Classes of Microbes

Fungi(yeast mold)

Protozoa

Bacteria Cell Stucture

Chromosomes: circular, attached to membranes, no histones

can carry plasmids

Cell Wall (peptidoglycan)

Shapes:

cocci (sphere)

bacilli(rod, e. coli)

spiral

Totipotentcy

-forms whole organism

Pluripotency

can form all cell types

Multipotency

-can only form cells in one type of tissue

Induced Pluripotent Cells

Come from normal somatic cells (fibroblasts)

Pluripotent

Not always safe for therapeutic uses

Provides embryonic stem cells

Wouldn't get rejected

Uses SCNT

-take nucleus out of egg

-put in nucleus of somatic cell

-zygote will be clone of donor

-take embryonic stem cells and use for therapy

Note:

In reproductive cloning, the ESCs would not be taken out but allowed to develop into an embryo

Pluripotent
Differentiate with chemical signals

(heart muscle, some tissure, neurons) 

Altered Nuclear Transfer

(ANT)

Haplodi egg + somatic cell = triploid cell

embryo is able to form

can't from a clone 

 addresses ehtical problems

Create and replenish immune system 

White blood cells and RBCs

Human Endothelial Growth Factor Reception

Important in cancer

-why? sit on edge of cell and look for growth factor to bind

-growth factors cause cell division

-in cancer, signals are going off even when without growth factor

-i.e. cell division happens

Important in breast cancer

-cancer is essentially uncontrolled cell division

tumor cells move to different parts of the body

-can happen when tumor cells get into the blood stream

-can result in several small tumors all over your body

Locate a chromosomal address 

ex. 5q12.3

Work on on own time, TA did poor job

Respectively: Chromosome, arm(q is long arm), region, band, suband

G0/G1--Cell at rest, not growing or dividing

Growth factor binds to growth factor receptor on surface of cell

-starts signaling cascade

RTKs

(know details)

Receptor Tyrosine Kinase

Dimer

Phosphoralated

Look for other than a pair of chromosomes

Down Syndrome(3 chromosomes #__)

Look at sex chromosomes

ex. "Trisomy 4"

Aerobic

-Grows well with O2

-dies without O2

Anaerobic

-grows well without O2

-doesn't grow well with O2

HAMA response

-what is it?

-how is it addressed in anti-body design?

Human Anti-Mouse Antibody

antibody design

take constant region from human antibody and fuse it to variable region from mouse antibody

RNAi

Antisense

-Goal for both: knock down expression of a gene

-Knock down: gene could theoretically get expressed

-limits expression

RNAi

long dsRNA
cut by dicer into 22 nucleotide fragments (siRNA)

Antisense

-insert gene complementary to target mRNA

-when its expressed, hybridization prevents translation

insert expression vector into bacteria

Goal: a lot of one protein

Shine-Delgado sequence enables ribosome to bind to the right part of the mRNA

-binds protein receptor on cell

-viral genome and enzymes are emptied into cell

-viral reverse transcriptase transforms RNA to DNA

-integrase integrates viral DNA into cell's genome randomly

-genome replication

-protein synthesis

-creates one long protein that is cut up into ind. pieces

Electron acceptor 

--> NADH

division every 20 min.

overnight 37 deg C, millions of  copies or original single cell

streak plates 

spread plates 

liquid culture

-Need to use competent cells 

-fragile, stored at -80 deg C

Process:

- add competent cells to plasmids +ice for 30 min.

-heat shock

-recover

-antibiotic + incubate overnight

another method of getting bacteria to take up plasmid DNA

-results in pores through which the DNA enters cell

Advantages over competent cells:

