BioTech and Cell Cycle Notes

Recombinant DNA

Dna in which genes from different sources are linked

Example of Recombinant DNA

the “green” mice

Genetic engineering

the direct manipulation of genes for practical purposes

Example of Genetic Engineering

using e.coli to produce insulin for diabetes treatment

Biotechnology

the use of living organisms or their components to perform practical tasks

Ex: use of bacteria to digest oil spills

Restriction Enzymes

cut dna at specific nucleotide sequences called “restriction sites”—> used to “cut and splice” dna obtained from bacteria

Plasmids

a small rig of independent replicating dna seperate from main chromosomes

• found in prokaryotes and yeast

• serves as a “vehicle” for transporting genes

Steps for plasmid use

1. get the dna for the trait

2. insert dna into the plasmid

3. bacterial transformation

4. identification of the new trait

insertion

placing foreign dna into a plasmid

• open plasmid with enzymes to create “sticky ends”

• splice the new dna and plasmid together

transformation

placing the plasmid into a bacterial cell

methods of transformation

template shock and salt treatment

• electric current

• injection

identification of transformation

screening the altered cells for the desired gene

Ex: antibiotic sensitivity(expression of a “new” trait-color or glowing)

Example of applications for transformation

1. insulin

2. human growth hormone

3. other proteins

DNA sources

1. organism- use section of their chromosomes

2. c DNA- complementary dna

   • created copy of dna from the mrna transcript to avoid introns

   • uses reverse transcriptase

Reverse Transcriptase

reverse transcribes mrna

• mrna—>dna

nucleic acid probes

used to find specific dna sequence in a mixture of dna pieces

Steps of Nucleic acid probes

1. dna is denatured to produce pieces

2. piece of complement dna is added as a “probe” the probe has been “labeled”

3. look for where the probe gets into the dna sample(DNA microarray)

dna microarray

used to separate pieces into wells

• well that is “marked” will hold the correct piece of dna

polymerase chain reaction

method for making many copies of a specific segment of dna

Method of polymerase chain reaction

1. seperate strands by heating(denature dna)

2. cool slightly

3. build new strand from primers and nucleotides

4. repeat

Importance of polymerase chain reaction

can amplify ANY dna with as little as one original copy

gel electrophoresis

used to seperate mixtures of dna or proteins according to size

• smaller pieces will move further

RFLP(restriction fragment length polymorphisms)

• method for detecting minor differences in dna structure between individuals

• common in dna fingerprinting

Method for Gels

1. digest dna with restrictive enzymes

2. separate pieces by gel electrophoresis

3. identify sequences with probes

Results of Gel

patterns of dna markers or dna fingerprint

• markers are inherited and can show relationships

  • pedigree studies

cloning of organisms

reproducing an organism by asexual means

• comaaly in plants

• concept of totipotency

totipotency

a single cell can develop into a new organism

cloning in animals

has been done by nuclear transplantation

steps of cloning

1. somatic cell is taken from donor parent and nucleus is removed

2. unfertilized egg is taken from surrogate parent and the nucleus is removed and replaces with the somatic nucleus

3. the egg is now grown in vitro until it is an embryo

4. embryo is placed in the womb or the surrogate mother

5. embryo develops

6. the new clone is born(identical to the donor mother)

therapeutic cloning

goal is to produce a group of certain cells not an organism

• start with embryotic stem cells

stem cells

• embryotic- from an embryo

• adult- found in various tissues of the adult body

research of stem cells

trying to use stem cells to replace damaged cells or body parts

dna tech applications

basic research, medical, forensics, agriculture

basic research

• dna and protein studies

• evolution

• gene structure and control mechanisms

medical research

1. diagnosis of diseases

2. gene therapy

3. vaccines

4. pharmaceutical products

forensics research

1. dna fingerprints for crime solving

2. dna identification records- standard for military

agricultural research

1. animal- increased milk production, feed utilization, and meat production

2. plants- herbicide resistance, retard spoilage of fruits, insect resistance(BT corn), nitrogen-fixation ability

genetically modified organism(GMO)

produced by direct genetic manipulation, not traditional

• from just approved sale of gmo animal products for consumption

“Omnis cellula e cellula”

every cell from a cell

roles of cell division

reproduction/ replace, growth, repair

must end up with

2 cells

genome

the cell’s hereditary endowment of dna

chromosomes

made of dna and protein complex called a chromatin

chromatids(sister chromatids)

