Chromosome Structure

In a post back in September, I quickly summarized the abnormalities that can occur with
chromosomes as a whole (such as deletions, insertions, transversions, etc). There is so much
more to learn (more than I could possibly put into one blog post), because the way
chromosomes behave, depends on their structure and DNA sequence. For instance, genes with
the same DNA sequence will behave differently depending on where they are located on a
chromosome as well as the effect of the surrounding DNA sequence.

So how exactly is the immense length of DNA compacted into a chromosome? Let’s take
a DNA sequence and see just how it makes up a chromosome. A single molecule of DNA spools
around histone protein cores forming bead like structures called nucleosomes. Between each
nucleosome is a sequence of DNA termed “linker DNA.” The amino acids associated with
histones are lysine and arginine. The super coiled form is compacted and can be visualized as a
karyotype in laboratory testing.

The centromere is the connection point of the duplicated chromosome, while telomeres
are the endpoints. The short arm of the chromosome is termed “p” and the long arm of the
chromosome is termed “q.” If we take these two chromosome arms into consideration, there
are three types of chromosome morphology:

1. Metacentric – Chromosome arms are equal in length

2.  Sub-centric – One arm is longer than the other
3.  Acro-centric / Telocentric – One arm is extremely small or even missing
Chromosomal Staining Methods

As I mentioned above the complete set of chromosomes for an individual can be visualized via a
karyotype. I’ve listed a few of the ways this can be accomplished:

1. G-Banding – Chromosomes are stained with giesma stains. The appearance differs based
on the treatment of chromosomes prior to staining.
2.  Q-Banding – Chromosomes are stained with fluorescent dyes, quinacrine or quinacrine
mustard. Q-Band staining is similar to G-banding in that the fluorescent regions represent
the AT-rich regions of the chromosome.
3.  R-Banding – Results from heat treatment in a phosphate buffer followed by staining with
Giesma dyes.
4. C-Banding – Centromere staining that results from alkali treatment.

TYPE OF BANDING STAINING SUMMARY

·     Geisma stain

G-Banding
·     AT-rich regions stain darker than GC-rich regions

Q-Banding ·     Quinacrine fluorescent dye stains AT-rich regions

R-Banding ·     Banding pattern is opposite G-banding

·      Stains heterochromatic regions close to the centromeres

C-Banding
·      Usually stains the entire long arm of the Y chromosome

So how do you exactly identify chromosomal location based on banding patterns?

In studying disease and mutation, we follow a specific type of nomenclature to

designate the regions that are of interest to us. Let’s take for instance something like
the 22q11.2 deletion. What do all of these numbers and letters mean? To quickly summarize,
22q11.2 deletion syndrome occurs from the deletion of a small piece of chromosome 22 at a
location: q11.2
 22q11.2 DELETION
So now, when we add in karyotope
information you might see something like the
following: 22 ·     Chromosome 22

46, XY, del(8)(q21)

q ·     Long arm of chromosome (q)
When you break it down, it states the
patient is male (XY) and has a deletion in the long
arm (q) of chromosome 8 at region 2, band 1 1 ·     Region 1

Translocation nomenclature can get a little

1 ·      Band 1
more confusing:

46, XX, t(3;12)(p12.1;p11) 2 ·      Sub band 2

This designates a female has a translocation

between the short arms (p) of chromosomes 3 and
12 and region 1, band 2, sub band 1; and region 1
band 1 respectively.

An example of Down syndrome: 47, XX + 21 (Female has an extra chromosome 21)

An example of Klinefelter Syndrome: 47, XXY (Male with extra X chromosome)