-more efficient

-plant, animal, and yeast cells

-can carry larger plasmids

Goal: a lot of one protein

needs: inducible promoter, Shine Delgarno seq, start/stop codons

Refer to notes and book

Encoded by Lux genes
Reporter: releases light under certain conditions

-determine location of fusion protein

what: reporter of the bacteria genus Vibrio 

Fermentation

Lactic

Alcohol

Lactic:

pyruvate-->lactic acid (single step)

coagulates milk proteins(cheese yogurt)

alcohol:

pyruvate-->CO2(released)--> ethanol

makes bread and alcohol

If NAD+ levels are low/NADH levels are high, glycolosis will not take place

(i.e. no puruvate made for fermentation)

produced by one microorganism that inhibits the growth of bacteria

can inhibit: transcription, translation, replication

can damage cell wall/membrane/metablic activity

Only counters bacteria, not viruses

Ice Minus

BT toxin

Ice Minus

Frost resistant plants

-frost is caused by a bacteria on the plants

Bt toxin

kills certain insects; usually root feeders

Smallpox Vaccine

Edward Jenner's work/effectiveness of vaccine

first produced 1796 using cowpox virus because it was very similar

very effective; eradicated smallpox by 1980

Antigens

Antigens

-non-self molecules present on surfaces of bacteria, viruses, fungi...

-elicit immune response

-usually proteins or large carbohydrates

-lymphocytes

B Cells

-activated by Helper T cells

-make Antibodies

Helper T cells

-excrete cytokines that stimulate T and B cells, activated by antigen

Cytotoxic T cells

-directly attack antigen containing cells

-bind to and destroy foreign invaders

Attenuated-use live viruses or bacteria (can't replicate efficiently in host)(chickenpox)

Inactivated-use inactivated viruses or dead microbes 

-flu, rabies

Subunit-use only part of virus or bacteria that elicit an immune response(often a protein) (HepB)

Influenza viruses

HA protein's role in infection

Flu

rapidly evolves

HA

-hemagglutinin

major surface protein

allows virus to get into host cell

Pulse-field gel electrophoresis

remove cell wall then mix cellular components together

-include genome from both plants

-use agrobacter to deliver gene

-remove Crown Gall gene from agrobacter and insert gene of choice

-remove Ti genes, replace with transgene
-add selectable marker 

can add more than one gene at once 

better rates of transgene activity in mature plants

no cross contamination(no pollen involved)

add gene for complementary sequence of mRNA gene of interest

interferes with translation

knock down gene(not knock-out) only limit expression

ex, flavr savr tomato

RNAi tech

(mechanism)

uses double stranded RNAs to elicit natural pathway in degredation of mRNA with matching sequence

mechanism:

-dsRNA recognized by Dicer protein complex

-RNAse within complex chops up dsRNA into smaller fragments(siRNA)

-siRNA trigger destruction of ssRNA with similar sequence

-siRNA associate with RNA-Induced Silencing Complex(RISC)

-"passenger" strand is destroyed

herbicide resistance

glyphosate

-blocks EPSPS enzyme required for photosynthesis

-transgenic plants have an altered EPSPS enzyme unaffected by gly

-chloroplast transgene

-inducible promoter= cauliflower mosaic virus promoter

Methods for engineering animals

(details)

pronuclear

-inject gene into male pronucleus 

-zygote is then implanted back into animal

ESC

-transform ESCs with transgene

(culture, select transfrormed)

-add transformed cells to inner cell mass of early blastocyst

-implant blastocyst

-offspring are crossed to eventually produce a true line of transgenic animal

improved taste

healthier/safer food

disease resistance

better grazers

ex. bio-reactors

(proteins in goats milk)

Knock-out Mice

(Details)

Uses:

-studying what happens when a known disease gene not working

-testing therapeutics

-studying gene function

made:

-transform ES cells; grow on media with gancyyclovir and neomycin

-place in gene you wish to knock out

-chimeric mouse- some transformed cells some not

-breed offspring to get pure germ line

has transgene in some of its cells but not all

ex. mouse that looks like a cow

(cartoon drawing to depict chimeric mouse)

Producing monoclonal antibodies in hybridoma cells

(details)

-inject target virus into mous to produce immune response

-get B cells and culture

Hybridomas are antibodies that do not experience senescence 

1. Myeloma cells(unlimited replication)

2. B lymphocytes(produces antibody)

HAMA response

How response it addressed?

