WEEK 5 CYTOGENICS DNA MUTATION Flashcards
1
Q
What is cytogenetics?
A
- the examination/visualisation of chromosomes
- “study of inheritance by visualising the structure and function of chromosomes”
2
Q
How many chromosomes do humans have?
A
- 46 chromosomes (23 pairs of homologues)
- 1 pair of sec chromosomes
3
Q
Where is the centromere in a matacentric chromosome?
A
- In the MIDDLE
4
Q
Where is the centromere in a acrocentric chromsome?
A
- Towards rthe end
5
Q
Where is the centromere in a telocentric chromosome?
A
- At the end
6
Q
Can you describe the chromosomes by centromere position and size?
A
- YES
- But this is NOT SUFFICIENT to distinguish them all
7
Q
What is one method of identifying individual chromosomes in mammals?
A
- Giemsa stain
8
Q
What is the process of G banding?
A
- Pre treat chromosomes on a slide with agent that differentially LOOSENS DNA-protein interaction
- It is then stained with Giemsa–> G bands (dark and pale)
9
Q
What is the function of Colcemid?
A
- Mitotic poison that stops cells at mitosis
10
Q
What are the proportions of Carnoy’s fixative that the Victoria Cancer Cytogenetics Service use?-
A
- 3 parts methanol and 1 part acetic acid
11
Q
In which stage of mitosis do you want to catch cells to observe chromosomes clearly?
A
- Metaphase –> So you can karyotype
12
Q
What is the order for the nomenclature of human karyotypes?
A
In order from: 1. Number of chromosomes in cell 2. Complement of sex chromosomes 3. Any abnormal chromosomes e.g. 46, XY normal male 46, XX, normal female 47, XY, +21 is male with extra copy of chr. 21 (Down syndrome)
13
Q
What is the nomenclature of human chromosomes in order?
A
- p-short arm; q-long arm
- Each arm is subdivided into regions by landmark G-bands
- Each band (dark and pale is numbered WITHIN regions starting FROM THE CENTROMERE
e. g. 7q36–> chr.7, long arm q , region 3, G band 6
14
Q
What is the most recent method for distinguishing between chromosomes?
A
- An extension of the FISH procedure
15
Q
What is the method of chromosome painting?
A
- Make probe with MANY sequences from individual chromosome
- Isolate the chromosome cytologically OR obtain DNA from library of clones from that chromosome
- Label probe CHEMICALLY with fluorescent dye
- HYBRIDISE probe to chromosome spreads IN SITU
- Chromosome of one type will fluoresce at many different sites along its length –> “painted”
16
Q
What are 2 examples of the wide range of reproductive modes found?
A
- Asexual organisms
- Some alternate b/w periods of sexual repro and lolng periods of asexual repro
17
Q
What type of reproduction do most DIPLOID organisms have?
A
- Only SEXUAL REPRODUCTION
18
Q
What does sexual reproduction require in terms of genetics?
A
- Sexual differentiation OR phenotypic dimorphism of the two sexes
19
Q
What type of chromosomes (what is the fancy name) distinguish the two sexes?
A
- heteromorphic chromosomes
20
Q
What are the basis of sex DETERMINATION?
A
- not just the genes on sex chromosomes, also normal chromosomes other than X and Y
21
Q
What is the proportion of genetic sex determining systems?
A
- Populations of females 50% to males 50%
- One sex is heterogametic, the other is homogametic
22
Q
Do non gametic systems have equal or unequal numbers of sexes?
A
- Unequal
23
Q
Is the ratio of males to females 1?
A
- NO
- Varies in DIFFERENT countries e.g. males 1.20
24
Q
What are 3 possible reasons as to why males are favored compared to females?
A
- May produce more Y bearing sperm than females
- Y bearing sperm might be more viable and motile
- Egg surface may be more receptive to Y bearing sperm
25
What did human karyotypes show that determines maleness?
- Presence of Y chromosome
26
Does the X or Y chromosome have more predicted genes?
