Gene Expression and Protein Synthesis

GENE EXPRESSION

• A gene's information is converted into the structures and functions of a cell.

• The process of producing a biologically functional molecule of either protein or RNA

(gene product) is made.

• Assumed to be controlled at various points in the sequence leading to protein synthesis.

Transcription

• RNA polymerase makes a copy of information in the gene (complementary RNA)

complementary to one strands of DNA.

Translation

• Occurs on ribosomes, messenger RNA decoded or translated to determine the sequence

of amino acid in the protein being synthesized.

Overview

• Genes in DNA contain information to make proteins.

• The cell makes mRNA copies of genes that are needed.

• The mRNA is read at the ribosomes in the rough ER.

• Protein is produced.

• mRNA carries the information from a gene in DNA.

• Ribosomes, made of rRNA, consist of subunits and carry out an enzyme-like role.

• tRNA carries specific amino acids to the ribosome.

Transcription

• RNA polymerase is crucial because it carries out transcription, the process of copying
DNA (deoxyribonucleic acid, the genetic material) into RNA (ribonucleic acid, a similar
but more short-lived molecule).

• Transcription is an essential step in using the information from genes in our DNA to
make proteins. Proteins are the key molecules that give cells structure and keep them
running. Blocking transcription with mushroom toxin causes liver failure and death,
because no new RNAs—and thus, no new proteins—can be made.

• Transcription is the first step of gene expression. During this process, the DNA sequence
of a gene is copied into RNA.

• Before transcription can take place, the DNA double helix must unwind near the gene
that is getting transcribed. The region of opened-up DNA is called a transcription
bubble.
 • Transcription uses one of the two exposed DNA strands as a template; this strand is
called the template strand. The RNA product is complementary to the template strand
and is almost identical to the other DNA strand, called the non
template (or coding) strand. However, there is one important difference: in the newly
made RNA, all of the T nucleotides are replaced with U nucleotides.

• The site on the DNA from which the first RNA nucleotide is transcribed is called the +1
site, or the initiation site. Nucleotides that come before the initiation site are given
negative numbers and said to be upstream. Nucleotides that come after the initiation
site are marked with positive numbers and said to be downstream.

• If the gene that's transcribed encodes a protein (which many genes do), the RNA
molecule will be read to make a protein in a process called translation.

RNA polymerase

• RNA polymerases are enzymes that transcribe DNA into RNA. Using a DNA template,
RNA polymerase builds a new RNA molecule through base pairing. For instance, if there
is a G in the DNA template, RNA polymerase will add a C to the new, growing RNA
strand.
 • RNA polymerase always builds a new RNA strand in the 5’ to 3’ direction. That is, it can
only add RNA nucleotides (A, U, C, or G) to the 3' end of the strand.

• RNA polymerases are large enzymes with multiple subunits, even in simple organisms
like bacteria. In addition, humans and other eukaryotes have three different kinds of
RNA polymerases: I, II, and III. Each one specializes in transcribing certain classes of
genes.

Transcription Initiation

• To begin transcribing a gene, RNA polymerase binds to the DNA of the gene at a region
called the promoter. Basically, the promoter tells the polymerase where to "sit down"
on the DNA and begin transcribing.

• Each gene (or, in bacteria, each group of genes transcribed together) has its own
promoter. A promoter contains DNA sequences that let RNA polymerase or its helper
proteins attach to the DNA. Once the transcription bubble has formed, the polymerase
can start transcribing.

Promoters in bacteria

• A typical bacterial promoter contains two important DNA sequences, the -10 and -35
elements.

• RNA polymerase recognizes and binds directly to these sequences. The sequences
position the polymerase in the right spot to start transcribing a target gene, and they
also make sure it's pointing in the right direction.

• Once the RNA polymerase has bound, it can open up the DNA and get to work. DNA
opening occurs at the -10 element, where the strands are easy to separate due to the
 many As and Ts (which bind to each other using just two hydrogen bonds, rather than
the three hydrogen bonds of Gs and Cs).

• The -10 and the -35 elements get their names because they come 35 and 10nucleotides

before the initiation site (+1 in the DNA). The minus signs just mean that they are
before, not after, the initiation site.

Promoters in humans

• In eukaryotes like humans, the main RNA polymerase in your cells does not attach
directly to promoters like bacterial RNA polymerase. Instead, helper proteins
called basal (general) transcription factors bind to the promoter first, helping the RNA
polymerase in your cells get a foothold on the DNA.

