BIMM 110     LECTURE 7

HUMAN METAPHASE CHROMOSOMES.
CYTOGENETICS
SLIDES

Textbook: Strachan and Read, Chapter 2 & Chapter 14.4
Pasternak: chapter 2

The Cell Cycle - Review

Inter phase (G1, S, G2) and mitosis
mitotic inhibitors: colcemid, nocodazole

metaphase spreads

mitotic chromosomes and sister chromatid exchange

THE CELL CYCLE AND MITOSIS (Review!)

TEXTBOOK: Strachan and Reed, Chapter 2    
this material is considered largely review, and coverage is fleeting;
the basic terminology and ideas should be familiar to all (from previous lectures in the Molecular Biology Course)

1. concepts and terms:

    - phases of the cell cycle: G1, S, G2, M
    - mitogens, inhibitors, and cell synchrony
    - events in S-phase; replicons; early and late replicating DNA (to be covered in connection with X inactivation
    - major events in mitosis;
             chromosome condensation (in prophase) and metaphase chromosomes;
             separation of sister chromatids beginning with anaphase
    - cytokinesis (to be discussed in Cell Biology)

Material in this chapter is central to the course, and close attention should be paid to the information in this chapter (it also covers material of later lectures)

Chromosomes
Technology I [commercial site: http://www.aicorp.com/ ; advertising reagents and equipment; good examples] 

A. The normal human karyotype

1. Overview

- history and early methodology (1920s to 1970)
- light microscopy and electron microscopy
- chromosome morphology; primary and secondary constrictions; sister chromatids
    centromeres (and telomeres); kinetochores; metacentric, acrocentric and telocentric chromosomes

- autosomes (homologues) and sex chromosomes;
- modern cytogenetics: G-bands, Q-bands, R-bands, C-bands (1970 - )
    chromosome arms, regions, bands; nomenclature (International)

2. Examples

a) a metaphase spread of human chromosomes(ignore the red dots for now):

b) a computer-assisted assembly of a karyotype from a female (46, XX)
[http://www.metasystems.de/index_noIE.htm]

c) metaphase and prometaphase G-banded human chromosome 1 and the standard nomenclature for labeling the bands;
short arm: p (petite); long arm: q;    
1 - 4 regions for each arm labeled from centromere towards telomere
each region has several bands, again numbered away from the centromere

d) the latest: chromosome painting with specific probes (1990s) (from MetaSystems)
     this methodology is based on in situ hybridization of probes to metaphase chromosomes; different probes can be conjugated to different fluorescent chromophores;
     whole chromosomes, individual bands of a specific chromosome, or even single genes can be detected (see FISH and gene mapping in a later lecture)

see also

RESOURCES FOR MOLECULAR CYTOGENETICS

3. Other cytological techniques and application

- silver staining and nucleolar organizer regions (NORs)
- staining polymorphisms as cytological markers
- fragile sites (more later in discussion of fragile X syndrome)
- BUdR labeling; Hoechst 33258 stain;
        measurement of sister chromatid exchange (SCE)
- mitotic chromosomes of male gametes (techniques; formation of "humster" cells)

4. Evolution of chromosomes of higher primates

- conservation of regional banding patterns
 

B. Chromosomal abnormalities

1. numerical abnormalities
    aneuploidy (deviations from 46 chromosomes)
    diploids and triploids (not viable)
    trisomy (eg. Down syndrome, 47, XY, +21) and monosomy (eg. Turner syndrome, 45, X)

2. structural rearrangements of chromosomes
    a) terminal and interstitial deletions
        examples:    46, XY, del(18) (pter -> q22:)
                           46, XX, del(1) (pter -> q21::q31 -> qter)
                           46, XY del(X) (p21); Duchenne muscular dystrophy
    b) pericentric and paracentric inversions
          example:    46, XX, inv(5) (pter -> p12::q23 -> p12::q23 -> qter)
    c) balanced (reciprocal) and unbalanced translocations
          example:    46, XY, t(8;14) (8pter -> 8q24::14q32 -> 14qter; 14pter -> 14q32::8q24 ->
                               8qter); Burkitt lymphoma
                        short version of the above:  46, XY, t(8;14) (q24; q32)

d) Robertsonian translocations (two telocentric chromosomes fused at centromeres)
e) Duplications
f) Isochromosomes (chromosomes with two identical arms)
g) Ring chromosomes

• Each human chromosome, and even fragments of human chromosomes, can now be unambiguously identified by cytogenetic techniques. Cells for study can be easily obtained by routine and harmless procedures. The level of resolution of such techniques is of the order of a few million basepairs.
• Some of the detectable abnormalities can be related to specific genetic diseases; others can be correlated with the expression of a phenotype. Structural abnormalities (breakpoints, deletions) have frequently been very helpful in mapping and eventually cloning a relevant gene.
• The techniques are enormously helpful in genetic screening and counselling, and in prenatal diagnoses.

3. Specific examples of chromosomal abnormalities and associated medical problems will be discussed inthe following lectures

4. Other Web Sites with useful cytogenetics resources

RESOURCES FOR MOLECULAR CYTOGENETICS
 

- Bickmore, W.A., and Sumner, A.T. (1989). Mammalian chromosome banding-An expression of genome organization. TIG 5, 144-148.
- Caspersson, T.O. (1989). The William Allan Memorial Award address: The background for the development of the chromosome banding techniques. Am. J. Hum. Genet. 44, 441-451.
- Korenberg, J.R., and Rykowski, M.C. (1988). Human genome organization: Alu, lines, and the molecular structure of metaphase chromosome bands. Cell 53, 391-400.
- Lichter, P., Ledbetter, S.A., Ledbetter, D.H., and Ward, D.C. (1990). Fluorescence in situ hybridization with Alu and L1 polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid cell lines. PNAS 87, 6634-6638.
- Therman, E., Susman, M. (1993).  Human chromosomes: Structure, Behavior, Effects. (Third Edition; New York: Springer Verlag).
- Weier, H.-U.G., Lucas, J.N., Poggensee, M., Segraves, R., Pinkel, D., and Gray, J.W. (1991). Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations. Chromosoma 100, 371-376.
- Speicher, M.R., Ballard, S.G., and D.C. Ward (1996).  Karyotyping human chromosomes by combinatorial multi-color FISH. Nature Genet. 12: 368-375
- Henegariiu, O., Heerema, N.A., Bray-Ward, P., and D.C. Ward (1999) Color-changing karyotyping: an alternative to M-FISH/SKY. Nature Genet. 23: 263-264.
- Ried, T., Schroek, E., Ning, Y., and J. Wienberg (1998). Chromosome painting: a useful art. Hum. Mol. Genet. 7: 1619-1626.