical Genetics

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74 Silver staining of NOR ( nucleolar organizing regions), N-Banding NORs appear darkly stained with AgNO 3 -As. Study for distribution and activation of 18S+28SrDNA 2012 年 4 月 6 日星期五 Medical Genetics

75 SCE (sister (itr chromatid exchange) xh staining tii Exchage between two sister chromatids during duplication of chromosomes--sce BrdU 1 exchage BrdU BrdU 2 exchages 1st cycle 2nd cycle

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77 High-resolution banding technique A newer technique allowing examination of chromosomes in prometaphase, at which time the chromosomes are more extended than in metaphase. More than 850 bands per haploid set can be distinguished by this technique 年 4 月 6 日星期五 Medical Genetics

78 Other staining and banding techniques Show of fragile sites on chromosomes FISH SKY

79 fragile site: Sites which appear as apparent breaks or gaps in special il region (or band) of chromosomes. Fragile X is associated to mental retardation (MR); Autosomal fragile sites are associated to cancer. When peripheral blood lymphocyte cultures are deprived of folate, or thymidine metabolism is perturbed, these sites can be observed 年 4 月 6 日星期五 Medical Genetics

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83 Chromosome Painting Spectral Karyotyping, SKY

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85 Chromosomal Polymorphism Polymorphism: a heritable difference between individuals in the same species

86 Polymorphism Presence in the general population of 2 or more alternative variants; the frequency of each of which cannot be maintained by recurrent new mutations alone. Polymorphism should follow the rules below: Having no remarkable heritable effect and cannot result in pathologic reaction According to Mendelian inheritance The rarest variant a needs to have a frequency 1%

87 For example: ABO blood groups Immunoglobulins Minor variants in chromosome structure DNA sequence polymorphism Exactly we should call these variants chromosome heteromorphism.

88 Common positions and forms of chromosome heteromorphism ht hi Size of Y long arm About 10% of longer or short Y. clinically normal males have a Size of centromeric heterochromatin relatively frequent for chromosome 1,9,1616

89 Common positions and forms of chromosome heteromorphism ht hi Satellite presence or absence, size mainly on 13, 14, 15, 21 and 22

90 Common positions and forms of chromosome heteromorphism ht hi Short arms and satellites of acrocentric chromosomes Length of short arms Satellite presence or absence, size Fluorescence and other staining variation Secondary constriction of chromosome 1, 9and 16 Presence or absence, length Some autosomes Centromere size and fluorescence intensity of chromosome 3, 4

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92 Chromosomal heteromorphism

93 Application: Individual Identification Detecting the origin of extra chromosomes Detecting the origin of cells Gene mapping

94 Gene mapping-- --Duffy blood group sex-linked inheritance it hemophilia and color-blindness In 1968, the first autosomal assignment of linkagewas made. Donahue, an associate of McKusick, observed a peculiar microscopically- visible stretch of chromatin on his own largest chromosome (Chromosome #1).

95 A pair of number 1 chromosomes fom from two different ent individuals These studies, done before the advent of chromosomes banding, detect a remarkable increase in length below the centromere, referred to as a heteromorphism, in one of the pair of chromosomes on the lft left. The chromosomes on the right show the more usual morphology 年 4 月 6 日星期五 Medical Genetics

96 Looking at a number of blood factors Donahue found that in both himself and his relatives, es an allele of the Duffy blood factor was linked to this observable physical change in their chromosome #1.

97 The pedigree shows the pattern of inheritance for the Duffy blood group and the chromosome 1 heteromorphism Ⅰ Ⅱ ab bb bb ab ab bb ab ab bb Ⅲ ab ab bb bb aa ab aa bb Ⅳ aa ab ab ab Presence of the heteromorphism on chromosome 1 a and b The Duffy blood group genotypes

98 Thus "Duffy" " was assigned to Chromosome #1.

99 2.2Mitosis and Meiosis

100 MITOSIS

101 Mitosis The form of cell division by which a eukaryotic somatic cell duplicates. Mitosis is asexual reproduction. Cell division is the continuation of life based on the reproduction of cells.

102 Mitosis Liver Cell 2n=46 2n=46 2n=46 Liver Cell two identical daughter cells Liver Cell

103 Cell Cycle The dividing and non-dividing stages in the life of a cell. Phases: 1. Interphase: growth and DNA replication 2. Prophase 3. Metaphase 4. Anaphase 5. Telophase Mitotic division

104 Cell Cycle S phase G 1 interphase G 2 Mitosis -prophase -metaphase -anaphase -telophase

105 Interphase Comprises about 90% of the cell cycle. Cellular growth: a. protein synthesis b. metabolic activities i i c. DNA synthesis Made up of three phases: 1. G 1 phase 2. S phase 3. G 2 phase

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114 Chromosome DNA

115 Question: A cell containing 20 chromosomes at the beginning of mitosis would, at its completion, produce cells containing how many chromosomes each?