• Many eukaryotic promoters have a sequence called a TATA box. The TATA box plays a
role much like that of the -10 element in bacteria. It's recognized by one of the general
transcription factors, allowing other transcription factors and eventually RNA
polymerase to bind. It also contains lots of As and Ts, which make it easy to pull the
strands of DNA apart.
Elongation

• Once RNA polymerase is in position at the promoter, the next step of transcription—
elongation—can begin. Basically, elongation is the stage when the RNA strand
gets longer, thanks to the addition of new nucleotides.

• During elongation, RNA polymerase "walks" along one strand of DNA, known as
the template strand, in the 3' to 5' direction. For each nucleotide in the template, RNA
polymerase adds a matching (complementary) RNA nucleotide to the 3' end of the RNA
strand.
 • The RNA transcript is nearly identical to the non-template, or coding, strand of DNA.
However, RNA strands have the base uracil (U) in place of thymine (T), as well as a
slightly different sugar in the nucleotide. So, as we can see in the diagram above, each T
of the coding strand is replaced with a U in the RNA transcript.

• DNA being transcribed by many RNA polymerases at the same time, each with an RNA
"tail" trailing behind it. The polymerases near the start of the gene have short RNA tails,
which get longer and longer as the polymerase transcribes more of the gene.

Transcription termination

• RNA polymerase will keep transcribing until it gets signals to stop. The process of ending
transcription is called termination, and it happens once the polymerase transcribes a
sequence of DNA known as a terminator.

Termination in bacteria

There are two major termination strategies found in bacteria:

a. Rho-dependent

• The RNA contains a binding site for a protein called Rho factor. Rho factor binds to this
sequence and starts "climbing" up the transcript towards RNA polymerase.

• When it catches up with the polymerase at the transcription bubble, Rho pulls the RNA
transcript and the template DNA strand apart, releasing the RNA molecule and ending
transcription. Another sequence found later in the DNA, called the transcription stop
point, causes RNA polymerase to pause and thus helps Rho catch up.^4

b. Rho-independent termination:
 • Depends on specific sequences in the DNA template strand. As the RNA polymerase
approaches the end of the gene being transcribed, it hits a region rich in C and G
nucleotides. The RNA transcribed from this region folds back on itself, and the
complementary C and G nucleotides bind together. The result is a stable hairpin that
causes the polymerase to stall.

• In a terminator, the hairpin is followed by a stretch of U nucleotides in the RNA, which

matches up with A nucleotides in the template DNA. The complementary U-A region of
the RNA transcript forms only a weak interaction with the template DNA. This, coupled
with the stalled polymerase, produces enough instability for the enzyme to fall off and
liberate the new RNA transcript.

What happens to the RNA transcript?

• After termination, transcription is finished. An RNA transcript that is ready to be used in

translation is called a messenger RNA (mRNA). In bacteria, RNA transcripts are ready to
be translated right after transcription. In fact, they're actually ready a little sooner than
that: translation may start while transcription is still going on!

• mRNAs are being transcribed from several different genes. Although transcription is still
in progress, ribosomes have attached each mRNA and begun to translate it into protein.
When an mRNA is being translated by multiple ribosomes, the mRNA and ribosomes
together are said to form a polyribosome.
3 Roles of RNA in Translation

1. Messenger RNA (mRNA) carries the genetic information copied from DNA in the form of a
series of three-base code “words,” each of which specifies a particular amino acid.

2. Transfer RNA (tRNA) is the key to deciphering the code words in mRNA. Each type of amino
acid has its own type of tRNA, which binds it and carries it to the growing end of a polypeptide
chain if the next code word on mRNA calls for it. The correct tRNA with its attached amino acid
is selected at each step because each specific tRNA molecule contains a three-base sequence
that cans base-pair with its complementary code word in the mRNA.

3. Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. These complex
structures, which physically move along an mRNA molecule, catalyze the assembly of amino
acids into protein chains. They also bind tRNAs and various accessory molecules necessary for
protein synthesis. Ribosomes are composed of a large and small subunit, each of which
contains its own rRNA molecule or molecules.

• Translation is the whole process by which the base sequence of an mRNA is used to

order and to join the amino acids in a protein. The three types of RNA participate in this
essential protein-synthesizing pathway in all cells; in fact, the development of the three
distinct functions of RNA was probably the molecular key to the origin of life.

