Download Esperimento di genetica 17.1

Robert J. Brooker - Genetica
Esperimento di genetica 17.1
The Staining of Harlequin Chromosomes
Can Reveal Recombination Between
Sister Chromatids
Our understanding of crossing over and homologous recombination
has come from a variety of experimental approaches, including genetic, biochemical, and cytological analyses. Chromosomal staining
methods have allowed researchers to visualize the genetic exchange
between eukaryotic chromosomes. In the 1970s, the Russian cytogeneticist A. F. Zakharov and colleagues developed methods that
improved our ability to identify chromosomes. They made the interesting observation that chromosomes labeled with the nucleotide
analog 5-bromodeoxyuridine (BrdU) bind certain types of stain to
a different degree compared to normal chromosomes. In 1974, Paul
Perry and Sheldon Wolff extended this approach to differentially
stain sister chromatids and microscopically identify SCEs.
Before we consider the experiment of Perry and Wolff, let’s examine how their staining procedure allowed them to accurately distinguish the two sister chromatids. In their approach, eukaryotic
cells were grown in a laboratory and exposed to BrdU for two
rounds of DNA replication. After the second round of DNA replication, one of the sister chromatids in each pair contained one unla-
beled strand and one BrdU-labeled strand. The other sister chromatid had two BrdU-labeled strands (Figure EG17.1.1). When
treated with two dyes, Hoechst 33258 and Giemsa, the sister chromatid containing two strands with BrdU stains very weakly and
appears light, whereas the sister chromatid with only one strand
containing BrdU stains much more strongly and appears very dark.
In this way, the two sister chromatids can be distinguished microscopically. Chromosomes stained in this way are referred to as harlequin chromosomes, because they are reminiscent of a harlequin
character’s costume with its variegated pattern of light and dark
patches. In these chromosomes, SCEs can be clearly identified as
exchanges between light and dark chromatids.
The steps in Perry and Wolff ’s protocol are shown in Figure
EG17.1.2. They began with Chinese hamster ovary cells, a commonly used mammalian cell line, and exposed the cells to BrdU for
two rounds of DNA replication. Near the end of the second round,
colcemid was added to prevent the completion of mitosis. The cells
were treated with KCl to spread out the chromosomes, which were
subsequently fixed and then stained with Hoechst 33258 and
Giemsa.
THE HYPOTHESIS
Crossing over may occur between sister chromatids.
FI GU RE EG1 7 .1.1 H arle quin c hrom osom e s.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
Starting material: A laboratory cell line of Chinese hamster ovary (CHO) cells.
FI GU RE EG1 7 .1.2 T he st a ining of ha rle quin c hrom osom e s re ve a ls sist e r c hrom a t id e xc ha nge .
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia
Robert J. Brooker - Genetica
THE DATA
INTERPRETING THE DATA
Reprinted by permission from Macmillan Publishers Ltd. Nature, New Giemsa
method for the differential staining of sister chromatids. Perry P. & Wolff S.
251:5471, 156–158, 1974.
A micrograph of their results is shown in the data of Figure
EG17.1.2. As seen here, the chromosomes show the classic harlequin appearance due to the differential staining of the sister chromatids. Furthermore, examples of SCE are clearly visible. The arrows depict regions where crossing over has taken place. In this
study, Perry and Wolff found that SCEs occurred at a frequency of
approximately 0,67 per chromosome. This method has provided an
accurate (and dramatic) way to visualize genetic exchange between
sister chromatids.
Many subsequent studies have used the harlequin staining
method to study the effects of agents that may influence the frequency of genetic exchanges. Researchers have found that DNA
damage caused by radiation and chemical mutagens tends to increase the level of genetic exchange. When cells are exposed to
these types of mutagens, the technique of harlequin staining has
revealed a substantial increase in the frequency of SCEs. In addition, certain genetic disorders that result in higher levels of chromosome breakage also show elevated SCEs. For example, Bloom syndrome is a rare autosomal recessive disorder characterized by short
stature, skin abnormalities, and a predisposition for developing certain forms of cancer. The defect is associated with a gene that is
involved with DNA replication. In the cells of Bloom syndrome
patients, chromosome breaks are more frequent during DNA replication. Likewise, SCE is typically 10- to 15-fold more frequent in
Bloom syndrome patients compared to unaffected individuals.
© 2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia