Chap. 6 Genes, Genomics, and Chromosomes (Part B)

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Chap. 6 Genes, Genomics, and Chromosomes (Part B)

• Topics
• Genomics Genome-wide Analysis of Gene Structure
  and Expression
• Structural Organization of Eukaryotic Chromosomes
• Morphology and Functional Elements of Eukaryotic
  Chromosomes

• Goals
• Learn about computer-based methods for analyzing
  sequence data.
• Learn how DNA and proteins are packaged in
  chromatin.
• Learn the large-scale structure organization of
  chromosomes.
• Learn the functional elements required for
  chromosome replication and segregation.

RxFISH-painted human chromosomes.
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Mining Sequence Data BLAST Searches
An enormous amount of DNA sequence information is
available from genome sequencing and sequencing
of cloned genes. This data is stored in data
banks such as GenBank at the NIH in Bethesda, MD
and the EMBL Sequence Data Base at the European
Molecular Biology Laboratory in Heidelberg,
Germany. Scientists working in the area of
bioinformatics use this data to find genes,
analyze their properties, and determine
phylogenetic relationships between organisms and
proteins. A common procedure in which this data
is used is the BLAST search (basic local
alignment search tool) which is used to compare
protein and DNA sequences. An example BLAST
search alignment is shown for the human
neurofibromatosis 1 (NF1) gene in Fig. 6.25. The
alignment shows NF1 is related to the S.
cerevisiae Ira GTPase-activating protein (GAP)
and suggests the disease is caused by aberrant
signal transduction.
Computer programs similar to BLAST are used to
identify protein sequence motifs (e.g., zinc
fingers) in unknown proteins. The identification
of structure regions with known function sheds
light on overall protein function and helps guide
experimental analysis of unknown proteins and
genes.
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Sequence Comparisons Establish Evolutionary
Relationships Among Proteins
BLAST search analysis can identify the members of
a protein family originating from gene
duplication and speciation mutations. As
illustrated for the a- and ß-tubulin protein
family in Fig. 6.26, an early gene duplication
event created the paralogous a- and ß-tubulin
genes. Later speciation mutations lead to
evolution of the orthologous members of the a-
and ß-tubulin subfamilies. Orthologous proteins
are most likely to share the same function.
x
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Genome Size vs Complexity
Genome sequencing has revealed that the
morphological complexity of an organism is not
strongly correlated with the size of its genome
(Fig. 6.27). Alternative splicing of RNAs and
post-translational modification of proteins are
thought to greatly increase the complexity of the
proteins encoded by the genomes of higher
organisms. In addition, the relative number of
cells formed in a tissue such as the cerebral
cortex can be important in increasing complexity
(e.g., mice vs humans). Genes can be identified
within the sequenced genomes of simple organisms
such as yeast and bacteria by searching for open
reading frames (ORFS). ORFs are long stretches of
triplet codons lacking stop codons. Gene
annotation (assignment of likely function) is
based on knowledge from biochemical studies
and/or alignments with known
sequences. In complex organisms such as humans
whose genes typically contain introns, more
sophisticated algorithms that ID intron splice
sites and compare cDNA and other sequence
information to genomic DNA sequences must be
applied to locate and annotate genes. Using such
methods 25,000 genes have been identified in
humans. However, conclusive evidence for
synthesis of protein or RNA products is lacking
for 10,000 genes.
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Extended and Condensed Chromatin
Human diploid cells contain about 2 meters of
DNA. To fit within nuclei, DNA must be condensed
by 105-fold. DNA exists in cells as a
nucleoprotein complex known as chromatin. During
interphase when cells are not dividing, chromatin
is relatively uncondensed compared to its state
in metaphase chromosomes. When released from
nuclei with low salt buffer, chromatin displays
an extended "beads-on-a-string" morphology, where
each bead is a nucleosome (Fig. 6.28). When
released in physiological salt concentrations,
more condensed fibers of 30 nm diameter are
observed. In general, extended chromatin can be
transcribed, whereas condensed forms cannot.
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Structure of Nucleosomes
Nucleosomes consist of 147 bp of DNA wrapped in
almost two turns around the outside of an octamer
of histone proteins (Fig. 6.29). In most
nucleosomes, the octamer has a stoichiometry of
H2A2H2B2H32H42. Histones are the most abundant
DNA-binding proteins in eukaryotic cells. The
sequences of the 4 histones that make up the
octamer are highly conserved across all
organisms, indicating their functions were
optimized early in evolution. Histones have a
large number of basic amino acids and bind to DNA
mostly by salt-bridge interactions to phosphates
in the DNA backbone. Another histone, H1, binds
to the linker DNA between nucleosomes. Linker DNA
is 10-90 bp in length depending upon the organism.