The Genetic Code

• Deciphering the genetic code required determining how 4 nucleotides (A, T, G, C) could
encode more than 20 amino acids.

• Francis Crick and Sydney Brenner determined that the DNA is read in sets of 3
nucleotides for each amino acid.
codon: set of 3 nucleotides that specifies a particular amino acid

reading frame: the series of nucleotides read in sets of 3 (codon)

o only 1 reading frame is correct for encoding the correct sequence of amino acids

o Marshall Nirenberg identified the codons that specify each amino acid.

o RNA molecules of only 1 nucleotide and of specific 3-base sequences were used to
determine the amino acid encoded by each codon.

o The amino acids encoded by all 64 possible codons were determined.

Stop codons: 3 codons (UUA, UGA, UAG) in the genetic code used to terminate translation

Start codon: the codon (AUG) used to signify the start of translation

o The remainder of the code is degenerate meaning that some amino acids are specified
by more than one codon.

Translation

• Translation involves “decoding” a messenger RNA (mRNA) and using its information to

build a polypeptide, or chain of amino acids. For most purposes, a polypeptide is
basically just a protein (with the technical difference being that some large proteins are
made up of several polypeptide chains).
 • In translation, the codons of an mRNA are read in order (from the 5' end to the 3' end)
by molecules called transfer RNAs, or tRNAs.

• Each tRNA has an anticodon, a set of three nucleotides that binds to a matching mRNA
codon through base pairing. The other end of the tRNA carries the amino acid that's
specified by the codon.

[What are 5', 3', and base pairing?]

• tRNAs bind to mRNAs inside of a protein-and-RNA structure called the ribosome. As

tRNAs enter slots in the ribosome and bind to codons, their amino acids are linked to
the growing polypeptide chain in a chemical reaction. The end result is a polypeptide
whose amino acid sequence mirrors the sequence of codons in the mRNA.

Stages of Translation

• Initiation ("beginning"): in this stage, the ribosome gets together with the mRNA and
the first tRNA so translation can begin.

• Elongation ("middle"): in this stage, amino acids are brought to the ribosome by tRNAs
and linked together to form a chain.
 • Termination ("end"): in the last stage, the finished polypeptide is released to go and do
its job in the cell.

Initiation

• A ribosome (which comes in two pieces, large and small)

• An mRNA with instructions for the protein we'll build

• An "initiator" tRNA carrying the first amino acid in the protein, which is almost always
methionine (Met)

• During initiation, these pieces must come together in just the right way. Together, they
form the initiation complex, the molecular setup needed to start making a new protein.

Inside the cells (and the cells of other eukaryotes), translation initiation goes like this:

1. The tRNA carrying methionine attaches to the small ribosomal subunit.

2. Together, they bind to the 5' end of the mRNA by recognizing the 5' GTP cap (added
during processing in the nucleus).

3. Then, they "walk" along the mRNA in the 3' direction, stopping when they reach the
start codon (often, but not always, the first AUG).
  In bacteria, the situation is a little different. Here, the small ribosomal subunit doesn't
start at the 5' end of the mRNA and travel toward the 3' end. Instead, it attaches directly
to certain sequences in the mRNA. These Shine-Dalgarno sequences come just before
start codons and "point them out" to the ribosome.

Why use Shine-Dalgarno sequences?

• Bacterial genes are often transcribed in groups (called operons), so one bacterial mRNA
can contain the coding sequences for several genes. A Shine-Dalgarno sequence marks
the start of each coding sequence, letting the ribosome find the right start codon for
each gene.
Elongation

• Elongation is when the polypeptide chain gets longer.

• Methionine-carrying tRNA starts out in the middle slot of the ribosome, called the P site.
Next to it, a fresh codon is exposed in another slot, called the A site. The A site will be
the "landing site" for the next tRNA, one whose anticodon is a perfect (complementary)
match for the exposed codon.

• Once the matching tRNA has landed in the A site, it's time for the action: that is, the
formation of the peptide bond that connects one amino acid to another. This step
transfers the methionine from the first tRNA onto the amino acid of the second tRNA in
the A site.

• The methionine forms the N-terminus of the polypeptide, and the other amino acid is
the C-terminus.

• Once the peptide bond is formed, the mRNA is pulled onward through the ribosome by
exactly one codon. This shift allows the first, empty tRNA to drift out via the E ("exit")
site. It also exposes a new codon in the A site, so the whole cycle can repeat.
Termination

• Polypeptides, like all good things, must eventually come to an end. Translation ends in a
process called termination. Termination happens when a stop codon in the mRNA (UAA,
UAG, or UGA) enters the A site.

