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Chapter 1

Tuesday, June 28, 2005

10:54 AM

Look at prokaryote cells to see how= we work

Prokaryotes= vs. Eukaryotes

There are two main types of cells, prokaryotes and eukaryotes<= /span>

Prokaryotes and Eukaryotes have both similarities and differences

Prokaryote<= /span>

Eukaryote

NO Nucleus

Nucleus

No membrane bound organelles=

Mitochondri= a

some sort of plasma membrane=

Membrane bo= und organelles

ribosomes

ER/Golgi Co= mpex

No cytoskeleton but have somethiung= like it

Plasma memb= rane

Cytoskeleton

Ribosomes

lysosomes

Peroxosomes=

Contractile Vacuole

Flagella/ci= llia

Circular DNA

Linear DNA<= /span>

Eukaryote

Cells that = have a nuclear envelope, cytoplasmic organelles, and a cytoskeleton

Screen clipping taken: 6/28/2005, 4:52 PM

Prokaryote

Cells lacki= ng a nuclear envelope, cytoplasmic organelles, and a cytoskeleton (bacteria)

Viruses are different, neither prokaryotes nor eukaryotes

Viruses

Have some kind of protein budding o= ut on the outside and some sort of genetic material usually DNA in the inside

Tightly packed DNA

Different are neither prokaryotes n= or eukaryotes

Not alive and very diverse

RNA as its genetic material =

RNA simpler than DNA and self repli= cating (via reverse transcriptase) but unstable and not a good model for permanent replication

All life has DNA as its genetic material

This is how genes are passed down f= rom one generation to the next

3 Types to Generate Metabolic Energy

Glycolysis

Break sugar down into say alc= ohol or lactic acid

Photosynthesis

Done by plants and provides e= nergy for complex organic molecules used in glycolysis and oxidative phosphorylation

Oxidative Phosphorylation

We are only 1000x more complex than= a simple prokaryote cell (E-coli) not all that much such that studying prokaryotes can help in understanding more complex Eukaryotes

Animal Cell= vs Plant Cell

Plasma Membrane not smooth

Animal Cell

Organelles

Nucleus

The most prominent organelle of eukaryotic cells; contains the genetic material

Mitochondria

Cytoplasmic organelles responsible = for synthesis of most of the ATP in eukaryotic cells by oxidative phosphorlatio= n

Two membrane system

Golgi apparatus

A cytoplasmic organelle involved in= the processing and sorting of proteins and lipids. In plant cells, it is also the site of the synthesis of cell wall polysaccharides

Lysosome

A cytoplasmic organelle containing enzymes that break down biological polymers

Peroxisome

A cytoplasmic organelle specialized= for carrying out oxidative reactions

Endoplasmic Reticulum

An extensive network of membrane-en= coded tubules and sacs involved in protein sorting and processing as well as lipid synthesis

Cytoskeleton

A network of protein filaments that extends throughout the cytoplasm of eukaryotic cells. It provides the structural framework o= f the cells and is responsible for cell movement

Plant Cell

Different from animal cell in that = it has a cell wall (two cell wall= s, a primary and a secondary)

Large central vacuole cell in the center that performs functions like a tr= ash car and closet and is responsible for the digestion of macromolecules and t= he storage of both wastes products and nutrients

Tonoplast: a membrane that encloses the central vacuole in a plant cell, separating the cytosol from the cell sap is a phospholipid bi-layer

Contains….

Nucleus, Golgi, ER near nucleus, go= lgi sacs, mitochondria

Chloroplasts=

The organ responsible for photosynt= hesis in the cells of plants and green algae

Have sacs of membranes

Golgi apparatus

A cytoplasmic organelle involved in= the processing and sorting of proteins and lipids. In plant cells, it is also the site of the synthesis of cell wall polysaccharides

Plant Cell basically =3D animal cell + Large central vacuole + Chloroplasts + Cell Wal= l

DNA: found in both prokaryote and eukaryotes

<= img src=3D"chap1-4_files/image017.gif" width=3D264 height=3D189>

Techniques = to study Cells

Microscopy

Light Microscope

Limited by resolution (the ability to distinguish objects separated by small distances=

Brightfield microscopy

The simples= t form of light microscopy, in which light passes directly through the cell and the ability to distinguish different parts of the cell depends on the contrast resulting from the absorption of visible light y cell components

Phase Contrast Microscopy

A type of microscopy used in which variations in density and thickness between parts = of the cell are converted to differences in contrast in the final image=

Common meth= od used for visualizing living cells, = stains are not used

Fluorescence Microscope

Type of microscopy in which molecules are detected based on emission of fluorescent light

Widely used= and very sensitive method for studying the in= tracellular distribution of molecules

Can't see much but linear stuff, usually will kill the specimen unless used with = GE

Confocal Microscope

A form of microscopy in which fluorescence microscopy is combined with electron image analysis to obtain images with increased contrast and detail.

Gets rid of anything except one plane focus, gets rid of out of focus light

Uses fluorescence, looking at a single thickness

A bunch of = 2D images can be put together to from a 3D image

Figure 1.30=

Electron Microscopy

A type of microscopy that uses an electron beam to form an image

The electron microscope can achieve a much greater resolution that that obtained with the light microscope because the wavelengths of electrons is shorter than that = of light.

Scanning EM

Electrons scattered from the surface of a specimen are analyzed to generate a three-dimensional image

Metals have to be coated with metal

Below the resolution of light, can look at single microtubules and single vesicles

3D Shape produced, restricted to studying whole cells rather than subcellular organelles or macromolecules

Differential Interference Contrast Microscopy

Can be used to look at microtubules over very low resolution

Transmission Electron Microscopy

Can see individual compartments and organelles

Beam of ele= ctrons in passed through a specimen stained with= heavy metals.

Totally dead cell but allows you to look= at certain sections like the organelles like nucleus but can also lead to many artifacts

Freeze Fracture

Used to study plasma membrane structure

Discovered = double layers of membranes, freeze the cell then hit it, exposing and breaking the cell along the membrane then coated with platinum and looked under electron microscope, of course cells are dead for this

Subcellular fractionation

Grind cells up and spin them at 800x G. Pellet out nucleus and things = that are very soluble remain in the supernate. Then take the subernate Break cell apart and cut it into a bunch of little pieces

Screen clipping taken: 7/10/2005, 5:57 PM

Velocity Centrifugation (density gradient)

The fractions obtained from the differential centrifugation corresponds to enriched, but still not pure preparations. A greater degree of purification can be achieved by density-g= radient centrifugation, in which organelles are separated by sedimentation through a gradient of a de= nse substance such as sucrose. In vel= ocity centrifugation, the starting material is layered on top of the sucrose gradient. Particles of different = sizes sediments through the gradient at different rates, moving as discrete bands. Following centrifugation t= he collection of individual fractions of the gradient provide sufficient resolution to separate organelles of similar size, such as mitochondria, lysosomes and peroxisomes.

Physical way to separate cell organ= lles

Tissue Culture

Grind of tissue and grind it up to = so to break the connections between the cells but not break up the individual cel= ls themselves

Can be used to= study a particular protein in a cell and see what happens

Help us learn cell structure and fu= nction

Heart Cell in culture

Figure 1.41=

Screen clipping taken: 6/29/2005, 11:38 PM

How do we s= tudy Cells?

We use model systems!