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Structure of 30-nm Chromatin Fibers
In 30-nm fibers, nucleosomes bind to one another
in a double helical arrangement (Fig. 6.30).
Histone H1 molecules bind to linker DNA between
nucleosomes and help stabilize the 30-nm fiber.
The stability of 30-nm fibers is modulated by
post-translational modification of the tails of
histones in the octamers (H4 in particular).
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Histone Tails and Chromatin Condensation
The N- and C-terminal tails of histones project
out from the nucleosome core (Fig. 6.31a). They
also contain numerous residues that can be
modified by acetylation, methylation, etc. (Fig.
6.31b). Acetylation of lysine side-chains by
histone acetylases (HATs) neutralizes positive
charge and promotes decondensation of 30-nm
fibers. Methylation, on the other hand, blocks
lysine acetylation, maintains positive charge,
and promotes 30-nm fiber condensation. Studies
have shown that chromatin condensation is not
controlled simply by the net acetylation state of
histones. Rather, the sites where acetylation and
other modifications occur also are important. The
combinations of modifications that specify
condensation/decondensation are referred to as
the "histone code".
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Interphase Chromatin
Interphase chromatin exists in two different
condensation states (Fig. 6.33a). Heterochromatin
is a condensed form that has a condensation state
similar to chromatin found in metaphase
chromosomes. Euchromatin is considerably less
condensed. Heterochromatin typically is found at
centromere and telomere regions, which remain
relatively condensed during interphase. The
inactivated copy of the X-chromosome (Barr body)
that occurs in cells in females also occurs as
heterochromatin. In contrast, most transcribed
genes are located in regions of euchromatin.
Common modifications occurring in histone H3 in
hetero- and euchromatin are illustrated in Fig.
6.33b.
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Formation of Heterochromatin
The trimethylation of histone H3 at lysine 9
(H3K9Me3) plays an important role in promoting
chromatin condensation to heterochromatin (Fig.
6.34a). Trimethylated sites are bound by
heterochromatin protein 1 (HP1) which
self-associates and oligomerizes resulting in
heterochromatin. Heterochromatin condensation is
thought to spread laterally between boundary
elements that mark the ends of transcriptionally
active euchromatin (Fig. 6.34b). Recruitment of
the H3K9 histone methyl transferase (HMT) to HP1
sites promotes heterochromatin spreading by
catalyzing H3 methylation.
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Structure of Interphase Chromosomes
FISH analysis performed with fluorescent probes
that bind to sequential sequence sites along DNA
supports a looped structure for interphase
chromosomes (Fig. 6.35). Loops range in size from
1 to 4 million base pairs in mammalian interphase
cells. The bases of the loops are located near
the center of the chromosome at
scaffold-associated regions (SARs), and
matrix-attachment regions (MARs). The DNA fibers
at the base of the loops are held together by
structural maintenance of chromosome (SMC)
proteins (Fig. 6.36c) and other non-histone
proteins. Transcription units containing
expressed genes are located in uncondensed loop
regions, away from the more condensed center of
the chromosome.
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Interphase Chromosome Territories
In situ hybridization of interphase nuclei with
chromosome-specific fluorescently-labeled probes
indicates that chromosomes reside within
restricted regions of the nucleus rather than
appearing throughout the nucleus (Fig. 6.37).
Interestingly, the precise positions of
chromosomes are not reproducible between cells.
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Structure of Metaphase Chromosomes
In metaphase chromosomes, the number of loops of
chromatin is increased and the lengths of the
loops are decreased compared to what occurs in
interphase chromosomes. In addition, more folded
structures called chromonema fibers and higher
order structures occur in prophase and metaphase
chromatids (Fig. 6.38).
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Microscopic Structure of Metaphase Chromosomes
Because interphase chromosomes are not easily
visualized by microscopy techniques, chromosome
morphology has been studied mostly using
metaphase chromosomes. Metaphase chromosomes are
duplicated structures formed after DNA
replication is complete. They contain two sister
chromatids joined at a structure called the
centromere (Fig. 6.39). The ends of chromatids
are called telomeres. Centromeres are required
for chromatid separation late in mitosis.
Telomeres are important in preventing chromosome
shortening during replication. The number, sizes,
and shapes of metaphase chromosomes constitute
the karyotype, which is distinctive for each
species.
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Chromosome Banding Patterns
A number of dyes, such as Giemsa reagent,
selectively stain different regions of
chromosomes forming distinctive bands. For Giemsa
reagent, banding is affected by G C content.
