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DNA Flashcards

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1
Q
A

Adenine

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2
Q
A

Thymine

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3
Q
A

Guanine

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4
Q
A

cytosine

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5
Q
A

Uracil

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6
Q

nucleoSides

A

5-carbon sugar pentose bonded to a nitrogenous base; formed by covalently linking the base to C-1’ of the sugar (base + sugar)

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7
Q

nucleoTides

A

phosphate group attached to C-5’ of a nucleoside; named according to the # of phosphates present; are the building blocks of DNA (base + sugar + phosphate)

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8
Q

Chargaff’s rule

A

dsDNA: %A = %T & %C = %G

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9
Q

what kind of bonds in the sugar phosphate backbone of DNA?

A

covalent phosphodiester bonds; phosphate group forms an ester bond to the 3’ carbon of one sugar molecule and the 5’ carbon of another

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10
Q

Describe DNA replication in prokaryotes

A

Circular chromosome, only 1 origin of replication

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11
Q

Describe DNA replication in eukaryotes (type of chromosome, origins, etc)

A

linear chromosome, multiple origins of replication, 25x more DNA than prokaryotic cell

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12
Q

Action of DNA gyrase (topoisomerase II)

A

alleviates supercoiling, working ahead of helicase, nicking the strand(s) relieving the torsional pressure

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13
Q

Action of DNA polymerase I (Pol I) in prokaryotes

A

okazaki fragments; excision repair (removes RNA primers and fills it with DNA)

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14
Q

Action of Pol II in prokaryotes

A

DNA repair

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15
Q

Action of Pol III in DNA repair

A

main process of DNA synthesis

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16
Q

DNA polymerase α in eukaryotes

A

initiates synthesis in replication in both the leading and lagging strand

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17
Q

DNA polymerase δ in eukaryotes

A

it takes over the synthesis role; operates more effeciently than DNA α and it adds nucleotides when the RNA primer is removd (fills in)

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18
Q

DNA polymerase ε in eukaryotes

A

extension in the leading strand; DNA repair

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19
Q

DNA polymerase β in eukaryotes

A

DNA repair

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20
Q

DNA polymerase y in eukaryotes

A

replicates mitochondrial DNA

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21
Q

RNase H in eukaryotes

A

removes RNA primers

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22
Q

role of helicase

A

uses hydrolysis of ATP to “unzip” or unwind DNA helix at replication fork to allow resulting single strands to be copied

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23
Q

role of primase

A

polymerizes nucleotide triphosphates in a 5’ to 3’ direction. Synthesizes RNA primers to act as a template for future Okazaki fragments to build on to.

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24
Q

role of DNA polymerase III

A

synthesizes nucleotides onto leading end in classic 5’ to 3’ direction.(adds nucleotides to growing daughter strand)

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25
Q

Role of DNA polymerase I

A

synthesizes nucleotides onto primers on lagging strand, forming Okazaki fragments. This enzyme cannot completely synthesize all the nucleotides.

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26
Q

action of ligase

A

glues together Okazaki fragments, an area DNA Pol I is unable to synthesize

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27
Q

action of telomerase

A

catalyzes lengthening of telomeres; enzyme includes molecule of RNA that serves as template for new telomere segments

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28
Q

action of nuclease

A

excises or cuts out unwanted or defective segments of nucleotides in DNA sequence

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29
Q

action of topoisomerases

A

introduces single-strand nick in the DNA, enabling it to swivel and thereby relieve the accumulated winding strain generated during unwinding of double helix

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30
Q

action of single strand binding proteins

A

holds the replication fork of DNA open while polymerases read the templates and prepare for synthesis (prevents reannealing)

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31
Q

action of telomerase

A

lengthens telomeres with repetitive sequences proteins the tellers from loss during replication

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32
Q

What enzyme and when does DNA proofreading occur?

A

DNA polymerase in the S phase of cell cycle

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33
Q

When in cell cycle does mismatch repair occur and by what enzymes?

A

G2; MSH2, MLH1; MutS and MuL in prokaryotes

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34
Q

When in cell cycle and by what does nucleotide excision repair occur?

A

G1/G2; done by excision endonucleases

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35
Q

In what phase of cell cycle and by what does base exision repair occur?

A

G1/G2; glycosylase, AP endonuclase

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36
Q

Mismatch repair

A

occurs during the G2 phase using MSH2 and MLH1. It cuts the strand that doesn’t have methylation

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37
Q

Nucleotide excision repair:

A

Fixes helix-deforming lesions of DNA like thiamine dimers. A cut-and-patch endonuclease; (requires an excision endonuclease)

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38
Q

base excision repair

A

Fixes non-deforming lesions of the DNA helix such as cytosine deamination by removing the base, leaving apurinic/apyrimidinic (AP) sites. AP endonuclease removes the damaged sequence which can be filled in with the correct bases.