- X chromosome
27
What are pseudo-autosomal regions in genes?
- Genes that are HOMOLOGOUS (shared) between the X and Y chromosomes (PAR1 and PAR2)
28
Are there any crossovers between the PARs?
- YES
| - Always one crossover in the paired pseudoautosomal region at the end of the short arm (red)
29
What does the SRY gene stand for and where is it found?
- Sex Determining Region
| - Found on Y chromosome (short arm-p)
30
How was SRY located?-
- Through rare and abnormal cases of XX males and XY females through rare crossovers BELOW the SRY gene at meiosis
31
Do individuals with 46, XX (male) or 46 XY (female) transmit the defect to the next generation?
- NO
| - Because they are sterile
32
What is the sex determining gene and how was it done?
- SRY
| - If inserted into transgenically normal XX female mice, they would turn male
33
Is the method for sex determination the same in Drosophila as is for humans?
- NO
34
What does the presence of the Y chromosome in Drosophila do?
- Only required for functional sperm
| - Sex is determined by the ratio of autosomes to sex*
35
What is the phenomena of dosage compensation?
- Most genes on X chromosome not involved in sex differences so there is compensation for the differences in gene dosage for males and females (bc. females have two X chromosomes and males only one)
36
What is an example of dosage compensation?
- G6PD (glucose-6-Phosphate dehydrogenase) is in RBCs and gene is on X chromosome
- Shows very similar levels of it b/w males and females despite females having XX
37
How is dosage compensation achieved in mammals?
- RANDOM INACTIVATION of one of the two X chromosomes in each cell EARLY in female dev. (15-16 days of dev)
- The inactivated state is propagated to all progeny cells
- Most genes on the inactivated X chromosome are SILECNED
- Then silenced X becomes REACTIVATED for oogenesis
38
What is a Barr body?
- An INACTIVATED X CHROMOSOME
| - Darkly stained, highly condensed structure (heterochromatin)
39
What does the number of Barr bodies per nucleus equal?
- The number of X chromosomes present -1 (n-1)
| - So all BUT ONE will be inactivated
40
What is the phenotype of a tortoiseshell cat due to?
- X inactivation in female mammals means they are NATURAL MOSAICS for X chromosome genes
- This phenotype is from the X-linked orange gene
41
If a female is heterozygous for a recessive allele of an X-linked disorder, will she be mosaic for that disorder?
- YES
| e. g. Red-green colour blindness
42
What are the three main steps involved in X inactivation?
1. Initiation (Xq13 required for initiation of X inactivation)
2. Spreading
3. Maintenance
43
What is a method for finding out which autosomal linkage group corresponds to which autosome?
- Gene mapping via somatic cell hybrids
44
What does genetic mapping via somatic cell hybrids involve?
- Assigning genes to individual chromosomes using tissue culture
- One or two human chromosomes were isolated in a mouse cell line and can correlate presence of human gene WITH presence of specific human chromosome
45
What are two requirements for gene mapping via somatic cell hybrids?
1. Human gene product identifiable in cell culture
| 2. Products of human and other species genes can be distinguished
46
Can data from linkage and somatic cell maps be combined?
- YES
47
Which stage of mitosis do the chromosomes condense making them visible?
- Metaphase
48
What is a karyotype?
- The number and structure of chromosomes within a cell
49
What does a karyogram have the chromosomes arranged in?
- Order of SIZE and centromere position
50
What are the 7 steps of the BASIC karyotyping method?
1. Blood, AFT (amniotic fluid), or bone marrow are common specimens
2. Cells must be cultured IN VITRO (3 days--> blood needs addition of PHA (potent antigen) to promote mitosis)
3. After incubation, COLCEMID (mitotic poison) is added --> arrests mitosis at metaphase
4. The cells are attached using trypsin (to burst cell membrane) and hypotonic KCl is added to lyse cells
5. Cells are washed in carnoy's fixative
6. 1 DROP of cell suspension is dropped from about 5cm onto slide
7. Slide is stained using Gimesa (now have made a G-banded metaphase spread)
51
what is a modern approach to detecting chromosome rearrangements?
