Biology study guide summary for quiz

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Biology study guide summary for quiz

Summary for Quiz

Monday, May 30, 2005

12:13 AM

Title

Identification of Some Macromolecules

Gist of Experiment

Use different tests to check for the existence of macromolecules in various substances

Iodine test checks for starch and/or glycogen
Benedict's test checks for reducing sugars
Biuret test checks for protein

Notes on Underlying Theory

Introduction

The most abundant elements in living material are:

Carbon
Hydrogen
Oxygen
Nitrogen
Sulfur
Phosphorus

There are 4 major types of biological macromolecules:

Carbohydrates

Monosaccharides (i.e. glucose, fructose)
Disaccharides (i.e. sucrose)
Polysaccharides (i.e. starch, glycogen)

Lipids
Proteins
Nucleic acids

Tests

Iodine test

Information on starch:

It is a polysaccharide used by plants to store glucose
Glucose is held together with glycosidic bonds
It is a mixture of 2 different polymers: amylose and amylopectin

Amylose

It is unbranched and helical molecule
The glucose is joined by alpha 1 -> 4 linkages

Amylopectin

It is straight and highly branched
The glucose is joined by alpha 1 -> 6 linkages

Information on glycogen:

It is a polysaccharide used by animals to store glucose
Glucose is held together with glycosidic bonds
It is heavier than starch
It is similar to amylopectin in overall structure, but is more highly branched

How does the test work?

Iodine solution is usually pale yellow
It turns blue-black in the presence of starch because of the amylose
It turns red-brown in the presence of glycogen because of the multi-branched components

Benedict's test

Information on sugar:

All sugars can exist as straight chains or in ring form
The straight-chain forms are called aldose sugars

They have a terminal aldehyde group (C single-bonded to H, double-bonded to O)

How does the test work?

Blue cupric ions (Cu++) in Benedict's solution are reduced to cuprous ions (Cu+) by the free aldehyde groups, and we get a precipitate of cuprous oxide (Cu2O):

4Cu+ + 2OH- + 2e- -> 2Cu2O + 2H+ + 2e-

The amount of cuprous oxide formed is proportional to the concentration of free aldehyde groups

The color of the precipitate varies depending on this as well (blue -> green -> orange -> red -> brown)

Ketose sugars (i.e. non-aldose, which means non-straight chain, which means no free aldehyde group) can ALSO reduce Benedict's solution

This happens because the basic conditions of the experiment isomerize a ketose to an aldose, and then the reduction happens with an aldose

Biuret test

Information on proteins:

They are composed of amino acids, which are connected by peptide bonds
A peptide bond is the carboxyl group of one amino acid covalently linked to the alpha-amino group of the next amino acid

How does the test work?

Biuret solution is a solution of sodium hydroxide (NaOH) and copper sulfate (CuSO4)
Under alkaline conditions (caused by the NaOH), the peptide bonds within proteins react with the Cu++ ions to form a purple complex
So we identify the presence of protein by looking for this purple color

Summary for Quiz

Monday, May 30, 2005

12:13 AM

Title

Isolation of Some Macromolecules

Gist of Experiment

Use a variety of techniques to isolate macromolecules from a starting mixture

Experiment Procedure and Justification Thereof

We start with a yeast-sand mixture

Yeast cells have:

Glucan (a polysaccharide) in the cell walls
Glycogen, proteins, and nucleic acids in the cytoplasm

Grind the yeast to rupture the cell walls and release all this stuff

Add TCA (trichloroacetic acid) and continue grinding

Polysaccharides (in this case glucan) are soluble in TCA, so they will go into solution
But the proteins and nucleic acids will stay suspended!

Centrifuge the suspension (so just the non-sand part)

When you do this on a liquid (remember the polysaccharides are suspended in the liquid) with particles suspended in it (remember these are the nucleic acids and proteins), all the suspended stuff goes to the bottom (it "sediments") and the liquid remains on top

The sediment is known as the precipitate, also known as "pellet"
And the top liquid stuff is the "supernatant"

Now we focus just on the pellet (i.e. the nucleic acids/proteins)

Add NaCl to the pellet

Nucleic acids are soluble in strong NaCl, so they go into solution!
But the proteins remain in suspension

Again we centrifuge this, and the proteins become the pellet, and the nucleic acids are the supernatant
Now we are going to SPLIT the nucleic acids and the proteins, and do stuff with each

Nucleic Acid Portion

Alright, first remember that we are dealing with a liquid here, because the nucleic acids were in solution!
But the first thing we'll do is to add chilled ethanol, which will cause the acids to precipitate out of solution to form a suspension
As before, we centrifuge to isolate the acids (which are in the pellet, of course)
But then we take the pellet and we add sulfuric acid, which makes the nucleic acids go into solution again
Then we boil the stuff - but only ONE of the test tubes! (We have 2 test tubes' worth of nucleic acid)

Boiling in acid is a "hydrolyzing process" - it breaks up the nucleic acid into the nucleotide subunits, and then even FURTHER into the base and sugar and phosphoric acid subunits!

