Lecture Test 3

Genetic Engineering

Adding/deleting genes from an organism’s genome

Transgenic Organism

transplanting genes from one organism to another

Arabinose Operon

Present in bacteria consists of genes that are required to metabolize arabinose

When NO arabinose is present

araC (repressor protein) binds to the arabinose operon → RNA polymers cannot bind so NO transcription

When arabinose is present

araC (repressor protein) binds to the arabinose operon →conformational change of ara C allows RNA polymerase to bind → which allows transcription to occur

Modified Arabinose Operon

araC protein binds to the operator → NO transcription, When arabinose binds to araC it changes conformation → GFP gene is transcribed and translated

PGLO Plasmid

small circular DNA; autonomously replicates

3 genes of interest

• GFP gene

• BLA gene

• araC gene

GFP gene

Codes for the GFP protein

Bla gene

• Codes for the enzyme b-lactamase

• B-lactamase destroys ampicillin

Bacterial Transformation

• Putting pGLO plasmid inside E. coli

• Add CaCl2 to make cells “competent”

• Ca+ shields –P of DNA and of phospholipids in membrane

  • This neutralizes the lipids and DNA that they do not repulse each other

    • This allows DNA to travel across membrane easily

•  Cl enters with water and swells cell

•  Incubate on ice

  • Slows fluid membrane

• Heatshock

  • Increases permeability of membrane creating small pores

  • DNA can cross membrane

• Recover at lower temperature in LB media

  • Allows membrane pores to close

  • Allows cells to grow and those that have pGlo plasmid DNA to express b-lactamase

Restriction enzymes

acts like molecular scissors, making cuts at specific sequence of base pairs that it recognizes 

• Used to open up a plasmid in preparation for the insertion of a gene

• In a follow-up reaction, gene fragments, cut plasmids and an enzyme that connects DNA fragments are mixed together

Sticky Ends

Uneven ends left by restriction enzymes

• Will be complementary if gene and plasmid are both cut with same enzyme

In this

(a) six nucleotide restriction enzyme recognition site, notice that the sequence of six nucleotides reads the same in the 5' to 3' direction on one strand as it does in the 5' to 3' direction on the complementary strand. This is known as a palindrome.

(b) The restriction enzyme makes breaks in the DNA strands, and

(c) the cut in the DNA results in “sticky ends”. Another piece of DNA cut on either end by the same restriction enzyme could attach to these sticky ends and be inserted into the gap made by this cut.

Ligase

attaches sticky ends in the solution to produce circular plasmids that now carry the gene that codes for the desired protein

Polymerase Chain Reaction (PCR)

Used to produce many copies of a specific sequence of DNA using a special form of DNA polymerase

Amplification of DNA segments makes possible the detection of pathogenic virus or bacteria. identification of individuals ( DNA fingerprinting), and several scientific research involving DNA manipulation

Target DNA, primers, polymerase, and nucleotides are combined in a tube, inserted into thermal cycler

Denaturation

First step of PCR

splits strands of DNA apart

Annealing

Second step of PCR

Primers (Starting point for DNA replication) bind to DNA

Extension

Third step of PCR

Taq polymerase (heat resistant enzyme) extends upon the primer

Adaptive Immunity

B Cells and T Cells fight microbes and record information about them, creating a memory of how they work to fight over future exposure

Live Attenuated Vaccines

• Made of pathogen itself but a much weaker and tamer version

• Difficult to make

• Can’t given to immunocompromised

Inactive Vaccines

Pathogens have been killed

Subunit Vaccine

Made from antigens (what triggers immune response)

DNA Vaccine

• Isolate very genes that make specific antigens the body needs to trigger immune response

• Genes instruct cells to make antigens 

• Doesn’t contain any other ingredients from rest of pathogen

Goals of Biotechnology in Agriculture

1. Create more nutritious foods

2. Increase the efficiency of farming

3. Reduce the ecological impact of farming

Potential Benefits of GMOs

• Many plants have had insecticides and herbicides engineered into them

  • Can reduce the amounts of pesticides and herbicides sprayed on your food

• These genetically modified plants can reduce the costs of producing food and the loss of topsoil to erosion

• May be better able to adapt to changes in the climate

• Feeding a global population

• May increase yield

Concern: They are bad for the environment

Many GMOs designed to actually mitigate problems like climate change and eutrophication

Concern: Organisms that we want to kill may become invincible

Concern for conventional farming too and byproduct of evolution

Concern: Organisms that we don’t want to kill may be killed inadvertently

Concern for conventional farming too

Concern: GMOs not tested or regulated adequately

Multiple agencies to regulate and test our crops

Long term studies possible without time?

What is toxic to one organism may be fine for another- Most people LOVE chocolate and it is dangerous for dogs.

Concern: Loss of Biodiversity

Some studies show increased biodiversity in fields where GMOs are used

Concern: No long term studies

Not possible until quite frankly more time has passed.

