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Molecular Biology

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Modified on 2012/12/05 00:26 by Ben Fulton Categorized as Uncategorized
L504 Notes

3 kinds of reversion mutations

bacteria acquire new DNA by transformation, conjugation, and transduction.

Transformation – A DNA strand is introduced into the cell and binds with existing DNA by a double crossover, causing it to replace a section of the existing DNA. With a repeated sequence in the DNA, as the double helix is attempting to be built, the first sequence in one strand may bind to the second sequence in another strand. This will cause two separated double helices, one looping and one straight. Or, with one looping and one straight strand, they may bind into a single straight strand. If there is a strand with an inversion in it, the inverted strands may bind together. If they do, and then there is a double crossover, the DNA in the middle ends up reversed. Two circular DNA with identical regions can bind and crossover in those regions, creating a Figure 8, which then untwists to form a larger circle. Since the identical regions remain intact, they might bind together again, crossover, and split the large circle back into the original two circles. During meiosis, direct repeats in a single strand might cause a mismatch. A->REPEAT->B->REPEAT->C Paired with A->REPEAT->B->REPEAT->C Should yield two identical strands, but it might match up as A->REPEAT->B->REPEAT->C A->REPEAT->B->REPEAT->C Then after crossover and separation we have A->REPEAT->B->REPEAT->B->REPEAT->C And A->REPEAT->C

Conjugation Plasmids – a separate DNA molecule, probably circular, probably inside a prokaryotic cell. It can be thought of as Parasitic DNA, with an origin of replication E. Coli has an F plasmid, which makes the cell male, in a way. It causes a tube (“F pilus”)to extrude from the cell, which can contact another cell and pass the F plasmid into the cell, causing that cell to become male as well. The F plasmid can recombine itself into the host DNA circle. When it does, the cell becomes able to replicate its entire DNA strand and send it, through the F pilus, into another cell. These are called HFR cells. (High Frequency of Recombination), since they work more effectively and often than F working on its own. The F plasmid is sent through last. The entire transfer takes about 100 minutes. These two facts make it possible to map genes on the chromosome, by interrupting the process and counting the recombinants. Mobile DNA Imagine DNA with a single gene, encoding a transposase enzyme. The ends of the DNA are reverse repeats. This is an Insertion Element, or IS. The transposase can recognize the elements ends and insert a copy elsewhere in the genome.

Prokaryotic genes are organized into operons. Operons contain cistrons (which are genes)

Lac operon

E. coli generally likes to get its energy from glucose. In the absence of that, it is able to get energy from lactose. How?

The Lac Operon has genes named lacZ, lacY, and lacA. LacZ encodes beta-galactosidase; LacY, permease. (LacA doesn't appear to be necessary). An independent gene, Lac I, produces Lac Repressor. It has a weak promoter so it doesn't produce very many.

Lac Repressor is a protein that binds to DNA. Specifically, it binds to a part of the lac operon that RNA polymerase would like to bind to. The polymerase is therefore unable to bind and create lac proteins. But, in the presence of lactose, the lac repressor changes shape and can no longer bind to the lac operon. This allows the polymerase to bind to LacZ and LacY, and the cell will generate beta-galactisodidase and permease. These two proteins allow the processing of lactose.

Ways this process will get messed up

  • Lac Repressor doesn’t bind DNA (I-). The polymerase can thus bind to the DNA and create LacZ and LacY all the time.
  • The repressor doesn’t bind lactose (the inducer) (Is). The repressor, then, always sticks to the DNA and LacZ and LacY will not be created.
  • The operator on the DNA doesn’t bind to the repressor (Oc) So the polymerase can, and will create.
  • The LacZ gene could just be busted (Z-)

So, consider the following gene patterns:

I+ P+ Oc Z+ / I- P- O+ Z- : The gene with the Oc doesn’t bind to the repressor, so it will bind to the polymerase, and lac proteins will be always be created (Constitutive)

I+ P- O+ Z- / I- P+ O+ Z+ : Even though the I- gene won’t create proper repressors, the I+ gene will, so lac proteins will be created in the presence of lactose (Normal, or Inducible)

Is P- O+ Z- / I- P+ O+ Z+ : The repressors that can’t bind to lactose will happily bind to DNA and block lac proteins from being created. (Uninducible)

I+ P+ O+ Z- / I+ P+ Oc Z+ : Oc gene doesn’t bind to repressor and will create lac (Constitutive)

Restriction

Enzymes exist for the sole purpose of cutting DNA. As a rule, these “restriction enzymes” look for a specific sequence of 4, 6, or 8 base pairs on the DNA, and then cut the DNA somewhere along that sequence. (E. Coli takes advantage of this by adding a methane block to its own DNA, preventing a particular restriction enzyme from cutting it. Then, an alien piece of DNA which gets incorporated, will not have a methyl cap and will be vulnerable to cutting.)