So now we have one test tube of "hydrolyzed nucleic acid" and another of "unhydrolyzed nucleic acid" Good times!
OK, remember we had sulfuric acid in there? Now we have to neutralize the solution! Details below…

We're going to use barium hydroxide, a base, to neutralize this solution
We're essentially going to perform a titration, where we use litmus paper to figure out when the solution is acidic, when it is basic, and when it is neutral
The chemical formula is this: H2SO4 + Ba(OH)2 -> BaSO4 + 2H2O

Note that the precipitate (salt) which forms is barium sulfate - we will filter this out later!

Protein Portion

OK, so remember that back in the day, we had protein and nucleic acid resulting from a centrifugation…Well, now we're dealing with the protein portion, which is solid
We take half the protein and add pancreatic enzyme

This enyzme will hydrolyze the protein into its amino acid subunits
This simulates how the hydrolytic process is carried out naturally, because in real life it is done with enyzmes!

And the other half of the protein we add phosphate buffer, which will not hydrolyze it at all!
To both we add thymol crystals, which prevent the growth of bacteria

Summary for Quiz

Monday, June 06, 2005

10:28 AM

Title

Characterization of Some Macromolecules

Gist of Experiment

Use the method of chromatography to separate the proteins and nucleic acids earlier into their individual components

Experiment Theory

Chromatography is a technique that separates mixtures into their individual components

For example:

If we put black washable ink onto a tissue, the ink will spread outwards from the place where we blotted it
However, the various components of the ink can't all move at the same speed as it spreads out - so the components will visibly separate
The pigment which moves the slowest will "stop" first, followed by the next slowest, and so on…

The stationary phase in any chromatogram is the "matrix" - it is the substance onto which we place the stuff to be measured (i.e. in the above example, it was the tissue - or more specifically, the cellulose in the tissue which was reacting with the ink in the fibers)

Note that the "matrix" has to be INERT - meaning that when we place the mixture we want to examine onto it, they can't react! Or else the whole point of it will be ruined!

And the mobile phase is the solvent - meaning that the mixture we want to study will DISSOLVE in this solvent, and then the solvent will move up the "matrix" (paper, in the above case), and like with the paper example provided above, certain parts of the mixture /solvent will stop based on how much the matrix slows them down

Or as the lab manual says, "separation depends on the relative tendencies of molecules in a mixture to associate more strongly with one or the other phase."

Here are some factors which affect how far a given substance (within a mixture) will travel:

How soluble is it in the solvent?

If it is COMPLETELY soluble, it'll just travel as far as the solvent does, and we won't see any separation
If it is NOT SOLUBLE at all…it won't travel anywhere at all!

How heavy is it? (What is the molecular weight?)
What is the overall polarity of the compound?

The thing we measure in chromatography is the difference between how far a substance (from the mixture) travels compared to how far the solvent travels

Rf = (distance traveled by a substance) / (distance traveled by the solvent)

Experiment Procedure and Justification Thereof

Well, this is a very general overview, but…
Put the mixtures on the chromatography paper (just a spot of each)
Sew the paper so that it forms a cylinder (but the ends of the paper should NOT overlap)
Put the "cylinder" into the solvent, making sure that the spots are just over the level that the solvent comes up to

Solvent:

For proteins, it is FORMIC ACID (10% formic acid, 70% isopropanol, 20% water)
For nucleic acids, it is ACETIC ACID (15% acetic acid, 60% butanol, 25% water)

Summary for Quiz

Sunday, June 12, 2005

11:19 PM

Title

Spectroscopy

Gist of Experiment

Measure the concentrations of unknown solutions using the concentration curves derived from the measurement of solutions of known concentration (all this using spectrophotometers).

