8/11/14

Review the nitrogenous bases & structure of DNA

DNA packaging: The composition of genes in the human genome, as well as
the determinants of their expression, is specified in the DNA of the 46 human
chromosomes in the nucleus plus the mitochondrial chromosome. Each
human chromosome consists of a single, continuous DNA double helix. Within
each cell, the genome is packaged as chromatin, in which genomic DNA is
complexed with several classes of chromosomal proteins.
o Histones: The DNA molecule of a chromosome exists in chromatin as
a complex with a family of basic chromosomal proteins called
histones and with a heterogeneous group of nonhistone proteins
o Packaging: Five major types of histones play a critical role in the
proper packaging of chromatin. Two copies of each of the four core
histones H2A, H2B, H3, and H4 constitute an octamer, around which a
segment of DNA double helix winds. This gives chromatin (protein +
DNA) the appearance of beads in a string. Each complex of DNA with
core histones is called a nucleosome, which is the basic structural
unit of chromatin, and each chromosome contains several thousand to
over a million nucleosomes.
o Condensation: During the cell cycle, chromosomes pass through
orderly stages of condensation and decondensation. However, even
when chromosomes are in their most decondensed state (during
interphase), DNA packaged in chromatin is substantially more
condensed than it would be as a native, protein-free double helix.
Further, the long strings of nucleosomes are themselves compacted
into a secondary helical chromatin structure that appears under the
microscope as a 30nm fiber (a solenoid fiber) that appears to be the
fundamental unit of chromatin organization.
Changes in chromosome structure throughout cell cycle:
Chromosome territory is the name given to decondensed chromosomes
during interphase
o Interphase: Chromosomes not present. DNA exists as chromosome
territory
o Prophase: Marked by gradual condensation of the chromosomes and
the beginning of the formation of the mitotic spindle (pair of
centrosomes)
o Prometaphase: Chromosomes continue to condense throughout this
stage. Very dense. Chromosomes are longest at this stage
o Metaphase: Chromosomes reach maximal condensation
o Anaphase: chromosomes migrate to poles
o Telophase: Chromosomes begin to decondense from their highly
contracted state & nuclear membrane reforms
DNA Methylation & DNA packaging (specialized histones):
o (** verifier) Methylated DNA may be bound by proteins known as
methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit
additional proteins to the locus, such as histone deacetylases and

other chromatin remodeling proteins that can modify histones, thereby

forming compact, inactive chromatin (termed heterochromatin).
Structural components of Chromosome:
o Centromere: A narrowing of pinching-in of the sister chromatids due
to formation of the kinetochore
o Arms: The centromere divides the p (petit) and q arms
Classification of Chromosomes (morphology & banding patterns):
o Morphology: Chromosomes are classified based on where along the
chromosome the centromere is located. Human chromosomes can be
metacentric (centromere in the center), submetacentric
(centromere slightly off center), or acrocentric (centromere far off
center)
o Banding Nomenclature: Each chromosome pair stains in a
characteristic pattern of alternating light & dark bands. The regions
immediately adjacent to the centromere are designated as 1 (p1 and
q1). Region numbers increase distally to the centromere. Regions are
divided into bands. The end of the p arm is 22.3 while the end of the q
are is 28. Ex: Xq21.2 denotes chromosome X, arm q, region 2, band 1,
sub band 2
Medical Indications for Chromosome Analysis (karyotyping):
Chromosome analysis is indicated as a routine diagnostic procedure for a
number of specific phenotypes encountered in clinical medicine. There are
also some nonspecific general clinical situations and findings that indicated a
need for cytogenetic analysis
o Problems of early growth & development: Failure to thrive,
developmental delay, dysmorphic facies, multiple malformations, short
stature, ambiguous genitalia, and mental retardation are frequent
findings in children with chromosome abnormalities
o Stillbirth & neonatal death: The incidence of chromosome
abnormalities is much higher among stillbirths than among live births,
as well as among infants who die in the neonatal period. Chromosome
analysis should be performed for all stillbirths and neonatal deaths that
might have a cytogenetic basis to identify a possible specific cause or
to rule out a chromosome abnormality as the reason for the loss. In
such cases, karyotyping.
o Fertility problems: Chromosome studies are indicated for women
presenting with amenorrhea (absence of menstruation) and for couples
with a history of infertility or recurrent miscarriage
o Family history: A known or suspected chromosome abnormality in a
first-degree relative is an indication for chromosome analysis under
some circumstances
o Neoplasia: Virtually all cancers are associated with one or more
chromosome abnormalities. Chromosome and genome evaluation in
the appropriate tissue sample (tumor itself or bone marrow in
hematological malignant neoplasms) can provide useful diagnostic or
prognostic information.

