Lec 2 - DNA & chromosomes

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Meiosis

i) Stages of meiosis

Interphase I

Replication of chromosomal DNA

Prophase I

​​​​​​​Chromatin condense to chromosomes

Homologus chromosomes pair up side by side in perfect alignment

​​​​​​​In males - X & Y chromosomes line up end to end!

Homologus chromosomes intertwine @ attachment points

2 homologous chromosomes bivalent (2 chromatids form 1 unit)

Tetrad (4 sister chromatids)

Chiasma formation occurs

​​​​​​​Attachment points @ which genetic crossing over occurs

Produces new possible gene combinations & new possible trait combinations

Bivalents move towards equitorial plate, spindle apparatus forms & nuclear membrane breaks down

Metaphase I

​​​​​​​Same as mitosis

Anaphase I

​​​​​​​​​​​​​​Chiasmata disappear

Homologus chromosomes pulled to opposite sides cell by spindle fibres

Unlike in mitosis, centromeres do not split (sister chromatids aren't seperated)

​​​​​​​Only 1/2 original number of chromosomes migrates towards each pole

i.e. In mitosis, if there are 4 chromosomes along equiplate - 4 chromatids migrate to each side of cell (splitting)

Whereas in meiosis, if there are 4 chromosomes along equiplate - 2 chromosomes migrate to each side

Telophase I

​​​​​​​Chromosomes reach opposite sides of cell

Same as mitosis

2 daughter cells formed in males

1 daughter cell in females + primary polar body

Haploid

Each chromosome = 2 sister chromatids

Meiosis II occurs after this stage - leads to:

Formation of 4 functional haploid daughter cells in males

Formation of secondary oocyte & 2nd polar body in females

​​​​​​​ Remember only occurs after fertilisation in females!

ii) Cross over

See prophase I above

In males:

X & Y chromosome are not the same size & therefore cannot align perfectly

Pair tip-to-tip

Cross over occurs @ tip of short arm of X & Y chromosomes (pseudo-autosomal region)

​​​​​​​These regions contain v similar DNA sequences

iii) Mitochondrial DNA inheritance

ONLY EVER INHERITED FROM MOTHER!!!

Due to fact that sperm only contribute a nucleus

Shed body containing mitochondria in order to enter egg

Ovum contains many copies of subcellular organelles of somatic body cells

All genes carried in mitochondrial genome passed on equally to offspring of both sexes

Mitochondria do not have DNA repair enzymes

High rate of spontaneous mutation

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Blood group antigens

i) Genotypes

Homozygous

AA - Group A

BB - Group B

OO - Group O

Heterozygous

AO - Group A

BO - Group B

AB - Group AB

ii) H antigen

Gene locus located on chromosome 19

Found on all 4 blood group types (A, B, AB, O)

Synthesises sequence of monomers which allow other RBC antigens to attach to RBC surface

iii) ABO group antigens

Gene locus located on chromosome 9

Group A = N-acetylgalactosamine glycosidically bonded to H/O antigen (see above)

Group B = D-galactose bonded to H/O antigen

Group O = Nothing bound to H/O antigen - caused by SND

iv) Homozygous blood group alleles

Homozygous AA blood group may have 2 different alleles of A:

Potential single nucleotide replacement which is 'silent'

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Cytogenetics

i) What is cytogenetics?

Cytogenetics = study of number & structure of chromosomes + associated abnormalities

Used to identify abnormalities that affect v small regions of genome

Identification DNA polymorphisms in parents & offspring

ii) Stages of karyotyping

Living tissue sample (usually blood) cultured in vitro for required length of time

Can also take:

T lymphocytes

Skin fibroblasts

Bone marrow

Chorionic villus cells

Metaphase arrest induced using colcemid

Cells are harvested & nucleus is ruptured

Chromosomes stained with designated nuclear stain & photographed

Images of 22 pairs of chromosomes arranged according to length

Karyogram

Sex chromosomes always put in right hand corner

iii) Nomenclature

Autosomes arranged according to size (where 1 = biggest)

Except 21 & 22!