• Stop codons are recognized by proteins called release factors, which fit neatly into the P
site (though they aren't tRNAs). Release factors mess with the enzyme that normally
forms peptide bonds: they make it add a water molecule to the last amino acid of the
chain. This reaction separates the chain from the tRNA, and the newly made protein is
released.

• After the small and large ribosomal subunits separate from the mRNA and from each
other, each element can (and usually quickly does) take part in another round of
translation.

Our polypeptide now has all its amino acids—does that mean it's ready to do its job in the
cell?

• Polypeptides often need some "edits." During and after translation, amino acids may be
chemically altered or removed. The new polypeptide will also fold into a distinct 3D
structure, and may join with other polypeptides to make a multi-part protein.

• Many proteins are good at folding on their own, but some need helpers ("chaperones")
to keep them from sticking together incorrectly during the complex process of folding.

• Some proteins also contain special amino acid sequences that direct them to certain
parts of the cell. These sequences, often found close to the N- or C-terminus, can be
thought of as the protein’s “train ticket” to its final destination. For more about how this
works, see the article on protein targeting.

Gene regulation in bacteria

• Cells vary amount of specific enzymes by regulating gene transcription

o turn genes on or turn genes off

• turn genes OFF example

if bacterium has enough tryptophan then it doesn’t need to make
enzymes used to build tryptophan
 • turn genes ON example
if bacterium encounters new sugar (energy source), like lactose, then it
needs to start making enzymes used to digest lactose

Regulation of Gene Expression in Prokaryotes

• Operons: a cluster of genes grouped together under the control of one promoter

 Remember that a promoter is where RNA polymerase binds to DNA to begin

transcription

 Occurs in prokaryotic genomes

• Genes that are involved in the same metabolic pathway are often found in the same
operon

 All under the control of the same promoter region

 Thus these genes are transcribed all together into one continuous mRNA strand:
polycistronic mRNAProteins are then synthesized from that mRNA

Bacteria group genes together

• Operon

o genes grouped together with related functions

• example: all enzymes in a metabolic pathway

o promoter = RNA polymerase binding site

• single promoter controls transcription of all genes in operon

• transcribed as one unit & a single mRNA is made

o operator = DNA binding site of repressor protein

So how can these genes be turned off?

• Repressor protein

o binds to DNA at operator site

o blocking RNA polymerase

o blocks transcription

Tryptophan operon

 What happens when tryptophan is present?

 Don’t need to make tryptophan-building enzymes

Tryptophan is allosteric regulator of repressor protein

Lactose operon

 What happens when lactose is present?

 Need to make lactose-digesting enzymes
Lactose is allosteric regulator of repressor protein

Positive Gene Regulation

o If glucose levels are low (along with overall energy levels), then cyclic AMP
(cAMP) binds to cAMP receptor protein (CRP) which activates transcription.

• If glucose levels are sufficient and cAMP levels are low (lots of ATP), then the CRP
protein has an inactive shape and cannot bind upstream of the lac promotor.
Control of Eukaryotic Genes
Points of control

• The control of gene expression can occur at any step in the pathway from gene to
functional protein

1. packing/unpacking DNA

2. transcription

3. mRNA processing

4. mRNA transport

5. translation

6. protein processing

7. protein degradation
1. DNA packing as gene control

• Degree of packing of DNA regulates transcription

o tightly wrapped around histones

• no transcription

• genes turned off

heterochromatin: darker DNA (H) = tightly packed

euchromatin: lighter DNA (E) = loosely packed

DNA methylation
• Methylation of DNA blocks transcription factors

o no transcription

 genes turned off

o attachment of methyl groups (–CH3) to cytosine

• C = cytosine

o nearly permanent inactivation of genes

• ex. inactivated mammalian X chromosome = Barr body

1. Transcription initiation

Control regions on DNA

- promoter

1. nearby control sequence on DNA

2. binding of RNA polymerase & transcription factors

3. “base” rate of transcription

- enhancer

1. distant control
sequences on DNA

2. binding of activator
proteins

3. “enhanced” rate (high level)

of transcription
Model for Enhancer action

• Enhancer DNA sequences

o distant control sequences

• Activator proteins

o bind to enhancer sequence & stimulates transcription

• Silencer proteins

o bind to enhancer sequence & block gene transcription

Post-transcriptional control

• Alternative RNA splicing

o variable processing of exons creates a family of proteins

Protein processing & degradation

• Protein processing

o folding, cleaving, adding sugar groups, targeting for transport

 • Protein degradation

o ubiquitin tagging

o proteasome degradation

Gene Regulation

Mutations

Are Mutations common?

• As scientists learn to read the instructions in our genes, they are discovering that much
of our DNA is riddled with errors.

• We each inherit hundreds of genetic mutations from our parents, as they did from their
forebears.
 • In addition, the DNA in our own cells undergoes an estimated 30 new mutations during
our lifetime, either through mistakes during DNA copying or cell division or, more often,
because of damage from the environment.

When are Mutations Harmful?

• During copying, bits of our DNA may be deleted, inserted, broken, or substituted.

• Most mutations affect only the parts of DNA that do not contain instructions for making
a gene, so we need not worry about them.

• Problems arise only when an error in DNA alters a message that tells certain cells to
manufacture a certain protein.

• Such messages are spelled out in varying sequences of the four chemical bases that
make up DNA: adenine (A), thymine (T), guanine (G), and cytosine (C).

Mutations

• Random chance events and cause changes in the DNA sequence

• If mutations occur in gametes, they can be passed on to offspring

• Mutations may be caused by exposure to toxins or radiation (mutagens).

• Some mutations cause disease, yet others may lead to variations in the organism that
make it better adapted for their environment

There are 2 kinds of Mutations:

1. Chromosomal Mutations
• An abnormal change in the structure of all or part of a chromosome, OR in the
number of chromosomes an organism has
• Ex: normal humans have 46 chromosomes
• Humans with Down Syndrome have 47

Down syndrome (Trisomy 21)

• Extra 21 chromosome

• Effects 1/700

• Alters child’s phenotype– characteristic facial features, short stature

• Usually some degree of mental retardation

 2. Gene Mutation
• A change that affects a gene on a chromosome
• Causes the cell to produce abnormal proteins

There are 2 types of gene mutations:

a. Point Mutation
• A gene mutation involving only a single nucleotide
• Ex: ACA mutates to read ACT
• mRNA codes ACT to be UGA which is a stop codon!
• This will cause the necessary protein to not be made
b. Frame-Shift Mutations
• This is the insertion or deletion of one or more nucleotides
• Ex: THE CAT ATE
• In a frame shift mutation it would read THE ATA TE which is meaningless!
• Which letter was deleted?
• C

Genetic Disorders

• Major types of genetic disorders:

o Autosomal

• Single genes

• Multiple genes

o Sex-linked

o Chromosome abnormalities

Autosomal Disorders

• Autosomal genetic disorders are caused by alleles on autosomes (chromosomes other

than the sex chromosomes)

• Most are recessive (need 2 recessive alleles to have the disorder)

o People with 1 recessive allele are carriers – they do NOT have the disorder but
are able to pass the allele on to their children

o Ex: Cystic fibrosis (CF), sickle cell anemia

 • Can also be dominant (need only 1 allele to have the disorder)

o Ex: Huntington’s disease

Cystic Fibrosis (CF)

• Cystic fibrosis is the most common genetic disorder among white people

• 1 in 2500 white babies are born with CF (4-5 born every day)

• It is estimated that 1 in 20 white people is a carrier for CF

• Caused by an abnormal gene on chromosome 7

• The gene is for a protein pump that uses active transport to regulate the movement of
sodium (Na+) and chloride ions (Cl-) into and out of cells
 • In healthy individuals, the normal protein allows movement of Na + and Cl- ions

o Keeps mucus thin and easily swept away

• With CF, not enough Cl- ions are pumped out

o Thickening of mucus in airways and pancreatic ducts

Symptoms of CF

• Buildup of mucus in the lungs/ respiratory system

• Difficulty breathing

• Infections

• Blocks digestive enzymes (produced by the pancreas) from entering the intestine

• Malnutrition

• Abnormal Na+ transport also results in salty sweat

Treatments for CF

• For respiratory symptoms:

o Physical therapy

o Breathing exercises

o Antibiotics

o Lung transplants in severe cases

• For digestive symptoms:

o Capsules containing pancreatic digestive enzymes

• Even with treatment, CF continues to be fatal, but patients live longer and have a higher
quality of life

Sickle-Cell Anemia (Sickle-Cell Disease)

• The most common genetic disorder among black people

• About 1 in 500 African Americans has sickle-cell anemia.