116 Answer: 20 chromosomes

117 MEIOSIS

118 Meiosis The form of cell division by which gametes, with half the number of chromosomes, are produced. Diploid id (2n) haploid (n) Meiosis i is sexual reproduction. Two divisions i i (meiosis i i I and meiosis i II).

119 Prophase I Longest and most complex phase (90%). Chromosomes condense. Synapsis occurs: together to form a tetrad. occurs: homologous chromosomes come Tetrad is two chromosomes or four chromatids (sister and nonsister chromatids).

120 Prophase I - Synapsis Homologous chromosomes sister chromatids Tetrad sister chromatids

121 Crossing Over - variation nonsister ste chromatids Tetrad chiasmata: site of crossing over variation

122 Sex Chromosomes XX chromosome - female XY chromosome - male

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124 Prophase I spindle fiber centrioles aster fibers

125 Shortest phase Mt Metaphase I Tetrads align on the metaphase plate. INDEPENDENT ASSORTMENT OCCURS: 1. Orientation of homologous pair to poles is random. 2. Variation 3. Formula: 2 n Example: 2n = 4 then n = 2 thus 2 2 = 4 combinations

126 Metaphase I OR metaphase plate metaphase plate

127 Question: In terms of Independent Assortment - how many different combinations of sperm could a human male produce?

128 Answer Formula: 2 n Human chromosomes: 2n = 46 n = = ~8 million combinations

129 Anaphase I Homologous chromosomes separate and move towards the poles. Sister chromatids remain attached at their centromeres.

130 Telophase I

131 Prophase II same as prophase in mitosis

132 Anaphase II same as anaphase in mitosis sister chromatids separate

133 Telophase II Same as telophase in mitosis. Nuclei form. Cytokinesis occurs. Remember: four haploid daughter cells produced. gametes = sperm or egg

134 Telophase II

135 Meiosis n=2 sex cell n=2 n=2 sperm 2n=4 diploid (2n) n=2 n=2 n=2 haploid (n) meiosis I meiosis II

136 Variation Important to population as the raw material for natural selection. Question: What are the three sexual sources of genetic variation? 1. crossing over (prophase I) 2. independent assortment (metaphase I) 3. random fertilization

137 Question: A cell containing 20 chromosomes (diploid) id) at the beginning of meiosis would, at its completion, produce cells containing how many chromosomes?

138 Answer: 10 chromosomes (haploid)

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140 mitosis i meiosis Funtion Somatic cell repreduction Producing germ cells No cells No chr

141 Spermatogenesis 46 XY Mitotic period 46 XY 46 XY 46 XY 46 XY 46 XY 46 XY 46 Primary spermatocyte XY Meiotic period X Y Secondary spermatocytes 23 X 23 X 23 Y 23 Y spermatids 23 X 23 X 23 Y 23 Y sperms

142 Oogenesis es s Mitotic period 46 XX 46 XX 46 XX Oogenesis takes place in ovary 46 XX XX XX XX Meiotic period Primary oocyte in 46 follicle XX Secondary oocyte Fertilization 23 X 23 X 23 X 23 X 23 X Suspended in prophase Ⅰuntil sexual maturity 1st polar body 23 X ovum 2nd polar bodies

143 Fertilization The fusion of a sperm and egg to form a zygote. A zygote is a fertilized egg n=23 egg sperm n=23 2 2n=46 zygote

144 2.3 Sex Chromosomes

145 Tortoiseshell cat

146 Human males are the heterogametic sex with two different sex chromosomes, (XY). Human H females are the homogametic sex (XX). 22 pairs of auto 1 pair of sex chro. XX or XY

147 Chromosome DNA content of human chromosomes Amount of DNA (Mb) Chromosome Amount of DNA (Mb) X Y 59

148 X chromosome dosage Sex Chromosomes: females XX, males XY Genes on X: females 2, males 1 How to create equal amount of X chromosome gene products in males and females? Shouldn t XX females produce twice the amount of X-linked gene products (proteins) as XY males?

149 Dosage Compensation decrease X gene products by half in females (e.g. humans called X-inactivation) XX females compensate by inactivating one of their X chromosomes to make a single dosage of X-linked genes.