Notes on Underlying Theory

The energy content of light depends on its wavelength (because light moves as waves)

The human eye can recognize light between 400 nm (violet) and 750 nm (red)

A spectrophotometer has a white light source which focuses on a prism that splits it up into the different portions of the spectrum…after this, we can focus each different "incident beam" on a sample specimen

The sample specimen is dissolved in a solvent, and it is housed in a tube called a cuvette

When the incident beam hits the sample, one of 3 things will happen:

It gets absorbed
It gets transmitted
It gets reflected

The part that gets transmitted goes through and hits the photoelectric cell, which generates an electric current - and the current tells us what the intensity of the transmitted beam is! Or in other words, it tells us how much got through…
The current is measured in 2 ways:

Percent transmittance - this is an arithmetic scale with equidistant units from 0% to 100% which tells us what percentage of the light was transmitted (i.e. how much got through)
Absorbance - this is a logarithmic scale with unequal divisions from 0.0 to 2.0 which tell us how much light was absorbed

Beer's Law says that the concentration of a light-absorbing solute is directly proportional to the absorbance over a given range of concentrations

So this means that as we vary the amount of solute we put in the solvent, obviously the absorbance readings we get from the spectrophotometer will change…but they will be LINEAR!

On the other hand, the relationship between the solute concentration and the percentage transmitted is NOT linear

Random note: Know that the photocolorimeter we use can measure the entire visible spectrum and slightly overlaps into the U.V. and infrared ranges, but other spectrophotometers can use the whole U.V. range (180 to 350 nm) and infrared range (780 to 300,000 nm)
So how do we analyze a substance?

First we dissolve the substance in a suitable solvent
Then we insert a cuvette containing ONLY the solvent into the electrophotometer (this is called the "blank" cuvette), and we zero the scale at this level so it acts as our baseline
Then we replace the solvent-only cuvette with a cuvette containing a solution with some solvent in it…And obviously the solute in this solvent will absorb some light so the reading will be different

Graphical analysis of a substance

Firstly, we have to plot an absorption spectrum for the substance - this means that you read the absorbance of that substance at many different wavelengths at one constant concentration (remember, this would mean that when you use the spectrophotometer and split it up into different wavelengths of light, you don't just use one of those…you use many!)

If you draw a curve that relates absorbance to wavelength, look for the highest point on that curve - it will tell you what the wavelength of maximum absorption is

Then, you make a concentration curve - so you set the spectrophotometer to use the wavelength of maximum absorption which you just figured out, and then take multiple measurements with different concentrations of the solute

Note that you only have to measure 2 or 3 different concentrations and then plot them - the rest of the points can be deduced by drawing a straight line, because according to Beer's Law, it is a directly proportional relationship!
The plot should go from 0.025 to 1.0

This entire process allows us to determine the concentration of an unknown sample! Because once we have the concentration curve, we can just take any other sample of that substance (even if we don't know its concentration) and figure out what the absorbance level is! And from there, we can check the curve to find what the associated concentration is…

Notes on Experimental Procedure

Experiment 1 - Congo Red

Basically, we are creating solutions of different concentrations of Congo Red first…
Then we get the maximum absorbance by measuring some of the cuvettes at varying wavelengths (from 400 nm through 600 nm, going up by 20 nm each time)

But when we narrowed it down to a wavelengths, re-measure the area going up by 5 nm each time to be even more precise

Then we use this wavelength and try all the different concentrations so that we can get our concentration curve

Experiment 2 - Chloroplast pigments

Information:

Within chloroplasts, there are different pigments, and they all absorb different parts of the visible spectrum…but the parts of the spectrum they can't absorb are reflected! And these are the colors we see!
The major pigments in a chloroplast:

Chlorophylls

Chlorophyll A
Chlorophyll B

Carotenoids

Carotenes
Xanthophylls

Procedure:

We get the chloroplast extract from spinach leaves and we dab it along the line of a paper which we will perform chromatography on (the procedure is much the same as last week)

Note that the solvent in this case is 90% petroleum ether, 10% acetone

When the chromatogram is finished it should look like so (starting from the distance the solvent traveled and going backwards):

A thin orange band (carotene)
2 distinct yellow bands (xanthophylls)
Green band (chlorophyll A)
Green band (chlorophyll B)

Then cut up the paper into these different strips and put them into test tubes of acetone, which will facilitate elution of the pigment into the solvent - we only want Chlorophyll A and Chlorophyll B!
Now we have different test tubes with different pigments in them! We now use these with the spectrophotometer and we find a maximum absorbancy wavelength for each of them

Summary for Quiz

Monday, June 20, 2005

2:28 AM

Title

Enzymes

Gist of Experiment

See the effect that enzyme concentration has on reaction time and the effect that substrate concentration has on enzyme reaction.