21 is smallest

Location of gene loci on short arm = p (petit)

Location of gene loci on long arm = q

Chromosome regions counted from centromere outwards!

p1 = near centromere - p2 - p3 = near tip

q1 = near centromere - p2 - p3 = near tip

Regions on chromosomes divided into bands

i.e. 14q32 = second band in the 3rd region of long arm of chromosome 14

Sub-bands designated by decimal points following band number

i.e. 14q32.3 = third sub-band of band 2

iv) Limitations of conventional cytogenetics

Limited resolution

Changes need to be around 5-10Mb DNA to be visible

Cannot detect very small chromosomal changes

v) Molecular cytogenetics

Fluorescence in-situ hybridisation (FISH)

MOA

Labeled single-stranded DNA segment (probe which has been labelled with a fluorescent dye) exposed to denatured metaphase, prophase or interphase chromosomes from a patient

Probe undegoes complementary base pairing only with complementary DNA sequence @ specific location on one of the denatured chromosomes

Chromosomes are viewed under a fluorescence microscope

What is FISH used for?

Used to determine if portion of chromosome has been deleted

In unaffected person

Probe will hybridise in 2 places (due to presence 2 homologus chromosomes in somatic cell nucleus)

In an affected person

Probe will hybridise in 1 place (deletion of one of chromosomes)

Can also detect if there is an extra copy of a chromosome

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Trisomy 21

i) Case study & genetics

D.K.

First child

Mother aged 38

Father aged 51

Observation of child detected:

Hypotonicity

Single transverse crease across palm

Large tongue

Short fingers

Flat distinct facial profile

D.K. has trisomy 21

What is trisomy 21?

Common autosomal aneuploidy - person has 3 pairs of chromosome 21 - instead of normal 2 pairs

Clinically important features of trisomy 21 include:

Intellectual disability (can vary in severity)

GIT obstruction

Congenital heart defects

Respiratory infections

Extra 21 chromosome is contributed by mother in ~90% of cases

Strong correlation between maternal age & Down syndrome risk

ii) Aneuploidy

Cells that contain missing or additional chromosomes

Number of chromosomes in cell is not multiple of 23

Consist of monosomies & trisomies:

Monosomy = presence of only one copy of chromosome

Very uncommon - most do not survive to full term

Trisomy = 3 copies of a chromosome

More common - see among live births

Illustrates that body can tolerate excess genetic material more easily than a deficit in genetic material

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Chimeras

i) Case study & genetics

Mr & Mrs X had a son in June 2014

Healthy male

Blood group = AB

Mr X's blood group = A (AA or AO)

Mrs X's blood group = A (AA or AO)

How is it Mr & Mrs X's baby has a different blood group to them?

Wrong child?

Mr X is not the father?

Wrong sperm? (IVF)

Mr X is a chimera

Essentially a combination of 2 people!

How does this happen?

Chimeras start out as fraternal twins

​​​​​​​2 seperate eggs have been fertilised by 2 seperate sperm around same time

If these 2 eggs develop seperately - you get fraternal twins!

In chimeras, instead of the two eggs developing seperately - one person ends up with both sets of cells

The 2 siblings fuse together to become one person

This means Mr X has some 'blue' cells & some 'red' cells -

When Mr X produces sperm

Some sperm will get his 'true' DNA...

Whilst others will get his 'twin' DNA

The sperm that fertilised Mrs X's egg to produce their son contained the 'twin' DNA

This led to the baby having some cells which carry the allele for attachment of N-acetylgalactosamine

& other cells which carry the allele that add D galactose!

http://genetics.thetech.org/node/313

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Acute promyelocytic leukemia

i) Case study

21 year old female

Presenting with extensive bruising & heavy vaginal bleeding

WBC count = low

Platelet count = low

Excess of promyelocytes in bone marrow

Patient has Acute promyelocytic leukaemia

ii) Genetics of APML

Translocation of genetic material between chromosome 15 & chromosome 17

Gene originally located 2nd band of 2nd part of long arm chromosome 15

Gene is transposed to 1st band of second part of long arm chromosome 17

iii) Use of FISH to detect AMPL gene

iv) Treatment

ATRA - vitamin A derivative

Interacts with RAR to induce cell differentiation & apoptosis

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Points to remember

Cros over during meiosis = homologus chromosome pairs mixing genetic information

Expression of genes can be monoallelic or biallelic - blood group antigens are biallelicaly expressed

Down's syndrome is an anueploidy where someone is born with 3 copies of chromosome 21

Trisomy 21 is the most common form of aneuploidy in live-born infants