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SBIU4 > Molecular Genetics > Flashcards

Molecular Genetics Flashcards

1
Q

What did Griffith do and when?

A

1928: carried out experiments on pneumonia bacteria in mice and found that something in the heat killed virulent bacteria could be transferred to the live, harmless bacteria to make them virulent.

He did this by injecting different bacteria into the mice. Rough nonvirulent resulted in a healthy mouse. Smoother virulent resulted in a dead mouse.And rough nonvirulent plus heat killed smooth virulent killed the mouse and found traces of viral smooth bacteria

He concluded that he observed phenomenon transformation which is a change in a cells function due to an unknown substance (or change in geno/pheno type)

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2
Q

What did Hammerling do and when?

A

1930: used a single cell green algae that had a foot with a nucleus, a stalk and a cap. he cut off the cap and the algae grew a new one, but when he cut off the foot the algae did not grow a new one. And when he grafted a different stalk onto a different foot, the cap that grew was the same as the foot.

Concluded that the nucleus controls the growth of the organism

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3
Q

What did Avery et al do and when?

A

1944: continued Griffith’s experiments by isolating various substancde to determine if they are the virulent factor –> determined that DNA is the hereditary molecule

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4
Q

What did Hershey do and when?

A

1952: experimented with bacteriophages with radioactive markers of phosphorus and sulphur on DNA and protein respectively, and agitated and centrifuged it and determined that because the protein cell was radioactive but not the pellet, it was not the protein that was the reprogrammer, while the pellet was radioactive w P.

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5
Q

What did Chargaff do and when?

A

1947: studied DNA to determine the structure and found that there were equal amount of AT and CG and proposed that they were base pairs

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6
Q

What did Franklin and Wilkins do and when?

A

1952: used x-ray crystallography to discover the structure and found out that there was a double helix structure with an alternating backbone of phosphates and sugar with the bases in the middle

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7
Q

What did Watson and Crick do and when?

A

1952-1953: built first model and determined that a deoxyribose sugar is used and hydrogen bonding exists between AT and CG, and showed that DNA was anti parallel

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8
Q

What did Meselson and Stahl do and when?

A

After W and C: discovered that DNA was a semi conservative model by using nitrogen isotopes to differentiate between the two DNA

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9
Q

Explain the semi conservative model

A
  • when DNA replicates the daughter strands are conserve half of the parent DNA strand
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10
Q

When does DNA replication happen?

A

during interphase

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11
Q

Explain initiation in DNA replication

A
  • DNA is unwound to expose the bases
  • starts at the origin of replication
  • several initiator proteins bind to the DNA molecule
  • Helicase cleaves the hydrogen bonds between the bases to create a replication bubble with replication forks to allow DNA to unravel in the opposite direction
  • single stranded binding proteins stabilise the the single strand sections
  • topoisomerase 2 relieves the strain on the helix sections
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12
Q

Explain elongation in DNA replication

A
  • complementary strands are assembled by joining individual nucleotide strans
  • DNA polymerase 3 reads in 3’ to 5’ and builds in 5’ to 3’
  • Leading strand: primase creates one RNA primer on 3’ end and DNA polymerase 3 constructs it continuously
  • lagging strand: primase creates multiple RNA primers so DNA polymerase 3 builds multiple okazaki fragments
  • DNA polymerase 1 removed RNA primer and fills in spaces by extending DNA fragment
  • 1 and 2 proofread
  • DNA ligase joins Okazaki fragments
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13
Q

explain termination in DNA replication

A

the 2 new DNA molecules seperate from each other and the replication machine dismantles, and the new strands revert back into their double helix form

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14
Q

explain error checking in DNA replication

A
  • in built proofreading via DNA Polymerase 1 and 2 that recognize if wrong nucleotide is added or omitted and will stall replication to remove or add bases to correct it, repairing most errors
  • mismatch repairs which is a mechanisms of other proteins recognize the errors and fix them
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15
Q

What is the purpose of DNA?

A

to code for protein production

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16
Q

Define a gene:

A

sequence of nucleotide bases that code for a specific protein and is inherited

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17
Q

Explain the central dogma of molecular genetics:

A
  • through the production of mRNA and the synthesis of proteins, the information from DNA is expressed
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18
Q

Explain the one gene one polypeptide hypothesis

A
  • organism genome is in the nucleus and proteins are synthesized in the cytoplasm by the ribosomes. As DNA cannot leave the nucleus, mRNA goes to the ribosomes that are copied into the RNA that become the proteins
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19
Q

Explain the triplet hypothesis:

A
  • because there are 20 amino acids and 4 bases, each codon codes for one amino acid, resulting in 64 combinations
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20
Q

What is a codon?

