19.01.02 Chromosome structure and organisation in human

Question 1

Chromosomes consist of

Answer 1

chromatin fibre (DNA and protein complex) folded and coiled into a compact arrangement.

Question 2

centromere is

Answer 2

Constricted point of a chromosome. Divides the chromosome into two arms, p arm (short) and q arm (long). A region of highly specialized chromatin that provides the foundation for kinetochore assembly and serves as a site for sister chromatid attachment.

Question 3

three major types of Eukaryotic chromosomes

Answer 3

· Metacentric: p and q arms are of roughly equal length
· Submetacentric: the arms are of unequal length
· Acrocentric: if the p arm is very short but still present. The human genome includes five acrocentric chromosomes: 13, 14, 15, 21 and 22.

Question 4

Centromere function

Answer 4

• essential for accurate chromosome segregation during cell division and also provides the foundation for assembly of the kinetochore.
• During mitosis and meiosis II the centromeres shared by the sister chromatids must divide so that the chromatids can migrate to opposite poles of the cell, while during the 1st meiotic division the centromere of sister chromatids must remain intact.

Question 5

Acentric fragments

Answer 5

chromosomes lacking centromere. fail to attach to the mitotic spindle, segregate randomly during mitosis and are eventually lost from cells.

Question 6

Dicentric chromosomes

Answer 6

are subject to fragmentation if the centromeres become attached to opposite spindle poles by way of their kinetochores.

Question 7

Centromere structure

Answer 7

centromeres are composed of constitutive heterochromatin that consists of various families of repetitive satellite DNA, comprised largely of hundreds of kilobases of alpha satellite DNA (which consists of a repeated 171bp sequence).

Question 8

How many centromeric DNA sub domains

Answer 8

3- CDEI, II and III

Question 9

Classes of proteins associated with human centromeres (CENPs – centromeric proteins):

Answer 9

1. first class comprises proteins that are constitutively associated with the centromere such as CENPA, CENPB and CENPC, which are thought to have structural roles in kinetochore formation.
2. The second class known as passenger proteins associate with the centromere transiently during the cell cycle and comprises proteins with diverse roles in cell division such as spindle capture, metaphase to anaphase transition, and sister chromatid cohesion.

Question 10

Premature centromere division (PCD)

Answer 10

age-dependent phenomenon occurring in women, characterised by rod-shaped X chromosome(s) without discernible centromeres, possible cause of age-dependent increase of X chromosome aneuploidy.

Question 11

Premature chromatid separation (PCS)

Answer 11

consists of separate and splayed chromatids with discernible centromeres and involves all or most chromosomes of a metaphase. Inherited in autosomal dominant mode

Question 12

Roberts syndrome

Answer 12

Disease associated with centromere malfunction. Mutations in ESCO2 (acetyltransferase).required for the establishment of sister chromatid cohesion in S phase, holding the two sister chromatids together until the chromosomes are ready to separate. Mutation results in delayed cell division and increased cell death. Phenotype includes pre/post growth retardation, Limb malformations (reduction), craniofacial (microcephally, clefting).

Question 13

Kinetochore

Answer 13

large multiprotein (>80) complex with a plate-like structure that assembles on a centromere and acts as a point of attachment for the microtubules/spindle fibres. It is essential for proper chromosomal segregation during mitosis.

Question 14

How many kinetochores per ceontromere

Answer 14

2 (inner and outer), formed during late prophase of mitosis

Question 15

Neocentromere definition

Answer 15

a new centromere that forms on a chromosome at a location that is normally not centromeric.

Question 16

Neocentromere classes

Answer 16

Class 1 marker chromosomes (inverted duplication of distal part of a chromosome) are the most common.
Class 2 marker chromosomes (from an interstitial deletion).

Question 17

Telomeres definition

Answer 17

caps at the end of each strand of DNA that protect our chromosomes

Question 18

Telomere function

Answer 18

• Maintain structural integrity – if lost the chromosome end is unstable (becomes ‘sticky’); it can fuse with other broken chromosomes, be involved in recombination or be degraded.
• Prevents shortening of the chromosomes at each round of cell division. If telomeres were to shorten progressively, this would result in cell death.
• Are important for chromosome positioning as they help to establish the 3-D architecture of the nucleus and aid chromosome pairing

Question 19

Telomere structure

Answer 19

consists of 3kb to 20kb of tandem TTAGGG repeats (well conserved during evolution), which are associated with a variety of telomere-binding proteins.

