Download CHAPTER 16 – THE MOLECULAR BASIS OF INHERITANCE

UNIT 5 – MOLECULAR GENETICS (CHAPTERS 16 – 20)
CHAPTER 19 – EUKARYOTIC GENOME
YOU MUST KNOW:
 The impact of DNA methylation and histone acetylation on gene
expression
 The role of oncogenes, proto-oncogenes, and tumor suppressor
genes in cancer.
I. CHROMATIN STRUCTURE
 Eukaryotes have substantially more DNA than prokaryotes. This DNA
must be organized and managed for cell specialization. Gene
expression in both groups is regulated mostly during transcription.
 Eukaryotic DNA is packed with proteins. The complex of these two
molecules is called chromatin.
 During interphase the chromatin is loose, extended. At the beginning
of cell division, this extended chromatin undergoes a coiling and
folding process that significantly shortens the chromatin into
chromosomes. Each chromosome contains a single, linear DNA
double helix. (The total length of DNA in each human cell is about 99
cm = 3 feet).
 First level of DNA packaging: histones, small, bead-shaped proteins
with positively charged amino acids can bind to the negatively
charged DNA molecule and wrap the DNA double helix up like a
spool. Each histone with the wrapped DNA is a nucleosome. The
nucleosome basically remains intact throughout transcription and
translation.
 Second level of DNA packaging: The neighboring nucleosomes and
the linker DNA between them will interact with each other. This pulls
the nucleosomes closer together. This is the form that is seen during
interphase.
 Next level of packaging: A long protein scaffold made up of
nonhistone proteins folds the second level protein fiber up by forming
loops. These loops may help to organize the chromatin that is being
actively transcribed. The organization of the chromatin in this phase is
visible under light microscope. The darker colored and compact
heterochromatin is not transcribed as opposed to the actively
transcribed euchromatin that is lighter in coloration.

During metaphase the chromatin folds further, resulting in the
maximally compacted chromosome with two chromatids.
Animation:
http://www.dnai.org/text/mediashowcase/index2.html?id=564
I.
GENE REGULATION DURING TRANSCRIPTION
 All organisms have mechanisms for turning genes on or off. This
process either activates the genes for transcription or inactivates them
into a dormant stage.
 Multicellular organisms also undergo long-term gene regulation that
result in cell differentiation (Think back to the genetic update
conference)
Animation (shows a brief review of what we learned so far in this unit and
how it relates to gene expression):
http://www.genomicseducation.ca/animations/gene_expression.asp
A. Differential Gene Expression
 Although all cells in a multicellular organism have the same genome,
each type of specialized cell expresses a different set of genes to carry
out its specific function – differential gene expression.
 Because only a small percentage of the genome is coding gene
(gene that codes the information for a polypeptide) transcription
proteins must locate the right genes at the right time to transcribe
them.
 Gene expression can be regulated at various points in eukaryotic cells.
On these points (shown below) gene expression can be turned on, off,
accelerated or slowed down.
Great overview of gene expression:
http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter18/animations.html#
B. Regulation on the DNA level (Chromatin Structure Regulation)
 The organization of the chromatin in the chromosome is important in
gene expression. Heterochromatin that is tightly packed, is usually not
expressed.
 Chemical modifications to the histones or to the DNA can influence
chromatin structure and gene expression:
a. Histone acetylation and deacetylation – adding
acetyl groups to histones does not allow them to bind
to each other any more. As a result, chromatin has a
looser structure and transcription factors have easier
access to the genes of the acetylated region.
Acetylation promotes transcription.
b. DNA methylation – methyl groups can be attached to
the DNA molecule at certain bases (usually cytosine).
Highly methylated DNA is usually inactive.
Methylation also seems to be responsible for genomic
imprinting – permanent inactivation of the mothers or
fathers genes for certain traits.
C. Regulation of Transcription Initiation
 The eukaryotic gene and additional control DNA elements are
responsible for adding the proper transcription factors to the
promoter region of the DNA molecule before the RNA polymerase II
can bind to it.
Figure 19.5
A. Post-Transcriptional Regulation:
 Once the gene has been transcribed, mRNA processing, mRNA
degradation and translation also can change the outcome of the
protein synthesis:
a. RNA processing – This type of regulation is only
available in eukaryotes. One example of alternating
the primary transcript is alternative splicing – different
RNA molecules are produced from the same primary
transcript, depending on which segments are treated
as exons and introns. Alternative splicing is controlled
by regulatory proteins.
b. mRNA degradation – mRNA molecules are short lived
and degrade very quickly in prokaryotes. However, in
eukaryotes mRNA molecules can survive for weeks
and translated repeatedly in the cytoplasm of some
cells. Eukaryotic mRNA molecules can be inactivated
by using other RNA molecules (miRNA, siRNA) and
protein complexes. These complexes can inactivate
or degrade the mRNA molecule.
c. Initiation of Translation –Regulatory proteins can bind
to the untranslated region of the mRNA and can
block translation.
d. Protein processing or degradation – phosphate
groups can be added to inactive forms of already
made proteins to activate them or phosphate groups
can be removed to inactivate proteins. Proteasome
molecules can degrade already existing proteins.
II.
THE GENETICS OF CANCER
A. Types of Genes Associated with Cancer
 Cancer can be caused by mutations that occur in genes that are
responsible for cell growth and division (genes that code for growth
factors, growth factor receptors and intracellular molecules of cell
signaling)
 These mutations can be random or caused by mutagens in the
environment or certain viruses (HPV cause cervical cancer, EpsteinBarr virus causes infectious mononucleosis).
 All tumor viruses transform cells into cancerous cells by integrating
viral DNA into the host DNA.
 Proto-oncogenes are normal genes that are responsible for cell
growth and differentiation in animals and humans. These genes
can become mutated and become oncogenes (cancer causing
genes). There are three main groups of genetic changes that can
convert proto-oncogenes into oncogenes:
a. A mutation in the gene can change the protein structure that
the gene codes for. The produced protein becomes
overactive or loses its sensitivity to regulators and does not
degrade fast enough.
b. The gene can have more than normal number of copies by
gene amplification. This result in an increased concentration
of proteins that are produced by the proto-oncogene.
c. Translocation or transposition of the gene can put it in new
locations under new genetic control. This can result in an
overproduction of the coded protein.
d. A point mutation can also occur in the control element of the
gene, “upstream” from the gene and can result in excess
amount of protein.
Figure 19.11
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