If we have a cell with a plasmid inside of it, we can perform DNA extraction on the cell. This will give us lots of small bits of bacterial DNA, with, hopefully, an intact plasmid. We can isolate the plasmid by looking at all the bits of DNA by length, and matching it up with the DNA length of our plasmid. Now we can cut the plasmid with a restriction enzyme. Typically a plasmid with 3-6kbp will have 10-15 sites that are cut with a 4-cutter. Vectors If we have a plasmid DNA, we can insert DNA fragments into it. They must both be cleavable by the same restriction enzyme. Put them together and cleave, then use a DNA ligase as glue to repair the damage. The ligase will match the plasmid DNA cleave with the foreign DNA cleave and glue them together.

Now we can insert our plasmid into a bacterial cell. As the cell reproduces, many copies may inherit the plasmid, and so we have lots of copies of our altered DNA. This is cloning.

A simple application of this is to create libraries of sequences. Take a bacterial genome, cut it with restriction enzymes into hundreds of pieces. Put each fragment into a plasmid, put the plasmid in a bacteria and let it reproduce. Cut it with a different enzyme, and you get the same result but different sequences. This allows us to get overlaps.

Sequencing – using four different cutting enzymes, one for each nucleotide.

Reverse transcriptase – initially found in retroviruses. Is able to create single strand complementary DNA (sscDNA) from RNA. Once we have that, can use mormal DNA polymerase to create double-stranded DNA.

Southern Blotting

Polymerase chain reaction (PCR)

hybridize: rna and dna together

primer - dna polymerase can[t start code so the primer is about 10-15 bases at 3' end. RNA polymerase doesn't need it.

PCR not just useful for cloning. Use PCR to engineer proteins. primer + tag.

polymorphisms can be thought of as markers. Can be found using restriction fragments. Pull out small piece of DNA and make it radioactive. From a bunch of people get DNA and probe using the radioactive. See if the lengths change. Restriction fragment LENGTH polymorphisms

prokaryotic mRNA - pancreatic cells which export proteins out of the cell

IgM - a cell creating antibodies. Sticks a protein in the membrane. If that's stimulated the acceptor site gets changed and th.e proteins can be secreted outside of the cell. Not possible without the splicing mechanism

ORFs are not in eukaryotes since they have introns and get spliced

mRNA transcripts are useful, but due to splicing the DNA might make multiple transcripts

Enhancers - clusters of binding sites.

Big Stuff:
  • Translation; poly-A; cap
  • Exons/Introns, splicing: effects on gene structure, annotation
  • Mobile elements

Splicing means you can get multiple transcripts from a single set of bps Genes within genes promoters, activation May take several proteins to create the proper regulator to activate a gene. Insulators - sit between two genes and keep a promoter from promoting the wrong one.

histone codes

different genes can be expressed based on the concentration of promoters in the cell. Kruppel promoter, chooses which stripe to express in a fly embryo.

how does a cell decide what kind of cell to be?

transcription factor - something that binds to DNA and modifies the transcription (repress, accelerate, etc.). The set of transcription factors in the cell is its "address" - tells it where it should be and what it should do. Then an Enhancer in the cell can respond and do the things - a sort of "Sensor" of the transcription factors.

Transgenics

Taking DNA from somewhere and putting it in a new organism.
  • Replace a gene
  • Insert a gene
  • Mutate a gene
  • Alter a gene

Getting DNA into cells is easy - electroporation, liposomes, ballistics. Something big like an egg, you can just inject DNA directly. But it won't replicate unless it gets onto the host chromosome somehow.

Still, you could inject a gene into a cell and it might express. You could study a particular promoter by creating a sequence that has the promoter promoting a highly visible gene (perhaps it flouresces, or looks blue under certain light, etc. A "reporter" gene.) Then if the gene expresses, the promoter must be working. These are called "transient expression assays".

Yeast has a plasmid, unlike most eukaryotic cells. We can clone DNA into this plasmid, creating a "Yeast Episomal Plasmid", or YEp.

A YEp that contains a centromere is a YCp. These are easier to convince to reproduce. Segregation works in higher cells due to centromeres. Make a library of the yeast genome, recover some clones. The ones that segregate well must have centromeres. This is practically a new chromosome - it only lacks telomeres at the ends. But we can then just take another random sequence and attach it to both ends. Since the sequence does have telomeres, we've just created a Yeast Artificial Chromosome (YAC).

Getting external DNA into a chromosome. Works only if the DNA has a region homoglous to the yeast genome.

A Selectable Marker is an introduced gene that can be selected for. So we create a sequence that encodes a pair of genes, say an antibiotic resistance gene and a gene we want to study. Now introduce these sequences into yeast and grow them with antibiotic. The ones that survive should also have our gene of interest.