Question 20

Located immediately adjacent to the TTAGGG repeats are

Answer 20

telomere associated repeats (TAR), also known as the subtelomeric repeats, which are 100-300kb in size. They contain regions of shared homology between subsets of certain chromosomes. These have not been conserved during evolution and their function is not yet understood.

Question 21

T loop

Answer 21

As a result of natural difficulty in replicating the lagging DNA strand, the extreme end of the telomere has a single-stranded overhand at its 3’ end around 150-200 nucleotides long. This can fold back to form a telomeric loop known as the T loop and protects chromosome ends.

Question 22

End-Replication Problem

Answer 22

Replication of linear DNA presents a problem in that DNA synthesis works in the 5’ to 3’ direction; this is ok for the leading strand but is opposite to the direction of the lagging strand. A succession of ‘back-stitching’ syntheses is required to produce a series of DNA fragments (okazaki fragments) whose ends are then sealed by DNA ligase to ensure continuity of synthesis along the lagging strand.

The RNA primer requires the presence of DNA ahead of the sequence to act as a template. However, at the extreme end of the linear DNA molecule there is no template and this leads to incomplete synthesis of the lagging strand and a 50-100bp shortening of the telomere per replication

Question 23

What does DNA polymerase require to extend synthesis

Answer 23

a free 3’-OH group

Question 24

How is a a free 3’-OH group generated for the DNA polymerase?

Answer 24

Uses an RNA polymerase to synthesise a complementary RNA primer to prime synthesis of each DNA fragment used to make the lagging strand.

Question 25

Telomerase

Answer 25

RNA-dependent DNA polymerase. Telomerase carries an 11 nucleotide sequence – 5’ CUAACCCUAAC 3’ – with internal hexanucleotide sequence which is an antisense sequence to TTAGGG telomere repeat sequence. This acts as a template to prime extended DNA synthesis of telomeric sequences on the leading strand, providing a template for DNA polymerase to complete synthesis of the lagging strand.

Question 26

Telomereopathies

Answer 26

Diseases associated with telomere malfunction

Question 27

Dyskeratosis congenital (DC)

Answer 27

Rare inherited disorder with increased incidence of cancer. Carry mutations in 3 main components of the telomerase holoenzyme complex resulting in decreased telomerase stability and shorter telomeres.

Question 28

Cri du Chat syndrome (CdCS)

Answer 28

Deletion of 5p (variable breakpoints). Characteristic phenotype including cat-like cry, microcephaly, distinct facies and palmar creases. Deleted region includes the hTERT gene – telomerase reverse transcriptase, which helps maintain telomere ends.

Question 29

Anaplastic anaemia

Answer 29

Characterized by hypocellular bone marrow and low blood cell counts. Affected patients have significantly shorter telomeres than age matched controls. Mutations identified in both protein and RNA components of telomerase7.

Question 30

Nucleolar organizing region (NOR)

Answer 30

responsible for organising the nucleolus structure and contain the approx. 200 rRNA genes necessary for protein synthesis.

Question 31

Nucleolar organizing region (NOR) location

Answer 31

satellite stalk (p12) of acrocentric chromosomes

Question 32

Nucleolar organizing region (NOR) structure

Answer 32

Contains ribosomal RNA (rRNA) genes 5.8S, 18S and 28S, which are organised on a 13kb transcription unit. A compound unit of 13kb transcription unit and an adjacent nontranscribed spacer is tandemly repeated about 50-60 times.

Question 33

Staining method for Nucleolar organizing region (NOR)

Answer 33

If the NOR is transcriptionally active it stains darkly with silver nitrate (Ag-NOR staining)

Question 34

Replication Origins (ORs)

Answer 34

• Cis-acting DNA sequences which bind proteins in preparation for DNA replication
• AT rich
• Recognised by a six protein complex = ORC (Origin of Replication Complex) ORC1-6