Chromosome Structure and Organisation in Humans

Question 1

Give a general definition and description of a chromosome.

Answer 1

• Chromosomes are structures consisting of chromatin fibre (DNA and protein complex) folded and complexed into a compact rearrangement. - Chromosome are constricted at a point called the centromere. This divides the chromosome into two arms, the p arm (short), and the q arm (long). - Chromosomes are only visible during cell division when the DNA becomes tightly packed.

Question 2

What two elements make up a chromatin fibre?

Answer 2

• Chromatin fibre = DNA + Protein

Question 3

How are Eukaryotic chromosomes classified?

Answer 3

• Eukaryotic chromosomes are classified into three major types based on the position of the centromere and further classified into groups according to size.

Question 4

Chromosomes can be classified into three different categories based upon the location of their centromere - what are the names of these three classifications?

Answer 4

1. Metacentric - p and q arms are roughly equal in length 2. Submetacentric - the p and q arms of unequal length 3. Acrocentric - the p arm is very short but still present

Question 5

List the acrocentric chromosomes.

Answer 5

chromosomes 13, 14, 15, 21, and 22.

Question 6

Outline the chromosome groupings by letter (A-G).

Answer 6

• Group A (Largest) - Chromosomes 1-3 - 1 and 3 are metacentric but 2 is submetacentric. - Group B (Large) - Chromosomes 4 and 5 - submetacentric chromosomes with two arms very different in size. - Group C (Medium size) - Chromosomes 6 to 12 and the X chromosome - Submetacentric chromosomes. - Group D (Medium size) - Chromosomes 13 and 14 - Acrocentric chromosomes with satellites. - Group E (Small) - Chromosome 16 is metacentric but chromosomes 17 and 18 are submetacentric. - Group F (Small) - Chromosomes 19 and 20 - Metacentric. - Group G (Small) - Chromosomes 21, 22, and Y - Acrocentric chromsomes. Chromosomes 21 and 22 have satellites whereas the Y chromosome does not.

Question 7

In what order of size are autosomes numbered?

Answer 7

• Autosomes are numbered from largest to smallest, except that chromosome 21 is smaller than chromosome 22.

Question 8

What is the centromere of a chromosome and what is its basic function?

Answer 8

• The centromere is a region of highly specialised chromatin that provides the foundation for kinetochore assembly and serves as a site for sister chromatid attachment. - It is easily visualised as the most constricted region of a condensed mitotic chromosome.

Question 9

What is the function of the centromere?

Answer 9

• The centromere is essential for accurate chromosome segregation durning cell division and also provides the foundation for the assembly of the kinetochore. Microtubules of the spindle attach to the centromere via the kinetochore. - For accurate mitoses, sister chromatids must remain attached until the spindle checkpoint has been passed. The attachment of sister chromatids is mediated by the cohesin complex of proteins. As the cell progresses into anaphase the cohesion is degraded allowing the sister chromatids to be separated to opposite poles of the spindle. - Chromatid separation occurs in mitosis and meiosis II. The centromere remains intact with attached sister chromatids in the 1st meiotic division. - Defects in any of the pathways that regulate centromere assembly and function can lead to chromosome missegregation, aneuploidy, and chromosomal instability. Acentric fragments fail to attach to the mitotic spindle, segregate randomly during mitosis and are eventually lost from cells. Dicentric chromosomes are subject to fragmentation if the centromeres become attached to opposite spindle poles by way of their kinetochores.

Question 10

Describe centromere structure. (Your answer should mention basic structure, and touch on any sub-domains, and associated proteins).

