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PLNT3140 Introductory Cytogenetics
Lecture 5, part 1 of 3

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September 18, 2018

CYTOGENETIC TECHNIQUES (cond.)


REFERENCES:

Chapter 2, The Handling of Plant Chromosomes .pp. 13- 16 Singh.R.J. 1993. Plant Cytogenetics. CRC Press.

Cremer et al. (1993) Role of chromosome territories in the functional compartmentalization of the cell nucleus. Cold Spring Harbour Symposia on Quantitative Biology, Volume LVIII. Cold Spring Harbour Press. 777-792.

Lawrence, J.B. (1993) Probing functional organization within the nucleus: Is genome structure integrated with RNA metabolism? Cold Spring Harbour Symposia on Quantitative Biology, Volume LVIII. Cold Spring Harbour Press. 807- 818.

Trask, B.J., S. Allen, H.Massa, A Fertitta , R. Sachs, G.van den Engh and M.Wu. 1993 Studies of metaphase and interphase chromosomes using fluorescence in situ hybridization. Cold Spring Harbour Symposia on Quantitative Biology, Volume LVIII. Cold Spring Harbour Press.


KEY CONCEPTS FOR TODAY

1. Local variations in chromatin coiling, and the consequent variations in chromatin density, result in distinct staining patterns that can be used to identify each chromosome.'
2. C-bands represent constitutive heterochromatin. G-banding patterns on mitotic chromosomes correspond very closely to the chromomere patterns of meiotic chromosome bivalents at pachytene.

3. Polytene chromosomes exhibit easily discernable bands without staining.
4. Individual loci on chromosomes can be visualized by fluorescence in-situ hybridization (FISH).
5. FISH allows 'chromosome painting' using chromosome-specific probes.
6. FISH reveals that chromosomes are not randomly dispersed in the interphase nucleus, but rather, each chromosome occupies a defined 'territory'.


I. Chromosome banding

A. Local variations in chromatin coiling, and the consequent variations in chromatin density, result in distinct staining patterns that can be used to identify each chromosome.

The discovery of techniques which produce banding patterns on chromosomes is one of the most significant developments in cytogenetics. These techniques told us that chromosomes have an identity and structure that is consistent in all cells within a species, supporting the chromosome theory of inheritance. Staining made it possible to construct the first chromosome maps, and  to distinguish between chromosomes of many different species.

Fixed chromosomes are not equivalent to chromosomes in vivo. Fixation does not appear to extract DNA from chromosomes but it does extract histones and nonhistone proteins to varying degrees. The fixative may not always have a quantitative, reproducible effect on these proteins. The net result is that when you look at fixed chromosomes, you are looking at chromosomes that are chemically and structurally different from chromosomes in living cells.

Some relevant definitions:

B. Giemsa Staining Methods

Mitotic metaphase is the best stage for studying chromosome morphology. What G-banding attempts to do is to accentuate the differences in high-level chromatin structure along the length of the chromosome. In other words, some regions of the mitotic chromosome are very densely coiled (heterochromatin) and others are less densely coiled (euchromatin).

G-banding is produced using Giemsa staining and usually pretreatment with a diluted trypsin solution, urea or protease.

There are many variants of the G-banding protocol in animals. Even for human cells, there are many different protocols. Below is an overview of the basic steps of G-banding to give an idea of what the main steps are. The precise details vary from lab to lab, and may need to be optimized for the types of cells used.

Based on Zahama M (2009) Human Chromosomes: Identification by G-banding. Karyotyping
http://site.iugaza.edu.ps/mona/files/Exp._4_Karyotyping1.ppt
 
This procedure assumes that cells have already been spread on slides and have undergone pretreatment and fixation.

Brief trypsin treatment will hydrolyze peptide bonds in proteins on the surface of the protein, but leave the overall protein/DNA structure of chromatin intact. Brief staining with Giemsa will result in heterochromatin taking up and retaining more dye than euchromatic regions. The net result is that heterochromatic regions show up as dark bands, and euchromatic regions as light bands.

Key point: Each banding method has a different chemistry affecting different aspects of chromatin structure.
 

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PLNT3140 Introductory Cytogenetics
Lecture 5, part 1 of 3

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