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39
Q

tumor suppressor genes

A

Code for proteins that reduce cell cycling or promote DNA repair = cutting the breaks.

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40
Q

Oncogenes

A

Proto-oncogenes + mutation → oncogenes = promotes cell cycling = can lead to cancer;

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41
Q

Meselson-Stahl experiment

A

Is DNA semiconservative, conservative, or dispersive? Used 15N heavy DNA - E coli

Initially all DNA was heavy; grew the cells in the absence of the heavy nitrogen so all of the new DNA made in subsequent cell divisions would be lighter. After one cell division, the DNA was half as heavy (half of the DNA molecule had heavy nitrogen and the other didn’t. This ruled out the conservative method, which if were true, would’ve produced one molecule that was all light and the other all heavy. After 2 cell divisions, the DNA molecule was now either half heavy and half light or all light. ruled out the dispersive method.

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42
Q

Hershey-chase experiment

A

Is protein or DNA the genetic material of the cell?

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43
Q

What does the stabilization of unwaound template strands?

A

single stranded DNA binding protein

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44
Q

RNA primer synthesis is

A
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45
Q

What does DNA synthesis in prokaryotic cells?

A

DNA polymerase III

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46
Q

What does DNA synthesis in eukaryotic cells?

A

DNA polymerase α, δ, ε

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47
Q

RNA primer removal in prokaryotic cells

A

DNA polymerase I (5’ -> 3’ exonuclease)

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48
Q

RNA primer removal in eukaryotic cells

A

RNase H (5’ -> 3’ exonucelase)

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49
Q

What does the replacing of RNA with DNA in prokaryotic cells?

A

DNA polymerase I

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50
Q

What does the replacing of RNA with DNA in eukaryotic cells?

A

DNA polymerase δ

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51
Q

What joins okazaki fragments?

A

DNA ligase

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52
Q

What removes positive supercoils ahead of advancing replication forks?

A

DNA topoisomerases

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53
Q

missense codon

A

mutated codon -> different amino acid

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54
Q

Nonsense codon

A

→ a stop codon (UAG, UAA, UGA)

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55
Q

Initiation codon

A

= starts translation = AUG; lies just downstream of the Shine Dalgarno sequence (Kozak sequence for eukaryotes)

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56
Q

Termination codon

A

(UAG, UGA, UAA) = ends translation; no tNA is involved; protein “release factor” comes along and terminates translation

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57
Q

5’ cap

A

modified nucleotide that protects the 5’ end from exonuclease degradation

7-methylguanylate triphosphate cap = recognized by the ribosome as the binding site

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58
Q

Poly-A tail

A

protects 3’ end of mRNA from exonuclease degradation

longer tail = more survival time

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59
Q

How do prokaryotes and eukaryotes differ when it comes to increasing gene variability?

A

prokaryotes increase it through polycistronic genes while eukaryotes increase it through alternative splicing

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60
Q

where does transcription take place vs where does translation take place?

A

Transcription takes place in the nucleus while translation takes place in the cytoplasm

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61
Q

Describe the 6 steps of transcription

A
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62
Q

Describe the 3 steps of translation

A
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63
Q

Initiation site

A

The site on the DNA from which the first RNA nucleotide is transcribed; +1 site

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64
Q

upstream

A

negative #s = Nucleotides that come before the initiation site

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65
Q

Downstream

A

positive #s = come after the initiation site

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66
Q

A site (aminoacyl)

A

provides space for a new approaching tRNA with attached amino acid and corresponding anticodon to match the next codon in the mRNA sequence. (Exception = start codon, which matches to the P site)

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67
Q

P site (peptidyl)

A

2 AA are held adjacent to each other, one to a tRNA in the A site and one in the P site → ribosome’s ribozyme-acting rRNA catalyzes peptidyl transferase activity which transfers the P site amino acid onto the A site. Simultaneously, the ribosome advances across the mRNA transcript, moving the tRNA P site into the E site, positioning the elongating polypeptide attached to a tRNA from the A site into the P site, freeing up the A site for a new tRNA to enter for the next codon.

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68
Q

E site (exit)

A

A tRNA moved into the E site, having just released its amino acid (and growing polypeptide chain if it was not the first tRNA), is then free to dissociate from the ribosome and mRNA (exit).