Notes on Underlying Theory

General

Enzymes are:

Biological catalysts (remember from CHEM 123 what a catalyst does)
Specific in their action (specificity is determined not only by amino acid order, but also by 3-D conformation)
Proteins (except for a small subset of enzymes which are called ribozymes)

Enzymes combine with a substrate to form a substrate-enzyme complex, which then breaks down into the enzyme again (which is UNALTERED!) and the product:

Substrate + enzyme -> substrate-enzyme complex -> product + enzyme

Here are some factors which affect the rate at which the enzyme converts the substrate into the product:

Temperature
pH
Enzyme concentration
Substrate concentration
Product concentration
Energy of activation

As for the direction of the reaction, all enzyme-mediated reactions are theoretically reversible, but the direction which the reaction actually goes in depends on the conditions under which the reaction is taking place

Experiment #1: Salivary amylase

Salivary amylase is a digestive enzyme found in saliva
It acts on starch molecules by breaking off maltose molecules from the end of the starch chain
Each time the chain is broken, a water molecule is consumed - thus this reaction needs water and is called a hydrolytic reaction

The general term for bond-breaking with water is hydrolysis

Experiment #2: Phosphorylase

Phosphorylase is an enzyme that acts on starch by breaking off glucose molecules
Instead of using water to do this, we consume phosphoric acid - and so the general term for bond-breaking with phosphoric acid is phosphorolysis
Here is the general reaction: (Glucose)n + HPO42- <--> (Glucose)n-1 + Glucose-1-phosphate

When the reaction goes -->, it is phosphorolysis
When the reaction goes <--, it is synthesis

Other notes on this reaction:

No energy is released from the system during phosphorolysis because the energy released in rupturing the glucose-glucose bond is used to create the glucose-phosphate bond
Water is not a reactant, so its concentration does not affect the direction of the reaction
The direction of reaction depends on the relative concentrations of substrates and products

Before 1960, we only thought that an enyzme called starch synthase could perform that reaction going backwards (that is, the synthesis of starch)
But now, we know that phosphorylase can do it too! It just needs the right concentration gradient…

The only thing is, all it can do is attach glucose molecules to a "starch primer" molecule that already exists - in other words, it can't make starch out of nothing!

So the number (or molar concentration) of starch won't increase -- it's just that they will be put into long chains instead of short ones

The other kerfuffle is that phosphorylase won't put together glucose molecules on their own…they have to be glucose-1-phosphate!

Notes on Experimental Procedure

Experiment #1: Salivary amylase

We make solutions of salivary amylase of different concentrations (these should FAIL the iodine test for starch/glycogen)
Then we make an equal amount of solutions of starch suspension, but each with the SAME concentration (these should PASS the iodine test for starch/glycogen)

The starch suspension is made up of 0.25% NaCl, because the Cl- ions specifically activate salivary amylase
We also add McIlvaine's buffer to the starch solution so that optimal pH levels are maintained

Then we mix each salivary amylase solution with one starch solution, and we periodically try the iodine test…as the enzyme finishes reacting with the starch, there should be NO starch left because it eats up starch!

We do this mixing in 37o water, because this is the optimal temperature for salivary amylase

So the idea is that the solutions were salivary amylase is in higher concentration should take LESS time to fail the iodine test!

Experiment #2: Phosphorylase

First we prepare the phosphorylase:

Homogenize potatoes along with sodium fluoride in a blender

The NaF is necessary to inhibit the action of potato phosphatase, which would break down glucose-1-phosphate into glucose and phosphate, thus making it impossible for the phosphorylase to perform synthesis

Centrifuge the result - phosphorylase is present in the supernatant

Note that we should test the phosphorylase with the iodine test to make sure it didn't get any starch from the potatoes!

Always handle the phosphorylase with a pro-pipette because the NaF is poisonous!

We make solutions of different substances:

Glucose
Glucose-1-phosphate
Potassium phosphate

Then we add either 1 drop of starch (i.e. primer starch) or 1.5 mL of starch to the tubes, and then we put phosphorylase on top of that…and watch what happens in terms of whether or not the solution passes the iodine test!
Based on what we know about the behavior of phosphorylase, and concentration gradients, etc. - we should be able to predict what happens!

Summary for Quiz

Sunday, June 26, 2005

11:11 PM

Title

Osmosis

Gist of Experiment

To demonstrate some aspects of the movement of water in and out of a cell.

Notes on Underlying Theory

Introduction

All the molecules in a substance are always moving…and the NET movement is from an area of higher concentration to one of lower concentration…this is called DIFFUSION
In biological systems, water is the solvent where all other substances (called SOLUTES) are dissolved in
The cell membrane is a semi-permeable membrane that allows water to go through freely in both directions…but NOT solutes!
In biological systems, the movement of water through the cell membrane is called osmosis

ISOTONIC: If the fluid outside a cell has the SAME concentration of solutes as the fluid inside the cell
HYPERTONIC: If the fluid outside the cell has a HIGHER concentration of solutes than the fluid inside
HYPOTONIC: If the fluid outside the cell has a LOWER concentration of solutes

Part 1: Osmosis in a Model System

None

Part 2: The Compound Microscope

A diagram which you might want to look at if you're bored!

