Biochemistry Lectures 1-3

Front 1

Living matter is characterized by (5)

Back 1

1. High degree of complexity/organization

2. Extraction, transformation and systemic use of energy to create and maintain structures and to do work

3. Interactions of individual components are dynamic and coordinated

4. Ability to sense and response to changes in surrounding

5. A capacity for fairly precise self-replication while allowing enough change for evolution

Front 2

realistic representation of proteins in cytosol

Back 2

Full of protein so all of these reactions are constantly taking place in a very crowded cell

Front 3

The Genome

Includes ____ chromosomes (humans)

Back 3

The complete genetic endowment of an organism (all DNA even mitochondrial)

46 chromosomes

Front 4

Correlation between genome size and complexity

Back 4

Poor, being more complex doesn't mean you have more chromosomes (some plants have more chromosomes or more DNA than humans)

Front 5

Of all DNA ______ codes for proteins

Back 5

1.5%

Front 6

Exons

Back 6

expressed sequences (translated into amino acid sequences)

Front 7

Introns

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regions of genes that are transcribed but not translated

Front 8

Introns are removed

Back 8

after transcription and the exon mRNA sequences are spliced together

Front 9

Almost _____ % of the genome are transposons

_____% of the genome is introns

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50%

26%

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What are transposons

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sequences that can move within the genome

(not all 50% are moving at once, 50% are transposons or transposon remnants)

Front 11

SSRs

characteristics

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simple sequence repeats

Short sequences (10bp or less)

repeated millions of times

Front 12

Much of SSRs are associated with _____

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centromeres and telomeres

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Centromeres are

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where proteins attach during mitosis

(region where two daughter chromosomes are held together during mitosis)

-after DNA replication but before cell division

essential for equal distribution of chromosome sets to daughter cells

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Function of telomere sequence

Carried out by _____

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caps the end of the eukaryotic chromosomes

added by enzyme telomerase (not present in some cells causes aging)

Front 15

Miscellaneous unique sequences

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multiple random things that silence other genes

Front 16

Eukaryotic DNA is organized ______

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with proteins into a complex called chromatin

Front 17

Describe the 46 chromosomes of humans

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22 pairs (diploid)

plus X or Y

length varies

Front 18

How is DNA found in the cell

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never naked always associated with proteins

Front 19

Humans have about ____ length of DNA per cell

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2 meters

Front 20

first step of DNA compaction

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DNA is supercoiled (coiled and then the coil is coiled)

Front 21

When not super coiled called ______

Supercoiling has great influence on _______

and is ______

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relaxed

has great influence on and is greatly influenced by transcription and replication of DNA

Supercoiling is highly regulated

Front 22

What regulates the coiling of DNA?

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Topoisomerases

Front 23

2 types of topoisomerases?

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Type I: makes a transient cut in one DNA strand

Type II: make a transient cut in both DNA strands

Front 24

______ are used as antibiotics and for _____

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topoisomerase inhibitors and for cancer because this enzyme is so important for replication. Without it, bacteria can't grow and cancer can't replicated

Front 25

How does topoisomerase function?

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twists the DNA and changes the tension on the DNA

Front 26

Partial unfolding on DNA reveals _____

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bead on a string

Front 27

What are the bead made of in DNA wrapping?

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146 bp of DNA wrapped around 8 histones.

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Linker between beads on a string DNA length

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54 bp bound to histone H1

Front 29

Purpose of histone tails

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Interact with other histones, proteins or DNA some of the most modified peptides in excistence. Methylated, carboxylated, glycosylated... ect. Modifications change the function of the histone.

Front 30

_____ has a profound effect on DNA- related processes

Back 30

Hostone location, histone variants, and histone trail modifications and have a profound effect on DNA-related processes

Front 31

Nucleosome formation compacts DNA ____fold

Back 31

7 fold

Front 32

Overall compaction of DNA is _____ fold

Back 32

10K fold

Front 33

After nucleosome formation what is the next level of structure?

then???

Back 33

30nm fiber strands of histones are then coiled

then these coils are arranged into higher ordered structures. Not totally understood

Front 34

How does the structure of 30 nm fiber forming a higher order structure appear to work?

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Appears to involve a loop of DNA associating with a scaffold of proteins

Front 35

How does the DNA appear in nondividing state, and interphase

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Chromatin is amorphous

Front 36

How does the DNA appear in mitosis?

Back 36

Chromosomes become condensed, pairs of sister chromatids

Front 37

3 characters of DNA metabolism

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A new copy of DNA is synthesized with high fidelity before each cell division

Errors arise during/after DNA synthesis are constantly checked for, repairs are made.

Segments of DNA are rearranged either within a chromosome or between two DNA molecules, giving a novel DNA (recombination)

Front 38

Polymerases

Functions?