150 The Lyon Hypothesis of X Inactivation In normal females, only one of the two X chromosomes is genetically active X chromosome inactivation occurs early in development t(lt (late blastocyst t stage of embryogenesis cell stage). Proposed by Mary Lyon and Liane Russell (1961)

151 X inactivation is random. The inactive X can either be maternal or paternal in origin and the choice is random in each cell and independent of the choice in other embryonic cells. X inactivation is irreversible in somatic cells - the inactive X in a particular cell remains inactive in all descendents of that cell. Inactive X late replicating 2012 年 4 月 6 日星期五 Medical Genetics

152 X-inactivation

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155 Barr Bodies Murray Barr (1949) chromatin (inactive X) appears as a dense object in the nucleus XX with one Barr body XY XY chromatin negative (no Barr body)

156 Inactive X chromosome is visible as Barr body

157 Barr Bodies are Inactivated X Chromosomes in Females Normal male, Turner female 0 1 Normal female, Klinefelter male 2 3 # Barr bodies= N-1 rule

158 Inactive X chromosome is visible as Barr body XXX female XXXX female 2012 年 4 月 6 日星期五 Medical Genetics

159 X-inactivation reveals alleles in cats heterozygous for the fur color gene Genotype is X yellow /X black Yellow patches: black allele is inactive Black patches: yellow allele l is inactive 2012 年 4 月 6 日星期五 X yellow /X black X yellow /X black Medical Genetics

160 Lyon-Hypothesis: X-inactivation A precursor cell to all coat color cells Random inactivation early in dev.

161 anhidrotic ectodermal dysplasia 无汗性外胚层发育不良 So if you get women with this "anhidrotic ectodermal dysplasia" to exercise vigorously and generate a sweat, you can see the skin broken up into thousands of alternating little patches, each about one centimetre across. One patch has skin with sweat, while the patch right next to it has skin without sweat - a mosaic pattern right across the whole body. This doesn't happen to men because they have only one X chromosome

162 Inconsistencies between syndromes and X inactivation If normal XX female has one X inactivated, why is a XTurner female not normal? Similarly, if XXY male has one X inactivated, why does he have Klinefelter syndrome?

163 Exceptions to Lyon hypothesis h Although X inactivation is usually random, a structurally abnormal X, e.g., an X chromosome bearing a deletion, is preferentially inactivated; i i di id l i h l i i i in individuals with X-autosome translocations, it is usually the normal X chromosome that is preferentially inactivated;

164 Although X inactivation is extensive, it is not complete, some genes are known to escape inactivation; While x inactivation is permanent in most somatic cells, it must be reversible in the development of germ cells.

165 Some genes on X are not inactivated Genes in pseudo-autosomal regions PAR1 and PAR2. XIST active only on the XIST, active only on the Inactive X.

166 Many Genes Escape X-Inactivation In a survey of 224 human X-linked genes, 34 (15%) escaped X inactivation. The genes escaping X-inactivation occur primarily on Xp. From: Carrel et al. Proc. Natl. Acad. Sci. 96(1999)

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168 Nonrandom X Chromosome Inactivation The cells with deleted X: The cells carry X-autosomal translocation ti

169 Nonrandom X Chromosome Inactivation i Normal X Deleted X

170 Nonrandom X Chromosome Inactivation Normal X from X from auto

171 Sex Chromatin X-chromatin Y-chromatin Origin Inactivated X Long arm of Y Feature Facultative Constitutive Heterochromatin Heterochromatin Number =No X -1 =N of Y

172 Selection of one active X chromosome Normal females : one Xa and one Xi. However, studies of individuals id with extra copies of the X chromosome show that in cells with more than two X chromosomes there is still only one Xa, and all the remaining X chromosomes are inactivated. Default state of the X chromosome in females is inactivation, but one X chromosome is always selected to remain active.

173 Blocking g factor model It is hypothesized that there is an autosomally- encoded 'blocking factor' which binds to the X chromosome and prevents its inactivation. The model postulates that there is a limiting blocking factor, so once the available blocking factor molecule binds to one X chromosome the remaining X chromosome(s) are not protected from inactivation.

174 This model is supported by : The existence of a single Xa in cells with many X chromosomes The existence of two active X chromosomes in cell lines with twice the normal number of autosomes.

175 Chromosomal component The X-inactivation center (XIC) on the X chromosome is necessary and sufficient to cause X-inactivation inactivation. Chromosomal translocations which place the XIC on an autosome lead to inactivation of the autosome, and X chromosomes lacking the XIC are not inactivated. t The XIC contains two non-translated RNA genes, Xist and Tsix, which are involved in X-inactivation. The XIC also contains binding sites for both known and unknown regulatory proteins.