Part 3: Osmosis in Animal Cells

The cytoplasm of any animal cell is surrounded by a thin and flexible membrane

The membrane is elastic, so it can accommodate very small increases in volume
But if too much water comes in, the membrane ruptures!

The bursting of an animal cell is called lysis
The bursting of a red blood cell specifically is called haemolysis

The hematocrit of whole blood is the percent of blood (by volume) that is made up of cells

So like, if someone's hematocrit is 40, that means that 40% of the blood volume is cells and the rest is plasma

Hematocrit is affected by: degree of bodily activity, altitude, and anemia (pathological deficiency in the oxygen-carrying component of blood)

Part 4: Osmosis in Plant Cells

Just like animal cells, plant cells have a thin and flexible cell membrane
But ALSO, the plant cells have a cell wall OUTSIDE the cell membrane
And AS WELL, there is a huge vacuole within the cytoplasm which plants have…so huge that there is only a little space between the cytoplasm and the cell membrane

In an ISOTONIC environment: the vacuole is filled with sap
In a HYPOTONIC environment: water REALLY wants to enter the cell because there is way more solute concentration inside the cell, and it wants to balance out…

So lots of water enters and pushes out the cell walls…but the cell walls respond by pressing back IN and squeezing water OUT!
When this happens, we call the cell TURGID

In a HYPERTONIC environment, water wants to LEAVE the cell to go outside because the solute is really concentrated outside! So water leaves the vacuole and the cell shrinks…

We call this cell PLASMOLYZED, and the phenomenon is called PLASMOLYSIS

Notes on Experimental Procedure

Part 1: Osmosis in a Model System

We're just preparing beakers with different concentrations of sugar, then putting dialysis bags with different concentrations of sugar inside the beakers…and noting the net direction of travel by the sugar by weighing the dialysis bags every so often

Part 2: The Compound Microscope

None

Part 3: Osmosis in Animal Cells

Put cow blood in 3 different flasks, then add solutions of different concentrations of sodium chloride, to simulate isotonic, hypotonic, and hypertonic environments

0 M NaCl
0.14 M NaCl
0.34 M NaCl

Also, collect samples from each flask with a hematocrit tube, and centrifuge to separate the cells from the plasma

Observe the approximation volume of each, and use this to calculate the hematocrit of the substance

Part 4: Osmosis in Plant Cells

Prepare 2 beakers - one with distilled water, and the other with 0.5 M sucrose solution
Place 2 "Elodea sp. leaves" in each beaker

Observe them…

Summary for Quiz

Monday, July 04, 2005

1:17 AM

Part A - Mitosis

Theoretical Notes from Introduction

The appearance of the nucleus and its chromosomes tell us a lot about what stage of the cell cycle a given cell is in
Nuclear division is called mitosis and cytoplasmic division is called cytokinesis, and they usually happen together, but they are NOT necessarily linked

The cell cycle consists of an interphase stage (takes up most of the cycle) and a mitosis stage (just a little bit)
The interphase stage has 3 parts:

G1 phase
S phase
G2 phase

You can't see the chromosomes in the nucleus during interphase because they are in a "diffuse" state

The granular chromatin (the stuff that eventually condenses to become chromosomes) is bounded by a distinct nuclear membrane, and sometimes you can see nucleoli within the chromatin

As for the nucleus, it looks the same through all 3 stages of interphase, so you can't tell what part of interphase a cell is in by looking at the nucleus
Now, recall that mitosis is the separation of the nucleus (and by extension, the chromosomes in the nucleus)…but interphase is the time when the chromosomes are duplicated

G1 phase: no chromosome synthesis happening here…chromosomes are still unipartite
S phase (synthesis phase): here the chromosome duplication happens, and now each is bipartite - each pair is held together by a centromere
G2 phase: the chromosomes are still bipartite…more specifically, they are two double-stranded DNA molecules called chromatids which are held together by centromeres, as stated earlier

Then mitosis happens…there are 5 steps in mitosis (PPMAT):

Prophase

Chromosomes condense (become shorter and thicker), and now we can see them

Prometaphase

The nuclear membrane disappeared, so now the chromosomes can go wherever they want…so they're spread out all around the cytoplasm, although they are starting to go towards the center of the cell
Here we form a mitotic spindle, and the chromosomes move along the microtubules towards the center of the cell

Metaphase

The chromosomes are all in a line on the equatorial region of the cell, and each chromosome is connected to a separate spindle fiber by its centromere