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69
Q

histones

A

responsible for compact packing and winding of chromosomal DNA

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70
Q

Single copy DNA

A

Long unique sequence of nucleoTides in the DNA; exons; coding regions; sites for transription; present in euchromatin; similar in many individuals; does not repeat; low mutation rate

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71
Q

Repetitive DNA

A

DNA sequence that does repeat; nucleoTides don’t code for proteins; centromeres; introns; noncoding; present in heterochromatin; higher mutaiton rate; Unique in different individuals

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72
Q

Heterochromatin

A

Dense, transcriptionally silent; dark; tightly coiled

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73
Q

Euchromatin

A

Majority of DNA in this form; only in prokaryotes; lesss dense, transcripationally active; light; uncoiled

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74
Q

Telomeres

A

capping regions on the ends of chromosomes; high GC content; protects chromosome from degradation during replication

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75
Q

Centromeres

A

single point region located in the middle of a chromosome that connects the two sister chromatids (the original chromosome and its replicated parnter) until they’re separated

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76
Q

operon

A

gene expression; they allow a bacterium to respond to changes in its environment by increasing or decreasing the expression of certain genes as appropriate.

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77
Q

Positive control

A

activator stimulates transcription

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78
Q

Negative control

A
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79
Q

Negative inducible operon

A

repressor is normally present and the genes aren’t expressed except under specific conditions

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80
Q

Negative repressible operon

A

genes are usually transcribed, but transcription can be halted by binding the repressor in appropriate conditions

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81
Q

Promoter

A

regulatory DNA sequence where the RNA polymerase can attach

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82
Q

Operator

A

regulatory sequence where a repressor can attach and keep the RNA polymerase from being able to perform the transcription

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83
Q

Regulatory gene

A

produces repressor protein that binds to operator to block RNA polymerase

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84
Q

Structural gene

A
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85
Q

Operator site

A

An operator is a regulatory region that is regulated by the binding of a repressor protein.

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86
Q

Promoter site

A

provides a place for RNA polymerase to bind

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87
Q

Inducible systems

A

Bonded to a repressor under normal conditions; they can be turned on by an inducer pulling the repressor form the operator site. Example: Lac operon.

88
Q

Repressible systems

A

Transcribed under normal conditions; they can be turned off by a corepressor coupling with the repressor and the binding of this complex to the operator site. Example: Trp operon

89
Q

Lac operon– what happens when lactose is absent?

A

repressor is bound to the operator and prevents RNA polymerase from transcribing the structural genes

90
Q

Lac operon– what happens when lactose is present?

A

allolactose (isomer) binds w/ the repressor; it dissociates from the operator which allows RNA polymerase to transcribe the structural genes. E coli now has the ability to metabolize lactose

91
Q

Aminoacyl tRNA synthetases

A

enzyme that binds tRNAs to the amino acid

92
Q

RNA interference

A

the process where RISC, a ribonucleoprotein, uses dsRNA fragments to target mRNA transcripts

93
Q

Describe the process of splicing

A

Splicing—accomplished by the spliceosome where small nuclear RNA (snRNA) molecules couple with proteins known as small nuclear ribonucleoproteins (snRNPs); the noncoding sequences are excised in the form of a lariat (lasso-shapped structure) and then degraded

94
Q

snRNPs

A

RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs

95
Q

snRNAs

A

complexed with proteins to form snRNPs to splice primary RNA transcripts.

96
Q

What situation occurs in the presence of low glucose when lactose is available?

A

lac genes strongly expressed

97
Q

What situation occurs in the presence of high glucose when lactose is unavailable?

A
98
Q

What situation occurs in the presence of low glucose when lactose is unavailable?

A

lac genes not expressed

99
Q

What situation occurs in the presence of high glucose when lactose is available?

A

very low (basal) level of lac genes expressed

100
Q

What is the outcome when both glucose and lactose are present?

A

low-level transcription of the lac operon occurs. The lac respressor is released from the operator b/c the inducer (allolactose) is present. .cAMP levels decrease because glucose is present. CAP remains inactive and can’t bind to DNA, so transcription occurs at a low leaky level

101
Q

What is the outcome when lactose is present but glucose is absent?

A

strong transcription of the lac operon occurs. The lac repressor is released from the operator b/c the inducer (allolactose) is present. cAMP levels are high b/c glucose is absent, so CAP is active and bound to the DNA. CAP helps RNA polymerase bind to the promoter, permitting increasing levels of transcription.

102
Q

What is the outcome when gucose is present but lactose is absent?

A

no transcription of the lac operon occurs. the lac repressor remains bound to the operator and prevents transcription by RNA polymerase. cAMP levels decrease, glucose level sincrease, so CAP is inactive and can’t bind DNA

103
Q

What is the outcome when both lactose and glucose are absent?