 • Carriers are said to have sickle-cell trait

• Caused by an abnormal gene on chromosome 11

• The gene is for one of the polypeptide chains in hemoglobin, a protein found in red
blood cells that is responsible for transporting oxygen through the bloodstream

• Sickle-cell anemia causes hemoglobin to clump within red blood cells, which distorts
their shape from the normal biconcave disc to a sickle shape.

• People with sickle-cell trait have some abnormal hemoglobin but do not have the
symptoms of sickle-cell disease.

Treatments for Sickle-Cell Anemia

• Treatments for sickle-cell anemia include:

o Blood transfusions

o Antibiotics
 o Drugs that increase oxygen-carrying capacity of RBCs

o Drugs that “switch on” the gene for fetal hemoglobin, which is normally switched
off after birth

o Living with sickle-cell anemia

Heterozygote Superiority

• Why are cystic fibrosis and sickle-cell anemia so common?

• Sickle-cell anemia is most common in areas of the world where malaria is prevalent

• Malaria is caused by a parasite that invades red blood cells

• These parasites do not thrive in people with abnormal hemoglobin, so people

with sickle-cell trait (who are heterozygous) are resistant to malaria.

• People who are heterozygous for the cystic fibrosis allele may be more resistant to
cholera

• When carriers have an advantage over people who are homozygous dominant, it is
called heterozygote superiority

Huntington’s Disease

• Caused by an abnormal dominant allele (unlike most human genetic disorders)

• Both men and women need only one Huntington’s allele to get the disorder.

Symptoms of Huntington’s Disease

Huntington’s disease affects a person’s brain cells

o Clumsiness

o Irritability

o Depression
 o Memory loss

o Loss of muscle coordination & ability to speak

• Symptoms normally appear by age 40

• Huntington’s disease is always fatal

o Death normally occurs within 20 years of the onset of symptoms

Living with Huntington’s

Multiple Genes

• Cystic fibrosis, sickle-cell disease, and Huntington’s disease are all caused by mutant
alleles for a single gene.

• Many other genetic disorders are believed to be the result of multiple genes:

o Diabetes mellitus

o Heart disease

o Some personality disorders

• Bipolar disorder, schizophrenia

• These are much more complicated to analyze than disorders caused by single genes

Sex-Linked Disorders

• Sex-linked disorders are almost always caused by mutant alleles on the X chromosome

o Hemophilia
 o Red-green colorblindness

• Women can be carriers, but men cannot

Hemophilia

• Hemophilia is caused by an abnormal gene for a blood clotting factor (clotting factor
VIII)

• Blood does not clot normally, so even a tiny cut can result in excessive bleeding

• Internal bleeding is a major concern

o Most common around joints

• Hemophiliacs bruise very easily

Red-Green Colorblindness

• Red-green colorblindness is caused by an abnormal gene for photoreceptors in the

retina

• The genes for both red and green photoreceptors are located on the X chromosome –
colorblindness can result from recessive alleles for either one or both of these genes

Photoreceptor Cells

Chromosome Abnormalities

• Autosomal and sex-linked genetic disorders are both caused by certain alleles – small
segments of DNA that make up part of a chromosome

• Other genetic disorders result from chromosome abnormalities caused by mistakes

made during meiosis.

o May change the number or structure of chromosomes within gametes

Nondisjunction

• Nondisjunction is the failure of a pair of chromosomes to separate during meiosis

o Results in one gamete having too many chromosomes and the other too few

o Trisomy – a zygote gets 3 copies of a chromosome

o Monosomy – a zygote gets only 1 copy of a chromosome

Translocation

• Translocation is when a piece of one chromosome breaks off and attaches to a different
chromosome

o Often happens to 2 chromosomes at once

Karyotypes

• Both nondisjunction and translocation can be detected in karyotypes

 • A karyotype is made from taking individual pictures of all of a human’s chromosomes
and matching up homologous pairs

Down syndrome

• Down syndrome - a genetic disorder caused by chromosome abnormality

• Nondisjunction – the person has an extra copy of chromosome 21

• Called trisomy 21

• Translocation – most of chromosome 21 breaks off during meiosis and fuses with
another chromosome, usually #14