Anaphase

The centromeres separate - each bipartite chromosome becomes two unipartite daughter chromosomes…they head to opposite poles of the cell

Telophase

The chromosomes start to uncoil again, and the nucleus begins to form…
Cytokinesis starts here:

For animal cells, it is a cytoplasmic constriction
For plant cells, a cell plate is formed

Theoretical Notes from Experimental Procedure

Plant cells

Onion (or hyacinth) root tip

There are 4 sections "A", "B", "C", "D" going away from the root tip

A: the root cap (no mitosis happening)
B: zone of cell division (lots of mitosis)
C: zone of cell elongation (no significant mitosis)
D: zone of cell differentiation (no significant mitosis)

Permanent onion root tip "squashes"

Animal cells

Ascaris sp. uterus

In general, animal chromosomes are much smaller than plant chromosomes, so the material is harder to see
We are looking at the uterus of a female Ascaris sp. worm (Ascaris sp. is a parasitic roundworm)
Within the uterus we are able to see spherical cells - these are fertilized eggs (i.e. zygotes) undergoing mitosis

There are many layers of extracellular membranes surrounding the actual "cell"
LEARN THE DIAGRAM ON PAGE 50!

Centrioles & asters

They are only present in animal cells
Asters are an array of microtubules in a flower-like arrangement surrounding each centriole pair at the poles of the spindle

They can be most easily seen in metaphase or anaphase
The center of the aster is the region of the centriole

Part B - Meiosis

Theoretical Notes from Introduction

Meiosis is the formation of gametes (sex cells) which have half the number of chromosomes of the parental nucleus

As with mitosis, cytokinesis usually happens at the same time…

Normal somatic human cells have 46 chromosomes, and they are called diploid cells

The chromosome can be paired up into "homologous" pairs, which are morphologically identical (i.e. they have the same shape, although they may not necessarily have the same genes on them)
We'll call this number "2N"…because there are N morphologically unique chromosomes, and the somatic cells have 2 of them each…so 2N in total

Human sex cells only have 23, or N, chromosomes - i.e. for every morphological/homologous pair, they only have one of the chromosomes

The stages in meiosis are as follows:

Interphase I

Same as in the mitotic cycle

Prophase I

This is very DIFFERENT than prophase in mitosis because the homologous chromosomes pair up…and the pairing is called synapsis

And the complex formed by the paired chromosomes is a "tetrad" or a "bivalent"

The 4 sub-stages of Prophase I are:

Leptotene (condensation of chromosomes)
Zygotene (pairing of homologous chromosomes)
Pachytene (recombination)

This part is important…it is when chromosomal exchanges called crossovers are made

Diplotene (transcription)
Diakinesis (recondensation)

Metaphase I

The homologous pairs line up along the equator…not the individual chromosomes, as in Mitosis metaphase
And the centromeres attach to the spindle fibers

Anaphase I

What this means is that when the pairs separate, you're going to have FULL BIPARTITE chromosomes (aka DYADS) going to each end…unlike mitosis when you have unipartite

i.e. The centromeres are not separated…they stay intact because their job is to hold together the bi-partite chromosomes, and right now the chromosomes are still bi-partite

Telophase I

So in mitosis you would have 46 unipartite on each end…now we have 23 bipartite on each end

Interphase II

This stage is like other interphase, with the important exception that there is NO G1 or S phase, since we are not re-duplicating the DNA

Prophase II

Just like a mitotic prophase!

Metaphase II

Just like a mitotic metphase, when you have bipartite chromosomes lining up along the equator

Anaphase II

Same as mitotic anaphase...

Telophase II

Same as mitotic telophase…except we make haploid cells now!

Theoretical Notes from Experimental Procedure

Plant cells

Male gametogenesis: lily anthers

Lily anthers contain pollen sacs

LEARN DIAGRAM ON PG. 52!
At the early stage of an anther's development, the young pollen sacs contain diploid cells called pollen mother cells
As the anther matures, the mother cells undergo meiosis I, then meiosis II and have haploid cells which we call microspores (immature pollen grains)

Note that pollean formation in the lily anther is SYNCHRONOUS, so in any given anther, most of the nuclei will be in the same stage of development!