Answer 10

• The centromeres of chromosomes are composed of constitutive heterochromatin that is made up of various families of repetitive satellite DNA. - Human centromere are built on a series of head-to-tail tandem repeats of 171 bp AT rich DNA named alpha satellites, that extend several Mb and make up 3% of the genome. - There are 3 centromeric DNA sub-domains (CDEI, II, and III). Mutations in the first two have no effect upon segregation, but those in CDEIII disrupt chromosome function completely. - There are approximately 20 known proteins associated with human centromeres (known as CENPs - centromeric proteins), which play a variety of roles. CENPs can be classified into two groups depending upon their spatial positioning throughout the cycle. The first class of CENPs comprise proteins that are constitutively associated with the centromere such as CENPA, CENPB, and CENPC, which are thought to have structural roles in kinetochore formation. The second class of CENPs, known as passenger proteins, associate with the centromere transiently during the cell cycle. This class comprises proteins proteins with diverse roles in cell division such as spindle capture, metaphase to anaphase transition, and sister chromatid cohesion. - Centromeric DNA sequence is not sufficient to maintain centromere position. Centromeres are epigenetically identified. The histone H3 variant CENP-A has been demonstrated to be the epigenetic mark for centromere identity and function. CENP-A forms a unique complex with other histones (H2A, H2B, and H4). CENP-A marks active centromeres independently from DNA sequence and mediates centromere assembly through tightly regulated complex processes.

Question 11

How many centromeric DNA sub-domains are there? What is the effect of mutations in each of these sub domains?

Answer 11

There are 3 centromeric DNA sub-domains (CDEI, II, and III). Mutations in the first two have no effect upon segregation, but those in CDEIII disrupt chromosome function completely.

Question 12

What are the two main groups of proteins associated with human centromeres?

Answer 12

• There are approximately 20 known proteins associated with human centromeres (known as CENPs - centromeric proteins), which play a variety of roles. CENPs can be classified into two groups depending upon their spatial positioning throughout the cycle. 1). The first class of CENPs comprise 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 of CENPs, known as passenger proteins, associate with the centromere transiently during the cell cycle. This class comprises proteins proteins with diverse roles in cell division such as spindle capture, metaphase to anaphase transition, and sister chromatid cohesion.

Question 13

What is the function of the protein CENP-A?

Answer 13

• Centromeric DNA sequence is not sufficient to maintain centromere position. - Centromeres are epigenetically identified. - The histone H3 variant CENP-A has been demonstrated to be the epigenetic mark for centromere identity and function. - CENP-A forms a unique complex with other histones (H2A, H2B, and H4). - CENP-A marks active centromeres independently from DNA sequence and mediates centromere assembly through tightly regulated complex processes.

Question 14

Name 3 disorders that are associated with centromere dysfunction.

Answer 14

1). Premature Centromere Division (PCD) - age-dependant phenomenon occurring in women, characterised by rod-shaped X chromosome(s) without discernible centromeres, possible cause of age-dependent increase of X chromosome aneuploidy. 2). Premature Chromatid Separation (PCS) - consists of separate and splayed chromatids with discernible centromeres and involves all or most chromosomes of a metaphase. It is found in up to 2% of metaphases in cultured lymphocytes from approximately 40% of normal individuals. When PCS is present in 5% or more of cells, it is known as the ‘heterozygous PCS trait’ and has no obvious phenotypic effect, although some have reported decreased fertility and possible increase of aneuploidy in offspring. 3). Roberts syndrome - a chromosomal breakage syndrome (an example of a disease associated with centromere malfunction). Autosomal recessive. The gene ESCO2 (8p21.1), 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. Metaphase spreads show characteristic (pathognomonic) premature centromere separation, and puffing of centromeres on 1, 9, and 16. Phenotype includes pre/post growth retardation, limb malformation (reduction), craniofacial (microcephally, clefting), intellectual disability, and renal and cardiac abnormalities.

Question 15

What is Premature Centromere Division (PCD)?

Answer 15

• Premature Centromere Division (PCD) is a disease associated with centromere dysfunction. It is an age related phenomenon that occurs in women. PCD is characterised by rod-shaped X chromosome(s) without discernibly centromeres and is a possible cause of age-dependent increase in chromosome X aneuploidy.

Question 16

What is Premature Chromatid Separation (PCS)?

Answer 16

• Premature Chromatid Separation (PCS) consists of separate and splayed chromatids with discernible centromeres and involves all or most chromosomes of a metaphase. It is found in up to 2% of metaphases in cultured lymphocytes from approximately 40% of normal individuals. When PCS is present in 5% or more of cells, it is known as the ‘heterozygous PCS trait’ and has no obvious phenotypic effect, although some have reported decreased fertility and possible increase of aneuploidy in offspring.