- Multiplex Quantitative Fluorescence PCR (QF-PCR)
52
What occurs in QF-PCR and which type of DNA can be collected?
- DNA markers (short tandem repeats) are chosen for regions common in aneuploidies
- Foetal DNA can be collected via cell free DNA
53
How can Foetal DNA be collected?
- Using traditional methods (CVS-chronic villus sampling or AFT-Amniocentesis)
- Or more modern method of cell free DNA
54
What is genetic analysis of the unborn fetus common for the diagnosis of?
- Autosomal aneuploidies
- Sex chromosome aneuploidies
- Chromosomal rearrangemetns
55
What techniques did pre natal screening previously rely on?
- AFT or CVS and karyotyping
56
What are the disadvantages of AFT or CVS for the fetus:
- Dangerous for the unborn child (spontaneous abortion)
| - Both require culturing of cells post collection (which means its pretty slow!)
57
What occurs in the Harmony test (cell free DNA)?
- Foetal DNA is shed during pregnancy in the maternal bloodstream (from apoptosis of placental cells during embryogenesis)
- Foetal DNA consists of less than 3-10% plasma derived DNA
- Purification of DNA is obtained by epigenetic patterns (Ch3 of DNA b/w mother and fetus)
58
What are the two main ways that fetal aneuploidies can be detected?
1. Quantitative PCR methods
| 2. Massively Parallel shotgun Sequencing (MPSS)
59
What does Quantitative PCR methods involve in the detection of fetal anueploidies and what is an example of one?
- Determination of the copy number of aneuploidic markers
- Directed towards common abnormalities (chr. 13,18,21, X and Y)
e. g. of test is HARMONY
60
What does MPSS involve for detection of fetal aneuploidies?
- Shows promise for the detection of chromosomal translocations
- Detection of common genetic mutations
61
What is an aneuploidy defined as and what are examples (3)?
- A loss or a gain of a SINGLE chromosome
| - e.g. Monosomy, Trisomy, Tetrasomy
62
What is a euploidy defined as and what are examples of names?
- Increase in the complete set of chromosomes (chromosome number doubles)
e. g. triploidy (3n), Tetraplodiy (4n), Polypoidy (3n, 4n, 5n, 6n)
63
What is the standard naming convention of anuploids and what would the examples be for:
a) Female with extra X chromosome
b) Person with an extra chromosome 21
- Chromosome number, genotype
a) 47, XXX
b) 47, +21
64
What are aneuploids the result of in general?
- The non-dysjuncton of chromosomes during meiosis
65
What happens to the gametes if non dysjunction occurs in meiosis I?
- gametes carry DIFFERENT recombinant chromosomes
66
What happens to gametes if non dysjunction occurs in meiosis II?
- Gamete carries the SAME recombinant chromosomes
| - Useful to determine when the ND occurred
67
How many copies of every gene do diploid, trisomy and monosomy individuals have respectively?
- Two, three and one
68
Is the abnormal phenotype characterisitic for each chromosome?
- YES
| - Determines which genes are on each chromosome
69
What are some rules regarding aneuplodies with regards to gene dosage?
- Abnormal phenotype is characteristic for each chromosome
- Monosomy results in the WORST phenotype (compared to trisomy)
- Aneuploidy of a larger chromosomes results in a more severe abnormal phenotype
- Severe imbalance of genes leads to inviability
70
Does monosomy result in the worst phenotype compared to trisomy?
- YES
71
Are sex aneuploidies generally better tolerated than autosomal aneuplodies?
- YES
72
How many different combinations of sex auneuplodies are possible technically BUT how many are more common?
- 18 possible BUT 4 are more common
73
What are the 4 most common sex aneuploidies?
- Monosomy X (turner)
- XXY (Kleinfelter syndrome)
- trisomy X (triple X syndrome)
- XYY (Double Y syndrome)
74
What are the two reasons as to why the sex aneuploids are better tolerated?