A

no transcription of the lac operon occurs. the lac repressor will also be bound to the operator (absence of allolactose) acting as a roadblock to RNA polymerase and preventing transcription. cAMP levels increase, glucose levels decrease, so CAP is active and will be bound to DNA

104
Q

Trp operon

A

essential for the production of tryptophan

The trp operon allows the expression of genes in the absence of tryptophan (W), but not when tryptophan is present because this would be energetically unfavorable.

The trp operon = a repressible negative operon b/c it can be induced by environmental conditions, but transcription is not repressed by default.

105
Q

Trp operon: In the absence of W, what happens?

A

the repressor doesn’t bind to the operator and W synthesis proceeds.

106
Q

Trp operon: In the presence of W, whatp happens?

A

it binds to the repressor protein and causes it to bind to the operator, thus inhibiting synthesis.

107
Q

trp repressor

A

The trp repressor is a regulatory protein that recognizes the operator (stretch of DNA). When the trp repressor binds to the operator DNA, it keeps the operon from being transcribed by physically blocking RNA polymerase.

The trp repressor doesn’t always bind to DNA; it binds and blocks transcription only when tryptophan is present.

108
Q

Corepressor

A

a small molecule that switches a repressor into its active state.

109
Q

When a repressor is bound to its operator, transcription is

A
110
Q

When an activator is bound to its DNA binding site, it

A

increases operon transcription

111
Q

what is an activator

A

Activators = enhances the interaction b/t RNA polymerase & a particular promoter, encouraging the expression of the gene.

112
Q

catabolite activator protein (CAP)

A

This protein activates transcription of the lac operon in E. Coli. Cyclic adenosine monophosphate (cAMP) is produced during glucose starvation. The cAMP binds to the CAP → conformational change that allows the CAP protein to bind to a DNA site located next to the promoter. The activator CAP then makes a direct protein-to-protein interaction w/ RNA polymerase that recruits RNA polymerase to the promoter.

113
Q

Repressors

A

proteins that bind to the operator and impedes RNA polymerase progress on the strand thus inhibiting expression of the gene.

114
Q

What’s the difference b/t acetylation and methylation?

A

acetylation is the covalent modification of histones while methylation is hte covalent modification of nucleotides. Acetylation increases transcription resulting in increased accessibility. Methylation deactivates genes resulting in decreased accessibility.

115
Q

GC box & CAAT box

A

∼10-150 bp upstream; function is to bind to proteins that help recruit RNA polymerase to initiate transcription

116
Q

Transcription factors

A

DNA binding proteins that bind to specific regions of the DNA (DNA-binding domain) to influence transcription

117
Q

Introns

A

sequences in the mRNA that are removed (staying in the nucleus)

118
Q

Exons

A

remain in the transcript (exiting the nucleus as part of the ).

119
Q

ncRNA

A

non-coding RNA = a functional RNA molecule that isn’t translated into protein. (Goes directly from transcription → RNA molecule then goes and performs a variety of functions in the cell).

120
Q

Small nucleolar RNA (snoRNA)

A

a class of small RNA molecules that guide covalent modifications of rRNA, tRNA, and snRNAs primarily through methylation or pseudouridylation (addition of an isomer of uridine)

121
Q

Small nuclear RNAs (snRNAs)

A

average length = ∼150 nucleotides; primary function = processing premRNA in the nucleus and aids in polymerase II and telomeres

122
Q

Restriction enzymes

A

(restriction endonuclease) = cut dsDNA @ palindrome sequences → restriction fragments

123
Q

Recombinant DNA

A

DNA composed of nucleotides from 2 different sources

124
Q

procedure of gene cloning

A

◦ Gene or other DNA fragment is inserted into a DNA vector plasmid using restriction enzymes, enzymes that “cut”, and DNA ligase that “pastes”. This produces a molecule of recombinant DNA

◦ The recombinant plasmid is introduced into bacteria. Antibiotic selection identifies the bacteria that took up the plasmid. Then they use a reporter gene.

◦ The bacteria w/ the plasmid are grown and used as factories to make the protein. They reproduce they replicate the plasmid and pass it on to their offspring. Harvest the protein from the bacteria and purify it.