Question 17

Describe Roberts Syndrome.

Answer 17

• Roberts syndrome is a chromosome breakage syndrome. This is an example of a disease associated with centromere malfunction. The disorder follows an autosomal recessive inheritance pattern. - The gene ESCO2 (8p21.1), 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. - ESCO2 mutation results in delayed cell division and increased cell death. - Metaphase spreads show characteristic (pathognomonic) premature centromere separation, and puffing of centromeres on 1, 9 and 16. - Phenotype includes pre/post growth retardation, Limb malformations (reduction), craniofacial (microcephally, clefting), intellectual disability and renal and cardiac abnormalities.

Question 18

What is the kinetochore and what is its function?

Answer 18

• The kinetochore is a large multi-protein (>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. - A pair of kinetochores is formed at each centromere during late prophase of mitosis. - A kinetochore is positioned on the side each sister chromatid, facing the spindle pole to which the chromosome will be drawn during anaphase. Multiple microtubules appear to insert into the kinetochore. - Subcomplexes of proteins within the kinetochore are required for the kinetochore’s numerous functions. Many of these functions involve microtubules (e.g., microtubule attachment, polymerisation, and motor-directed movements). Other kinetochore proteins are part of the spindle checkpoint that allows only cells with correctly assembled spindles to pass into anaphase. All of the kinetochore, except for the inner layer, disassembles at the end of mitosis.

Question 19

What is Mosaic Variant Aneuploidy (MVA)?

Answer 19

• Mosaic Variant Aneuploidy (MVA) is a rare autosomal recessive syndrome associated with various trisomies and monosomies involving different chromosomes and tissues. - Mosaic Variant Aneuploidy causes severe IUGR, microcephaly, dysmorphysm, and increased risk of malignancy such as rhabdomyosarcoma, Wilms tumour, and leukemia. - MVA can be caused by mutations in BubR1, a gene involved in the mitotic spindle checkpoint.

Question 20

What is a neocentromere?

Answer 20

• A neocentromere is a new centromere that forms on a chromosome at a location that is normally not centromeric, usually as a result of disruption of the natural centromere. - Can spontaneously form on acentric chromosome fragment preventing them being lost during cell division. - Whereas most natural centromeres contain highly repetitive sequences, neocentromeres usually possess unique sequences - this supports the idea that centromeres are specified by sequence-independent epigenetic mechanisms in most organisms. - There are two forms of neocentromeric chromosomal rearrangements: 1). Class 1 marker chromosomes (inverted duplication of distal part of a chromosome) are the most common. 2). Class 2 marker chromosomes (from an interstitial deletion. - There is a similar organisation of centromeric chromatin in neocentromeres to that of normal centromeres. They form a primary constriction and assemble a functional kinetochore, but lack repetitive α satellite DNA sequences and are C-band negative. Neocentromeres also lack CENP-B protein.

Question 21

How does the organisation of neocentromeres differ to that of normal centromeres?

Answer 21

There is a similar organisation of centromeric chromatin in neocentromeres to that of normal centromeres. They form a primary constriction and assemble a functional kinetochore, but lack repetitive α satellite DNA sequences and are C-band negative. Neocentromeres also lack CENP-B protein.

Question 22

What are the two forms of neocentromeric chromosomal rearrangements?

Answer 22

There are two forms of neocentromeric chromosomal rearrangements: 1). Class 1 marker chromosomes (inverted duplication of distal part of a chromosome) are the most common. 2). Class 2 marker chromosomes (from an interstitial deletion.

Question 23

What are telomeres?

Answer 23

Telomeres are highly conserved gene-poor, DNA-protein complexes that cap the ends of eukaryotic chromosomes and are required to maintain the normal structure and function of chromosomes.

Question 24

What function do telomeres serve?

Answer 24

• Telomeres 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. - Telomeres prevent shortening of the chromosomes at each round of cell division. If telomeres were to shorten progressively, this would result in cell death. - Telomeres are important for chromosome positioning as they help to establish the 3-D architecture of the nucleus and aid chromosome pairing.