176 Xist and Tsix RNAs The X-inactive specific transcript (Xist Xist)geneis located in the X inactivation center of Xq13 and is transcribed only from the inactive X chromosome. XIST mrna transcripts are only detected in normal females, not normal males. It is a non-coding RNA, not translated into a protein.

177 The inactive X chromosome is coated by Xist RNA, whereas the Xa is not. The Xist gene is the only gene which is expressed from the Xi but not from the Xa. X chromosomes which lack the Xist gene cannot be inactivated.

178 Tsix gene Like Xist, the Tsix gene encodes a large RNA which is not believed to encode a protein. The Tsix RNA is transcribed antisense to Xist, meaning that the Tsix gene overlaps the Xist gene and is transcribed on the opposite strand of DNA from the Xist gene. Tsix is a negative regulator of Xist; X chromosomes lacking Tsix expression (and thus having high levels of Xist transcription) are inactivated much more frequently than normal chromosomes.

179 Uses in experimental biology Stanley Michael Gartler used X chromosome inactivation to demonstrate the clonal origin of cancers. Examining normal tissues and tumors from females heterozygous for isoenzymes of the sex- linked G6PD gene demonstrated that tumor cells from such individuals express only one form of G6PD, whereas normal tissues are composed of a nearly equal mixture of cells expressing the two different phenotypes. This pattern suggests that a single cell, and not a population, grows into a cancer

180 Question What are the possible mechanisms for a female being an XR affected?

181 Sex SxDetermination

182 What determines gender? Sex Chromosomes XX (female) XY (male) Number of X chromosomes, or presence of Y chromosome?

183 What determines gender? Individuals with unusual chromosome combinations provide a clue: X0 female XXY male => Y chromosome determines gender

184 Y Chromosome mostly inert, very few genes, mostly repeat sequence DNA (high and middle).

185 PAR: pseudo-autosomal a regions on tips of X and Y: homologous, contain some genes. PAR1 has a required cross over for successful sperm development.

186 Genes on the Y chromosome There are three classes of genes on the Y. Genes es shared with X chromosome define the pseudoautosomal regions (PAR) Genes similar to X chromosome genes are X-Y homologs Genes unique to the Y including SRY gene

187 Mapping the gene responsible for maleness Experimental paradigm: sex-reversed individuals XY females (missing critical bit of Y) XX males (possessing critical bit of Y) Deletion mapping of Y coupled with analysis of sex-reversed individuals and chromosome walking to get new sequences

188 Mapping the gene responsible for maleness In 1990, Sinclair and colleagues narrowed the region to a 35,000 base-pair domain of the small arm of the Y chromosome.

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190 Sex-determining region Y 2012 年 4 月 6 日星期五 Medical Genetics

191 Sex determining region Y Gene symbol : SRY Location : Yp11.3 SRY encodes a 223 amino acid zinc finger transcription factor that is a member of the high mobility group (HMG)-box family of DNA binding proteins. The protein is expressed during testis development for only 2 days.

192 Sequencing revealed a conserved motif that could have DNA-binding function SRY = TDF XY sex-reversed females have deletions or mutations of SRY transgenic mouse model dl - XX + Sry leads to testis development SRY expressed in gonad, but only transiently, at the onset of differentiation

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194 SRY gene on the Y chromosome was identified as the gene that codes for TDF: SRY is translocated to X in rare XX males SRY is absent from Y in rare XY females The home run experiment by Koopman et al. used transgenic mice.

195 Genotypically Female Mice Transgenic for SRY are Phenotypically y Male XY male XX male

196 Anne McLaren. What makes a man a man? Nature. 1990,19;346(6281):

197 Sexual development At the beginning of human development either male or female development is possible. Unspecialized gonads and two sets of reproductive ducts exist until week 6. An embryo develops as a male or female using information from the Y chromosome.

198 Effect of Y Chromosome appearance of structures that will give rise to external genitalia appearance of uncommitted duct system of embryo at 7 weeks 7 weeks Y Y Y Y present absent present absent testes ovaries 10 weeks ovary 2012 年 4 月 6 日星期五 birth approaching testis Medical Genetics

199 Downstream sex-determining genes Puzzling XY sex-reversed females without detectable mutation in SRY provide evidence for additional genes, including: DAX1 - on X, can suppress testis- formation in a dosage-sensitive sensitive manner SOX9 -on 17q, required with SRY for normal testis formation

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201 The End