Practical 1 Feedback 4th March 2013 Ms G Schutte

Unbanded metaphase 46 chromosomes Acrocentric G group – 5 Acrocentric D group – 6 Long A group – 6 Probably 46,XY

There is one crossover – this is nothing to do with recombination during meiosis.

Unbanded chromosomes are used for detecting chromosome breaks and fragile sites such as the fragile-X – see arrow. Also easier to detect very small marker chromosomes which may stain with a pale giemsa band.

Unbanded versus G-banded metaphase cells

• Centromeres very clear on unbanded • Chromatids clearly visible • Y not identifiable with certainty

•  Centromeres more subtle • Chromatids not visible • Y distinguishable using banding

Difficult to karyotype unbanded chromosomes

C-banding

o  note the dicentric chromosomes in a)

o Note the ring chromosomes in b)

Cytogenetics Essay Review

Tuesday 5th March 2013 Ms G Schutte

Essay Title   Research the function of a) centromeres and

b) telomeres. Write an essay explaining the function of these two chromosome areas. Do a literature search for “marker chromosomes”. Relate your knowledge found in a) and b) above, and your knowledge of chromosome aberrations occurring during meiosis, to your discussion of the pathology of marker chromosomes

Marking Criteria   Best results from those essays written in your own

words. Copy and Paste not good especially when you do not understand the principle.

  Diagrams good but you must explain them.

  Wiki not always right or relevant to your essay question – check Wiki with at least one other source.

Centromeres   What and where?

  Used in the microscopic identification of chromosomes in metaphase.

  Methods used to visualise centromeres.

  Spindle attachment during meiosis.

  Presence in marker chromosomes.

  Revision of the cell cycle.

  Chromatids and chromosomes.

  Normal variants.

Centromeres in Chromosome identification

  Metacentric, Submetacentric, Acrocentric

  (Telocentric, holocentric,- are not human)

  Made of heterochromatin containing alpha centromeric protein. Controls crossing over.

  Can be variable in length (with no consequences because there are no transcribed genes). Known as qh+ variants which are inherited polymorphisms

  Normal polymorphisms are commonly found in chromosomes 1,9, 16, andY

9qh+

qh+ variants

Chromosome 1

Staining techniques

• G-banding: • Harvest metaphase cells • “Age” with heat 100oC • Treat with enzyme – trypsin • Stain with giemsa

• C-banding: • Treat with Hydrochloric acid • Heat slides in Barium Hydroxide • Soak in 2 X SSC (Sodium Citrate)

• Stain in giemsa

Centromere staining techniques continued:

Q-banding is a fluorescent stain using quinacrine dihydrochloride.

  The bands resemble those seen in G-banding. It is quick and reliable without having to age the slide or pretreat. Good for visualising:

  Centromeres

  Satellites – found on acrocentric chromosomes

  The polymorphic region on the Y chromosome …ie quick method for sex determination in pre natal samples.

Centromere detection using FISH (fluorescent in situ hybridisation)

  Harvested cells pre-treated (chemically with SSC) to increase permeability of the cell nucleus.

  DNA denatured by heat into single strands

  Commercial probe labelled with fluorophores hybridised to sample DNA

  Slide visualised using a fluorescent microscope

Centromere detection using a pancentromeric FISH probe

Alpha satellite DNA binds to the centromeres (repetitive sequence DNA)

The centromere fluoresces with the colour of the commercial probe, while the chromosomes are stained with DAPI, a nucleic acid stain.

Chromosome variants involving the centromere (cont)

I.   Pericentric inversion

Does not affect centromere function. Does not interrupt gene function.

2. Paracentric inversion – does not involve the centromere

3. Dicentric – when a chromosome has more than one centromere – can be seen with Robertsonian translocations. This presents a problem for the spindle identification of the chromosome if the Robertsonian involves two different acrocentric chromosomes such as rob(14;21)

4. Neocentromere does not contain alpha satellite DNA and does not stain with c-banding – can form a functional kinetochore

Acrocentric Chromosomes Regions within the "stalks" of chromosomes 13,14,15,21, and 22 (the acrocentric chromosomes) contain genes for 18S and 28S rRNA. The silver nitrate stain used in this method binds to proteins near these regions during active transcription. The staining intensity varies from person to person and chromosome to chromosome. NOR Stain (Nucleolar organising region)

Cell Cycle and function of the Centromere

  G1 to S to G2 to Division including metaphase.

  Chromosomes replicate and the two resulting chromatids are joined at the centromere.

  Identification of chromosomes done so that one homologous copy of each chromosome goes to the correct side of the nuclear spindle

  Correct function avoids errors such as triploidy and monosomy

Metaphase: Mitosis and Meiosis

  Homologous pairs separated to opposite ends of the cell before cell division. Process hinges around the centromere proteins.

  Errors can result in trisomy, monosomy

  Multiple miscarriages can be caused by syndrome called Robert’s syndrome, also known as premature centromere division, growth retardation, symmetric limb deficiencies, absent thumbs, facial anomalies and borderline to mild mental retardation.

Premature Centromere Division

• Note how the centromere have allowed the chromatids to separate before being pulled by the spindle into the daughter cells. • Note unusual breaks where chromosome markers may result

Telomeres –at Last!   To understand the protective function of the

Telomere we must look at DNA replication

A telomere is a repeating DNA sequence (for example, TTAGGG) at the end of the body's chromosomes. The telomere can reach a length of 15,000 base pairs.

DNA replication   Because DNA replication does not begin at either

end of the DNA strand, but starts in the centre, and considering that all known DNA polymerases move in the 5' to 3' direction, one finds a leading and a lagging strand on the DNA molecule being replicated.