Observation + Reductionism

Reductionism is where we break down things and then try to figure out how individual aspects work and then end up putting them back together

Model Syste= ms

To determine how cells function, model systems are studied by a varie= ty of techniques

= E. coli

Rapidly divide in 20 minutes=

Advan= tages

Very simple, small/simple genome, replicat= es very quickly, know all the genes and sequence, good for looking at D= NA and replication, simple nutritional requirements

Investigation of the basic mechanis= ms of molecular genetics

Most of our understanding of DNA replication= , the genetic code, gene expression, and protein synthesis derive from studies of E. coli

Disad= vantages

Simple, single cell, prokaryotic (no membrane bound organelles like mitochondria or chloroplasts) would NOT woul= d to use it to look at secretion

Can not be used to study aspects of= cell structure and function that are unique to eukaryotes

Yeast

S. cerivisia (more common and one used = in book)

S. pombe

Advan= tages:

Single cell, prokaryotic, secretion= s of alcohol fundamental aspects of eukaryotic cell biology, simple genome compa= red to more complex eukaryotes

Disad= vantage

Bad for cell to cell interaction, f= ungus that has a cell wall so not too much like humans

Studies of the= ir DNA sequences indicate that the archaebacteria and eubacteria are as different = from each other as either is from present day eukaryotes

S. cerivisia are very different S. pombe but they are as different from each o= ther as we are from them!

= Drosophila Melanogaster

Advan= tages:

Replicates quick, 7 day life cycle, developmental experiments, fundamental concepts of genetics, such as the relationship between genes and chromosomes.

Disad= vantages

Has cytoskeleton, 4 chromosomes (sm= all genome), not to close to us

Dictyostelium (slug movie) (D. Discoideum)

Individual amoeba aggregate to one another to from multicellular units which can now look for new conditions a= s a single organism (slug)

Culmination setup fruiting body, an= terior cells form stalks

Use of proteins to adhere to one an= other and "walk"

Advan= tages:

Observe single cells moving around = and eating bacteria and cell to cell adhesion like when healing a wound/cut

Can be single cell or multi-cell or= ganism can stream into slugs

Good metafit cancer cells, neutrophillis

Disad= vantages

Makes cellulose which we don't make= , not a good model for cell to cell interaction, has no backbone or nerves=

Clamydomona= se (flagella movie)

Green Algae. Pond scum, can have mu= ltiple flagella

Advan= tages:

Flagella movement similar to mammal= ian sperm has same components, can be grown by the liters

Disad= vantages

Arabadopsis

Advan= tages:

Good plant model

Disad= vantages

= C. Elegans

Worm, simple genome, know the linea= ge

Advan= tages:

Model system for animal development, smaller genome compared to more complex animals, relatively simple multicellular organism

Disad= vantages

NO nervous system or blood vessels<= /span>

Xenopus

Frog, eggs

Advan= tages:

Early vertebrate developmental stag= es

Disad= vantages

Zebra Fish<= /span>

Advan= tages:

Transparent, embryo develop in four= days, early embryology

Disad= vantages

Fish, hard to take care of, difficu= lt to work with

Mice

Advan= tages:

Great model system with many similar genetic mutations, pigmentation, a lot of similarities, entire genome has b= een sequenced, can do experiments on mice that you can not do on humans, genetic mutations, similar homologous gene defects

Disad= vantages

Expensive to keep, short generation= time for a mammal but long when compared to E-coli, can't really see what is goi= ng on them in early development but still a good model system for humans

The Scale of Evolution

A long time ago we had molecules fo= rming on earth, then prokaryotes being made, then eukaryotic, then evolving into multicellular organisms

Taking the cell breaking them open, then isolating different factions by spinning = them different amounts of time. And ea= ch time you get a different organelle or protein

Chapter 2 Molecular Composition of = the Cell

Sunday, Jul= y 10, 2005

6:13 PM

Molecular Composition of the Cell

Cells are m= ade of water, I

.norganic i= ons and organic molecules

Water makes= up 70%of the cell mass

Interactions between water and everything else are extremely important

Polar interactions

Water is a polar molecule
Interactions between water and oth= er polar molecules for example carbohydrates help hold the cell together<= /span>
Polar molecules are soluble in wat= er - hydrophilic

Carbohydrates, DNA, protein

Non-polar molecules are NOT solubl= e in water – hydrophobic

Lipids, plasma membrane

Polar interactions - Hydrogen Bonds

Inorganic Components of the cell

Sodium, magnesium manganese, calcium, phosphate chloride and bicarbonate are less than 1% of the ce= ll mass

Important as co-factors and c= ell transduction

Organic Molecules that make up the cell

Nucleic Acids, Carbohydrates and Proteins are polymers made up of hundreds or thousan= ds of low molecular weight subunits (monomers) Dehydration Condensat= ion Reactions

Carbohydrates
- Formed via dehydration conden= sation reaction
- Polymers (many subunits)

Nucleic Acids
- Formed via dehydration conden= sation reaction
- Polymers (many subunits)

Proteins

Formed via dehydration conden= sation reaction
Polymers (many subunits)

Lipids

Not big enough to be polymers= TAG's with glycerol backbone

Carbohydrates

Sugars - if= > 5 C, then can cyclize

Depending o= n the structure of the sugar it may be involved in different things

Figure 2.2<= /span>

Formation o= f a glycosidic linkage

A dehydration condensation rea= ction
Occurs between sugar molecules=

Different glycosidic linkages give rise to different polysaccarides

Amylopectin (starch) = Storage (α 1-4 bonds with <= /span>α 1-6 branching)
Glycogen Storage (α 1-4 bonds with <= /span>α 1-6 branching)
Cellulose Structural Not Edible by us = (β 1-4 bonds)

Shows us how selective enzyme= s are and how they will only break certain bonds
Specificity is important

Screen clipping taken: 7/10/2005, 7:39 PM

Lipids

Fatty acids=

Saturated have as many Hydrogens as possible

Very flexible

Unsaturated have at least 1 C= =3DC double bond and are kinky

Triglycerid= es

Note Glycerol Backbone<= /span>
Note 3 Fatty acids hanging off= it!
Building blocks for the all the phospholipids found in plasma membranes

Lipid Compo= sition of Membranes

The composition of the lipids = in the membranes is important in how the protein work
Many enzymes embedded in prote= ins and if the wrong phospholipids are present the enzyme will not work
All membranes are not equal and depending on their function they will have different phospholipids and also different proteins

Phospholipi= ds - main component of membranes

Phosphate and two FA's bound to Glycerol
All except sphingomyelin have a glycerol backbone (it has a serine backbone)

Figure 2.7

Phosphatidic = Acid, Phosphatidyl-ethanolamine, Phosphatidyl-choline, Phosphatidylserine, Phosphatidyllinositiol, Sphingomyelin

Cholesterol= and Steroid Hormones

Flat and planar, 4 ring struct= ure
Figure 2.9

Components = of Nucleic Acids (figure 2.10)

DNA - a pol= ymer (like sugars) held together by phosphodiester bonds

Another dehydration condensation reaction
Figure 2.11
Bases play no part in the back= bone (involved in base pairing)

DNA is held together by Hydrogen bonds

Figure 2.12
Much more dif= ficult to sequence that GC rich than DNA that is AT rich because you have to = heat the DNA much higher if GC rich

Structure o= f an Amino Acid

Figure 2.13=

Amino Acids= 1 (Nonpolar Amino Acids/Hydrophobic)

Amino Acids= 2 (Polar, Basic, Acidic)