125
Q

plasmid qualities

A

◦ Has a restriction site

◦ Has an origin of replication

◦ Has antibiotic resistant genes which let you kill the bacteria w/o the plasmid

◦ Replicates independently of the Genomic DNA of the bacteria

126
Q

Process of recombination

A
    1. 2 different sources of DNA in a Petri dish
    1. Digest sequences w/ restriction enzyme
    1. Treat fragments w/ DNA ligase
127
Q

Reporter gene

A

Distinguishes bacteria with recombinant plasmids from those with non-recombinant plasmids

128
Q

Antibiotic resistance gene

A

Allows selection of bacteria that have taken up the plasmid

129
Q

Plasmid vectors

A

useful in amplifying DNA sequences & generating large amounts of gene products

130
Q

To generate recombinant DNA plasmids

A

◦ 1. human DNA & plasmid DNA are digested w/ restriction enzymes

◦ 2. DNA ligase reseals

◦ 3. E coli cells are treated w/ the plasmid/DNA mixture → diverse set of bacteria

◦ How to distinguish b/t the bacteria that haven’t taken up any plasmids, those that took up the nonrecombinant plasmids & those with recombinant plasmids? = 4. use of antibiotics

◦ 5. Then to distinguish b/t non-recombinant & recombinant plasmids = use a reporter gene (codes for a product leading to an obvious phenotypic change & contains sites for the restriction enzyme that’s used in the restriction)

131
Q

DNA library

A

a collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them

132
Q

Genomic libraries

A

have large fragments of DNA in either bacteriophages or bacterial P1derived artificial chromosomes (BACs and PACs).

133
Q

cDNA libraries

A

made with cloned, reverse-transcribed mRNA. Lack DNA sequences that correspond to genomic regions that aren’t expressed. They generally have much smaller fragments than genomic DNA libraries and are usually cloned into plasmid vectors.

134
Q

Hybridization

A

a technique that harnesses the base-pairing of complimentary strands to ascertain the presence of particular mRNA transcripts in a sample by exposing the sample to known complimentary mRNA and measuring the amount of binding.

135
Q

Southern blotting

A

DNA probes hybridize onto the DNA fragments that have the target sequence

136
Q

RNA sequence cloning

A

accomplished through the generation of cDNA (complementary DNA)

  • Step 1. Synthesize a DNA copy of RNA using reverse transcriptase. This makes cDNA
    1. cDNA ligated to vector DNA
  • cDNA cloning allows the mRNA corresponding to a single gene to be isolated as a molecular clone.
137
Q

The key ingredients of a PCR reaction are

A

Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks.

138
Q

PCR Basic steps:

A
  1. Denaturation (96˚C) — heat the reaction to separate the DNA strands → makes single-stranded template.
  2. Annealing (55-65˚C) — cool the rxn so the primers can bind to their complementary sequences on the ss-template DNA
  3. Extension (72˚C) — raise the rxn temp so Taq polymerase extends the primers, synthesizing new strands of DNA
  4. Cycle repeats 25-35 in a typical PCR reaction; generally takes 2-4 hours
  5. Results of PCR are visualized using gel electrophoresis
139
Q

Gel electrophoresis steps

A
  • A technique where fragments of DNA are pulled through a gel matrix by an electric current
  • Separates DNA fragments according to size; Smaller fragments move faster than larger fragments
  • DNA ladder = a standard that’s included so the size of the fragments in the PCR sample can be determined
  • DNA fragments of the same length form a “band” on the gel, which can be seen by eye if the gel is stained with a DNA-binding dye
  • DNA (- charge from its phosphate groups) is attracted to the + charged anode
140
Q

What is the southern blot used for?

A

used to detect the presence & quantity of various DNA strands in a sample

141
Q

Steps of Southern Blot

A

◦ 1. DNA is cleaved by transcription enzymes

◦ 2. The DNA fragments are run through gel electrophoresis, separating them based on size

◦ 3. DNA fragments are transferred to a filter membrane, still separated

◦ 4. The filter membrane is exposed to radiolabeled DNA probe. The radio-labeled DNA will be complement to the gene of interest. This will anneal the gene of interest.

‣ So far we have a (1) radio labeled piece of DNA stuffed to a (2) DNA fragment that’s complement to the gene of interest

◦ 5. We need to be able to visualize the DNA so we expose the filter to an X-ray film. B/c the piece of DNA is radiolabeled, it pops up on the x-ray film.

142
Q

Flow Cytometry

A

single cells either from lab cell culture or tissue samples are stained for certain protein markers using specific antibodies. Flow cytometry detects the fluorescent-tagged antibodies. The labeled cells are suspended in a stream and are passed through a beam of light. As the labeled cells pass through the laser, they emit light. The emitted light is measured giving information on cell size and how many of the cells express the markers that were labeled. The cells can also be sorted based on the different markers detected by the laser.