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Synthesize DNA by adding nucleotides to a strand

Different polymerases perform different tasks

Leading strand, okasaki fragment synthesis, primer synthesis, DNA repair

Front 39

Nucleases

functions?

Types?

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Degrade DNA by cutting the backbone

Also called Dnases or Rnases

2 types: Exonucleases and endonucleases

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Exonucleases:

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remove nucleotides from the ends of DNA

Front 41

Endonucleases

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Cleave bones within a DNA sequence

Front 42

DNA helicases

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Enzyme that separates DNA strand

Front 43

DNA ligases

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Seal nicks in the DNA backbone

Front 44

DNA synthesis is _____

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semi conservative

(each new DNA strand has one old parent strand and one new daughter strand)

Front 45

Synthesis of DNA always occurs by addition of new nucleotides to the ____ end

Back 45

3'-OH

synthesis is done 5' to 3' end (of parent)

Front 46

Synthesis of leading vs lagging strand

Back 46

The leading strand is made continuously as the replication fork advances

The lagging strand is made discontinuously in short pieces that are later joined together

Front 47

Incoming DNA is a ______ which is needed for _____

Back 47

triphosphate needed for engery of addition

Front 48

______ enzyme is involved in DNA synthesis

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DNA polymerase

Front 49

Add new nucleotide to the _____ which ______

Back 49

3' hydroxylgroup which attacks the phosphate and the other 2 phosphtes leave as pyrophosphate

Front 50

How is the right pair formed in DNA synthesis

Back 50

Bonding is favored and geometery of polymerase favors correct pairs

Front 51

How many lesions in DNA? how many become mutations?

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thousands per day but only 1/1000 becomes mutation because of DNA repair

Front 52

Majority of DNA lesions are corrected using ____

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the undamaged strand as a template

Front 53

Human genome contains genes for over _____ repair proteins

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130

Front 54

What is a mutation?

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Bases escape repair and an incorrect base serves as template in replication, now both strands have incorrect base pair

Front 55

What can happen with mutations?

Back 55

Nothing (many silent)

Compromise cellular function

change cellular function

change regulation

Front 56

Location of mutation matters

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germline cell (will get passed to offspring)

somatic cell (will maybe effect organism but not get passed on)

Front 57

Cancer caused by _____

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mutations (usually multiple)

Front 58

Are all mutations as dangerous or risky?

example?

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not all mutations are equally cancerous

BCRA1 &2 (breast cancer causing agent)

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Ames test

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Uses salmonella strain with a mutation that makes bacterium unable to synthesize histidine add compound and see if bacteria grows in histidine free medium (means mutation occured)

Ring occurs if strong carcinogen because causes too many mutations in high concentration

Front 60

Mismatches

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incorporation of incorrect nucleotides

Front 61

Abnormal bases

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spontaneous deamination, chemical alkylation or exposure to free radicals

Front 62

Pyrimidine dimers

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UV exposure

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Backbone lesions

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Exposure to ionizing radiation, free radicals

Front 64

Types of DNA repair

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Proofreading

Mismatch repair

Direct repair

Base excision

Nucleotide excision

Error-prone translesion DNA synthesis

Recombination repair

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1st line of DNA for damaged DNA

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DNA repair proofreading

Without exonuclease activity DNA polymerase

is far less accurate

Front 66

DNA polymerase proof read from ___ to ___

called _____ activity

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3' to 5'

called exonuclease activity

Front 67

If polymerase adds wrong base _________

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translocation to next position is inhibited, 3' to 5' exonuclease activity removes incorrect nucleotide and polymerase begins again.

Front 68

How does DNA mismatch repair work?

Back 68

Special proteins recognize and bind mismatched base pairs (due to changed geometry)

Nucleotides from the newer strand and DNA is resynthesized using the parent strand as a template

Front 69

How does the cell know which strand is the parent strand during mismatch repair

Back 69

In bacteria parent strand is methylated

In humans not quite sure but it has a method

Front 70

Direct repair

Back 70

Directly repairs abnormal bases

Specific enzyme for each repairable base

Fixes bases that have been methylated

these proteins are sacrificed (one time use)

Front 71

Base Excision Repair

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DNA glycosylases recognize specific lesions (cleave bond between surgar and base, leaves the sugar and phosphate)

Then DNA polymerase adds a few new bases, and DNA ligase seals the strand

Front 72

Nucleotide excision

Back 72

Large distortions in DNA are repaired by nucleotide excision

Exinuclease come in and nick both sides, helicase removes the small section with the lesion, DNA polyermase adds right base pair and DNA ligase seals

Front 73

In nucleotide excision _____ base nicks in bacteria

____ in eukaryotes

Back 73

13 in bacteria

29 in eukaryotes

Front 74

What happens when there is no undamaged DNA to use as a template

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1. Repair using another chromosome as template (recombination)

2. Error prone translesion synthesis

Front 75

What happens if DNA is already separated and find lesion in single stranded DNA?