Pregnancy in a woman of advanced age: There is an increased

risk of chromosome abnormality in fetuses conceived by women older
than about 35 years. Fetal chromosome analysis should be offered as a
routine part of prenatal care in such pregnancies
Cytogenic Assays (study more brief, slide 83): The 24 types of
chromosome found in the human genome can be readiy identified at the
cytological level by a number of specific procedures
o Karyotyping: A blood test extracts cells used for karyotyping. The
condensed chromosomes of a dividing human cell are most readily
analyzed at metaphase or prometaphase. At these stages, the
chromosomes are visible under the microscope as a chromosome
spread. Each chromosome consists of its sister chromosomes, although
in most chromosome preparations, the two chromatids are held
together so tightly that they are rarely visible as separate entities.
Karyotyping refers to the common procedure of cutting out the
chromosomes from a photomicrograph and arranging them in pairs in a
standard classification.
o G Banding (Giemsa banding): The most common method used in
clinical laboratories. Dark bands are gene poor regions while light
bands are gene rich regions
o Q Banding: This method requires staining with quinacrine mustard or
related compounds and examination by fluorescence microscopy. The
Q bands (bright & dim banding pattern) correspond almost exactly to
the dark bands seen after G banding. Q & C banding are useful for
detecting variants in chromosome morphology or staining, called
heteromorphisms, differences are usually benign and reflect
differences in amount or type of satellite DNA sequences
o R banding: After special treatment (such as heating) before staining,
the resulting dark & light bands are the reverse of those procured by G
or Q banding and are referred to as R bands. Strength: When regions
that stain poorly by G or Q banding are examined, R banding gives a
pattern that is easier to analyze than that given by G or Q banding
o C banding: Involves staining the centromeric region of each
chromosome and other regions containing constitutive hematocrin.
Strength: Hematocrin is the type of chromatin defined by its property
of remaining in the condensed state and staining darkly in nondividing
(interphase) cells.
o High-Resolution Banding (prometaphase banding): Achieved
through G-banding or R-banding techniques to stain chromosomes that
have been obtained at an early stage of mitosis (prophase or
prometaphase), when they are still in a relatively uncondensed state.
Strength: High resolution banding is especially useful when a subtle
structural abnormality of a chromosome is suspected. Prometaphase
chromosomes reveal 550-850 bands or even more in a haploid set,
whereas standard metaphase preparations show only about 450.
o Fluorescence In Situ Hybridization (FISH): Used to examine the
presence or absence of a particular DNA sequence or to evaluate the
o

number or organization of a chromosome or chromosomal region.

