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PLNT3140 Introductory Cytogenetics
Lecture 5, part 1 of 3

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September 19, 2017

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. Giemsa stain is specific for constitutive heterochromatin adjacent to centromeres and telomeres.

1. Geimsa C-banding technique has been used to identify individual chromosomes in many species by showing the position of constitutive heterochromatin.

Somatic mitotic metaphase chromosomes of from a human female. Displayed by hypertext link to
University of Washington Cytogenetics Gallery http://www.pathology.washington.edu/galleries/Cytogallery/

 

Geimsa staining methods were first described in a paper by Purdue and Gall (1970).  However, it is important to recognize that staining methods typically need to be fine-tuned for each species, to give the optimal number of bands. We will concentrate on the general treatments and the basis for their application in terms of what each treatment is doing to produce the banding pattern.

Steps of C-banding:

Other Geimsa banding techniques include G banding which provides more detail than C-banding. G banding provides identification of almost every chromosome in a complement and structural variation can be detected.

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

The Human Chromosome Study Group has published a diagrammatic representation of chromosome bands observed with G-,Q-, and R-staining methods. The nature of these bands appears to be stronger chromosome condensation, but the bands could also be interpreted as the result of alteration of histones and other proteins of the chromosomes. G bands cannot be produced in plant chromosomes although the underlying reason is not clear. N-banding was originally developed to stain the nucleolar organiser region of the chromosomes, plant and mammals.

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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