Tumor-suppressor genes – Genes in cells that normally inhibit cell
division. The proteins that these genes code, help to prevent
uncontrolled cell growth. Any mutation that results in the decrease
of the activity of the tumor-suppressor genes can cause cancer.
According to today’s theory of cancer development, more than
one somatic mutation is needed to produce all the changes that
result in cancer. These mutations usually accumulate over time, so
the rates of cancer increases as a person ages.
Steps of developing colorectal cancer:
o First, the loss of a tumor-suppressor gene can result in small
benign tumors (polyps)
o Second, a new mutation results in the overproduction of a
protein that stimulates cell division. The result is a larger
benign growth (adenoma) of the same cells.
o Third, a serious mutation in a tumor-suppressor gene can result
in a malignant tumor (carcinoma) in the same cells.
Figure 19.13


Certain cancers can run in families because a person can inherit an
already mutated gene that starts the person on the pathway of
many other mutations that can lead to cancer sooner.
Examples of cancer types that be the result of inherited mutations
are some colorectal cancers and some breast cancers.
Movie segment:
http://www.pubinfo.vcu.edu/secretsofthesequence/playlist_frame.asp
III.
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
NONCODING DNA SEQUENCES, TRANSPOSONS
The bulk of most eukaryotic chromosomes are made up of
noncoding DNA sequences, often described as “junk DNA”.
However, there is evidence today that these sequences play
important roles in the cell.
Trends in genomic complexity shows that as we move up on the
phylogenetic tree, more complex organisms usually have more
DNA but fewer coding genes in a given length of DNA.
In prokaryotes, most genes code for proteins, tRNA and rRNA and
there are a few noncoding, regulatory sequences, such as
promoters. Coding occurs continuously without interruptions.
In eukaryotes, most of the DNA does not code proteins or RNA and
it includes more complex regulatory sequences. Some of these
noncoding sequences are present as introns within genes, a large
portion of DNA is also made up of gene fragments, mutated genes
that are not functional. Most of the intergenic DNA is repetitive
DNA, sequences that are present in multiple copies in the genome.