Banding patterns are very important in chromosome
ID and in looking for chromosomal abnormalities
and mapping the locations of genes. The most
detailed staining is achieved via multicolor FISH
chromosome painting. In this technique, staining
is performed using a mixture of DNA probes
coupled to several fluorescent dyes (See Slide
1). In Fig. 6.40 below, FISH staining patterns
have been converted to false-color images to
visualize chromosomes. Standard terminology is
used for naming band and gene locations in
chromosomes. The short arm is designated "p", and
the long arm "q". Arms are further divided into
major sections and subsections that are numbered
consecutively out from the centromere.
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Detection of Translocations
The analysis of chromosome banding patterns is
used to detect anomalies such as truncations and
translocations associated with certain genetic
disorders and cancers. In chronic myelogenous
leukemia, leukemic cells contain a shortened
chromosome 22 and a longer chromosome 9 resulting
from a translocation event in the q arms of these
two chromosomes (Fig. 6.41). The shortened
chromosome is distinctive and is referred to as
the "Philadelphia chromosome". Multicolor FISH
staining (right) is useful in identification of
such chromosomes in a chromosome spread.
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Evolution of Human Chromosomes
Through the determination of locations of common
chromosomal segments in modern primate
chromosomes, investigators have calculated the
most likely karyotype of the common ancestor of
all primates (Fig. 6.42c). In addition, they have
proposed a model for how the human karyotype
evolved from that ancestor. Major events in the
evolution of the human karyotype include 1)
formation of chromosome 2 by fusion of ancestral
chromosomes 9 and 11, 2) formation of chromosomes
14 and 15 by breakage of ancestral chromosome 5,
and 3) formation of chromosomes 12 and 22 by
translocations between ancestral chromosomes 14
and 21. In other cases (e.g., chromosome 1), no
significant rearrangements have occurred over
time.
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ID of Functional Chromosomal Elements (I)
Studies with yeast have demonstrated that all
chromosomes must contain 3 functional elements to
replicate and segregate correctly 1) replication
origins, 2) a centromere, and 3) telomeres. Yeast
replication origins were identified in plasmid
cloning studies. Only yeast plasmids containing a
copy of a sequence referred to as the
autonomously replicating sequence (ARS) could be
transfected into yeast cells (Fig. 6.44a). The
haploid S. cerevisiae genome contains many ARSs
distributed among its 16 chromosomes.
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ID of Functional Chromosomal Elements (II)
While only ARSs are needed for plasmid
replication, an additional sequence identified by
cloning procedures was found to be required for
efficient segregation of plasmids to yeast
daughter cells (Fig. 6.44b). This DNA proved to
contain chromosomal centromere sequences (CEN
sequences). Yeast CEN sequences are relatively
simple (Fig. 6.45, not covered). In humans, they
consist of 2-4 x 106 bp of simple sequence DNA
composed of a 171 bp repeat unit. The human
centromere sequence is bound by specialized
nucleosomes containing a centromere-specific
histone H3 variant (CENP-A). A large complex of
non-histone proteins (the kinetochore) binds to
centromeres and attaches them to microtubules of
the mitotic spindle apparatus.
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ID of Functional Chromosomal Elements (III)
Yeast transfection studies also showed that
linearized plasmids containing ARS and CEN
sequences could be maintained in cells only if
telomere (TEL) sequences were attached at their
ends (Fig. 6.44c). The function of TEL sequences
in replication of chromosome ends is illustrated
in the next two slides.
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Function of Telomeres
A special mechanism is needed to complete the
replication of DNA in DNA strands that have their
3 ends located at the ends of chromosomes. DNA
polymerases cannot complete synthesis of this
region of DNA, and without synthesis, chromosomes
become shortened with each round of replication
(Fig. 6.46). Shortening results in the loss of
binding sites for proteins that protect the ends
of linear chromosomes from attack by
exonucleases. As illustrations of the importance
of telomere replication, knockout mice lacking
the enzyme that synthesizes DNA at telomeres,
telomerase, cannot produce viable offspring after
six generations. In addition, telomerase often is
switched on in cancer cells.
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Mechanism of Action of Telomerase
Telomere sequences typically consist of tandemly
repeating sequence units with a high G content in
the strand that has its 3' end at the end of the
chromosome. In humans and other vertebrates, the
repeating sequence is TTAGGG. This sequence unit
repeats over a few thousand base pairs in humans.
The mechanism of replication of this DNA is
illustrated in Fig. 6.47 for a protozoan species.
Replication is carried out by the enzyme known as
telomerase. Telomerase is a reverse transcriptase
that carries its own internal RNA template which
binds to the ssDNA at the chromosome 3 end and
allows this strand to be elongated. Ultimately,
DNA Pol ?/primase can synthesize a primer on this
strand, which is elongated by DNA Pol ?. Some
organisms rely on a different mechanism for
replication of telomeric DNA. For example, flies
lack telomerase and maintain telomere length by
regulated insertion of non-LTR retrotransposons
into telomere DNA.