Chapter 7 and 8: Microbial Genetics and Recombinant DNA Technology

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1

Genetics:

Study of inheritance and inheritable traits

(nucleotides A,T,C,G)

2

Characteristics eukaryotic DNA:

wrapped around histone proteins to generate the chromosome, open-stranded molecule, found in the NUCLEUS

3

Characteristics of prokaryotic DNA:

mostly circular, associated with proteins, no membrane bound NUCLEUS; DNA material is found in a floating region of cytosol called the NUCLEOID

4

Chromosomal DNA:

DNA essential for that organism/cell

5

Plasmids can also be called:

Factors

6

Fertility factor (F)

plasmid that contains a gene on it for making pilli

7

Resistance factor (R)

when a plasmid contains antibiotic-resistance genes

8

Bacteriocin factor

allows the bacteria to make certain types of toxins

9

Virulence plasmid/factor

(hodge-podge) lol

plasmids that contain, sometimes a combination, of all of the above. can allow the bacteria to make fimbrae (sticky protein that sticks off the surface to allow the bacteria to stick to a host cell).. basically contains anything that can allow the bacteria to invade a host

10

MRSA:

Gram(+) bacteria, on the surface of our skin, these staph bacteria have plasmids/factors in them that allow them to move deeper into the host cells/tissues. Normally staph live on the skin, what MRSA does is it can gain the extrachromosomal genes that benefit the bacteria by allowing them to grow in new environments that they couldn't grow in normally.. it's how they become resistant to antibiotics

11

DNA replication

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In order for DNA to replicate (which happens every time the cell divides),

12

DNA polymerase:

binds to both strands and moves along the strand. for every A it sees on the original DNA, DNA polymerase will make a T -- helping build a new DNA strand. (if we have 2 strands originally, we will now have 4 strands)

13

DNA helicase:

enzyme that prevents the DNA from twisting and tightening and the end; it helps break apart the two strands

14

Topoisomerase:

prevents further twisting from the DNA... very important for both prokaryotic and eukaryotic cells

15

DNA needs to make proteins; transcription and translation

In a eukaryotic cell, RNA is made in the nucleus. In a prokaryotic cell, it all happens in the cytoplasm; the DNA is still used for RNA and the RNA is still used to make the protein -- but in a prokaryotic cell, these two things can sometimes occur at the same time! The key is to control which proteins are being made/are there; the cell is dependent on this!

16

Bacterial lag phase, further explained:

bacteria has to CHANGE what proteins it's making, and ADJUST to the media

17

In order for DNA-->mRNA-->protein, we need:

RNA polymerase, making RNA for the transcription process. We cannot get RNA unless the RNA polymerase molecule binds to the DNA. Once we get our RNA made, our RNA will bind to ribosomes, which will then make a chain of amino acids (primary protein structure)

18

Transcription factors:

(method of controlling transcription/translation)

Proteins that bind to the DNA and either prevent or help the RNA polymerase to bind. If they help, then that means that the RNA will be made and the protein will be made (it's called turning the gene on); if they prevent, RNA polymerase won't bind, and we won't get a protein (turning a gene off)

19

Methylating the DNA:

(another way the cell an control )

Methyl is a carbon group bound to four hydrogens, what it does is it binds to the nucleotides on our DNA. not every nucleotide will have this methyl group. this methylation will affect how the DNA is turned, twisted, and coiled. the methylated DNA can take the DNA and coil it a little bit tighter, making it harder for the RNA polymerase to bind to the DNA (it will determine whether that part of the DNA will be on or off)

**methylation comes from what the parent cell had on their DNA

20

Environmental factors w/ methylation:

Some chemicals promote/take away methylation, determining which sections of DNA/which proteins will be used.

21

Why are baby bottles BPA free?

BPA is a form that can be used to methylate DNA! Anyone exposed to BPA could be victim to changing the methylation of their DNA and which proteins they make! The cause of genetic disorders are problems with a protein!

**Folic acid, found in prenatal vitamins, can ALSO change methylation patterns

22

Riboswitch:

something that affects the RNA. in order for the ribosome to bind to the RNA, it needs to recognize it. the ribosome will recognize the shape the RNA makes (insert picture, looks like rectal thermometer). the ribosome recognizes the loops, binds to it, and the protein will be made.

with the riboswitch, it binds to the secondary structure (loops in RNA) and prevents the RNA polymerase from binding. it is one quick way for bacteria/prokaryote to control which proteins are made

23

siRNA:

(both eukaryotic cells and prokaryotic cells use this)

"si" --> short interfering
another name: anti-sense RNA
it is RNA that the cell makes itself, and this RNA doesn't code for a protein; instead, it is a compliment for other RNA's that DO code for a protein. siRNA compliments mRNA and makes a double-stranded RNA molecule, exposing to to be destroyed (others will think it is dangerous/viral)

**the only time you'll get a double stranded RNA molecule is when you have a viral infection!

24

Operons:

series of genes all under the control of one promoter. Promoter region is important because you can have 5 or 6 genes all under the control of one promoter region (thus, an operon!)

**you typically only see operons in prokaryotic cells

25

Regulatory gene:

will either turn on or turn off an operon (either binds or doesn't bind to the operator)

26

Inducible operon:

type of operon that's always off, but the cell can turn it on

**(lac operon makes enzymes that help breakdown lactose so the cell can use the lactose as a carbohydrate source) since there is usually no lactose, these genes are normally OFF (by default)

27

Repressory operon:

opposite of inducible, it is always on -- the cell can choose whether or not to turn it off from time to time

**trp operon (tryptophan operon), tryptophan is a required amino acids for the cells to make proteins. cell needs this most of the time. when enough tryptophan is around, the operon can be turned off because no trp synthesis is needed

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Horizontal gene transfer:

when prokaryotic cells get genes from other prokaryotic cells. DONORS

29

Transformation:

takes DNA up from its environment, not found from another cell. this exists/is seen when a cell lives in a population where the cells around it are dying and releasing their contents.

**we can force bacteria to take up genes that we give them in the lab, because we know this method exists in nature

common transforming prokaryotes are: neisseria, streptococcus, staphylococcus, bacillus, pseudomonas

30

Competent cell:

cells that do transformation

31

Transduction:

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DNA is transferred from one cell to another via a VIRUS. This is the most common form of horizontal gene transfer. It's a random event, there's no controlling which nucleic acid/gene will be transferred.

32

Bacterial conjugation:

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donor cell is still alive as genes are transferred to the recipient, pilli are required, it doesn't occur as often because you need the same type of cell next to each other for conjugation -- they need eHarmony! compatibility! lol

33

Transposons:

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"jumping genes".. short segments of DNA that can move from one location in a DNA molecule to another location in the same or different DNA molecule...

**transposition is a result of the transposon movement

34

Complex transposons:

still need the inverted repeats and the conjugase enzyme... in addition to all of this, the complex transposons can have other genes in between all of this

35

Recombinant organisms:

an organism that has DNA from different sources. when DNA has been mixed from different sources and put into one cell

36

Why recombinant DNA technology? (on notes)

1. to eliminate undesirable phenotypic traits
2. to combine beneficial traits to create valuable organisms
3. to create organisms that synthesize products humans need (vaccines, hormones, antibiotics)

37

What do we need to make a recombinant organism?

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1. Original bacteria/organism
2. gene of interest
3.mix them together to get a plasmid that has our gene of interest
4. need restriction enzymes & DNA ligase to help cut DNA to get our gene of interest into the "vector"
5. DNA ligase

38

Applications of RDT

1. protein synthesis
2. vaccines
3. genetic screening (WHODUNIT!?)
4. DNA fingerprinting
5. gene therapy
6. agricultural applications (herbicides, resistances, improvements)

39

PCR:

polymerase chain reaction. polymerase represents the DNA polymerase required to make DNA.. and it keeps amplifying itself, one reaction after another

**PCR is a quick way of amplifying, making more, DNA