About 44% of these sequences are transposable elements and
related sequences.
Eukaryotic transposable elements are two kinds:
o Transposons – sequences that move around the genome by
means of a DNA intermediate. These sequences can move
by “cut-and-paste” mechanisms that remove them from one
site of the genome and inserts them at another site.
Transposons can cause damage to functioning genes but
also can give rise to new variations of proteins by changing
the arrangements of genes.
o Retrotransposons – these are mobile genetic elements that
always leave a copy at the original site during transposition
because it is copied into an RNA molecule. The RNA
molecule than copied into a DNA by reverse transcriptase
and than inserted back into the genome as DNA at another
location. These can also result in new genetic arrangements
that are usually harmful but can have evolutionary
significance.
Figure 19.16
CHAPTER 20 – DNA TECHNOLOGY AND GENOMICS
YOU MUST KNOW:
 The terminology of biotechnology
 The steps in gene cloning with special attention to the
biotechnology tools that make cloning possible.
 The key ideas that make PCR possible
 How gel electrophoresis can be used to separate DNA fragments or
protein molecules.
I. Understanding and Manipulating Genomes:
 Biotechnology is a booming field of science with constantly improving
technology and new discoveries weekly.
 Some key terms to know:
o Recombinant DNA – DNA in which nucleotide sequences from two
different sources, often different species, are combined in vitro into
the same DNA molecule.
o Genetic engineering – the direct manipulation of genes for
practical purposes
o Biotechnology – the manipulation of organisms or their components
to make useful products (from wine and cheese making to
analyzing personal genomes and fixing mutations)
Radiolab:
http://www.wnyc.org/flashplayer/player.html#/play/%2Fstream%2Fxspf
%2F92351 – 35:00 min
II. DNA Cloning:
A. The Method of DNA Cloning:
 Gene cloning – methods for preparing well-defined, gene-sized
pieces of DNA in multiple identical copies.
 Most commonly bacteria and their plasmids are used:
o Plasmid is isolated
o Foreign DNA is inserted into the plasmid – recombinant DNA
o Plasmid is returned into the bacterium
o Bacterium reproduces to form clones of identical cells

Cloned bacteria can make many copies of a certain gene and
can produce certain proteins.
B. Using Restriction Enzymes:
 Restriction enzymes – enzymes that cut DNA molecules at a limited
number of specific locations. In nature, these enzymes protect the
bacterial cell against intruding DNA from other organisms, by
cutting this DNA segments up.
 Restriction sites – short segments of DNA that are recognized by the
restriction enzyme.
 The bacterium’s own DNA is protected by methylation from
restriction enzymes.
http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html#
C. Cloning a Eukaryotic Gene in a Bacterial Plasmid
 The original plasmid is called a cloning vector – this plasmid has the
ability to carry foreign DNA into a cell and replicate it there.
Bacterial plasmids are widely used cloning vectors, because they
are easy to isolate, manipulate and can be reintroduced back into
the bacterium after isolation. Because bacterial cells reproduce
quickly, the inserted gene or its proteins can be obtained in large
quantities. Use: http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.htm
l#

After genes are inserted into bacteria by using DNA cloning, the
success of the experiment can be analyzed by two methods:
a. Looking for the inserted gene in the bacterial colonies
b. Looking for the synthesized proteins in the new
bacterial colonies

Nucleic acid hybridization – is the process that detects certain
sequences of the DNA molecule by using nucleic acid probes that
are radioactively labeled. These probes can bind to denatured
DNA molecules (two strands of DNA separated) and radioactively
label the colonies that contain the inserted gene.
Animations:
DNA cloning:
http://www.sumanasinc.com/webcontent/animations/content/plasmidcl
oning.html
http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.htm
l
Restriction enzymes:
http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html#
III. THE POLYMERASE CHAIN REACTION (PCR)
 When the source of DNA molecule is impure or there is only a small
amount of DNA present, PCR is the most effective way to amplify one
or many segments of DNA.
 This method can make millions of copies of a segment of DNA in a few
hours.
 PCR is a three-step cycle that brings about a chain reaction that will
produce an exponentially growing number of copies of identical DNA
molecules.
 Steps:
a. first the target sequence is denatured (separated to
individual polynucleotide chains) by heat
b. second, cooling allows short segments of DNA primers
to attach by hydrogen bonding at the 5’ → 3’
direction
c. Heat-stable DNA polymerase is used to assemble the
nucleotides of the new strands
Animations:
PCR -- http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html#
http://www.dnalc.org/ddnalc/resources/pcr.html
PCR song:
http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/
IV.
GEL ELECTROPHORESIS
 This process uses a gel to separate various segments of DNA
molecule or protein based on their size and electrical charge.
 A mixture of DNA segments of different sizes can be injected into
the wells of the gel than put in an electric current. The electric
current is running from the – to the + electrode and drags the
molecules with it.
 The smallest pieces of molecules run the furthest. A fluorescent dye
can be used to dye the DNA segments and make them visible.
 Gel electrophoresis can be used to locate mutations on various
DNA molecules, separate certain segments of DNA from the others
for further examination, purify DNA, the band pattern can help in
identifying a person etc.
Interactive lab: http://learn.genetics.utah.edu/content/labs/gel/
Animation:
http://www.dnalc.org/ddnalc/resources/electrophoresis.html
V.

SOUTHERN BLOTTING
This technique combines gel electrophoresis and DNA hybridization
to allow researchers to find a specific human gene. It can be used
to identify individual nucleotide differences (mutations) in the DNA
molecule. It can also compare particular DNA fragments from
different sources that were digested by restriction enzymes.
http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapter16/animations.html#
Biotechnology movie clips:
http://www.pubinfo.vcu.edu/secretsofthesequence/playlist_frame.asp
THIS IS THE END OF THE MOLECULAR BIOLOGY UNIT