143
Q

DNA sequencing

A
  • Used to determine the sequence of nucleotides in a strand of DNA
  • Uses dideoxynucleotides (ddNTPs). These are missing the OH group on the 3’ carbon so they’re unable to create a new 5’ → 3’ phosphodiester bond putting us in control of terminating the replication
  • Process:

◦ 1. DNA strand of interest is denatured using an NaOH solution to create a ssDNA strand that we can use for replication

◦ 2. ssDNA strand of interest is added to a solution containing:

‣ A radiolabeled DNA primer that is complementary to the gene of interest ‣ DNA polymerase ‣ All four dNTPs (dATP, dTTP, dCTP, dGTP) ‣ A very small quantity of a single ddNTP (e.g., ddATP) ‣ This is done once for each of the four nucleotides in separate solutions

◦ 3. Each solution containing a specific dNTP and ddNTP are placed in their own lane of a gel and ran under gel electrophoresis. The gel is transferred to a polymer sheet and autoradiography is used to identify the strands in the gel.

◦ For each respective nucleotide, the insertion of a ddNTP will terminate replication and create various strands of different length that correspond to that specific nucleotide. Therefore, the gel can be read from bottom-to-top to determine the nucleotide sequence. The smaller the fragment, the further it travels in the gel.

144
Q

Northern blot:

A

detects a specific RNA in a mixture of RNAs. Uses:

◦ Electrophoresis- separates RNA molecules by size

◦ Blotting- transfer molecules from one membrane to another

◦ probing (hybridization)- label the target molecule with a radioactive or fluorescent tag

Steps:

◦ 1. RNA isolation

◦ 2. Gel electrophoresis ‣ Formaldehyde agarose gel electrophoresis (Formaldehyde is able to the bonds and denature the RNA) ‣ The RNA backbone = lots of phosphates = negative charge ‣ Charge is run (horizontally) and the molecules will separate from one another by size; each band = 1 RNA molecule.

Large RNA molecules are closer to the (-) and smaller ones, since they run faster, they’re closer to the (+)

◦ 3. Blotting— transfer the RNA molecules from the gel electrophoresis to a nitrocellulose membrane.

‣ Salt solution < sponge < gel w/ the bands w/ the RNA molecules < nitrocellulose membrane (filter) < paper towels < weight

◦ 4. Hybridization w/ labelled probes ‣ Chose a probe with a similar sequence to the RNA of interest. Usually uses a cDNA. ‣ the cDNA can bind to the RNA molecule ‣ Probe either has a radioactive label or a fluorescent dye

◦ 5. washing off excess probe

◦ 6. Visualization through autoradiography which allows us to find out exactly where in the bands the RNA of interest is present

145
Q

Western blot

A

detects a specific protein in a sample

◦ 1. Proteins from a sample are loaded into an SDS-PAGE gel and separated based on size

◦ 2. They’re transferred to a polymer sheet and exposed to a radiolabeled antibody (sometimes using two antibodies; one specific to protein of interest and another radiolabeled antibody that binds to first antibody) that is specific to protein of interest

◦ 3. The polymer sheet is viewed using autoradiography. The protein of interest that is bound to the radiolabeled antibody will be visible.

146
Q

In-Situ Hybridization

A

studies gene expression in tissue or embryo.

    1. A thin slice of tissue is fixed to a slide and permeabilized to open the cell membrane
    1. A labeled probe is added to the selection of tissue and it binds to the transcript of interest
    1. An enzyme-linked antibody is added to the tissue selection and it binds to the probe
    1. An enzyme substrate is added and the transcript-probe-antibody complex is detected by the substrate
  • This process is used to detect where transcripts are expressed
147
Q

Immunohistochemistry:

A

uses an antibody to detect a specific protein and measure its expression. The antibody used in this process is recognized by a second antibody. The second antibody is linked to either an enzyme or a fluorescent molecule. • Ex- This technique is often used in clinical studies to detect certain diseases like breast cancer. The HER2 gene is targeted in breast tissue biopsies to see if it is highly expressed..

148
Q

Protein domains

A

Distinct protein structural units, typically b/t 25-500 AA long & contributes to the protein’s function.

◦ Ex- zinc fingers: small protein domains commonly found in transcription factors.

◦ Ex- pleckstrin homology domains: functions in lipid binding, targets proteins to cellular compartments; involved in signaling pathways

149
Q

Protein interactions: Immunoprecipitations-

A

used to find protein binding partners.

‣ 1. Here, cell lysates are collected & incubated w/ an antibody of a specific protein of interest.

‣ 2. Result = complex forms & a microscopic bead is added. This bead has an antibody binding protein liked to it.

‣ 3. For analysis, the solution is centrifuged, pulling the beat out of the solution allowing it to collect @ the bottom.