Amino Acids= - Building Blocks of Proteins

Peptide Bond Formation

another dehydration condensati= on reaction
Figure 2.15

Sugars held together by Glycosidic Bonds

Proteins held together by peptide bonds

DNA/RNA held together by phophodiester bonds

Protein Str= ucture

1º =3D Amino Acid Sequence
2º =3D Local Fol= ding α-helices and β-sheets (H- bondi= ng)
3º =3D Long distance folding = (3-D shape, interactions between R groups that form ionic bonds or interact= ions between polar/nonpolar groups or disulfide bonds)
4º =3D multiple subunits fold= ing together to give an active protein stabilized by the same interactions that stabilize 3º structure

Protein str= ucture continued

Enzymes

Are proteins

Enzymes are always proteins

Act to lower the activation en= ergy of a reaction
lock and key model =3D perfect= fit
induced fit - initial binding = of the substrate causes the enzyme to bind more tightly wet jeans

Enzymatic A= ction Video

Enzymes Str= ucture

◗ Active site - where substrate bind= s

◗ Allosteric site - where another, regulatory molecule/ion can bind

Coenzymes

Small organic molecules that c= an be involved in enzymatic catalysis
NAD--> NADH+ -->NAD in oxidative-reduction reactions – mitochondria

Coenzymes a= nd Vitamins

Coenzymes derived from vitamin= s

Lipids form a water impermeable bi-layer, allowing for the cell to be isolated from the surrounding environment and even for the portioning of organelle components from the rest of the cell

Cell Membra= nes

separate the inside of the cell fr= om the outside
create environments where norm= ally “energetically unfavorable” reactions can take place=

Outside environment usually oxidizing (protein have disulfide bridges)
Inside usually reducing (prot= eins usually do not have disulfide bridges)

Those endothermic reactions ca= n take place in the cell at the expense of the sorrounding
a phosopholipid bilayer with embedded proteins

Membrane Li= pids

◗ fatty acid tails of phosopholipids= stick together to form a hydrophobic bilayer

◗ phosphate groups on the phospholip= ids are very hydrophillic

= • point away from the fatty acid tai= ls into an aqueous environment (inside the cell or outside the cell)

Phospholipi= ds

VERY mobile
Can move around very quickly
BUT only in the plane of the membr= ane (Diffuse rapidly in 2 dimensions)
CAN NOT flip-flop from one lea= flet to the other,
unless a “flipase” is invo= lved

Membrane fl= uidity

Mostly due to phospholipids=
Longer fatty acid chains are more = “rigid” or “stable” to high temperatures (think of drag in long ones, can'= t move around as well)
Unsaturated fatty acid chains = are “kinky” membranes so they don’t pack as tightly against the other phospholipids, resulting in a more “fluid” membrane<= /li>

More on Mem= brane Fluidity

Cholesterol can also affect me= mbrane fluidity

At = high temperatures, Ch= olesterol by preventing the phospholipids from moving around very quickly
At = low tempera= tures, Cholesterol increases membrane fluidity by preventing the phospholipids from packing very tightly together

Membrane fl= uidity and membrane proteins

Many membrane proteins need the right phospholipid mix to function properly
SO…. Membrane fluidity is critical to the cell’s ability to carry out basic cellular functions - for example
- transport of molecules across= the membrane
- signal transduction (cell signaling)
- Endocytosis (picking up nutri= ents from outside of the cell)
- cell motility, etc…(mitocho= ndria transport)

Membranes a= s a fluid mosaic of proteins in a lipid bilayer Fig.2.48

2 main classes of membrane pro= teins
Integral (transmembrane usuall= y)

embedded in the lipid bilayer=
can only be removed by additi= on of detergent (destroy plasma membrane)

Peripheral

associated with either the in= side or outside of the membrane (charge to charge interactions)
can be removed by high salt
can be removed by high or low= pH
Removed without destroying the lipid bilayer

Figure 2.48

Modification of the Fluid membrane

Not all parts of the membrane are the same

Does behave like a mosaic in that phospholipids are fluid

But there are micro-domains called = lipid rafts

Probably all Eukaryote cells have them (have cholesterol)
These domains are different from the surrounding domains in t= hat they are cholesterol and sphingomyelin rich
Have different lipids in them compared to the surrounding are= a
These rafts are rich in proteins that have a lipid anchor GPI (post trans mod)
Contain proteins that are involved in signal transduction (ce= ll signaling the cell) and adhesion (cells going together to form a tiss= ue or crawl along)
Not all lipid rafts are equal and not all are stable, exist v= ery shortly

Lipids Rafts (online info)

Lipid rafts are cholesterol-rich microdomains in cell membranes.

Since 1972, it has been believed that, in cell membranes, phospholipids and membrane proteins are ubiquitously distributed accor= ding to a fluid mosaic model. However, in 1988, Kai Simons at the European Molecular Biology Laboratory (EMBL= ) in Germany suggested the novel idea that there= exist microdomains, which are enriched with many kinds of lipids such as choleste= rol, glycolipids, and sphingolipids, present in cell membranes. He called these microdomains "lipid rafts". The concept of lipid raf= ts has been related to the liquid-liquid immiscibility observed within model membranes. This immmiscibility has been reported between two liquid crystal= line phases, the liquid ordered phase (Lo phase) and the liquid disordered phase= (Ld or Lalpha phase).

The evidenc= e for lipid rafts is still very controversial and has yet to be confirmed despite many experiments involving several different methods e.g. detergent extract= ion and fluorescence cell imaging. Furthermore, recent studies of cell membranes reported that lipid rafts contain not only lipid components but also numero= us signaling molecules such as GPI-anchored proteins and receptor or non-receptor type tyrosine kinase, which modulate various physiological processes in cells. Therefore, lipid rafts are thought to act as a platform= for protein segregation and signaling in cells. However, the functional role of lipid rafts still remains very uncertain because it has not been determined whether they have a positive effect or negative effect on signal transduction in cells. Investigating the ef= fect of the lipid components on cellular signaling pathways may provide a novel understanding of the roles of lipid rafts in cells.

Premeabilit= y of the Membrane

Passive Tra= nsport

Small non polar molecules can diffuse across the membrane (Oxygen, Carbon dioxide, etc.) down their concentration gradient
Charged or Large molecules can= NOT diffuse across the membrane because of hydrophobic core<= /li>
They cross via Channel protein= s or carrier proteins

How semi-bi= g or charged stuff gets into the cell (fig. 2.50)

Channel Proteins - form pores = or channels in the membrane through which small charged things through - = Ion channels
Carrier Proteins - allow things through by undergoing a conformational change upon the molecule bindin= g to the carrier protein

Active Tran= sport

See fig. 2.51
How to get stuff into the cell= even if the concentration gradient is going the other direction
USE ENERGY of some sort (usual= ly ATP) to drive things through a transport protein

against the concentration gradient

Chapter 3 Fundamentals of Molecular Biology

Thursday, June 30, 2005

3:40 PM

= DNA

The genetic material
Genes are e= ncoded in the DNA
Inherited i= n a Mendelian Fashion

Unless the= y are near each other on a chromosome, then they appear to be “linked”<= /span>

A polymer of deoxyribonucleotides - a double helix

= Information flow - not quite right -- sometimes the info goes the other way!

Central Dog= ma states that DNA → RNA → protein

Retroviruse= s are an exception to this though… they flow RNA → DNA → RNA → protein

"Mad Cow" protein based disease (not a DNA based disease)- does not go thro= ugh DNA at all! causes proteins in our body to misfold

= Bacteria Transformation - Avery and MacLeod

DNA contained/encoded whatever= it was for genes to get transferred or traits
The purified DNA therefore con= tains the genetic information responsible for transformation of R to S bacte= ria
DNA can transform cells=
Figure 3.4 (pg 94)

= The Structure of DNA

Nucleic acids are the genetic material

Nucleic Acids

(DNA and RNA) are the principal molecul= es of the cell and are the genetic material

= Purines and Pyrimidines

2 kinds of nitrogenous= bases

purines

2 ring structures
Adenine and Guanine

pyrimidines

1 single ring structure
Thymine (DNA), Cytosine (DNA/RNA) and Uracil (= RNA)

Double helix with two major gr= oves, DNA binding found in these groves
Sugar-phosphate backbones on t= he outside of the molecule
The bases are on the inside, oriented such that hydrogen bonds are formed between purines and pyrimidines on opposite chains.

DNA and RNA are assembled from nucleotides with phosphodiester bonds. The phosphate group on the= 5' end (of the ribose or deoxyribose) of one nucleotide binds to the hydroxyl grou= p on the 3' end (of the ribose or deoxyribose) of the last nucleotide in the DNA= or RNA chain

The fact th= at there is a phosphate group on one end and then a hydroxyl group on the end = of the strand parallel to it shows that these two strands are different.

The Structure of Nucleotides (8:00)

Pentose: 5 = Carbon Sugar

DNA and RNA are polymers of nucleotides, which consist of purine and pyrimidine bases linked to phosphorylated sugars.
DNA contains two purines (aden= ine and guanine) and two pyrimidines (cytosine and thymine)<= /li>
RNA also contains adenine, gua= nine, and cytosine but RNA contains uracil in place of thymine.
The bases are linked to sugars (2'-deoxyribose in DNA, or ribose in RNA) to for nucleosides.
Nucleotides additionally cont= ain one or more phosphate groups linked to the 5' carbon of nucleoside sug= ars

Nucleoside

A Purine or pyrimidine base linked to a sugar (ribose or deoxyribose)

Nucleotide

A phosphory= lated nucleoside

The building block of a nucleic acid, consisting of a five-carbon sugar covalently bonde= d to a nitrogenous base and a phosphate group.

DNA is double stranded (with deoxyribose) and RNA is single stranded (with a ribose)

DNA - deoxyribose sugar

Deoxyribose

Requires a = primer

(2'-deoxyribose) The five carbon sugar found in DNA

RNA - ribose sugar

Ribose

The five carbon sugar found in RNA

linked to a nitrogenous base at the 1 carbon
with a phospha= te group on the 5 carbon
Does not require a primer<= /span>

Figure 2.10, 2.11, 2.12

Each tRNA is going to carry its own amino acid

Nucleotides grow in the 5'= → 3' direction, adding at the 3' end (hydroxyl)

Read in the= 5' → 3' direction as well

Oligonucleotides

Small polym= ers containing only a few nucletides

AMP<= /p>

GMP<= /p>

TMP<= /p>

CMP<= /p>

UMP<= /p>

= Structure of DNA

Phosphate s= ugar backbone
Held togeth= er by phophodiester bonds

The double helix is held together by hydrogen bonds = (10:20)

G always pairs with C, A always pairs with T in DNA

3 H bonds

2 H bonds

= DNA structure - space filling model

= DNA - antiparallel strands held together by hydrogen bonds

Figure 3.5<= /span>

DNA replication - in detail = (12:00)

DNA and RNA are assembled in the 5' to 3' direction

If you know= one strand of DNA you can always regenerate the other strand

When we clo= ne stuff we take advantage of this machinery to copy stuff

DNA replica= ted in the 5' → 3' direction

= DNA replication is semi-conservative

DNA is replicated in a semi-conservative manner

Figure = 3.6

DNA 5' ---------------------------- 3'

Protein N (amino) ---------------------------<= span style=3D'mso-spacerun:yes'> C (carboxyl)

Experimental evidence that DNA replication is semi-conservative (15:12)

Figure = 3.7

= continued

DNA and Protein are co-linear (17:05)

The DNA sequence and the protein sequence for a gene are colinear

Figure = 3.8

= RNA is copied from DNA in a 5’-->3’ direction (more in a later chapter!!)

= Dissecting the genetic code

= The genetic code is Redundant

Nearly the = same in all organisms with a few exceptions

= Each Amino acid is encoded by a set of 3 nucleotides (codon) on the mRNA - tRNA�= ��s

Know how to= use Table 3.1!

Stop Codon

Trigger the ribosome to fall apart at the end of every codon sequence to stop making protein

Codons are = groups of 3 nucleotides that code for an amino acid

Many codons= can code for the same amino acid

= carry the correct amino acid (stay tuned!)

= Frameshift mutations

Three readi= ng frames

If you don'= t know what strand is the coding strand there is a total of 6 different peptides f= or any DNA sequence. 3 (3 different = reading frames) x 2 (double strand) =3D 6

Figure 3.11!=

Insertion or deletion of one or 2 nucleotides

= Missense mutation - movie

Single point mutation, change of 1 nucleotide, <= span style=3D'font-weight:bold'>NOT an addition or deletion

= Nonesene mutation –movie

Termination= of polypeptide synthesis at the effected codon, a type of a point mutation, re= sult of premature stop codon, if an addition or deletion causes a stop codon to = be inserted early

= Molecular Techniques or more fun with DNA

Restriction Mapping (38:25)

DNA can be cut= by restriction enzymes
restriction en= zymes are like molecular scissors

By recognizing certain specific sequences
Palindromes because they read the same from either direction
Written in 5' →3' direction

they cut at sp= ecific sites

Single strand= s, isolated from bacteria

sometimes they= leave sticky ends, sometimes blunt ends

Re= striction mapping can be used to tell if you generated mutation and to cut DNA f= or cloning!
Why would we want things that cut up DNA?

Restrictions enzymes restrict the entry of bacteriophage

Each Restriction enzyme has a speci= fic site at which it cleaves

Recombinant= DNA

<= span style=3D'position:relative;left:.125in'>Recombinant DNA - movie (42:34)

A host DNA,= for example from a mammal is cut with an restriction endonuclease which recogni= zes a specific base sequence or recognition site, same endonucleases is used to= cut a foreign DNA from a different organism. If there are multiple recognition sites on the DNA it will be cut in= to pieces of various lengths. If the= cut host and foreign DNA are then mixed in presence of DNA ligase some of them = will splice to form recombinant DNA. A= host DNA with a segment of foreign DNA incorporated.

Staggered e= nds =3D sticky end because they have an overlap or 2-4 nucleotides that can hydrogen bond to 2-4 nucleotides on the other side

Vectors:

Carries som= ething and transmits it to something else, mosquitos are vectors of West Nile Viru= s, DNA plasmids cab be vectors, viruses can be vectors if they pick of DNA

= Cloning - sticky end ligation

Restriction Maps of Adenovirus and Bacteriophage <= /span>λ

= How to clone a piece of DNA

Campbell Ac= tivity 20C on CD-Rom

We can use = the techniques of DNA technology to recombine and copy genes. A restriction enzyme is used to cut open a plasmid, a small circular DNA molecule obta= ined from a bacterium. The plasmid serves as a cloning vector. The restriction enzyme cuts only at= a certain DNA base sequence, called a restriction site. The same enzyme is us= ed to cut up DNA obtained from a human cell.

The restriction enzyme produces DNA fragments t= hat have matching sticky ends that join by complementary base pairing. An enzyme called DNA ligase forges covalent bonds that join human and plasmid DNA, creating recombinant DNA. The = human gene is inserted in the middle of the plasm= id lacZ gene. The plasmid also carries a gene for resistance to the antibiotic ampicillin.

The human gene must be cloned to produce copies for u= se or study. A bacterium is induced to take up the recombinant plasmid from the surrounding solution in a process called transformation. (This isn't as clear-cut as it sounds. Some bacteria do not take up the plasmid, and some take up plasmids whose lacZ g= enes lack the inserted human DNA.)

A bacterium is placed on a growth medium. It replicates the plasmid at the same time as its own DNA. Its descendants form a clone of bacteria that all have copies of the recombinant plasmid. The ampicillin in= the medium limits growth only to plasmid-containing cells, but which cells have plasmids with a human gene? Bacteria without human genes have intact lacZ genes; this allows them to br= eak down a substance in the medium that stains their colonies blue. Thus, the bacteria with human genes are in the colorless colonies.

Her Discussion at 48:20!

2 kinds of vectors

= That has been cut and has been self-ligated without the insert DNA being put in
= Vector of DNA that has had for= eign DNA inserted in the middle of the lax z coding region and so this gene will not work, non-functional lax z gene

The only ba= cteria you see growing are those bacteria carrying a plasmid

The ones containing a plasmid may have picked up an insert and therefore will be whi= te

Or.. It may= not contain an insert and the protein will be made the colony will be blue

Of the colo= nies that are white they could have a bunch of different pieces of it

= Cloning - some more

= Selection of E.coli carrying recombinant plasmids

Chapter 3 Day 2

= Phage Library

You have one gene or sequence = of amino acids and you know the sequence but don't know what it does or w= here it is located in relation to the entire genome
Isolate one strand of DNA then= use restriction enzymes to cut the DNA into pieces
Each piece now contains a diff= erent sequence
Take the mixture of pieces and= add them to vector DNA (phage DNA)
Each piece now ends up in a different bacteria phage
Each piece/sequence is kind of= like a book, each chunk of DNA as a story
All the different sequences as= a collection can be considered a library
Libraries are collections of p= ieces of DNA and contain all the sequences contained in a genome and are cal= led a genomic library
Figure 3.19, Figure 3.20,

= Screening a Genomic DNA phage library - Plaque Lifts

Figure= 3.31

= Reverse Transcriptase - from Retroviruses

HIV good exam= ple
Have an RNA g= enome that gets into the host cell and the RNA is copied into DNA
Information f= lows from RNA → DNA →RNA → protein
The enzyme th= at does this is reverse transcriptase
Reverse transcriptase is carried as a protein linked to the RNA is the virus particle, primed to do its thing
Integrated in= to the host DNA as a provirus, viral DNA incorporated into the host cell that= at any time can make virus or not, stability incorporate into the host DNA and if it gets there its not coming out, and only comes out through the lytic cycle.

= HIV Mechanism of Action (movie)

= How Retroviruses work

cDNA - Complementary DNA made by reverse transcriptio= n of mRNA

Can't replicate RNA by itself! (20:00)
How to Identify the gene from = mRNA must first convert to DNA Figure 3.18

Use reverse transcriptase iso= lated from a retrovirus
Make sure you have a primer b= ecause all DNA polymerase needs a primer to copy RNA to DNA (retroviruses ha= ve their own primer)
mRNA (poly A tail at the end)= is then primed by Oligo-dt
Then reverse transcriptase wi= ll copy the first strand of DNA off of that (complementary to RNA)
Add another polymerase which = uses other stuff as the primer and then makes the 2nd strand of cDNA
We now have double stranded complementary DNA that is complementary to mRNA
But we have blunt ends, which= does not clone easily, so we add linkers that has a restriction site in it=
Clone it into a piece of DNA (plasmid or bacteriaphage)
Transfer to E-coli and then d= o a screen to see what gene we are interested in

Making cDNA. Generati= on of cDNAs can also be done by a wide variety of processes, but, in virtually all cases, cDNA is generated by the enzyme reverse transcriptase* (RT) which has the ability to use the informatio= n in an RNA to generate a complementary DNA. Thus, reverse transcriptase is a RNA-dependent DNA polymerase. Like all DNA polymerases it cannot initiate synthesis de novo but depends on the presence of a primer. Since many mRNAs have a poly-A tail at the 3' end (see polyadenylation*), oligo-dT is frequently used to prime DNA synthesis (it is also possible, and frequently essential, to generate cDNAs= by using either random primers or primers designed to amplify a specific mRNA). Once the initial cDNA has been generated it is generally necessary to produ= ce a second strand of DNA. Again, there are many strategies for doing this, but a convenient mechanism involves exposure of the DNA/RNA hybrid to a combinati= on of RNAase-H and DNA polymerase. RNAase-H has the ability to cause single-st= randed nicks in the RNA, and DNA polymerase can then use these single-stranded nic= ks to initiate " second strand" DNA synthesis. This two-step procedu= re has been optimized to maximize fidelity and length of cDNAs.

<= span style=3D'position:relative;left:.125in'>cDNA Library construction

= Why cDNA libraries?

◗ cDNA is = made from mRNA using reverse transcriptase

◗ This mea= ns ONLY genes being expressed in a tissue will be represented by mRNA

molecules<= /span>

◗ Also, cD= NA’s contain the sequence that a ribosome would translate into protein

◗ NO intro= ns (we’ll cover these later)

Why do all = this?

Human genome is very complex a= nd will have 90% of which you know you don't want in a genomic DNA=
Increase your odds of getting = the sequence you want by only looking at genes that are being made in the tissues you know where the genes you are interested in being expressed= .
cDNA contain sequences that the ribosomes will translate into protein (no introns)
Increase ours odds of finding = the sequence we are interesting in and having it being of use

= Cloning REALLY Big Pieces of DNA - Cosmid Libraries

Figure 3.19=

Also PAC

= YAC (yeast artificial chromosome) libraries

Why do this= ?

The gene fo= r the protein for which you are interested may be big and not fit into a plasmid = thus need to do other methoids

Temperature sensitive mutation: = (pg 124)

Mutation in= a protein that causes the protein to unfold at high temperatures

BAC<= /p>

Small DNA v= ia phage and or plasmid vector

PCR the movie (38:16)

= Polymerase Chain Reaction

◗ How to a= mplify a specific gene

= Heating
= Add primers (hybridization)
= DNA synthesis (DNA polymerase)=

Have to kno= w the sequence at the ends in order to choose the correct primers.

= PCR continued

= So what do you do once you have cloned a piece of DNA???

Try to get the nucleo= tide sequence

◗ Sequenci= ng (43:41)=

◗ Southern= Blot (DNA probed with the gene)

◗ Northern= Blot (RNA probed with the gene)

◗ Express = RNA in E.coli as a fusion protein

◗ Make an antibody to fusion protein - Western Blots

◗ Gene disruptions - homologous recombination

◗ Transgen= ic animals/plants

= DNA sequencing - dideoxy chain termination method

Figure 3.21=

Not done th= at much anymore

= DNA sequencing continued

= DNA sequencing - Using Fluorescent Dyes instead of Radioactivity

More common= now than gel

= Detection of Fluorescent Products

Chemically = using DNA polymerase figure out the sequence of DNA that you have cloned

= Expression of foreign genes in E.coli -fusion proteins

Insert the = coding strand that you have sequenced (hopefully cDNA) into E-coli

◗ How to m= ake LOTS of a gene product

Figure 3.23=

= What can you do with a fusion protein?

◗ Use it f= or = in vitro st= udies of the protein you are interested in (by isolating gram quantities)<= /span>

◗ Use it to generate antibodies to the protein you are interested in

Something e= lse you can do with a gene you are interested in

= DNA melting and reanealing we take advantage of this in doing Southerns and Northerns

= Making a DNA probe - for blots

Have double stranded DNA, heat to 95º and melt/denature. DNA now opens up and is available to H bond to something else. Add a stretch of DNA that you know (ge= ne you have isolated) lable it with a radioactive tag, then it will only base pair with the one it matches. <= /p>

Figure 3.25=

Southern Blots (55:47)

A way to determine the size of= the piece of DNA you are looking at and what is around it.
Take genomic DNA and cut it wi= th a restriction enzyme (2-3) run it on a argerus gel and transport the DNA= by capillary blotting to a piece of filter paper to which has DNA on it.<= span style=3D'mso-spacerun:yes'> Have DNA fragments at different b= ands, then add NaOH soultion to denature the DNA. Add your probe which is going to = only bind to the strand that it is complementary to; one of the fragment ba= nds then can be observed on X-ray film.
Figure 3.26

Northern Blot (58:10)

◗ Just like Southern BUT instead of DNA you use RNA

◗ Gather RNA from several different tissues RNA is separated on a Formaldehyde gel to keep everything linear<= /p>

◗ Can be u= sed to determine

�= � if a gene is being expressed in a tissue<= /span>

�= � the size of the mRNA/coding region=

�= � very useful if you only have a small gene fragme= nt and don’t know the size of the protein

encoded by= the gene

�= � 3 bases/codon so if mRNA is 900 bases long the p= rotein should be less than 300 amino acids or

about 30 k= Da (an amino acid =3D about 110 Da)

= Western Blot analysis

◗ Proteins= are separated by molecular weight

◗ transfer= red to nitrocellulose

= Western blots continued

◗ The blot= is probed with an antibody to a specific protein

�= � perhaps made to a fusion protein????

◗ A second= ary antibody is used to visualize the band

�= � radioactivity

�= � enzyme linked to antibody

Figure 3.29=

By having t= he DNA sequenced or even part of it sequenced you can find out…

Where the protein is made
What tissues it is expressed i= n
Size of it
Identify it in a cell lysase o= r in a cell

= Immunoprecipitation

◗ Isolatio= n of a protein from a complex mixture using an antibody

Radioactive labeled proteins

= Ways to determine gene function (what the gene is doing!)

◗ Transfection of cells in culture

Put the protein/gene back into the cell and see what it does

◗ Generation of transgenic animals

Animals that carry a foreign gene

◗ Generation of transgenic plants

Plants carr= ying a foreign gene

◗ Generation of a series of mutants

deletion mutag= enesis
site-directed mutagenesis

◗ Disruption of the gene (get rid of the gene)

homologous recombination
antisense

�= � microinjection - mutant fusion protein or antibo= dy

= Transfection of Tissue Culture cells

Figure 3.33= - can ask yourself what does this cell now do that it didn't do before

= Retroviruses as vectors

Figure 3.34= - replication deficient that can only transfer DNA to one cell and won't make virus, gotten rid of viral sequence.

Have mechan= isms for getting the DNA incorporated = into the genome at very high frequencies

= Generation of Transgenic Mice by micro-injection

Figure 3.35=

= Transgenic Mice - using ES cells

Figure 3.36=

= Transgenic mice (ES cells) continued

= Transformation of Plants

= Transformation in Plants cont.

= One way to do deletion mutagenesis

= Site Directed Mutagenesis

= Homologous Recombination

= Switching out an endogenous gene with a mutagenized gene

= Antisense disruption of gene expression

= Gene disruption by Microinjection

= The NCBI database

◗ How to r= ead a data file

�= � see handout

Does not have huge am= ounts of information on environmental issues or ecology

Lots of information a= bout medicine, genetics, development and very up to date!

◗ we all h= ave access to NCBI through the internet - courtesy of Al Gore - REALLY!<= /span>

Definitiions

Gene:

A segment o= f DNA that encodes a polypeptide chain or an RNA molecule

Chapter 4 The Organization and Sequ= ence of Cellular Genomes

Tuesday, Ju= ly 05, 2005

4:47 PM

Chapter 4 Organization of the Genome

Intro

The initial applications of recombinant DNA were directed toward the isolation and anal= ysis of individual genes.

Genome size= s

The genome of Eukaryotic cells contains a lot of DNA that does not co= de for a gene product (protein, rRNA or tRNA). In higher eukaryotes only about 3% of the genome codes for proteins<= /span>

Figure 4.1

The genomes of most eukaryotes= are larger and more complex that those of prokaryotes
However, the gen= ome size of many eukaryotes does not appear to be related to genetic complexity

Lilies and salamanders have m= ore DNA (amount) but are not 100x more complex

Resolved by discovery that the genomes of most eukaryotic cells contain not only functional genes but large amounts of DNA sequence that do not code for protein (introns)
Much of the comp= lexity of eukaryotic genome thus results from the abundance of several differ= ent types of noncoding sequences, which constitute most of the DNA of high= er eukaryotic cell and the larger number of genes.

Structure of Eukaryotic Gene

Gene: a segment of DNA that is expressed = to yield a functional product, which may be either an RNA (e.g. ribosomal and transfer RNA's) or a polypeptide.

Genes often contain introns which must be removed (spliced out) of the RNA before its product - protein, rRNA, or tRNA - can be made (protein) or = is functional (rRNA, tRNA)

Figure 4.2<= /span>

Gaps at 5' end and 3' end beca= use it is a eukaryotic gene

If a prokaryote gene all the = genes may be smashed together

Most <= span style=3D'font-family:"Times New Roman"'>prokaryote genes do not have i= ntrons

Introns are sections of DNA within a gene that do not encode part of t= he protein= that the gene produces, and = are spliced= out of the mRNA that is transcribed= from the gene before it is exported from the cell nucleus. Introns exist mainly (but n= ot only) in eukaryotic<= /a> cells. The regions of a gene that = remain in the spliced mRNA are called exons.
Introns sometimes allow for <= /span>alternative splici= ng of a gene, so that several different proteins that share some sections in common can be produced from a single gene. The control of mRNA splicing, and hence of which alternative is produced, is performed by a wide variety of signal molecules. Introns also sometimes contain "old code," secti= ons of a gene that were probably once translated into protein but which a= re now discarded.

Some intron= s such as Group I and Group II introns are actually ribozymes that are capable of catalyzing their own splicing out of the pr= imary RNA transcript. They remove themselves on their own.

The amount = of intron DNA varies widely between species. The pufferfish species Fugu rubripes has a very low amount of intron DNA, whereas related species have higher amounts. Introns are not to be confused with junk DNA, which is all DNA without known fu= nction that is not part of a gene.

Identificat= ion of introns

How many introns and extrons a= gene has is highly variable
The entire gene is transcribed= to yield a long RNA molecule and the introns are then removed by splicing= , so only exons are included in the mRNA
3' end used for regulation of = mRNA

Duplex of D= NA w/ RNA – adenovirus

Took adenovirus DNA, made it s= ingle stranded, then added it to mRNA from the adenovirus, and found consist= ent loops
First indication that mRNA is = not an exact duplicate of the genome and that there were these integrating sequences
Adenovirus mRNAs do not hybrid= ize to only a single region of viral DNA
Instead a single mRNA molecule hybridizes to several separated regions of the viral genome
Thus the adenovi= rus mRNA does not correspond to an uninterrupted transcript of the template DNA; rather the mRNA is assembled from several distinct blocks of sequences that originated from different parts of the viral DNA=

Why have in= trons at all?

Introns may be evolutionary remains of formerly important sequences or from retroviruses

Reminiscent of retroviruses that have been integrated into the genome
Play roles in controlling gene expression

Exon shuffling=

Process by w= hich the evolution of proteins with multifunctional domains could be accelerated. If exons each encoded individual functional domains, the= n i= ntrons would allow= their recombination to form new functional proteins with minimal risk of da= mage to the sequences encoding the functional parts.
Evolutionary a good thing bec= ause of exon shuffling
Introns played = an important role in evolution by facilitating recombination between protein-coding regions (exons) of different genes; a process known as exon shuffling

Exons frequently encode functionally distinct protein domains, so recombination between intr= ons of different genes would result in new genes containing novel combinations of protein coding sequence.

This is for exons that code f= or a particular domain of a protein…this is not true for all exons
New genes can be formed by recombination between intron sequences

sometimes int= rons fall between functional domains of proteins
this may give= the cell a selective advantage
the sequences encoding one domain of the protein may be duplicated and added to a different gene
resulting in = a new gene-->protein
if evolutiona= rily GOOD will be come fixed in the population

Exon shuffling would have then played an important role in the further evolution of genes in higher eukaryotes but would not account for the initial assembly of protein-coding sequence prior to the evolutionary divergence of prokaryotic and eukaryotic cells.

Is exon shu= ffling the real reason why we have introns?

Real answer= s is probably we don't really know where they came from or why

Structure o= f an mRNA

5’ Untransl= ated Region (UTR)

UTR =3D untranslated region
Where ribosomal subunits will= bind
Especially small ribosomal will bind to 5' untranslated reg= ion

Move down to where imitator AUG codon is

May contain regulatory elements

Figure 4.4<= /span>

3’ UTR

After Stop codon
Ribosomes fall of
PolyA+ Tail

Beginning of mRNA does not correspond to the beginning of the protein, same with= the end

Bunch of sequence that other proteins interact with for regulation
Also does not get shift out of nucleus

Protein coding region is in between the two

Gene Famili= es and Pseudogenes

One of the reasons we have a l= ot of DNA is not just for introns, a lot of our DNA has been duplicated over time
Some sequences of DNA are pres= ent in multiple copies and therefore reassociated more rapidly that those sequences that were represented only once per genome

Over evolutio= nary time scales many genes have been duplicated

With time, their sequences di= verge due to mutations

The sequence is going to cha= nge from the original gene, mutation occurs and a new gene is created?

These are Gene families they = encode related proteins with related structures and functions<= /li>

Pseudogenes look like duplica= te copies of a gene, BUT they are missing all or part of the promoter
SO NO protein is expressed

Increase the size of eukaryot= ic genomes with out making a functional genetic contribution

or they contain mutations that prevent the expression of functional protein

Globin Gene Family

During evolution genes are sometimes copied. This is one way of getting more of a p= rotein expressed at a single time. The c= opied genes accumulate mutations with time (evolutionary scale time) and may resu= lt in the copies genes encoding related proteins which have a different functi= on, activity, or time of expression. = (see the example in the text of globin genes) These copied, related genes are ca= lled "gene families"

Figure 4.9<= /span>

Text Below:=

Gene Family

A group of related genes that have arisen by duplication of common ancestor

Repetitive = DNA sequences

2 populations= of DNA
repetitive - anneals quickly
non-repetitiv= e - anneals more slowly

Actually we= only have about 30,000 genes. Our DNA contains lots of repeats!

Satellite D= NA - some of that non-coding DNA repetitive stuff

Simple sequence repeats=

Consists of tandem arrays of = up to thousands of copies of short sequences (1-500 nucleotides)\
Can be separated by equilibri= um centrifugation fig. 4.7 (see below)
Simple Seq= uence DNA are not transcribed and don not convey functional genetic materia= l

Equilibrium Centrifugation of Drosophila DNA
Main band at = 1.701 g/ml
“Satellite�= �� bands at 1.705, 1.687, 1.672 g/ml
AT-rich simple repeats 5-200 bp long

SINES, LINE= S

Scattered throughout the genome rather that being clustered in tandem repeats
Major contributor to genome si= ze

Sines

short interspersed elements about= 10% of genome
300 bp long Alu sequences the= best example
Are transcribed into RNA but = they do not encode proteins and their functions are unknown<= /li>

Lines

long interspersed elements
L1 6000 bp long, 500,000/geno= me
Transcribed into and some enc= ode proteins but their function in cell physiology is not known

Both Alu (SIN= E) and LI (SINE) are transposable elements (transposons)

Capable of moving to different sites in genomic DNA

Transposase an enzyme helps the transposon jump around in the genome
Both SINES and LINES are retrotransposons

Their transposition is mediat= ed by reverse transcriptasezasd

DNA is contained in structures called chromosomes

The genome of prokaryotes are contained in single chromosomes, which are usually circular DNA molecu= les
The genomes of eukaryotes are composed of multiple chromosomes, each containing a linear molecule of= DNA
Although the numbers and sizes= of chromosomes vary considerably between different species, their basic structure is the same in all eukaryotes
The DNA of eukaryotic cells is tightly bound to small basic proteins (histone) that package the DNA i= n an orderly way in the cell nucleus

Movie of Chromosome Structure

Chromatin -= Wound up DNA on a nucleosome core particle

Transcription= ally active

The complexes between eukaryotic DNA and proteins are called = chromatin, which typically contains twice as much protein as DNA.
The major proteins of chromat= in are the histones

Histones are small proteins containing a high proportion of basic amino acids (arginine and lysi= ne) that facilitate binding to the negatively charged DNA molecules
There are five major types of histones called H1, H2A, H2B, H3, and H4, which are very similar amo= ng different species of eukaryotes

So why care= about chromatin packing?

If the DNA wa= sn’t properly packaged, it would be a mess during division - think of tryin= g to separate a bunch of thread without breaking any
Turns out the regulation of DNA packaging by histones is a very important pa= rt of regulating gene expression - what genes get turned on, in which cells, and when.

Amino Acid Composition of Histones - positively charged amino acids interact w/ negati= vely charged DNA

Chromatin structure - see nucleosomes - beads on a string

Agarose gel electrophoresis of chromatin after cleavage

Treat chromat= in with micrococcal nuclease - an enzyme that cuts DNA
Run on an aga= rose gel
a “ladder�= � of DNA protected from digestion by nucleosome core proteins – histones

Figure 4.11c *<= /li>

Structure o= f a chromatosome (has H1)

Figure 4.12=

Chromatin Structure

The basic structural subunit of chromatin is the nucleosome

In non-dividing cells (Interphase), DNA is package= d by histone particles in to 10 nm filaments, 30 nm filaments, or heterochromatin

Euchromatin

"true" DNA, what y= our DNA looks like during interphase
10 nm

Can be actively transcribed=
During this period of the c= ell cycle, genes are transcribed and the DNA is replicated in preparati= on for cell division
During interphase most of t= he chromatin (called euchromatin) is relatively decongested= and distributed throughout the nucleus (30 nm)

During this period of the = cell cycle, genes are transcribed and the DNA is replicated in preparat= ion of cell division

30 nm more tightly wound-up

Not transcribitally active<= /span>
RNA polymerase can not get = in

Heterochromatin

10% of interphase chromatin<= /span>
Highly condensed state that resembles the chromatin cells undergoing mitosis
Not Transciptionally Inactiv= e
Contains highly repeated DNA sequences, such as those present at centromeres and telomeres=

Genes that are actively transcribed are in a more decondensed state that mak= es the DNA more accessible to the transcription machinery

Euchromatin
Can be active= ly transcribed
10 nm =

Figure 4.13

30 nm more ti= ghtly wound-up

DNA structu= re during Mitosis

In dividing cells (Mitotic), DNA is tighltly wound (condensed) into chromosomes (≈10,000 x more tightly wound) so they can be transferred to daughter cells

Chromosomes during Mitosis highly condensed

Not transciptionally active

Figure 4.18

Screen clipping taken: 7/9/2005, 9:01 PM

Condensed chr= omatin can no longer be used as template for RNA synthesis, so transcription ceases during mitosis

Genes can be localized to specific chromosome bands by in situ hybridization, indicating that= the packaging of DNA into metaphase chromosomes is highly ordered and reproduci= ble process

Despite being highly order and condensed they will always fold up the same!
One chunk of DNA is always goi= ng to get folded into a chromosome with a particular appearance and not a different one even if the two have the same amount of DNA, the locatio= n of their genes on one chromosome will make it fold different?

Chromosomes=

Karyotype -= (we are diploid) -line up those Chromosomes

Structure o= f a Chromosome on the Metaphase Plate

3 Important Regions each with its own separate function
1. Telomers
2. Centreomere
3. Proteins at centromere to form kinetichore

What does a centromere do?

Makes chromosomes segregate properly into daughter cells after division

Where sister chromatids are gl= ued together by protein

All centromeres do the same thing But they don’t have to look alike!

Telomers

Keep linear D= NA molecules like Chromosomes - Stable
Chromosomes w= /o Telomers become shorter and shorter …..
They are crit= ical for the accurate replication of linear chromosomes --- chapter 5 -telomerase
They are repe= at sequences, and apparently can be in a loop!

Proposed Structure of a Telomer!!

YAC libraries - Yeast Artificial Chromosomes =

have centrome= re, origin of replication and telomers, selectable marker
Act like mini chromosomes

Polytene chromosomes - note banding pattern - from salivary glands of Drosophila

FISH=

Fluorescent <= /span>in situ hybridization on polytene chromosome
How to determ= ine where a sequence of DNA is on a chromosome
pink band
Cytological M= aps

More FISH

On normal met= aphase chromosomes Where’s the gene?

Genome Proj= ects - Completely Sequenced (more or less)

E.coli
Arabadopsis
C. elegans
S. cerivisea
Rhodobacter spheroides
D. melanogaster
H. sapiens
All are avail= able at NCBI!
Each one give= s us more information

Bioinformat= ics

A new field of Biology
Takes DNA seq= uences from high-throughput projects - 10,000s DNA sequences per week<= /span>
Put in a user friendly database
Annotate as t= o what each sequence is likely to encode, e.g., a kinase, a phosphatase, etc.

Proteomics<= /span>

So what we re= ally want to know is what proteins are made and how are they modified in different cell types
Isolate all proteins in a cell type, using analytical methods, MALDI-TOF (matrix assisted laser desorption ionization time-of-flight mass spectroscopy) etc.
query the NCBI database, to identify all of the proteins
A new technol= ogy - LOTS of info coming out!!

Functions of Predicted Genes from Arabidopis

Maps of the= Human Genome

Banding patte= rn from cytogenetic staining
> 90% of t= he genes have been sequenced - BUT still more to learn

Restriction Fragment Length Polymorphism (RFLP)

Can be used to identify inheritance patterns
or an individ= ual!

Detection of Microsatellite Markers by PCR

Another way to identify inheritance

Transcript = Map of Human Chromosome 1

Check out OMI= M map viewer at NCBI!!!

8321 =3D FZD1=

Gene Therap= y

Now that it is possible to identify genes that cause disease
We can attemp= t to carry out gene therapy
The replaceme= nt of a defective gene with a functional gene in the cell
Work in progr= ess

Because of advances in molecular biology, it is now possible to map complete genomes using RFLP, Microsatellite sequences, and FISH

Because of advance in molecular biology, it is now possible to map and sequence complete genomes

Note noncod= ing spacer sequence found at 5' end and 3' end located between genes

Members of the human gene α and <= /span>β-globin families are clustered on chr= omosomes 16 and 11 respectively. Each fami= ly contains genes that are specifically expressed in embryonic, fetal, and adu= lt tissues, in addition to nonfunctional gene copies

Terms

Bioinformat= ics

a new field of biolo= gy endeavoring to identify genes and protein structures from DNA sequences.

Allele

One copy of a gene

Alleles are different forms of the same gene

Diploid

An organism or cell that carries two copies of each chromosome=

Haploid

An organism or cell that has one copy of each chromosome

Noncoding DNA

Spacer sequ= ences

The DNA sequences between genes

Introns

A noncoding sequence that interrupts exons in a gene

RNA splicng/spliceosome

Splicing: The = joining of exons in a precursor RNA molecule

Splicesome:= Large complexes of snRNAs and proteins that catalyze the splicing of pre-mRNAs

Exons

A segment of a gene that contains a coding sequence

Gene famili= es

A group of related genes that have arisen by duplication of co= mmon ancestor

Pseudogenes=

A nonfunctional gene copy

Repetititve DNA sequences

Satellite D= NA

Simple-sequence repetitive DNA with a buoyant density differing from the bulk of genomic DNA

Chromosomes (Eukaryotic and Prokaryotic)

The carriers of genes, consisting of long DNA molecules and associated proteins

Chromatin

The fibrous complex of eukaryotic DNA and histone proteins

Histones H1= , H2A, H2B, H3, H4

Proteins that package DNA in eukaryotic chromosomes

Nucleosome<= /span>

The basic structural unit of chromatin consisting of DNA wrapp= ed around a histone core

Euchromatin=

Decondensed, transciptionally active interphase chromatin

Heterochrom= atin

Condensed, transciptionally inactive chromatin

Telomeres

Repeats of single-sequence DNA that maintain the ends of linear chromosomes

Centromeres=

A specialized chromosomal region that connects sister chromati= ds and attaches them to the mitotic spindle

Mitosis

Nuclear division

In situ hybridization

a tec= hnique that is used to locate genes to specific bands/location on a chromosome. The probe is RNA or DNA --FISH

The use of radioactive or fluorescent probes to detect RNA or = DNA sequences in chromosomes or intact cells

Kinetochore=

A specialized structure consisting of proteins attached to a centromere that mediated the attachment and movement of chromosomes at their centromeres

S. cerivisiae vs. S. pombe centromeres

The centrom= eres of S. pombe span 40 to 100 kb of DNA; they are approximately a thousand tim= es larger that those of S. cerevisae (125 base pairs)

Although S cerevisiae and S pombe are both yeasts, they appear to be as divergent from each other as either is from humans and are quite different in many aspects= of their cell biology

Teleomers

YAC =

(yeast artificial chromosome) a minichromosome with its own telomere and centromere for cloning large pieces of genomic DNA from other organisms, e.g., humans.

A vector that can replicate as a chromosome in yeast cells and= can accommodate very large DNA inserts (hundreds of kb)

RNAi (interference RNA)

The degradation of mRNAs by short complementary double stranded RNA molecules

Lipid Rafts as modification of the = fluid mosaic membrane model

Created with Microsoft Office OneNo= te 2003
One place for all your notes

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