• This cause of Down syndrome is most likely to occur in children born to mothers over
age 40
 • Symptoms of Down syndrome include:

o Mild to severe mental retardation

o Short stature

o Heart, vision, and intestinal problems

o Susceptibility to infections and leukemia

Congenital Disabilities

• Congenital disabilities are different from genetic disorders

o Not inherited

o Occur during fetal development

• Both genetic disorders and congenital disabilities can often (but not always) be detected
before a baby is born

Genetic Counseling
 • Genetic counseling can help parents determine the likelihood of their child being born
with a genetic disorder

o Genetic counselors study the family histories of both parents

• Create pedigree charts to trace the passage of traits

o Medical geneticists analyze blood tests to determine if parents are carriers of

certain genetic disorders

• Genetic counseling usually can NOT determine whether or not a child will be born with a
genetic disorder

Diagnosing Genetic Disorders

• There are several ways to determine whether a child will have a genetic disorder

• Two main ways to diagnose:

o Analysis of fetal cells

• Amniocentesis

• Chorionic villus biopsy

o Imaging techniques

• Ultrasonography (computerized image)

• Fetoscopy (direct observation)

Amniocentesis

• Amniocentesis

o Amniotic fluid is the fluid that surrounds a fetus inside the uterus

• Also contains fetal cells

o A sample of amniotic fluid is taken and cells are grown in a lab

• Can be used to make a karyotype – takes 10 days to grow enough cells

o Detects chromosome abnormalities

• Can be analyzed for defective alleles

 o Detects other genetic disorders

o Cannot be conducted until the 14th week of pregnancy

Chorionic Villus Biopsy

• Chorionic villus biopsy

• Chorionic villi are structures that help maximize the surface area for nutrient and
gas exchange between a mother and developing fetus (they are part of the
placenta)

• The villi develop from fetal cells and therefore have the same chromosomes as
the fetus & amniotic fluid

• A sample of these cells can be taken and analyzed as in amniocentesis

• Karyotyping

• Tests for recessive alleles

  Can be done as early as the 9th week of pregnancy

Ultrasonography

• Uses high-frequency sound waves which bounce off of tissue

• Depending on the density of tissue, waves “echo” back at different wavelengths and are
used to produce a computerized image called an echogram

• Used in most pregnancies to detect the position and anatomy of the fetus

• Used with amniocentesis to reduce risk of injury

• Can also help doctors detect abnormalities such as congenital heart defects

Fetoscopy

• A small incision is made in a pregnant woman’s abdomen

• An endoscope tube is inserted through the incision

o Has a camera on the end that shows an image on a monitor

o Instruments can be inserted through the endoscope to perform additional

procedures
Developing Cures for Genetic Disorders

• Gene therapy

o Introducing normal genes into the cells of people with defective alleles

• Using viruses to inject alleles into cells

• Enclosing alleles in droplets of fat, which are taken into cells by

endocytosis

o Currently these are still experimental procedures and have had limited success

RECOMBINANT DNA

INTRODUCTION

• Recombinant DNA (rDNA):  DNA molecules formed by laboratory methods of genetic

recombination (such as molecular cloning) to bring together genetic material from
multiple sources, creating sequences that would not otherwise be found in the genome.

• rDNA is possible because DNA molecules from all organisms share the same chemical
structure. They differ only in the nucleotide sequence within that identical overall
structure.

• rDNA is the general name for a piece of DNA that has been created by the combination
of at least two strands. rDNA molecules are sometimes called chimeric DNA, because
they can be made of material from two different species, like the mythical chimera. R-
 DNA technology uses palindromic sequences and leads to the production of sticky and
blunt ends.

What is Recombinant DNA

• This is DNA that has been formed artificially by combining constituents from different
organisms.

Recombinant DNA Technology

• Using Recombinant DNA technology, we can isolate and clone single copy of a gene or a
DNA segment into an indefinite number of copies, all identical. These new combinations
of genetic material or Recombinant DNA (rDNA) molecules are introduced into the host
cells, where they propagate and multiply. The technique or methodology is called
Recombinant DNA technology.

Obtaining rDNA

• Step 1: The DNA fragment containing the gene sequence to be cloned (also known as
insert) is isolated.

• Step 2: Cutting DNA.

• Step 3: Joining DNA

• Step 4: Insertion of these DNA fragments into host cell using a “vector” (carries DNA
molecule).

• Step 5: The rDNA molecules are generated when the vector self replicates in the host
cell.

• Step 6: Transfer of the rDNA molecules into an appropriate host cell.