‣ 4. Complexes are washed & purified w/ lysate solution.

‣ 5. Precipitated proteins are analyzed by western blot or mass spec.

150
Q

Cellular expression

A

cell location can help identify its protein’s unknown function.

◦ 1. A reporter system is used to tag the gene of a certain protein to see where in the cell it’s expressed.

◦ 2. The gene can be cloned in an expression vector and linked to a fluorescent molecule like GFP.

◦ 3. The cellular location can be ascertained using a fluorescent microscope

◦ 4. This will determine if the gene is expressed in the plasma membrane, cytoplasm, or nucleus

151
Q

Hematopoietic stem cells

A

differentiate into types of blood cells

152
Q

Intestinal stem cells

A

basis for the constant renewal of cells lining the intestines

153
Q

Mesenchymal stem cells

A

capable of differentiating into a wide range of cell types — adipocytes, osteoblasts, & hepatocytes

154
Q

Totipotent:

A

able to differentiate into any cell type; zygote → morula

155
Q

Pluripotent:

A

differentiates into any of the germ layers (ectoderm, mesoderm, endoderm); obtained from the internal cell mass of the blastocyst

156
Q

Multipotent:

A

adult stem cells; differentiates into several types of cells w/in a relatively limited functional scope

157
Q

Oligopotent:

A

can only derive into a few types of cells

158
Q

Phenotype:

A

physical manifestation of a genetic trait; characteristics visible to the naked eye; usually appearance, but could be something like the efficiency w/ which cells carry out a certain metabolic pathway is an example of a phenotype.; ie. The flower is red

159
Q

Genotype

A

combination of genes responsible for the phenotype; I.e. RR and Rr are genotypes for the red flower color

160
Q

gene

A

DNA sequence that codes for a given trait

161
Q

locus

A

a specific place on a chromosome

162
Q

Allele

A

variations of a gene

163
Q

Wild-type (w⁺):

A

the default phenotype or genotype that’s present in most members of a species; doesn’t have the mutation

Majority/most prevalent genotype or phenotype

164
Q

Complete dominance:

A

one copy of a dominant gene is enough to induce the dominant phenotype (I.e. RR and Rr both produce red flowers)

165
Q

Co-dominance

A

two dominant alleles can be expressed at the same time

166
Q

Incomplete dominance

A

a heterozygote displays a blended phenotype

167
Q

Leakage

A

genes traveling b/t species

168
Q

Penetrance:

A

the likelihood that the carrier of a given genotype will manifest the corresponding phenotype

169
Q

Expressivity

A

the intensity or extent of the variation in the phenotype

170
Q

Hybridization: Viability

A

◦ Process of two complementary, single-stranded DNA or RNA combining together, producing a double-stranded molecule through base pairing

◦ Technique is used for interbreeding between individuals of genetically distinct populations

171
Q

Gene pool:

A

the combined set of all genes/alleles in a population; describes the genetic status of a population

172
Q

DNA synthesis occurs during

A

S phase of interphase

173
Q

Synapsis of homologous chromosomes and crossover occurs during

A

prophase I of meiosis; crossing over -> genetic recombination

174
Q

Homologous chromosomes line up @ metaphase plate during

A

interphase I of meiosis

175
Q

outcome: # of genetic composition of daughter cells mitosis

A

Mitosis: 1 division - 2 diploid (2n) cells that are identical to parent cells

176
Q

outcome: # of genetic composition of daughter cells meiosis

A

1 round of replication & 2 rounds of division = 4 haploid (n) cells @ end of meiosis II that are different from parent cells

177
Q

daughter cells DNA in mitosis

A

DNA int he form of sister chromatids

178
Q

daughter cells DNA meiosis

A

after meiosis I: DNA in the form of homologous chromosome

179
Q

Mendel’s 2ⁿᵈ law of independent assortment

A

the inheritance of one gene does not affect the inheritance of another gene; also applies to genes on the same chromosome b/c of crossing over.

180
Q

phenomenon of linkage.

A

Genes close to each other are less likely to undergo recombination than genes farther away from each other; if genes are close enough to each other, this could violate Mendel’s 2ⁿᵈ law,

181
Q

Recombination frequency (θ)

A

how often a single crossover will occur b/t 2 genes during meiosis

182
Q

If θ > 50%

A

genes obey the law of independent assortment

183
Q

crossing over

A

occurs during prophase I; @ the chiasma (points of crossing over, essentially random); made possible b/c of pairing of homologous chromosomes = tetrads; which is formed by a process called synapsis

184
Q

Genes that are closer together are (more/less) likely to have a double crossover between them than a single crossover (and also more/less likely if farther apart).