4. CpG islands

 Short CpG-rich areas defined as >0.5 kb stretches of DNA with a G + C content ≥55%, and
an observed:expected frequency of at least 0.6
 CpG islands are typically located in the promoter region, 5’ to the TSS
 CpG islands have been demonstrated to influence local chromatin structures and simplify
the regulation of gene activity.
 CpG Islands reports potential CpG island regions using the method described by Gardiner-
Garden and Frommer (1987).
 The calculation is performed using a 200 bp window moving across the sequence at 1 bp
intervals.
 CpG islands are defined as sequence ranges where the Obs/Exp value is greater than 0.6
and the GC content is greater than 50%.
 The expected number of CpG dimers in a window is calculated as the number of 'C's in the
window multiplied by the number of 'G's in the window, divided by the window length.
 CpG islands are often found in the 5' regions of vertebrate genes, therefore this program
can be used to highlight potential genes in genomic sequences.
 5. Flourescent Binding-Quinacrine
Fluorescence of quinacrine in the presence of different polynucleotides was studied to
attempt to identify the specific nucleotides responsible for the fluorescence of stained
chromosome preparations. A marked enhancement of fluorescence was seen in the presence
of bihelical polynucleotides, such as poly(dA-dT), poly(dA)·poly(dT), and poly(rA)·poly(rU), but
not in the presence of single-stranded polynucleotides, such as poly(dA), poly(dT), poly(rA), or
poly(rU) alone. The higher was the GC content of natural DNAs, the more they quenched.
Quenching was also seen with poly(dG) or poly(rG) alone, but not with poly(dC) or poly(rC)
alone. Native and denatured DNA were both effective in quenching fluorescence. Thus, a
bihelical conformation is not required for fluorescence quenching. Nearly all of these
properties are shared with proflavine. In contrast, acridine orange, which stains most areas of
chromosome preparations, shows enhanced fluorescence in the presence of all members of a
series of natural DNAs. These data suggest that base-pairs composed of AT (rather than GC)
residues are responsible for the observed fluorescence of specific chromosome regions after
treatment with quinacrine, and support the proposal of Ellison and Barr (Chromosoma, in
press) that the highly localized quinacrine fluorescence in their cytological preparations
reflects the presence of DNA that has a high (A + T)/(G + C) ratio.

The first method to be used to identify all 46 human chromosomes was Q-banding
(Figure 1b), which is achieved by staining the chromosomes with quinacrine and examining
them under UV light. This method is most useful for examining chromosomal translocations,
especially ones involving the Y chromosome. Taken together, these banding techniques offer
clinical cytogeneticists an arsenal of staining methods for diagnosing chromosomal
abnormalities in patients.

6-8. Replication Banding (B—pulse, T-pulse, and BrdU)

Replication banding is most useful in identifying the early and late replicating X
chromosomes in females or in patients with sex chromosome abnormalities. It is well known
that one of the X-chromosomes in females is inactive, resulting in dosage compensation. It is
also known that X chromosome inactivation is random and that the inactive X chromosome
initiates and completes DNA synthesis later than the active X and other chromosomes
Replication banding, obtained by incorporation of 5-bromodeoxyuridine (BrdU) and
subsequent staining with Giemsa or other stains, allows distinction of the active and inactive X
chromosomes. Variations in replication banding can also be achieved. In the “T pulse”
procedure, BrdU is made available at the beginning of the cell cycle and then replaced with
thymidine for the last 5–6 h before the harvest. With the RBG technique (R-bands by BrdU and
Giemsa), the active or early replicating chromosome regions that inactive X chromosome, stain
dark. The “B pulse” is the opposite. Thymidine, made available at the beginning of the cell
cycle, is replaced with BrdU the last 5–6 h before harvest. Subsequent Giemsa staining will
result in early-replicating chromosome regions appearing dark because they have incorporated
thymidine, while the inactive or late-replicating chromosome regions appear pale due to the
BrdU-incorporation.
Banding patterns:
The equivalent of Q- and G- or R-banding patterns is achieved depending on whether a B
or T pulse is used. If a B-pulse is used, a Q or G-banding pattern is achieved and if a T-pulse is
used, an R-banding pattern is achieved. Subtle changes in pattern toward the earliest R-bands
or latest G-bands can be achieved by shortening the length of the BrdU pulse. A short T-pulse at
the very end of the S-period can produce what are referred to as T-bands (bright or dark bands
at the terminal ends of some chromosome arms). These bright bands with a T-pulse also
correspond to early replicating, GC-rich regions, whereas dull bands correspond to late
replicating AT-rich regions. The exception to this is the late replicating X chromosome whose
bright bands do not differ in AT: GC content from the less intensely stained bands at the same
locations on the early-replicating X