Female gametogenesis: lily ovaries

Lily ovaries contain ovules…and we are interested in the contents of these ovules

Within each ovule, the nucleus of one diploid megaspore mother cell (MMC) goes through meisois I and forms 2 nuclei…when it's doing meiosis we call it an embryo sac
After meiosis finishes, we have 4 haploid cells which…

All share the same cytoplasm (since no cytokinesis occurred)
And are not mature yet (we need 2 more mitotic divisions and some nuclear re-arrangement to get this to be mature)

So of course, the MMC has 3 stages:

Uninucleate stage (just one nucleus, probably in interphase)
Binucleate stage (two nuclei, probably in interphase)
4-nucleate stage (4 haploid nuclei, probably in interphase)

Animal cells

Male gametogenesis (grasshopper testis)

So we're going to study grasshopper testis…
Testis are composed of tubules, which are composed of compartments or cysts, which are composed of cells

All cells in one cyst develop synchronously, so they are all in the same meiotic stage

We can study these cells and find all the following:

Spermatogonia are mitotically active diploid cells which are preparing for meiosis

Usually found at the apex of a tubule

Then right before they do meiosis, they are primary spermatocytes
After meiosis I, we get 2 secondary spermatocytes
And then the spermatocytes undergo meiosis II and we get spermatids

These things are immature sperm

Female gametogenesis (Ascaris sp. uterus)

Female gamete formation is unique because only one of the four products of meiosis II becomes a functional gamete…
In general…

The diploid cells in the ovaries which eventually undergo meiosis are called oogonia
Then, they are primary oocytes right before they stop mitotically dividing and start meiosis
The first 2 products of meiosis are the secondary oocyte and the first polar body

The secondary oocyte is way bigger than the first polar body (due to the way cytokinesis occurred)

Then both of them undergo meiosis II…and again, we have the secondary oocyte splitting up into 2 products where one is way bigger than the other

The larger one is the ootid, and the smaller one is a second polar body
The products the first polar body's meiosis II division are also second polar bodies

The ootid eventually becomes a mature gamete called an ovum (or egg)
All polar bodies eventually disintegrate and are reabsorbed

In Ascaris sp., the primary oocytes don't begin meiosis until they have been fertilized - i.e. the sperm enters its cytoplasm…

Then karyogamy also needs to happen, which is a separate event - it's when the 2 nuclei fuse together
BUT…there is a significant time lag between fertilization and karyogamy…because obviously…you can't fuse the nuclei until they are each haploid! And…the ascaris sp doesn't become haploid (i.e. perform meiosis) until it is fertilizer…

So we go 1) fertilization 2) meiosis (triggered by fertilization) 3) karyogamy

Three different slides were examined:

Ascaris sp sperm entrance: here we should see primary oocytes (because the sperm JUST entered, so meiosis either hasn't been triggered yet or was JUST triggered)
Ascaris sp maturation: now we have primary/secondary oocytes because we are undergoing meiosis…and we might be able to see polar bodies, too!
Ascaris sp pronuclei: now, most of the nuclei have completed meiosis II…we should be able to see 3 polar bodies associated with 1 ootid!

Remember that the nuclei at this point should have an interphase appearance, because they just finished telophase…
And now the sperm nucleus is called the male pronucleus, and the ootid nucleus the female pronucleus
And NOW we start karyogamy, after which mitosis can occur...

Summary for Quiz

Sunday, July 24, 2005

1:10 AM

Title

Agarose gel electrophoresis

Gist of Experiment

Estimating the size of unknown DNA fragments (?)

Notes on Underlying Theory

Electrophoresis is a technique for separating and analyzing mixtures of charged molecules

The term electrophoresis means "to carry with electricity"

Nucleic acid molecules (both DNA and RNA) are negatively charged because the phosphate group in their sugar-phosphate backbone

So…if we put DNA in an electric field, it'll migrate towards the anode (positive electrode)!

However…when we're putting DNA in an electric field, we first suspend it in AGAROSE GEL…the purpose of agarose gel is to be like a semi-solid matrix for the DNA

Agarose is a natural polysaccharide of galactose and 3,6-anhydrogalactose (derived from agar, which in turn comes from marine red algae)

And we make the agarose gels by dissolving it while DRY in a boiling buffer (remember this for later!) then pouring the gels into casting trays and allowing them to solidify

Why do we need a buffer? Well, I'm glad you asked…

The IONS in the buffer allow the current to flow through the electrophoresis gel
The buffer SYSTEM in general controls the pH (so that the sample molecules don't get damaged) and controls the ionization state of the sample molecules

The RESOLVING POWER of an agarose gel (i.e. how far the nucleic acid molecules go) depends on:

Gel pore size

This is determined by how concentrated the agarose is!