each half of a duplicated chromosome

centromere

point where 2 sister chromatids are connected

goal of cell division

to split the sister chromatids and give one to each new cell

cell cycle parts

1. interphase

2. mitotic phase

Interphase

90% off the cell cycle— when the cell grows and duplicated the chromosomes

Mitotic Phase

when the chromosomes are split into separate cells

Interphase parts

G1:cell grows and carries out regular biochemical functions

S: when dna is replicated or synthesized and chromosomes are replicated

G2: cell completes preparations for division

mitosis

division of replicated chromosomes

cystokenesis

division of the cell’s cytoplasm

cytokinesis in animals

• cleavage furrow forms

• microfilaments contract into 2 parts

cytokinesis in plants

• cell plate develops from Golgi vesicles

• new cell wall develops around the cell plate

purpose of mitosis

to divide the 2 copies of dna equally

Mitosis Steps

prophase, prometaphase, metaphase, anaphase, telophase

prophase

• chromatin condenses into the chromosome

• centrioles separate to opposite ends of the cell

• mitotic spindle begins to form

Pro-metaphase

• nuclear envelope dissolves

• spindle fibers join with the kinetochore of the centromere

metaphase

• centrioles now at the opposite ends of the cell

• chromosomes line up on the metaphase plate

• spindle apparatus fully developed.

anaphase

• centromeres break: duplicate chromosomes are pulled toward opposite ends of the cell

• cell elongates, poles move slightly further apart

telophase

• chromosomes uncoil back to chromatin

• nuclear envelope reforms

• spindle fibers disappear

• cytokinesis usually starts.

kinetochore

• complex where the spindle fibers connect

• protein structure on the chromosomes appear to “ratchet” the chromosomes down the spindle fiber microtubule with a motor protein

• Microtubules dissolve behind the kinetochores.

cell divison must be ___

controlled

Rate depends on cell type:

Skin: frequently

Liver: as needed(when damaged)

Brain: rarely or never

checkpoints

a critical control point in the cell cycle. cells must receive a go ahead signal before proceeding to the next phase

G1 checkpoints(aka restriction point)

places cells in a non-dividing phase called go phase

go phase

• non dividing state

• some cells can be reactivated back into Mitotic phase

MPF(Mitotic phase promoting factor)

protein complex required for a cell to progress from g2 to mitosis

2 parts of active mpf

cdk and cyclin

cdk

• amount remains constant during the cycle

• inactive unless bound with cyclin.

cyclin

• protein whose concentration builds up over G1, s, and G2

• When enough cyclin is present, active mpf is formed.

active mpf

• triggers mitosis

• activates a cyclin-degrading enzyme which lowers the amount of cyclin in the cell

Result of mpf

no active mpf to trigger another mitosis until the cycle is repeated

growth factors

external signals that affect mitosis

PDGF(Platelet-derived growth factor)

stimulates cell division to heal injuries

density dependent inhibition

the number of cells in an area force competition for nutrients, space, and growth factors

cell density is high=

no cell division

cell density is low=

cells divide

anchorage dependence

• inhibition of cell division unless the cell is attached to a substrate

• prevents cells from dividing and floating off in the body

cancer cells

• do not stop dividing

• the control mechanisms for cell division have failed

regulation of cell division is a balance between:

• mitosis(making new cells)

• apoptosis(cell suicide or death)

• cancer can result if either processes do not work

plasmodesmata

• channels between cells through adjacent cell walls

• allows communication between cells

• also allows viruses to travel rapidly between cells

tight junctions

• very tight fusion of the membranes of adjacent cells

• seals off the area between cells

• ex. lining of the digestive tract

anchoring junctions

• doesn't close off the area between adjacent cells

• coordination of movement between groups of cells

• ex. tissue subject to stretching(skin and muscle)

gap junctions

• open channels between cells, similar to plasmodesmata

• allows “communication” between cells

Signal transduction pathway steps

reception, transduction, response

reception

target cell is going to detect ligand from another cell

transduction

conversation to start the signal for response

response

respond to the signal—> do something

phosphorylation cascades

reaction where one response leaders to a bigger response, and then a bigger one, and so on until there is a widespread response

local regulators

target cell is close to signaling cell

long distance regulators

target cell is elsewhere from singaling cell— release ligand through blood system

receptors

what holds the ligands

ligands

protein, carb, or lipid that sends message

signal transduction pathway

steps of how the cell sends messages to other cells

amplification

The amplification of signals can be defined as an increase in the intensity of a signal through networks of intracellular reactions. It is considered one of the essential properties in many cell signaling pathways.

positive feedback

Positive feedback occurs to increase the change or output: the result of a reaction is amplified to make it occur more quickly.

Ex. labor

negative feedback

Negative feedback occurs to reduce the change or output: the result of a reaction is reduced to bring the system back to a stable state.

ex. regulation of body temperature

centrosome

Centrosomes are structures found inside of cells. They are made from two centrioles. Centrioles are microtubule rings. The main purpose of a centrosome is to organize microtubules and provide structure for the cell, as well as work to pull chromatids apart during cell division.

somatic cells

Somatic cells are the cells in the body other than sperm and egg cells (which are called germ cells). In humans, somatic cells are diploid, meaning they contain two sets of chromosomes, one inherited from each parent.