• Step 7: Selection of the host cells carring the rDNA molecule using a marker.

• Step 8: Replication of the cells carrying rDNA molecules to get a genetically identical
cells or clone.

Isolation

• The first step in making recombinant DNA is to isolate donar and vector DNA. The
procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial
plasmids are commonly used vectors, and these plasmids must be purified away from
the bacterial genomics DNA.
Ultracentrifugation

• A protocol for extracting plasmids DNA can be achieved by ultracentrifugation Plasmids

DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient
containing ethidium bromide. The plasmid band is collected by punching a hole in the
plastic centrifuge tube.

Alkaline Lysis

• Another protocol relies on the observation that, at a specific alkaline pH, bacterial
genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the
genomic DNA, but plasmids stay in solution. Phages can also be used as vectors for
cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of
phages recovered from a phage lysate.

Cutting DNA

• The restriction enzymes EcoRi cuts a circular DNA molecule bearing one target
sequence, resulting in a linear molecule with single stranded sticky ends.

Insertion
 • Choosing a gene Cloning Vector

• A vector is any DNA molecule which is capable of multiplying inside the host to which
our gene of interest is integrated for cloning. In this process restriction enzyme function
as scissors for cutting the DNA molecule. Ligase enzyme is the joining enzyme that join
the vector DNA with the gene of interest this will produce the recombinant DNA.

Introducing Vector DNA into Host Cell

• Plasmid Vector

• The vector is added to a flask containing a culture of E.coli.

• Calcium ions usually in the form of calcium chloride are added to the flask followed by a
brief heat shock.

• This allows holes to briefly appear in the cell surface membrane of the E.coli making it
permeable to DNA and allowing the plasmids to enter.

Phage Vectors

• Introduced by infection of bacterial lawn growing on an agar plate.

• The culture or growth of viruses is made more difficult than the culture of bacteria or
fungi by the fact that viruses will only grow.

Placing the Gene in the vector

• Plasmid DNA

• DNA molecule are small and can be easily separated based on the size.

• Bacterial cells are broken open and chromosomal DNA is centrifuged down.

• This leaves the plasmid DNA in the liquid above the pellet.

• The plasmid are purified before cutting with a restriction enzyme.

• Restriction fragments from donar DNA are mixed with plasmid DNA and joined by their
sticky ends, the initial attraction is due to the hydrogen bonds, but the sugar phosphate
backbone is then joined using and enzyme called DNA ligase.

Example of use of Recombinant DNA Technology

• Insulin Production
• The DNA for insulin is first isolated

• A plasmid made of DNA is removed from a bacterial cell

• A restriction enzyme cuts the plasmid DNA open, leaving sticky ends.

• The insulin gene, with complementary sticky ends is added.

• DNA ligase enzyme splices (joins) together the plasmid DNA and the insulin DNA.

• The plasmid (now genetically modified) is inserted back into the bacterium.

• The bacterium host cell, divides and produces copies of the plasmid.

• The Bacterium makes human insulin using the gene in the plasmid.

• The insulin is extracted from the bacterial culture.

Joining
DNA
 Production of an Insulin

Applications

• Preparation of gene maps.

• In revealing details of various infections, diseases such as "inborn errors of metabolism."

• Finding out the complete nucleotide sequence of genome of an organism and

identification of genes.

• Detecting cytogenetic abnormalities e.g. Down's syndrome, multifactorial disorders,

atherosclerosis, coronary artery disease etc.

• Preventing various genetic disorders e.g. inherited haemoglobin disorders,

phenylketonuria, retinoblastoma etc.

• Understand a molecular event is biological processes like growth, differentiation, ageing

etc.

• Replacement or correction of deleterious mutation by transfer of clone gene in a

patient.

• Production of genetically modified organisms (GMOs) or transgenic organisms for

providing particular product and nutrient.

• Gene Therapy: Removal and replacement of defective genes with normal healthy
functional genes is known as gene therapy e.g. Sickle cell anaemia, Severe Combined
Immuno-Deficiency (SCID). SCID is due to a defect in the gene for the enzyme adenosine
deaminase (ADA) in 25 per cent of the cases.

• It has several negative features as well: extensive erosion and genetic destruction of
plant Germplasm; ecological imbalance; production of monsters; production of
dangerous toxic chemicals, production of highly lethal microbes and their use in
microbiological warfare to kill humans, animals and plants.