9. RE Banding
The sequence-specific DNA cleavage activity of restriction endonucleases (REases),
combined with other enzymatic activities that amplify and ligate nucleic acids, have enabled
modern molecular biology. After more than half a century of research and development, the
applications of REases have evolved from the cloning of exogenous DNA and genome mapping
to more sophisticated applications, such as the identification and mapping of epigenetic
modifications and the high-throughput assembly of combinatorial libraries. Furthermore, the
discovery and engineering of nicking endonucleases (NEases) has opened the door to
techniques such as isothermal amplification of DNA among others.
Restriction enzymes are traditionally classified into four types on the basis of subunit
composition, cleavage position, sequence specificity and cofactor requirements. However,
amino acid sequencing has uncovered extraordinary variety among restriction enzymes and
revealed that at the molecular level, there are many more than four different types.
Type I Enzymes
Type I enzymes are complex, multisubunit, combination restriction-and-modification
enzymes that cut DNA at random far from their recognition sequences. Originally thought to be
rare, we now know from the analysis of sequenced genomes that they are common. Type I
enzymes are of considerable biochemical interest, but they have little practical value since they
do not produce discrete restriction fragments or distinct gel-banding patterns.
Type II Enzymes
Type II enzymes cut DNA at defined positions close to or within their recognition
sequences. They produce discrete restriction fragments and distinct gel banding patterns, and
they are the only class used in the laboratory for routine DNA analysis and gene cloning. Rather
than forming a single family of related proteins, Type II enzymes are a collection of unrelated
proteins of many different sorts. Type II enzymes frequently differ so completely in amino acid
sequence from one another, and indeed from every other known protein, that they exemplify
the class of rapidly evolving proteins that are often indicative of involvement in host-parasite
interactions.

IN SITU HYBRIDIZATION BANDING

1. In situ Hybridization (ISH)
In situ hybridization (ISH) is a means of identifying where mRNAs are present in fixed
tissue samples; as IHC identified proteins in fixed tissue, ISH identifies mRNA. ISH is performed
by designing an antisense probe to your mRNA target, allowing your probe and mRNA to bind,
and visualizing where your probe is in the tissue slice. In situ is Latin for “in location,” and so
you could consider ISH and IHC to be in situ techniques. ISH and IHC can be run together in the
same tissue sample as an elegant control to show that your ISH probe and your IHC antibody
are specific, but the two techniques have different requirements and so this control is difficult
to pull off.

To get an ISH probe, you can design it much the same way as a PCR primer, except
you’re only interested in the antisense probe. For ISH, you can use cDNA
probes, oligonucleotide probes, or RNA probes. RNA probes are reputed to be the best,
followed by oligos, followed by cDNA. RNA probes are typically generated by
introducing plasmids into bacterial cultures (like Escherichia coli) and purifying the probe. They
are very sensitive (in every sense of the word) and require everything to be RNase free at every
step before probe binding (after binding, the mRNA/probe is considered by RNases to be
double stranded and therefore not a target). Oligonucleotide and cDNA probes are not as
sensitive but don’t come with the “everything must be RNase free and why won’t you cater to
my every whim” mentality that RNA probes require. Oligonucleotide probes are typically
ordered from companies, unless you have a nucleic acid synthesis machine available. cDNA
probes are generated from PCR; the PCR product is followed by asymmetric PCR (differing
concentrations of forward and reverse primers) to generate a concentrated amount of probe.

2. Fluorescent in situ Hybridisation (FISH)

In 1986, Pinkel et al. (39,40) developed a method to visualise chromosomes using
fluorescent-labelled probes called fluorescent in situ hybridisation (FISH). FISH technology
permits the detection of specific nucleic acid sequences in morphologically preserved
chromosomes, cells, and tissues. These techniques are useful in the work-up of patients with
various congenital and malignant neoplastic disorders, especially in conjunction with
conventional chromosome studies. Using FISH, cytogeneticists can detect chromosomal
abnormalities that involve small segments of DNA if their probe is situated, fortuitously or by
design, in the affected chromosomal segment (41). FISH can be used to establish the order of
DNA clones relative to bands, naturally occurring breakpoints, and other clones. Even more
importantly, FISH permits karyotype analysis of nuclei in non-dividing cells. FISH has been used
for the detection of t(2;5) (p23;q35) translocation in anaplastic large-cell lymphoma (42), for
minimal residual disease in hematopoietic stem cell assays from peripheral blood stem cells of
acute myeloid leukemia (AML) patients with trisomy 8 (43), and for analyzing chromosomal
abnormalities of tumors in children (44).