The more agarose we have, the more dense it is, and the smaller the pore sizes

Usually if we are using smaller DNA molecules, we will use high percentage agarose gels (0.2 - 1 kilobases)…and for bigger molecules we use lower-percentage gels (5 - 10 kb)

However…beware of the extremes! Low percentage gels are very soft and can easily break, while high percentage gels solidify fast and don't set evenly, are brittle, and can crack easily…

Size/conformation of nucleic acid moleculese

OK, by "size", we mean the number of base pairs in the nucleic acid in question

For double-stranded molecules, we go by base pairs
For single-stranded molecules, we go by nucleotides

Note that the CONFORMATION of a nucleic acid molecule can also influence how fast it goes through the gel!

Circular, twisted DNA > Linear, relaxed DNA > Circular, relaxed DNA
As a result…we can only compare the sizes of nucleic acid molecules if they have the same conformation…but this isn't bad because we can always control the conformation by cutting the DNA, etc.

One really cool application of electrophoresis is that we can estimate the molecular size (i.e. how many kilobases) of an unknown piece of nucleic acid by comparing the distance it travels with something called molecular weight standards

These things are a set of DNA fragments whose molecular size we KNOW…and so if we run them at the same time as the unknowns, we can figure out what how big the unknowns by comparing them with the ones we know!
This works because there is a linear logarithmic relationship between the distance traveled and the size of the piece…so if we make a graph based on the DNA fragments we know, we can find the spot on this graph where our unknowns fall, and figure out their sizes that way…

We also STAIN the gel with ethidium bromide because it allows us to see the nucleic acids afterwards when we put the gel on a UV trans-illuminator

Ethidium bromide…

Is a carcinogenic chemical
Contains a PLANAR GROUP that inserts itself (intercalates) between stacked bases in a double-stranded DNA molecule
Emits in the red-orange region of the visible spectrum when it is exposed to UV irradiation
Can be used for both single-stranded and double-stranded DNA, although it is WAY BETTER for double-stranded DNA because the bases are stacked, whereas they are not stacked for single-stranded DNA

And LASTLY…we have to mix the DNA (or RNA) with a "LOADING BUFFER" (this is different than the buffer which the gel is submerged in!) even BEFORE we insert the DNA/RNA into the gel walls

Loading buffers contain dye (usually bromophenol blue) and glycerol or sucrose…and we use them to help monitor the progress of the gel
Since the dye (remember, bromophenol blue!) is negatively charged in neutral buffers, it will move in the same direction as the nucleic acids during electrophoresis, so we can have a *rough* idea of where how the nucleic acids are progressing…
ALSO…the glycerol/sucrose make the nucleic acid more dense so it doesn't diffuse out of the gel well before beginning electrophoresis

Notes on Experimental Procedure

Alright…well first of all, what does it look like? See this diagram:

Notice that…

We have the gel tank

Electrodes at two sides
Casting tray
A set of combs (containing the wells we will use…)
A power pack

OK…now, remember how we have to dissolve the gel in a "running buffer"? Here is how we make it…

48.4 g of tris (a base)
11.4 mL of glacial acetic acid
7.44 g of EDTA
Add enough distilled water to get to 1 L

Now…dilute it in a 1:10 ratio so we get 1X TAE

Now we have to make the gel!

3 g agarose dissolved in 300 mL of 1X TAE buffer
Heat this to boiling in a microwave oven and cool to 50o C, stirring constantly
At 6 ul of ethidium bromide and stir slowly to mix it in there…
Now we pour the contents into the gel rig, put the comb in place and allow everything to solidify

This takes about 30 to 45 minutes…and we know it's ready when the clear agarose solution becomes opaque and semi-solid

Now pour more of the 1X TAE buffer over the surface (to a depth of 1 mm)
Now remove the comb…
And fill the gel rig with 1.8 more liters of 1X TAE buffer

And prepare the samples of DNA…

1:10 dilution of DNA (1 ul DNA, 9 ul water, 2 ul loading dye gives us 12 ul in all)
1:50 dilution of DNA (1 ul DNA, 49 ul water, 10 ul loading dye gives us 60 ul in all)
And our MARKER DNA…is called "Lambda DNA/Hind III marker", and we'll put 10 ul of it into a well…

And now we have to load the DNA in there!

We use a micro-pipettor…and this works by pressing down on the plunger through TWO stops (the second one is to blow out any remaining dye from the tip)

And now we run the gels!

You'll notice that there are tiny bubbles at the bottom edges of the gel tank, where the electrodes are…these indicate that electric current is going through the box

There are bubbles at both ends
But there are more at the negative end (the left side…where the DNA starts)
The bubbles are there because the charge causes hydrolysis of the water molecules, and the H and O separate…the O (gas) molecules rise up through the water and we see them as bubbles!
AND…there are more at the negative end because the water molecules are attracted to the negative end because they are slightly positive, having 2 (positive) hydrogen's and only one negative oxygen

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