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DNA denaturing

To split apart two strands of a double helix
--> absorbs more UV light

Nucleic acid hybridization

Separated strands coming together in weird ways: DNA-DNA, DNA-RNA, RNA-RNA
--> hybridization lets scientists figure out what mystery substances are

Restriction enzymes

cut nucleic acids at certain nucleotide sequences along the chain --> restriction sites are usually palindromic sequences

Endonuclease rules

-two DNA fragments cleaved by the same endonuclease can be joined together regardless of origin of the DNA
--> recombinant DNA

How to make a DNA library

1. take DNA fragment
2. use vector to inset into bacterium
3. grow that bacteria! and you have a little DNA clone library

Polymerase Chain Reaction (PCR)

-faster method of "cloning" DNA
-target DNA is denatured (heat style) and mixed with complementary primers
-primers make two new DNA fragments
-add polymerase, target DNA, and RNA primers and you've got an exponential reaction baby!

Southern Blotting

technique to identify target fragments of known DNA sequence in large populations of DNA
-fragments are separated according to size through gel electrophoresis
-large particles = slow, small particles = fast
--> Northern Blot does the same for RNA
--> Western blot does the same for proteins (uses antibody binding specificity)
--> Eastern blot? I guess it's not important

Restriction fragment length polymortphisms (RFLP) anlaysis

--> identifies individuals as opposed to identifying specific genes
--> each human has a unique pattern of restriction sites and this is the famous DNA fingerprint that everyone keeps talking about

What's RNA made up of?

-four different nucleotides
- U, C, A, G
-it takes three nucleotides to code for one amino acid (there are 20 total)
-Since 4^3 is 64, there is more than one triplet that will code you up the same amino acid
--> nearly every living organism uses the same code

Codon

three consecutive nucleotides on a strand of mRNA
--> every codon except for three code for amino acids
--> the three left-over codons are stop codons (termination codons)

Stop Codon

UAA
UGA
UAG

Start Codon

AUG

Translation?

protein synthesis: directed by mRNA (all three types of RNA have a role)

1. mRNA is the template that carries the genetic code from the nucleus to the cytosol in the form of codons
2. tRNA contains a set of nucleotides that is complimentary to the codon, called the anticodon
3. rRNA + protein makes up the ribosome: place of translation

Ribosomes, what's up with those?

Ribosomes: small subunit, large subunit = rRNA + other proteins
Note: prokaryotic ribosomes are smaller than eukaryotic ribosomes

Types of RNA: mRNA

-Template that carries the genetic code from nucleus to cytosol
-

Types of RNA: tRNA

Transfers anti-codons
Anti-codons are amino acid units

Types of RNA: rRNA

...

The story of how mRNA hits the ribosome

1. enters from 5 to 3
2. Ribosomes have those three sites , starting with A (like the alphabet), then P site (placement site), then E site (exit site)
3. When tRNA hits the P site (tRNA runs APE), --> signal for large subunit to join the small one (big + smal = intitiation complex

Phases of making new proteins

1. Initiation (small + big subunits)
2. Elongation (making that polypeptide)

Post-translational modifications can include:

-adding sugars, lipids, phosphate groups to amino acids

Ribosomes attaching to rough ER

--> attach because of characteristics of the polypeptide ("signal peptide") which hits a signal recognition particle (SRP --> 20 amino acids) which attaches to an SRP-receptor, which allows the polypeptide to be made directly into the ER lumen

Mutation

1. Gene mutation
--> alteration of DNA nucleotide sequence in a single gene
2. Chromosomal mutation
--> when the structure of the chromosome is changed

(3. somatic mutation: mutation of a somatic cell (duh), but also really unimportant because they just slough off after very short amounts of time)

Point mutation (transition, transversion)

changing a single base-pair

--> base-pair substitution (transition mutation, AT replaced by GC and vice versa, transversion mutation GC becomes CG)
--> missense mutation:

Neutral mutation, silent mutation

Neutral mutation: no effect on the functioning of the protein
silent mutation: amino acid turns out the same

Frameshift mutation

type of point mutation
insertion of deletion of base-pair
--> happen when mutations happen in anything but multiples of three
--> all nucleotides downstream will be shifted

Nonsense mutation

mutation results in the creation of a stop-codon
--> very serious since they prevent translation of the functional protein in its entirety

Aneuploidy, Polyploidy

Aneuploidy: deletion of entire chromosome
Polyploidy: insertion of additional chromosome

Chromosomal mutations - Translocation

Segment of DNA from one chromosome is inserted into another chromosome

Chromosomal mutations - Inversion

orientation of the section of DNA is reversed

Transposons

-Transposons cause translocation and inversion
-Transposons: transposable elements
--> this is how cells can alter their genetic makeup without meiosis

Forward mutation vs. backward mutation

We're talking about things that have already experience a mutation, if they experience a second mutation and that makes them wildtype again, that's backwards mutation. If you get the second mutation and it just makes more nonsense, then we've got a forward mutation (always moving forward into nonsense)

Oncogenes

Normal cell growth: proto-oncogenes
Cancer cell growth: mutation of proto-oncogenes

How many codons are there? (hint: 4^3)

64 different codons, fewer amino acids

Histones

Since stretched out DNA is about 5 feet long, DNA not in use is kept in globular proteins called histones

Nucleosome

8 histones

histones --> nucleosomes --> solenoids --> supercoils

Chromatin

DNA + protein complex (some RNA in there too)

--> so many amino acids in the histones that they absorbs dyes easily (hence the name)

Basicity of histones: net positive charge

How many chromosomes are there?

46 chromosomes!

--> 46 double stranded DNA molecules

Diploid vs. haploid

Diploid: cells that contain homologous pairs of chromosomes
Haploid: cells that do not have homologous pairs of chromosomes

Cell life cycle

0. G₀ = non-growing phase
1. G₁ = First growth phase
2. S = Synthesis
3. G2 = Second growth phase
4. M = mitosis or meiosis
5. C = cytokinesis

Interphase

G1 + S + G2
--> making and growing (interlude)

G₀ determines what?

How often cells divide. Some divide every hour, some every two weeks, longer G0s means that you've got more hang-time.

G1

-cell has just split, begins growing in size
-produces organelles and proteins
-G1 checkpoints makes sure that the cell is large enough (DNA to cytoplasm ratio) --> can only enter S-phase after passing this test

S

-DNA replicates
-not so much focus on organelle and protein production

G2

-prepare for division
-checkpoint checks for mitosis promoting factors (MPF)

Mitosis

nuclear division WITHOUT genetic change
[interphase -->] prophase --> metaphase --> anaphase --> telophase (PMAT)

Prophase

-condesation of chromatin into chromosomes
-movement to end of cell
-spindle apparatus forms
-

Spindle apparatus

-aster (microtubules radiating from centrioles)
-spindle microtubules (connect the two centrioles)
--> kinetochore is at the centromere

Metaphase

chromosomes align on the equator

Anaphase

sister chromatids split apart along the equator --> disjunction

Telophase

reforming of the nuclear membrane
chromosomes recondense

Mitosis vs. Meiosis

Mitosis = regular cells (otherwise known as somatic cells, you moron)
Meiosis = double nuclear division resulting in four gametes

Gametes = also called germ cells

What cells in the human body undergo meiosis?

1. Spermatogonium
2. Oogonium

Meiosis in detail

After S (replication): cell is called primary spermatocyte or primary oocyte

-->Two rounds of division:
--> genetic recombination occurs
--> daughter cells are haploid

Meiosis I

-two chromosomes --> one chromosome
-From 46 chromosomes to 23

prophase I --> metaphase I --> anaphase I --> telophase I

How many chromosomes do the primary vs. secondary spermatocytes have?

Primary: 46
Secondary: 23

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