Concern: Loss of genetic diversity among crop plants

What happens when we rely on one type of crop?

Concern: Patenting Genes

The business side is something we should always keep an eye on!

Concern: Unforeseen consequences

Always going to be true. We can not predict the future.

CRISPIR

Clustered regularly interspaced short palindromic repeats

CAS

• Chop DNA like scissors

• When virus invades bacterium, CAS proteins cut out a segment of the viral DNA to stitch into bacterium’s CRISPR region

• Viral codes then copied into short pieces of RNA, binding to CAS9, latching to free floating genetic material and looks for a match to the virus, recognizes and destroys viral DNA

CRISPIR

• Scientists design a “guide” RNA to match the gene to what they want to edit and attach to CAS 9

• Guide RNA directs CAS9 to target gene and snip the DNA

• When gene is cut, cell tries to repair it

• Nonhomologous end joining: nucleases trim broken ends and join them together

  • Prone to mistakes with extra or missing bases

  • Resulting gene is usually unusable and turned off

• Scientists add a separate sequence of template DNA, cellular proteins can perform homology directed repair. Template DNA guides rebuilding process

Cell division takes place because

• Cells die and need to be replaced 

• To allow organisms to increase in size

  • Cells can only grow so large before they become dysfunctional

• There are times in which an organism needs quantities of new above “replacement” level, e.g. injury

Why do cells need to divide?

Prokaryotic Chromosomes

Double stranded DNA

Single, round chromosome

Replicate through binary fission

Step 1 Binary Fission

Replication of the circular prokaryotic chromosome begins at the origin of replication and continues in both directions at once

Step 2 Binary Fission

The cell begins to elongate. FtsZ protein migrate toward the midpoint of the cell

Step 3 Binary Fission

The duplicated chromosomes separate and continue to move away from each other toward opposite ends of the cell. FtsZ proteins form a ring around the periphery of the midpoint between the chromosomes

Step 4 Binary Fission

The FtsZ ring directs the formation of a septum that divides the cell. Plasma membrane and cell wall materials accumulate

Step 5 Binary Fission

After the septum is complete, the cell pinches in two, forming two daughter cells. FtsZ is dispersed throughout the cytoplasm of the new cells

homologous

that is same in size and function, but not exactly alike

Somatic Cells

cells forming the body of the organism

Gametes (Reproductive Cells)

S3x cells

Key Players of Binary Fission

• A replication fork is formed when helicase separates the DNA strands at the origin of replication

• Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up

• Single-strand binding proteins (ssb) bind to the single-stranded DNA to prevent the helix from re-forming 

• Primase synthesizes an RNA primer

• DNA polymerase III uses this primer to synthesize the daughter DNA strand

• DNA polymerase I replaces the RNA primer with DNA 

• DNA ligase seals the gaps between the Okazaki fragments, joining the fragments into a single DNA molecule

Telomerase

• The ends of linear chromosomes are maintained by the action of the telomerase enzyme

• Telomerase has an associated RNA that complements the 3’ overhang at the end of the chromosome 

• The RNA template is used to synthesize the complementary strand 

• Telomerase shifts and the process is repeated 

• Primase and DNA polymerase synthesize the complementary strands

Editing

Proofreading by DNA polymerase (a) corrects errors during replication. In mismatch repair

(b), the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base. Nucleotide excision

(c) Repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

Interphase

G1 involves cell growth and protein synthesis

S phase involves DNA replication and the replication of the centrosome,

G 2 involves further growth and protein synthesis.

Mitosis

Nuclear division during which duplicated chromosomes are segregated and distributed into daughter nuclei.

Cytokinesis

The cell will divide after mitosis in a process called in which the cytoplasm is divided and two daughter cells are formed.

Prophase

• Chromosomes condense and become visible

• Spindle fibers emerge from the centrosomes 

• Nuclear envelope breaks down

• Centrosomes move toward opposite poles

Prometaphase

• Chromosomes continue to condense 

• Kinetochores appear at the centrosomes 

• Mitotic spindle microtubules attach to kinetochores

Metaphase

Chromosomes are lined up at the metaphase plate

Each sister chromatid is attached to a spindle fiber originating from opposite poles

Anaphase

• Centromeres split in two

• Sister chromatids (now called chromosomes are pulled toward opposite poles

• Certain spindle fibers begin to elongate the cell

Telophase

• Chromosomes arrive at opposite poles and begin to decondense 

• Nuclear envelope material surrounds each set of chromosomes

• The mitotic spindle breaks down

• Spindle fibers continue to push poles apart

Cytokinesis

Animal Cells

• A cleavage furrow separates the daughter cells

• a cleavage furrow forms at the former metaphase plate in the animal cell. The plasma membrane is drawn in by a ring of actin fibers contracting just inside the membrane. 

• The cleavage furrow deepens until the cells are pinched in two.

Cytokinesis

Plant Cells

• A cell plate, the precursor to a new cell wall, separates the daughter cells

• Golgi vesicles coalesce at the former metaphase plate in a plant cell.