Question 25

Describe the structure of telomeres?

Answer 25

- Each human telomere consists of 3kb to 20kb of tandem TTAGGG repeats (well conserved during evolution), which are associated with a variety of telomere-binding proteins. - Located immediately adjacent to the TTAGGG repeats are the 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. - Proximal to the telomere associated repeats lies unique chromosome-specific DNA. The most distal region of unique DNA on a chromosome arm, distinct from the repetitive DNA sequences, is commonly referred to as the subtelomere.

Question 26

What is the 'T-loop' on the chromosome end?

Answer 26

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

Question 27

Outline the main problem created by only being able to synthesise linear DNA in the 5' to 3' direction.

Answer 27

- 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.

Question 28

During the synthesis of linear DNA DNA polymerase absolutely requires a free 3’-OH group to extend synthesis - how is this achieved for the DNA fragments used to make the lagging strand?

Answer 28

- DNA polymerase absolutely requires a free 3’-OH group to extend synthesis and this is achieved by employing an RNA polymerase to synthesise a complementary RNA primer to prime synthesis of each DNA fragment used to make the lagging strand.

Question 29

Explain the mechanisms that result in the incomplete synthesis of the lagging strand an a 50-100bp shortening of the telomere per replication? How is this overcome?

Answer 29

- 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 are 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. - DNA polymerase absolutely requires a free 3’-OH group to extend synthesis and this is achieved by employing an RNA polymerase to synthesise a complementary RNA primer to prime synthesis of each DNA fragment used to make 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. - This problem is solved by extending the synthesis of the leading strand using a specialised form of reverse transcriptase (RNA-dependent DNA polymerase) provided by a specialised RNA-protein enzyme called telomerase.

Question 30

Describe the 2 subunits that make up Telomerase.

Answer 30

Telomerase includes 2 subunits: 1) . TERT (protein subunit) 2) . TERC (RNA subunit) - this subunit consists of an 11nt 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. In humans telomere length is known to be highly variable and telomere activity is absent in adult cells with the exception of highly proliferative cells (for e.g. germ cells). Cells that lack telomerase shorten progressively. This shortening can be used as a way of counting cell divisions and has been related to cell senescence and aging. Cancer cells find a way of activating telomerase, leading to uncontrolled replication.

Question 31

Give 3 examples of diseases associated with telomere malfunction.

Answer 31

**1). Dyskeratosis congenital (DC)** 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. Phenotype has similarities to premature ageing and is characterized by abnormal skin pigmentation, nail dystrophy, and leukoplakia of the oral mucosa. **2). Cri du Chat syndrome (CdCS)** 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. **3). Anaplastic anaemia** 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 telomerase.

Question 32

What is the function of the Nuclear Organising Region (NOR)?

Answer 32

- The nucleolus is the most prominent structure in a cell nucleus. It is the site of ribosomal RNA (rRNA) transcription, pre-rRNA processing and ribosome subunit assembly. - The nucleolar organising regions (NOR) are responsible for organising the nucleolus structure and contain the approx. 200 rRNA genes necessary for protein synthesis. - Human NORs are positioned on the short arms of the acrocentric chromosomes. - NOR contain 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 non-transcribed spacer is tandemly repeated about 50-60 times. - If the NOR is transcriptionally active it stains darkly with silver nitrate (Ag-NOR staining), although intensity can vary.

Question 33

Where are human NORs positioned in the genome (in terms of chromosomes)?

Answer 33

- Human NORs are positioned on the short arms of the acrocentric chromosomes.

Question 34

What are Replication Origins (OR)?

Answer 34

- Cis-acting DNA sequences which bind proteins in preparation for DNA replication. - Multiple sites along each chromosome, appear to be AT rich. - Recognised by a six protein complex = ORC (**_O_**rigin of **_R_**eplication **_C_**omplex) ORC1-6. - In higher eukaryotes, large chromosomal domains replicate at characteristic times during S phase. The density, distribution, and activation of replication origins define this temporal DNA replication program.