Human Anti-Mouse Antibodies(made in humans, attack mouse)

made becasue mouse antibody is seen as foreign and is attacked

lowers efficacy of treatment

Addressed by antibody design

-take constant region from humabn antibody and fuse to variable region from mouse antibody

Somatic Cell Nuclear Transfer

-fuse somatic cell nucleus to enucleated ocyte(egg w/out nucleus)

-allow to grow into early embryo

-omplant embryo into surrogate

-implant embryo into surrogate

Therapeutic

-produces ESCs that match patient

obligate aerobe- O2

obligate anaerobe-no O2

faultative anaerobes- can grow in either

makes use of indigenous organisms that have ability to degrade pollutants

treats environment in situ

fertilizer is important; organism needs nutrients, makes pollutant limiting facor

adding bacteria to environment to adi indigenous species in cleanup

removing polluted materials and treating off-site

ex. (composting)

carbon dioxide

methane

chlorofluorocarbons

nitrous oxide

phase 1- is it safe(20-80)

phase 2-effective(100-300)

phase 3-side effects?(1000-3000)

approval by FDA

phase4-has it been working well and safely?

preimplantation genetic diagnosis

-used when one or both genetic parents has a known genetic abnormality

-tests embryo to see if it also carries abnormality

does not hard embryo

identifies single gene disorders and chromosomal aberrations

useds to test if embryo carries genetic abnormality

takes amniotic fluid to test 

used to test if embryo carries genetic abnormality

8-10 weeks

collects fetal tissue from developing placenta

risk of chromosomal mosaicism

-(presence of two or more cell lines which differ from each other in chromosome # or DNA)

• 5 = chromosome number
• P = short arm, Q = long arm
• 1 = number of region on that arm; count up from the
• 2 = band number within that region
• .3 = sub-band

Polyploidy

Aneuploid

has extra set of chromosomes 

not compatible with human life

addition or deletion of a single chromosome

trisomy- one extra chromosome(21 = down)

Monosomy-one fewer

CML

Philadelphia chromosome

chronic myelogenous Leukemia

-crossing over of genetic material between two chromosomes

smaller of two resulting chromosomes

use FISH to detect

RFLP anaylsis

how it diagnoses Sickle Cell Anemia

Restriction Fragment Length Polymorphism analysis

-analyzes variation of fragment sizes from cutting w/ restriction enzyme

-distuption of MstII restriction site in Sickle Cell Anemia

-creates a varied fragment length from the normal copy

Receptor Tyrosine Kinases

(Details)

Growth factor-protein that signals to devide/differentiate

Ligand- molecule that specifically binds a receptor(gf)

Kinase-enzyme that adds a phosphate group

Tyrosine- amino acid

1. growth factor binds receptor

2. receptors dimerize(crosslink)

3. receptors autophosphorylate

4. target protein binds receptor

-only binds to phosphorylated domain

-signal cascade results in cell division

associated w/ breast cancer

targeted: herceptin w/ chemo

serious side effects

four subunits, each with heme group(Fe) that binds up to 4 O2

Hemoglobin carries oxygen through bloold to tissues

made by biopure

increases lifetime of therapeutic in blood

used to deliver a normal copy of a gene to a patient with defective copy

delivery usually carried out by viral vector that contains transgene, inserts into host cell

1999- death of Jesse Gelsinger(severe immune response-urea cycle)

02-03---two children developed Leukemia like condition

immune response

-less effective

-can be severe

convert ssRNA to dsDNA and incorporate its DNA into random location in chromosome of host

-reverse transcriptase and integrase

add dsDNA genomes into nucleus where it is used to produce transcripts that yield viral proteins

multiple copies of viral genome produced in replication

not integrated into chromosome

transgene is transient, can cause severe immune response

add ssDNA genome to a specific site within chromosome 19

adds dsDNA to chromosomes in neruronal cell nuclei

infects epithelial cells 

take over and kill cell

can infect non dividing cells

self-renewing

unspecialized

give rise to specialized cell types

Basics of early embryo development

Characterizing adult and embryonic stem cells

take induced pluripotent stem cell and direct it to desired tissue cell type

current status:

know some but not all 

hematopoietic stem cells(multipotent)

-from bone marrow,peripheral blood, umbilical cord blood

-give rise to cells of bllod

Graft Verses Host Disease

bone marrow match

would cause whole immune system to attack whole body of recipient