• The vesicles fuse and form the cell plate. The cell plate grows from the center toward the cell walls. New cell walls are made from the vesicle contents.

G1 Checkpoint

Integrity of the DNA is assessed

G2 Checkpoint

Proper chromosome duplication is assessed at the checkpoint.

M Checkpoint

Attachment of each kinetochore to a spindle fiber

Benign

• Masses of normal cells 

• Self-contained; localized 

• Can be removed 

• Ex: moles and warts

Malignant

Cancerous cells

Continuously replicate

Metastasis

Spreading of cancer

Cancer cells shed and spread to other parts of the body

Apoptosis

• The apoptotic program is hardwired into every single cell in our body. It is like a cyanide capsule, swiftly delivering death if the circumstances require cellular suicide

• If a cell detects that it has damaged DNA, it can activate apoptosis to remove itself from the population

• It is an entirely normal function of cells 

• Apoptosis is an extremely tidy process; the dying, shrinking cell is swallowed up by a neighboring cell or a patrolling immune cell, leaving no trace of the cellular suicide behind.

Haploid

Cells that have 1 copy of each chromosome

Human gametes

1 set of chromosomes= 23 chromosomes

Diploid

• Cells that have 2 copies of each chromosome

• Human somatic cells 

• 2 sets of chromosomes = 46 chromosomes 

DNA Synthesis: Both

Occurs in S phase of interphase

Synopsis of homologous chromosomes: Meiosis

During prophase I

Synopsis of homologous chromosomes: Mitosis

Does not occur

Crossover: Meiosis

During prophase I

Homologous chromosomes fire up at metaphase plate: Meiosis

During metaphase I

Homologous chromosomes fire up at metaphase plate: Mitosis

Does not occur

Sister chromatids line up: Meiosis

During metaphase II

Sister chromatids line up: Mitosis

During metaphase

Number and generic comparison of daughter cells: Meiosis

Four haploid cells at the end of meiosis II

Number and generic comparison of daughter cells: Mitosis

Two diploid cells at the end of mitosis

Crossover

occurs between non-sister chromatids of homologous chromosomes. The result is an exchange of genetic material between homologous chromosomes.

Recombinant

The chromosomes that have a mixture of maternal and paternal sequence

Non-recombinant

the chromosomes that are completely paternal or maternal

Crossing Over

In prometaphase I, microtubules attach to the fused kinetochores of homologous chromosomes. In anaphase I, the homologous chromosomes are separated. In prometaphase II, microtubules attach to individual kinetochores of sister chromatids. In anaphase II, the sister chromatids are separated.

Meiosis Main Points

The homologues separate into two new cells

These two new cells divide again, separating the sister chromatids into two more cells

Karyotype

A display of an individual’s complete set of chromosomes

Nondisjunction

occurs when homologous chromosomes (meiosis I) or sister chromatids (meiosis II) fail to separate during meiosis.

Genotype

Internally coded, inheritable information carried by all living organisms

phenotype

Outward, physical manifestation of the organism

Allele

Alternative form of a gene (one member of a pair) that is located at a specific position on a chromosome.

These DNA codings determine distinct traits that can be passed on from parents to offspring

Homozygous

Having identical alleles for a single trait

Heterozygous

Having two different alleles for a single trait

Dominance

A relationship between two alleles of a single gene, in which one allele masks the effects of the other in influencing some trait

Recessive

an allele that causes a phenotype that is only seen in a homozygous genotype and never in a heterozygous genotype.

Independent Assortment

genes don’t affect each other

Incomplete dominance

one allele does not completely mask the effects of another when both are present

A cross between organisms with two different phenotypes produces offspring with a third phenotype that is a blending of the parental traits.

Codominance

The heterozygote displays characteristics of both homozygotes

If someone is heterozygote for sickle cell disease some blood cells will display the sickle cell shape and others will not.

A cross between organisms with two different phenotypes produces offspring with a third phenotype in which both of the parental traits appear together

Multiple Allelism

A single gene has more than two possible alleles

Each individual still carries only two alleles

Ex: Blood Type

Antigen

Does antibodies or antigens match the blood type (ex: Does Blood Type A have A antigens or A antibodies

Polygenic Traits

A trait that is influenced by many genes

• Height, skin color, eye color, intelligence

Pleiotropy

One gene influences multiple, different traits

Most, if not all, genes are pleiotropic

Sickle cell trait not only impacts the shape of your blood cells but also your resistance to malaria

Sex-Linked Traits

• genes associated on one of the sex chromosomes (X or Y) but not the other

• This mode of inheritance is in contrast to the inheritance of traits on autosomal chromosomes, where both sexes have the same probability of inheritance. Since humans have many more genes on the X chromosome than the Y, there are many more X-linked traits than Y-linked traits.

• Examples: color blindness, Duchenne’s muscular dystrophy, hemophilia.