Back 75

the other side is already replicated, there is no template strand to work with, template fork will stall and will sometimes collapse

1. repair using another chromosome (recombination)

2. Error prone translesion

Front 76

Error prone translesion synthesis

Most ______

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Most recognize specific types of damage and have appropriate response

Most are limited to short regions of DNA minimizing mutagenic potential

These unqiue polymerases are good at adding nucleotides to damaged DNA

Front 77

2 ways segments of DNA can rearrange their location

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Within a chromosome

From one chromosome to another

Front 78

3 classes of recombination

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Homologous/General recombination

Site Specific recombination

DNA transposition

Front 79

Homologous/general recombination

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Exchange between two DNAs that share an extended region of similar sequence (homology) site of recombination does not matter

Front 80

Site specific recombination

Back 80

exchange DNA only at a particular sequence

Front 81

DNA transposistion

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Jumping genes: short DNAs that can move from one DNA location to another

Front 82

3 functions of Homologous Recombination

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Repair DNA when no template strand is available

Segregation of chromosome pairs in meiosis

Enhance genetic diversity in meiosis

Front 83

Meiosis

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when diploid germ cells divide into produce haploid cells

Front 84

benefits of recombination in sister chromosomes during meiosis

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Creates tension when chromosomes are pulled apart and ensures proper chromosome segregation

Front 85

Aneuploidy

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wrong # chromosomes

Front 86

What can cause anupoloidy

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Improper alignment

Front 87

Meiosis in females

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long term process with an extended suspended animation phase

Start meiosis in fetal stage, chormosomes align and cross over then stop (more likely cross over will not work and have anupolidy with age)

Front 88

Inversion or deletion _____

Back 88

occurs if recombination sites are on the same DNA

Front 89

Intermolecular recombination occurs _____

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Occurs if recmobination sites are on different DNAs

Front 90

Transposons carry _____

Back 90

genes for transposases, but some can contain extra genes (cut and paste or copy and paste)

Front 91

Transposases

Back 91

enzymes that binds end of transposon and catalyzed movement to another part of the genome

Front 92

Transposaes usually makes ______ and then ____

Back 92

makes staggered cuts in the target sits

so not only do you add transposon but you add gaps and polymerase will come fill in.

Replication fills in the gaps, duplicating the sequence flanking the transposon

Front 93

Immunoglobin genes assemble by ____

Back 93

recombination

Front 94

Each chain of antibody has

Back 94

variable and constant regions

Front 95

_____ produces different antibodies

Back 95

recombination

Front 96

Gene duplication benefit

Back 96

duplicate so one can mutate and gain extra function

Front 97

Evolution of Eukaryotes

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First were anerobic then engluf bacteria that is aerobic genes from this travel to nucleus and endosymbiont becomes mitochondria then engulf cyanobacteria then DNA travels to nucleus and endosymbiont becomes choloroplast

Front 98

How was the human genome sequenced?

Back 98

Used BAC plasmids and sanger sequencing

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What are BAC vectors?

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A special plasmid that can accommodate up to 300,000 base pairs

Front 100

After incorporating the DNA into the BAC then what?

Back 100

introduced into bacteria where they can be screened and replicated

Front 101

Plasmids are

Back 101

small DNA circles that can replicate in bacteria

Front 102

How do you get DNA into plasmids/

Back 102

DNA fragments from genomic DNA are mixed with cut plasmids and ligated together

Front 103

What is needed for sanger sequencing reaction?

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DNA template (DNA want to sequence)

Primer (to start polymerase going)

DNA polymerase (to replicate new DNA)

dNTPs

ddNTPs (fluorscently labeled)

Front 104

How do you ensure that the right part of the plasmid with be sequenced with sanger?

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Come up with special primer that matched the BAC DNA right where you made the insertion so polymerase will start right where the inserted DNA starts

Front 105

why do ddNTPs stop sequencing

Back 105

each base is fluorescently labeled with different color. You add to the 3' OH carbon, ddNTP doesn't have this hydroxyl group on the 3' carbon so nothing can be added

Front 106

What is the point of the primer for sanger sequencing?

Back 106

Most polymerases cannot replicate a single strand of DNA needs a little start of double strand (primer) special polymerases make the primer and then other polymerases extend from the primer

Front 107

how is the majority of genome sequencing now done?