  However, each time a cell divides, some of the telomere is lost (usually 25-200 base pairs per division). When the telomere becomes too short, the chromosome reaches a "critical length" and can no longer replicate. (Hayflick Limit)

  Cellular aging, or senescence, is the process by which a cell becomes old and dies – called “apoptosis”

  If the cell does not die at this stage the there will be loss of DNA to areas containing functional genes. This can lead to the development of cancer.

  On the leading strand, DNA polymerase can make a complementary DNA strand without any difficulty because it goes from 5' to 3'. However, there is a problem going in the other direction on the lagging strand. To counter this, short sequences of RNA acting as primers attach to the lagging strand a short distance ahead of where the initiation site was. The DNA polymerase can start replication at that point and go to the end of the initiation site. This causes the formation of Okazaki fragments. More RNA primers attach further on the DNA strand and DNA polymerase comes along and continues to make a new DNA strand.

  Eventually, the last RNA primer attaches, and DNA polymerase, RNA nuclease, and DNA ligase come along to convert the RNA (of the primers) to DNA and to seal the gaps in between the Okazaki fragments. But, in order to change RNA to DNA, there must be another DNA strand in front of the RNA primer. This happens at all the sites of the lagging strand, but it does not happen at the end where the last RNA primer is attached. Ultimately, that RNA is destroyed by enzymes that degrade any RNA left on the DNA. Thus, a section of the telomere is lost during each cycle of replication at the 5' end of the lagging strand.

Telomerase   Active Telomerase is found in fetal tissues, adult

germ cells, and also tumor cells. Telomerase activity is regulated during development and has a very low, almost undetectable activity in somatic (body) cells. Because these somatic cells do not regularly use telomerase, they age. The result of aging cells is an aging body. If telomerase is activated in a cell, the cell will continue to grow and divide. This "immortal cell" theory is important in two areas of research: aging and cancer.

Immortal Cells for Scientific work   On the human front, at least one human being possessed immortal

cells -- and they were found in a tumor. In 1951, Henrietta Lacks went in for a routine biopsy in Baltimore, Md. While a portion of her tumour cells went to a lab for diagnosis, another was sent, without her authorization, to researchers at Johns Hopkins University Medical School [source: Highfield]. Lacks died of cervical cancer in 1951, but her cells live on in laboratories around the world. Called HeLa cells, they divide indefinitely. Before this discovery, cells used in laboratories always carried a shelf life linked to telomere shortening.

  Why were these immortal cells found in a fatal tumor? While telomerase production decreases almost entirely in healthy adult cells, it increases in cancerous cells. In fact, 90 percent of human tumours exhibit more telomerase activity. Remember, cancer is essentially uncontrolled cellular replication. As older cells are most likely to turn cancerous, telomere shrinkage may have actually evolved as a means to repress tumour growth [source: Biever].

Telomerase in tumour cells   Cancer cells are a type of malignant cell. The malignant cells

multiply until they form a tumor that grows uncontrollably. Telomerase has been detected in human cancer cells and is found to be 10-20 times more active than in normal body cells. This provides a selective growth advantage to many types of tumors. If telomerase activity was to be turned off, then telomeres in cancer cells would shorten, just like they do in normal body cells. This would prevent the cancer cells from dividing uncontrollably in their early stages of development. In the event that a tumor has already thoroughly developed, it may be removed and anti-telomerase therapy could be administered to prevent relapse. In essence, preventing telomerase from performing its function would change cancer cells from "immortal" to "mortal”.

Dolly the sheep and

her telomeres

• Dolly should have lived for about 12 years but at the age of six years she developed arthritis and a progressive lung disease. • Why?

Dolly had been made from a somatic cell that was six years old. Was she born old?

When she died her telomeres were found to be 80% percent shorter than other sheep of her own age.

Terminology   Chromosomes do not mutate – gene sequences do

  A marker may look like a small X chromosome but they can be derived from any chromosome.

  “peer review” – what does this mean?

  Note references with a key throughout your text

Marker Chromosomes   Small unidentified supernumery chromosomes

(SMC)

  Karyotype will be abnormal – the phenotype can be variable depending on the working gene content of the marker.

  True trisomies such as Edwards, Patau and Down syndrome cannot be called marker chromosomes

  Try to use recent references – genetics is a fast moving field

How does a marker chromosome form?

  Many theories:

  Trisomic rescue after non disjunction.

  Abnormal chromatid separation resulting in dicentric chromosomes – these may not be able to separate properly on the spindle and be broken in the process of cell division, forming a marker chromosome.

  Multiple marker chromosomes caused by radiation exposure

Dicentrics and rings will be able to replicate and will be found in the divided cell. The fragments will be lost.

Ring Chromosome (marker chromosome or derived chromosome?)

Ring chromosome with one clear telomere – visualised using FISH

Pallister Killian   “floppy baby” – hypotonia

  Developmental delay

  Learning difficulties

  Karyotype 47,XX,+marker

  Found as a mosaic mainly in skin cells (fibroblasts), ie not in lymphocytes

Pallister Killian 47,XX,i(12)(p10) Tetrasomy 12p

conclusion   Centromeres mainly involved in managing the

segregation of chromatids during metaphase.

  Errors such as non disjunction and mal segregation can give rise to many abnormal phenotypes.

  Telomeres act as a mechanism to prevent DNA damage to working genes during DNA replication where small triplets of DNA are lost on the lagging strand

  Marker chromosomes can result from errors in meiosis, exposure to teratogens, …unknown. Their effect is highly variable. When found during prenatal diagnosis it is difficult to know how the phenotype will be affected.