Primers can be ordered over the Internet. Just write out your sequence.

Reverse Genetics - we introduce our own mutation rather than waiting for one randomly.

Changing the gene sequence

In yeast:

Insert a new combination, such that occasional recombinations will have the new gene and ALSO not have URA3. Then, put the yeast in an environment such that HAVING URA3 will cause it to die: Result: a population of yeast with the new gene. (URA3 is a selectable marker here)

In plants: In plants, unlike animals, soma cells can be used to create a new plant. Thus, the transformation problem becomes one of transforming a single cell. A agrobacterium has a plasmid that replace the T-DNA with its own stuff.

In flies:

P elements - mobile. Carries a repressor gene. Inject DNA into a single-cell embryo where the germ cells will form. Some of the cells that will be germ cells may use P to hop the DNA into the chromosome.

Stuff we can do with transgenes

Enhancer traps - send in a sensor that shows where a gene is

rescuing transgene

reporter transgene

ectopic expression transgene - fiddle with when a gene expresses

-- Gal4 System

yeast 2 hybrid screens

Insects

GAL binding - can be used to switch a specific gene on. Also, with a dominant negative, can switch off a gene.

Developmental Biology

The development of the embryo was documented more or less by the 1950's. Differentiation. But with the understanding of DNA people began to understand how it worked. Cells at different times and places express different genes. Differentiation is changing patterns of gene expression.

Signals can control "stickiness" of the cell (what cells it sticks to), how big it grows, when it can divide.



Developmental Biology

The development of the embryo was documented more or less by the 1950's. Differentiation. But with the understanding of DNA people began to understand how it worked. Cells at different times and places express different genes. Differentiation is changing patterns of gene expression.

mosaic or regulative organisms - depending on whether the cells can be predicted by their lineage. Nematodes are mosaic - we can say what each cell will become depending on its parent cell. Not necessarily the same type, either. Most animals are regulative though.

Cells in flies have gender. In some situations a fly can have a dividing line between male and female cells, so part of the fly is male and part female. This is caused by crossover in somatic cells. This sort of animal is called chimeric because it is one animal with two different genotypes. You can learn a lot by studying chimeric animals. Say a fly has white-eye genes - but only in the gut. It should have normal red eyes.

Compartments - areas are compartmentalized and cells will not cross the compartment boundaries. It's part of the cell address. Boundaries have signal molecules that tell the cells what to do.

Notch-Delta system - cells attach, a notch matching to the other delta. One cell chooses to be something and tells the other to back off. "I'm a liver, you go do something else."

Pathways - Receptors on the cell surface - phosphorylation inside the cell - create transc factors. Learned by looking at dead embryoes - say, one with two back ends instead of a front and a back. Mother lays down molecules in the egg that gradiate along the anterior/posterior and that tells the cells which genes to express and thus how to grow.

Cells can provide their own gradients by secreting molecules in a particular direction.

Then: Dorsal/Ventral information as well.

Making Transgenic Mice

We can insert genes into mice to test all the usual things - add a gene, knock out a gene, mutate a gene. Remember we did this in flies by injecting genes into multinuclear embryos and hoping some of them stick.

In mammals cell division is more controlled. How do we get around it? Could use cancer cells - they have a habit of growing without controls.

Transient expression

illegitmate recombination - cut the genome, stick in a piece of cut DNA, they will zip together. This is unlike yeast, which insists on homologous combination.

But we want homology! We can create it by a anti-selectable marker. We kill off all the cells that haven't recombined into just the right way.

Inner cell mass. Cells are totipotent (they can be made into any cell). Pull out a few from a developing embryo and grow them in a dish. Then you can dink with them just like yeast. When the cells are the way you want them, inject them back into a developing embryo, and they'll grow. Chimeric. Hopefully some of the cells will make it into germ cells - then, offspring from those cells will have our new gene.

Lac operon

E. coli generally likes to get its energy from glucose. In the absence of that, it is able to get energy from lactose. How?

The Lac Operon has genes named lacZ, lacY, and lacA. LacZ encodes beta-galactosidase; LacY, permease. (LacA doesn't appear to be necessary). An independent gene, Lac I, produces Lac Repressor. It has a weak promoter so it doesn't produce very many.

Lac Repressor is a protein that binds to DNA. Specifically, it binds to a part of the lac operon that RNA polymerase would like to bind to. The polymerase is therefore unable to bind and create lac proteins. But, in the presence of lactose, the lac repressor changes shape and can no longer bind to the lac operon. This allows the polymerase to bind to LacZ and LacY, and the cell will generate beta-galactisodidase and permease. These two proteins allow the processing of lactose.

