Continuing in our cytogenetics part two series, here we're going to talk about
chromosome analysis and karyotypes. So, this is an example of a chromosome
spread from a human female 2N = 46, XX and the arrow points to the X
chromosomes. There 4C DNA content here is 14 picograms. So, this individual, we would
say 2N = 46, XX, because she's a female and the DNA content 4C =
14 picograms of DNA. So, this is a chromosome spread at metaphase. So, how is
this done? You usually collect living cells. They're usually cultured from
blood. Culture the white blood cells for 48 to 72 hours. You disrupt the
spindle, usually with a drug like Colcemid. You swell the cells hypotonicly.
You rupture the cells and spread the chromosomes on a slide. You can actually
stain the chromosomes, photograph them, cut out the images, and arrange the
chromosomes by size. And when you do that, you can arrange the chromosomes into
distinct groups. So, we talked about the A group, which is chromosomes 1, 2, & 3. The B
group, chromosome 4 & 5. The C group, you can see the chromosomes in the C group,
which also includes the X chromosome. The D group, the E group, the F group and the
G group. You'll note that the chromosomes are arranged by size and by position of
the centromere. The D group chromosomes 13, 14, and 15. And the G group chromosomes
21 and 2 happened to be the chromosomes that in humans contain the nuclear or
organizer regions, and we'll talk more about those later. So, as we said, for a
karyotype you display the chromosomes by length and you display the chromosomes
by the position of the centromere. And we can talk about metacentric chromosomes,
sub-metacentric chromosomes, acrocentric chromosomes, and telocentric chromosomes.
Metacentric chromosomes, the centromere tends to be in the middle and the arm
lengths are about the same length. Sub-metacentric chromosomes, one arm is
clearly longer than another arm, so the centromere is not in the middle. Acrocentric,
the centromere is near the end. So, you've got a really small arm and
then a long arm. Telocentric chromosomes, which don't exist in humans but do exist
in, for example, rodents. The centromere is right at the end of the chromosome so
there's only one arm. But again, this is not something to worry about in humans.
So, here's a metacentric chromosome with a long arm and a short arm. It's a little
bit odd to call a long arm and a short arm with a metacentric chromosome. And a
metacentric chromosome, we usually think of the arm lengths as being equal, but by
tradition, one talks about a short arm, that is the P arm, so called petite arm,
and the long arm, the Q arm. By convention, we represent the chromosomes with the
short arm or the P arm on the top. Here's a sub-metacentric chromosome, clearly a
short arm and a long arm. And in acrocentric chromosome with a much shorter
arm and a longer arm. Now, oftentimes you'll see a chromosomal locus. We, for
example, say 18 Q 1.12. That's the locus for the genes that cause colorectal
cancer. We may say the p53 tumor suppressant gene is on the short arm of
chromosome 17, we would designate that as 17 P 13.1. I'm not going to test you on
these numbers right now, it's just to give you an example of how we would
designate loci on chromosomes. Now, let's get back to these acrocentric
chromosomes. What's the significance of the satellite region on the acrocentric
chromosomes. These satellites contain the ribosomal genes the so-called nuclear
organizer regions. Chromosome banding can be
useful to ID individual chromosomes and to find structural abnormalities on
chromosomes, and we can use normal banding and high-resolution chromosome
banding and some common chromosome banding techniques. These include Q bands, G
bands, reverse bands, C bands and NOR bands. So Q bands are just staining the
chromosomes with a stain called Quinacrine mustard. G bands uses Giemsa
staining. For reverse bands is just the negative image of either Q or G bands. C
bands is a stain specifically for the centromere regions of chromosomes. And
the NOR banding is a staining specifically for the nuclear
organizer regions on chromosomes. Here is a karyotype. Here is an ideogram. An
ideogram is a schematic representation of the chromosomes and it combines data
from various banding techniques. Go back here and you see in the karyotype you
see both pairs of the homologous chromosomes. In the ideogram, more than often times,
you just representing one of the chromosomes but these would be clearly
chromosomes that have replicated their DNA and they're metaphase chromosomes as
they're prepared. The red arrows on chromosomes 13, 14, 15, 21, and 22 are
highlighting the nuclear organizer regions. These are ideograms prepared
from G and our abandoned karyotypes. And they're just some to give you some
examples, there's nothing specific about the fact that I pick these. I just picked
them to show you how chromosome banding is designated. Here is a much more
complicated slide and it's just showing, for example, chromosome number one and
it's from the National Center of Bioinformatics from the National
Institute of Health. And basically, chromosome banding for each human
chromosome, it's very very complex and you can go to this database and you can
find what genes are located on each chromosome band. This is not something
you have to do for our course but it just gives you a
sense of how chromosome banding is used to identify individual human chromosomes
and where specific genes are located on each region of human chromosomes. You
were studying a case, in one of your other courses here, that was looking at
karyotypes and ideograms from an individual. Remember, the Misty White case.
The fetal karyotype for Misty's child in question was 46 XY with a derivative
chromosome 18 showing a translocation between chromosome 1 and 18 here showing
the position of the translocation. Here would be the derivative chromosomes, as a
derivative chromosome, derivative chromosome. The fetus received a normal
chromosome 1 from the father and a normal chromosome 1 from the mother,
and a normal chromosome 18 from the father and a derivative chromosome 18
from the mother. And this is the mother's ideograms in relation to these two
chromosomes. The fetus is a partial trisomy for chromosome number one and a
partial monosomi for chromosome number 18. We'll talk more about this later in
the course. What I'd like to do is talk about fluorescence in situ hybridization
here because you can use fluorescence in situ hybridization, so call FISH analysis
to monitor chromosome rearrangements. You can pick up deletions, additions, or
duplications, translocations. And basically, FISH uses label probes that
hybridize to interphase nuclei or two chromosomes at prometaphase and
metaphase. And so, here's a FISH analysis to show an individual that's got a
trisomy for chromosome 21. So, the probe was for chromosome 21 and here you can
see three fluorescent dots and an interphase nucleus. Here's a fluorescence
in situ hybridization for telomere sequences, as you can see the fluorescent
dots at the ends of the chromosomes. Here's a FISH analysis for the dystrophin
genes, they're located on the short arm of the x-chromosome. Here's a
fluorescence in situ hybridisation with two probes to monitor deletion of the
elastin gene from one copy of chromosome 7. This would be characteristic of
Williams syndrome. Here's an example highlighting chromosome 7 here and the
other probe here chromosome 7. Here is the probe to the elastin gene. It's on
this chromosome 7 but missing from this chromosome 7. That happened to be more
than 25 different genes from the long arm of chromosome 7 that can be deleted
in individuals with Williams syndrome. Here I just show the chromosomal locus,
I'm not going to ask you to remember it for an exam, but the chromosomal locus for
the William gene deletion here would be at chromosome 7, the long arm at band
11.23. Back to this case we talked about, the Misty White case. Here they were
doing pre-implantation genetic diagnosis of embryos. So, the green probe is to the
telomeres on the short arm of chromosome 18, so you can see the green probe here,
the red probe is the telomeres in the long arm of chromosome 18, so you can see
the red probe here. These are blastomere biopsies of a 118 balanced
translocation. There's an even number of probes. Here's blastema biopsies from a 118
unbalanced translocation. There's an odd number of probe color. Here you see
three green and one red. Here a true red one green. We'll talk about the
significance of balanced and unbalanced chromosome translocations in a later
podcast. This will help you to understand the significant
of this case that you've looked at in another one of your courses.