Back 107

reversible terminator sequencing

Front 108

Reverisble terminor sequencing also called/

Back 108

Illumina sequncing or next generation sequencing

Front 109

Describe next generation sequencing

Back 109

Take all fragments of DNA and bind one end to a slide with other end floating around then take primer. Only use fluorescent dNTPs with blocking groups, polymerase only adds one base to each strand. take picture of slide, read put chemicals in to clear blocking group and each round get one more basepair

Front 110

Each dNTP in next gen sequencing has ____

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dNTP fluorophore and blocking group that can be removed

Front 111

Original cost of sequencing vs now?

price change due to

Back 111

2.7 billion now $999

due to next generation sequencing

Front 112

What did we learn about protein coding genes when sequenced human genome

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Originally thought we had more genes that we had

thought 35,000-100,000

Now know 21,000

Front 113

Why can we make so many proteins with a much smaller amount of genes

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90% of protein coding regions are subject to alternative splicing

Front 114

What we learned about transposons when sequencing the genome

Back 114

Before thought junk DNA

Sequenced and found a whole lot of "junk DNA"

now know that transposons might drive some evolution, jumping around and sometimes taking other DNA with them may change regulatory elements and lead to evolution

Front 115

What we learned from non-coding regions of DNA when sequencing

Back 115

Before: protein coding regions were conserved

Now know 6% of sequences well conserved but only 1.5% of that is protein coding

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Issue with conserved DNA

Back 116

Conserved DNA is DNA that is very similar between species. We though that protein sequences would be the most conversed before the human genome project but only 1/4 of conserved is protein coding.

These other areas could be promotors, regulatory regions, insulators, noncoding RNA

Front 117

What we learned about non-coding RNA after genome sequence

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RNA that gets transcribed but not translated, after sequencing realize much more than we thought

Front 118

Micro RNA

Back 118

Regulate transcription of coding RNAs

Front 119

Linc RNA

Back 119

ling intergenic noncoding RNA : unclear function many smaller parts with important function?

Front 120

What we learned about evolution after sequencing the genome

Back 120

Before thought protein coding mutations lead to evolution now

More "evolution in conserved non-coding regions

Front 121

What can we do with genome known?

Back 121

Comparitive genomics

Improve methodologies (gene mapping and discovery, cloning, primer design, genetic manipulation)

Create new methodologies (genome wide association studies, RNA-seq, CHIP-seq, chromosome conformation capture

Make maps

Front 122

Comparing genomes

Conserved vs. homolog vs. paralog vs. ortholog

Back 122

Conserved: very similar across many species

Homolog: gene wither easily detectable sequence similarity

Paralog: homolog within same species (think gene duplication)

Ortholog (homolog in a different species, could have the same function in each species)

Front 123

How does knowing genomes of humans and other animals allow us to figure out what makes us human

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Figure out the unique parts of our DNA that are different from the majority of our ancestors

Front 124

______ help identify changes within the human lineage

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outgroups

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Outgroup______

Back 125

out groups such as orangutan because if you just compare human and chip and get to a different base pair then you don't know if the T makes humans unique or if the G is what makes chimps unique and everyone else has a T

Front 126

Accelarated evolution identified in _____

Back 126

regulatory sequences

Most other mammals have similar sequence different by 1 or 2 BP and for humans changed a lot (which changed the way our brain developed)

Front 127

Humans accelerated genome

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regions of genome that are much different between humans and other species

Front 128

Humans differ from each other by

Back 128

1/1000 bp

Front 129

Vast majority of human genetic variation

Back 129

are inherited they do not arise spontaneously in an individual

Front 130

SNPs

Back 130

Single nucleotide polymorphism (variation in single bp in genome)

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Allele

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Particular version of a gene

Front 132

Most human genome variants with frequency of _____ have been discovered

Back 132

>5%

Front 133

____% of SNPs in an individual are represented in current databases

Back 133

95%

Front 134

Haplotypes

Back 134

many times genes are inherited together from a parent. Basically you can look at a few SNPs and infer what most of your variations in that region are because of recombination things that are close together are usually inherited together

Front 135

Genome project and mendelian diseases

Back 135

Compared haplotypes of non effected people vs effected people which help determine where a particular gene that causes disease is located

Front 136

Linking SNPs to disease can

Back 136

identify genes

Front 137

genome wide association studies

Back 137

Take all common SNPs and you look t what SNPs all different individuals have. 4000 have disease, 6000 don't you can figure out for multifactorial diseases if 55% of people who have G have disease and 40% of non infected people have G then know having G somewhat increases risk

Front 138

Pharmacogenomics

Back 138

analysis of how genetic makeup affects an individuals response to drugs

Front 139

Discovery of a new cancer gene helps us

Back 139

understand mechanisms and direct therapeutic efforts