Strength: In FISH, DNA probes specific for individual chromosomes,
chromosomal regions, or genes can be used to identify particular
chromosomal rearrangements or to rapidly diagnose the existence of
an abnormal chromosome number in clinical material. Gene-specific
probes can be used to detect the presence, absence, or location of a
particular gene, both in metaphase chromosomes and in interphase
cells. Routinely used to diagnose specific deletions, duplications, or
rearrangements, both in prometaphase or metaphase preparations and
in interphase.
o Microarrays: With the availability of resources from the Human
Genome Project, chromosome analysis can also be carried out at a
genomic level by a variety of array-based methods that use
comparative genome hybridization (CGH). To assess the relative copy
number of genomic DNA sequences in a comprehensive, genome-wide
manner, microarrays containing either a complete representation of
the genome or a series of cloned fragments, spaced at various
intervals, from throughout the genome can be hybridized to control
and patient samples. I.E: To compare karyotypes of various individuals
simultaneously. Strength: This approach complements conventional
karyotyping and has the potential to provide a much more sensitive,
high-resolution assessment of the genome. Weakness: Array-based
CGH methods measure the relative copy number of DNA sequences
but not whether they have been translocated or rearranged from their
normal position in the genome. Therefore, they can analyze copy
number but not whether the situation is benign or pathogenic.
Mitosis vs Meiosis: Mitosis gives rise to daughter cells that are identical to
the parent cells and is found in the majority of cell types, whereas meiosis
gives rise to haploid daughter cells from diploid parent cells. Meiosis occurs
only in the gametes. Note: At meiosis homologous chromosomes pair (and
recombine), neither of which occurs in mitosis. Chromosomes do NOT pair in
mitosis
o Meiosis I is known as reduction division because it is the division in
which the chromosome number is reduced by half. Meiosis I is also
notable because it is the stage at which genetic recombination (or
crossing over) occurs. This is when homologous segments of DNA are
exchanged between non-sister chromatids of a pair of homologous
chromosomes. Meiosis has multiple contributions to genetic diversity:
Crossing over &Independent assortment: maternal & paternal
chromosomes assort independently Ex: maternal chromosome 1 and
paternal chromosome 2 in one cell as supposed to both maternal
chromosome 1s.
Types of recombination: 1) Somatic recombination functions
in dna replication and stalls the replication fork. Occurs between
non-homologous sister chromatids. This can occur during mitosis
and may cause cancer if wild-type allele encodes a tumor
suppressor gene. 2) Site-specific recombination refers to

integration of viral DNA or bacterial DNA into genome. 3)

Homologous (meiotic) recombination refers to crossing over of
maternal & paternal (homologous) chromosomes
*Prophase (review steps): Leptotene: Replicated chromosomes are
decondensed and are closely aligned so that they cannot be
distinguished. Zygotene: Homologous chromosomes are quite
condensed and begin to align along their entire length. Pachytene:
Chromosomes become much more tightly coiled (homologues are
bivalents or tetrads). Synapsis is completed in pachytene. Note:
crossing over occurs during pachytene. Diplotene: Synaptonemal
complex begins to break down. Chromosomes begin to separate, but
are still held together at the chiasmata. Diakinesis: Chromosomes
reach maximal condensation

Note: G-C base pairs are harder to break (3 hydrogen bonds) than A-T base pairs (2
hydrogen bonds)
Different forms of double helical DNA are A-DNA, B-DNA & Z-DNA. A-DNA forms
under conditions of high salt concentration & minimal water. B-DNA is the Watson &
crick form. Z-DNA is the left-handed double helical form. Function is not clear,
torsional strain relief (supercoiling) and possibly active during dna transcription
One copy of the H1 histone sits outside of each nucleosome and functions to help
form solenoid shape of chromosomes
DNA is packaged more tightly in sperm cells, where they are super condensed. In
sperm, histone proteins are replaced with specialized protamines at late
spermatogenesis
Review Cohesins, which are present during mitosis & meiosis
Women have more recombination events because oocytes sit around arrested in
meiosis for so long
DNA isnt simply crossed over at chiasmata, some dna is lost and then repaired by
adding new dna at holiday junction. Watch video about recombination &
holiday junction. Animation links at the end of the video
Telomeres have specialized ends (TTAGGG repeats) that function to prevent fusion
with other chromosomes. I.E. the difference between sticky and blunt ends

Autosomes are chromosomes 1-22 while gonosomes are chromosomes 23 & 24

Chromosome nomenclature: cen (centromere), ter (telomere)

Most chromosomally abnormal fetuses are dead before term. Therefore the
likelihood of such abnormalities declines are pregnancy progresses

In CGH (comparative genome hybridization), we expect all dna to be yellow (fully

hybridized). Green or red regions signify gain or loss of material, i.e. differences in
sequence