A

Genes that are closer together are even less likely to have a double crossover between them than a single crossover (and also more likely if farther apart).

185
Q

Synaptonemal complex

A

protein complex that glues the tetrad together

186
Q

Tetrad

A

a pair of four chromatids (synaptic pair)

187
Q

Sex-linked characteristics

A

inheritance that takes place for genes on the X chromosome

188
Q

SRY (sex-determining region Y)

A

codes for the transcription factor that initiates testis differentiation (male gonad formation); absence of the Y chromosome, all zygotes = female; Y chromosome present = male

189
Q

Inversion mutation

A

a stretch of DNA (segment or chromosome) breaks off, the reattaches in the opposite orientation

(can occur when a mistake takes place in the directionality of a chromosome; a segment is reversed from end to end)

190
Q

insertion mutation

A

a segment of DNA is moved from one chromosome to another

191
Q

deletion mutation

A

1 or more nucleotides removed from the genome; also deletion of large chromosomal regions

◦ Leads to either a loss of heterozygosity or a reduction in gene dosage

192
Q

amplification

A

generally ↑ the gene dosage by leading to more transcription of the genes in question

193
Q

Translocation

A

chromosomal regions are moved around; a sequence of genes switches places from one chromosome to another; involves a reciprocal switch

◦ Balanced translocation = the exchange of genetic material is even

◦ Unbalanced translocation = exchange is unequal

194
Q

Cytoplasmic/extranuclear inheritance

A

gene transmission outside of the nucleus; (inheritance of things other than genomic DNA)

◦ All cellular organelles like mitochondria is inheritsd from the mother

◦ found in most eukaryotes; commonly occurs in the cytoplasmic organelles like mitochondria and chloroplasts or from cellular parasites like viruses or bacteria

195
Q

2 things that increase genetic diversity include

A

synapsis and crossing over

196
Q

Hardy-Weinburg equilibrium equation

A

p² + 2pq + q² = 1

197
Q

test cross

A

In a test cross, an individual with a dominant phenotype is crossed w/ an individual w/ the recessive phenotype.

  • IF the dominant phenotype organism is homozygous → F1 generation won’t have any individuals w/ the recessive phenotype
  • IF the dominant phenotype is heterozygous → ∼ 50% will manifest the recessive phenotype.
198
Q

Backcross

A

a hybrid is crossed with a parent or something close to the parent genetically

◦ Goal = obtain offspring more similar to the parent

◦ Mating b/t the offspring and the parent; preserves parental genotype

199
Q

Genetic recombination— occurs b/t

A

• Genetic recombination— occurs b/t maternal & paternal sister chromatids

200
Q

Stabilizing selection

A

both extremes are selected against

201
Q

• Directional selection

A

only one extreme phenotype is selected against and the other extreme is favored

202
Q

Disruptive selection

A

the median phenotype is selected against

203
Q

Speciation

A

the evolutionary process of forming new and distinct species out of a shared ancestral population.

204
Q

Polymorphism

A

naturally occurring differences in form b/t members of the same population

205
Q

Adaptation & speciation

A

a process where certain individuals or subpopulations develop evolutionary strategies specific to certain microenvironments, or niches; sets the stage for speciation

206
Q

Adaptive radiation

A

the rapid rise of a # of different species from a common ancestor

207
Q

Inbreeding:

A

breeding b/t genetically closely related individuals; can → ↑ recessive mutation manifestations

208
Q

Outbreeding

A

breeding among genetically distant members of a population

209
Q

Bottlenecks

A

an external event dramatically ↓ the size of a population in a way that is random in regard to alleles

210
Q

The founder effect:

A

The founder effect: results from bottlenecks that suddenly isolate a small population → inbreeding & ↑ prevalence of certain homozygous genotypes

◦ Causes: migration

◦ Population w/ a non-random sample of genes

211
Q

Genetic drift-

A

random chance; more likely w/ small populations.

212
Q

Divergent evolution

A

Same lineage, evolving apart to be more different

◦ Produces homologous structures (bat’s wing & horse’s hoof)

213
Q

Parallel evolution

A

◦ Same lineage, evolving closer together to be more similar, using similar mechanisms.

◦ I.e. feeding structure in different crustacean species. The feeding structure came from mutation of pair of legs, turning them into mouth parts

214
Q

Convergent evolution:

A

◦ Different lineage, evolving closer together to be more similar, using different mechanisms.

◦ Produces analogous structures (bat’s wing & butterfly’s wing)

215
Q

Coevolution

A

◦ Two species evolve in response to each other.

◦ I.e. predator/prey or host/parasite species

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