1. X inactivation
| 2. Y chromosome only encodes a FEW genes
75
What are the results of X inactivation in terms of why sex aneuplods are better tolerated than autosomal ones?
- XXX individuals will have TWO BARR BODIES instead of one
- XXY will have ONE barr body
- then imprinting RETAINS inactivated X chromosomes in SUBSEQUENT cellular generations
76
What is required for the X and Y chromosome to pair up during meiosis?
- Pseudo autosomal region (PAR1)
77
Where do the abnormalities (aneuplodies) come from in the X chromosome?
- Excess/deficient gene dosage with PSEUDO-AUTOSOMAL regions (PAR)
78
Is the entire X chromosome activated?
- NO
79
Does an alteration in sex chromosome number necessarily make the individuals sterile?
- NO as long as they are balanced its all g
80
How come triple X (XXX) AND Double Y (XYY) are fertile
- During embryonic dev. NORMAL genotype is RESTORED (Oocytes, 46, XX and Spermatogonia 46, XY)
- One sex chromosome must be LOST to develop germline (non-dyjunction or lagging during early meiosis)
81
What are the phenotypes of turner syndrome XO?
- Female (45, XO)
- Mild phenotpye
- Near NORMAL intelligence
- Short stature
- Webbed neck
- STERILE (ovaries degenerate) but can still do IVF
82
What is mosaic turner syndrome?
- Missing X chromosome may NOT occur in the germline
- X chromosome could be lost in EARLY FETAL DEV.
- Leads to SOME cells being monosomic (45, XO) whilst others are normal (46, XX) --> mosaicism
- THERE IS ERROR IN MITOSIS AFTER FERTILISATION
83
Why is the Kleinfelter phenotpye only mild?
- Due to gene dosage
84
What are the phenotypes of Kleinfelter snydrome?
- Appears as male (47, XXY)
- Slightly LOWER IQ
- TALLER than average (long legs)
- 30% show breast development
- STERILE (developed tetes but no spermatogonia)
85
How many barr bodies does triple X have?
- 2 instead of 1
86
what are the phenotypes for triple X?
- MILD phenotype (appear as female)
- 47, XXX
- MILD reduction in IQ
- Tend to be very tall
- Occasionally behavioural problems reported
- Fertile
87
What are the phenotypes of double Y syndrome?
- Appear as male
- 47, XYY
- VERY MILD phenotype
- Very tall
- RARELY see a slight reduction in IQ
- Learning difficulties reported (delayed speech)
- RARE: antisocial behavior
88
What is a uniparental diploidy?
- generation of diploid set of chromosomes from a single parent --> this is very rare event
- Foetuses DO NOT develop correct
89
What is uniparental disomy and what are the symptoms?
- Inheritence of BOTH chromosomes from a single parent
- Chromosome lost during early mitotic division in foetus (only 1/3 chance)
- RANGE of symptoms --> many are undiagnosed --> possibly imprinting errors
90
What is prader Willi syndrome in terms of the genetics?
- Deletion of paternal 15q11-13 OR uniparental disomy where BOTH copies of the Chr. 15 are inherited from the mother --> maternal copies of region are SILENT due to imprinting
- Can also be deletion of the SAME region in the mother (maternal) --> results in angelman syndrome
91
What are the symptoms of Prader Willi syndrome?
- Poor muscle tone
- Insatiable appetite --> obesity
- Cognitive delays
92
In X inactivation, what does the gene XIST stand for, where is it expressed and what is the product of it?
- X-inactive specfic transcript
- Found within the Xic (X inactivation centre) and expressed only on the INACTIVE X
- XIST gene product is a LONG NON CODING RNA (lnc RNA)
93
How is X inactivation maintained?
-Inactivation is MAINTAINED by continued XIST expression in Barr body interphase
94
Which part of the X chromosome escapes X inactivation?
- PAR1