3. Chromosomal In situ Hybridization (CISS)

DNA libraries from sorted human gonosomes were used selectively to stain the X
and Y chromosomes in normal and aberrant cultured human cells by chromosomal in
situ suppression (CISS-) hybridization. The entire X chromosome was stained in
metaphase spreads. Interphase chromosome domains of both the active and inactive X
were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific
decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part
 (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact
domain in interphase nuclei. This approach was applied to amniotic fluid cells containing
a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained
by library probes from the gonosomes, thereby suggesting its autosomal origin. The
sensitivity of CISS-hybridization was demonstrated by the detection of small
translocations and fragments in human lymphocyte metaphase spreads after irradiation
with 60Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by
CISS-hybridization with Y-library probes. In both cases, metaphase spreads
demonstrated a translocation of Yp-material to the short arm of an X chromosome. The
translocated Y-material could also be demonstrated directly in interphase nuclei. CISS
hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a
known balanced translocation t(7;13) in the father. The same translocation was
observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes
involved in such translocations will be particularly important, in the future, in cases that
cannot be solved reliably by conventional chromosome banding alone.

4.Whole Chromosome Painting Probes (WCP)

Chromosome painting refers to the hybridization of fluorescently labeled,
chromosome-specific, composite probe pools to cytological preparations. Chromosome
painting allows highly sensitive and specific visualization of individual chromosomes in
metaphase or interphase cells and the identification of both numerical and structural
chromosomal aberrations in human pathology. In addition to human chromosome-
specific probe pools. ASI offers a comprehensive range of whole chromosome painting
probes for human, mouse and rat chromosomes. These probes are designed for use in
fluorescence in situ hybridization analysis. Whole Chromosome Paint (WCP) probes are
labeled with FITC, Rhodamine and Aqua. A combination of two or three colors can be
visualized simultaneously. They are ready to use in hybridization solution and supplied
in an economical 10 test format, or as a complete paintbox set. The painting probes are
visible using single band pass filters or a standard triple DAPI/FITC/Texas Red filter. Due
to their excellent stability, they are shipped at room temperature and should be stored
at -20℃ upon arrival. All ASI probes are manufactured in compliance with ISO
9000:2003 under rigorous quality control and strict GMP standards.
 5.Direct Visual Hybridization (DIRVISH)
The direct visual hybridization (DIRVISH) protocol is to stretched DNA fibers, it is
possible to label the intragenic and intergenic sequences with different fluorescent dyes and to
count the total number of visual pigment genes using direct microscopic inspection.
The procedure referred to as direct visual hybridization (DIRVISH) DNA mapping involves the
simultaneous hybridization of multiple probes and the fluorescent colors, red, green and blue
to produce images that convey high‐resolution mapping information. The images appear as
long strings of fluorescent signals positioned as they are in the genome. A visual multi‐color
map is generated within 2 days. Cosmid probes span a distance of 10 μm or more and have
been observed to contain patterns within the strings of signals. We have developed computer
imaging programs to scan through the strings of signals and plot the intensities. Scans through
multiple signal strings for one cosmid probe revealed consistent patterns. We have interpreted
the patterns as the result of suppression of repetitive DNA sequence hybridization. These
patterns may prove useful as fingerprints for regions of DNA.