Ways this process will get messed up

Lac Repressor doesn’t bind DNA (I-). So the repressor fails to function and lac proteins always get created. The repressor doesn’t bind lactose (the inducer) (Is). If this happens, the repressor never realizes there is lactose in the cell and always inhibits the production of the proteins. The operator on the DNA doesn’t bind to the repressor (Oc). Again the repressor fails to function.

So consider the following gene combinations:

I+ P+ Oc Z+ / I- P- O+ Z- : The gene with the Oc doesn’t bind to the repressor, so it will bind to the polymerase, and lac proteins will be always be created. This is called Constitutive

I+ P- O+ Z- / I- P+ O+ Z+ : Even though the I- gene won’t create proper repressors, the I+ gene will, so lac proteins will be created in the presence of lactose, as wanted. This is the normal, or Inducible state.

Is P- O+ Z- / I- P+ O+ Z+ : The Is repressors can’t bind to lactose. They will therefore happily bind to DNA and block lac proteins from being created. Uninducible. The I- repressors just sort of float around.

I+ P+ O+ Z- / I+ P+ Oc Z+ : Oc gene doesn’t bind to repressor and will create lac (Constitutive)

Restriction Enzymes exist for the sole purpose of cutting DNA. As a rule, these “restriction enzymes” look for a specific sequence of 4, 6, or 8 base pairs on the DNA, and then cut the DNA somewhere along that sequence. (E. Coli takes advantage of this by adding a methane block to its own DNA, preventing a particular restriction enzyme from cutting it. Then, an alien piece of DNA which gets incorporated, will not have a methyl cap and will be vulnerable to cutting.)

If we have a cell with a plasmid inside of it, we can perform DNA extraction on the cell. This will give us lots of small bits of bacterial DNA, with, hopefully, an intact plasmid. We can isolate the plasmid by looking at all the bits of DNA by length, and matching it up with the DNA length of our plasmid. Now we can cut the plasmid with a restriction enzyme. Typically a plasmid with 3-6kbp will have 10-15 sites that are cut with a 4-cutter. Vectors If we have a plasmid DNA, we can insert DNA fragments into it. They must both be cleavable by the same restriction enzyme. Put them together and cleave, then use a DNA ligase as glue to repair the damage. The ligase will match the plasmid DNA cleave with the foreign DNA cleave and glue them together.

Now we can insert our plasmid into a bacterial cell. As the cell reproduces, many copies may inherit the plasmid, and so we have lots of copies of our altered DNA. This is cloning.

A simple application of this is to create libraries of sequences. Take a bacterial genome, cut it with restriction enzymes into hundreds of pieces. Put each fragment into a plasmid, put the plasmid in a bacteria and let it reproduce. Cut it with a different enzyme, and you get the same result but different sequences. This allows us to get overlaps.

Sequencing – using four different cutting enzymes, one for each nucleotide.

Reverse transcriptase – initially found in retroviruses. Is able to create single strand complementary DNA (sscDNA) from RNA. Once we have that, can use mormal DNA polymerase to create double-stranded DNA.

Southern Blotting

Polymerase chain reaction (PCR)

hybridize: rna and dna together

primer - dna polymerase can[t start code so the primer is about 10-15 bases at 3' end. RNA polymerase doesn't need it.

PCR not just useful for cloning. Use PCR to engineer proteins. primer + tag.

polymorphisms can be thought of as markers. Can be found using restriction fragments. Pull out small piece of DNA and make it radioactive. From a bunch of people get DNA and probe using the radioactive. See if the lengths change. Restriction fragment LENGTH polymorphisms

prokaryotic mRNA - pancreatic cells which export proteins out of the cell

IgM - a cell creating antibodies. Sticks a protein in the membrane. If that's stimulated the acceptor site gets changed and th.e proteins can be secreted outside of the cell. Not possible without the splicing mechanism

ORFs are not in eukaryotes since they have introns and get spliced

mRNA transcripts are useful, but due to splicing the DNA might make multiple transcripts

Enhancers - clusters of binding sites.

Big Stuff:
  • Translation; poly-A; cap
  • Exons/Introns, splicing: effects on gene structure, annotation
  • Mobile elements

Splicing means you can get multiple transcripts from a single set of bps Genes within genes promoters, activation May take several proteins to create the proper regulator to activate a gene. Insulators - sit between two genes and keep a promoter from promoting the wrong one.

histone codes

different genes can be expressed based on the concentration of promoters in the cell. Kruppel promoter, chooses which stripe to express in a fly embryo.

how does a cell decide what kind of cell to be?

transcription factor - something that binds to DNA and modifies the transcription (repress, accelerate, etc.). The set of transcription factors in the cell is its "address" - tells it where it should be and what it should do. Then an Enhancer in the cell can respond and do the things - a sort of "Sensor" of the transcription factors.

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