6.Primed in situ labeling (PRINS)

Primed in situ labeling (PRINS) is a fast and sensitive technique for sequence specific in situ
detection of DNA. An unlabeled oligonucleotide probe is hybridized and used as primer for
chain elongation in situ catalyzed by a DNA polymerase. Thus, the oligonucleotide primer binds
by sequence-specific base pairing to its target sequence, which is subsequently labeled when
labeled nucleotides are incorporated by the DNA polymerase, using the oligonucleotide as
primer and the cellular DNA as template. Using unlabeled probes (primers) in the PRINS
reaction means that high concentrations can be used, because probe bound to cell-structures
cannot function as a primer and therefore does not give rise to background signals—only probe
hybridized correctly to DNA can function as primer for chain elongation. Thus, using high probe
concentrations the hybridization is very fast. A PRINS reaction normally runs for only 5–30 min.
Owing to the speed of the reaction the morphology of chromosomes is very well preserved,
which makes it possible to obtain chromosome banding of good quality after a PRINS reaction

7. FICTION (Fluorescence Immunophenotyping and Interphase

Cytogenetics as a Tool for Investigation of Neoplasms)
Technique will be detailed. This method has been described in 1992 by Weber-
Matthiesen et al. as the first technique to combine immunophenotyping and FISH. In contrast
to previous methods like the morphology-antibodychromosome (MAC) technique (Teerenhovi
et al. 1984; Schlegelberger et al. 1992), the FICTION technique can be applied on interphase
cells, which is of great advantage especially for tumor cytogenetics. To briefly introduce the
technique: In general, conventional unstained peripheral blood or bone marrow smears,
imprints and cytospin slides from fresh tissue and cryostat sections are applicable for FICTION.
In order to preserve cellular antigens, preparing high quality slides is one of the critical steps for
successfully performing FICTION. Freshly prepared slides should be air dried overnight at room
temperature. Thereafter, slides can be processed immediately or stored airtight at -80 aC for
years. For the FICTION procedure, slides are fixed in acetone and then immunophenotyped by a
direct or indirect fluorescence detection system. Immunophenotyping is followed by a fixation
step to preserve the fluorescent immunostaining during the harsh hybridization procedure.
After fixation, standard direct or indirect fluorescence in situ hybridization (FISH) is performed
using appropriately modified DNA probes. Using appropriate fLlter sets, the immunophenotype
and the hybridization signals can be evaluated simultaneously under a fluorescence microscope
(for review see Siebert and Weber-Matthiesen 1997; Siebert and ScWegelberger 1997).

For example, the following objectives can be studied using the FICTION
technique:

 The immunophenotypic characterization of tumor cell clones with defined

chromosome aberrations (Haferlach et al. 1996, 1997; Zhang et al.
1999, 2000)
 The definition of the differentiation state present in the tumor cell
clone (Weber-Matthiesen et al. 1995a);
 The identification of the most immature precursor cells within the tumor
clone representing the cell population from which the tumor clone
may have originated (Zhang et al. 1996);
 The immunophenotypic characterization of bystander cells with a normal
 chromosome constitution (Weber-Matthiesen et al. 1995b);
 The chromosomal characterization of small tumor cell populations
with a selective immunophenotype within a background of normal
cells (Weber-Matthiesen et al. 1995c, 1996).

REFERENCES:

1. https://www.nature.com/scitable/topicpage/karyotyping-for-chromosomal-
abnormalities-298/
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3336168/
3. https://www.sciencedirect.com/topics/neuroscience/cpg-island?
fbclid=IwAR0sGwVGcxhsWxKb_BsBxBSDZ20G-6xetk5_z1UKkRvfKmAqF9Vk1jauBdE
4. https://labmedicineblog.com/2016/01/04/chromosome-structure-staining-and-naming/
5. https://pdfs.semanticscholar.org/ad6a/1252390102b816ce34aba5b7fa8267d8595a.pdf
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC426522/
7. sciencedirect.com/topics/neuroscience/in-situ-hybridization
8. https://link.springer.com/chapter/10.1007/978-3-642-56404-8_34
9. https://link.springer.com/article/10.1007/BF00193186
10. http://www.tokyoinst.co.jp/en/products/detail/AS05/index.html
11. https://www.researchgate.net/figure/FISH-results-with-whole-chromosome-
painting-WCP-for-chromosomes-3-and-7-A-B-and-7-and_fig3_269172143