A
  • sequence of three nucleotides that code for one amino acid
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21
Q

What is the start codon?

A

AUG

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22
Q

what are the stop codons?

A

UAG, UAA and UGA

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23
Q

Define transcription in protein synthesis:

A
  • process where DNA gets copied into mRNA
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24
Q

Explain initiation in transcription

A
  • RNA polymerase binds to a promoter region upstream of the gene
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25
Q

What is the promoter region?

A
  • characteristic area upstream of the gene with At base pattern, recognized by RNA polymerase
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26
Q

Explain elongation in transcription

A
  • RNA polymerase builds mRNA in 5’ to 3’ direction after reading the template strand in the 3’ to 5’ direction
  • mRNA is complementary to the template strand and identical to the coding strand, except T is replaced with U
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27
Q

explain template vs coding strands

A

template: what RNA polymerase reads and is opposite to what is actually wanted (anti sense)

coding: original of what RNA polymerase is actually making (sense)

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28
Q

explain termination in transcription

A
  • at the end of the gene, RNA polymerase recognizes the terminator sequence and stops transcribing, by which then mRNA dissociates from the template strand
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29
Q

Explain post transcriptional modifications in eukaryotes

A
  • 5’ cap is added to 5’ start of primary transcript to protect mRNA fom being broken down by nucleases and phosphatases, and plays a role in the initiation in translation
  • a Poly-A tail is added to the 3’ end by Poly A polymerase made of 200 A bases
  • eukaryotes have exons and introns and the introns are removed by spliceosomes that rejoin the exons
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30
Q

what are exons and introns?

A

exons: coding regions
introns: non coding regions

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31
Q

explain why introns need to be removed

A

bc the protein will not fold properly otherwise

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32
Q

Explain translation in protein synthesis:

A
  • synthesis of proteins using ribosomes and RNA as a template or blueprint
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33
Q

Explain initiation in translation

A
  • ribosomes recognize the 5’ cap and bind to the RNA
  • ribosomal unit: large subunit and small subunit with mRNA clamped in between, moving in 5’ to 3’ direction
  • tRNA delivers amino acid to building site
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34
Q

what is tRNA?

A
  • single stranded nucleic acid in a clover shape
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35
Q

what is an anticodon?

A

branch on one arm of tRNA that recognizes mRNA codon while the opposite arm carries the corresponding amino acid

36
Q

what is amino-acyl tRNA?

A
  • molecule with corresponding amino acid attached to acceptor site at 3’ end
37
Q

what is aminoacyl-tRNA synthetase

A
  • charges cast out tRNA by adding an amino acid
38
Q

explain elongation in translation

A
  • codon first read is always AUG that codes for methionine
  • tRNA with methionine enters P site and next tRNA enters A site
  • methionine bonds with second amino acid and ribosome translocates (shifts one codon over)
  • process repeats as first tRNA exits
  • polypeptide chain is created and tRNA recycled are released
39
Q

what are the parts of the ribosomal subunit? the different sites are:

A
  • Acceptor Site
  • Peptide Site
  • Exit Site
40
Q

explain termination in translation

A
  • ribosome reaches stop codon that is read in the A site
  • release factor recognizes stalled ribosome and subunits disassemble
  • mRNA and polypeptide chain is released
    -protein is folded and modified before sent to target
41
Q

What are mutations?

A

errors made in DNA sequences that are inherited

42
Q

what are point mutations?

A
  • erros specific to one base pair
43
Q

explain frameshift mutations

A
  • change in the reading frame of RNA due to the insertion and deletion of less than or more than 3 base paits
44
Q

explain substitution mutations

A
  • one base pair is replaced with another resulting in the wrong amino acid placement
45
Q

explain silent, missense and nonsense mutations:

A

silent: no effect on cell because it occurs in introns or final base pair
missense: changes codon that changes amino acid that affects protein folding
nonsense; change in DNA creating stop or start codon earlier so translation stops early

46
Q

define chromosomal mutations:

A

affects multiple genes

47
Q

define translocation mutations

A

relocation of base pairs from one part of genome to another

48
Q

define inverse mutations

A

section of genes switch order resulting in a reversed orientation in chromosomes

49
Q

define spontaneous mutations

A

errors from genetic machinery

50
Q

define induced mutations

A

comes from mutagenic agents like UV rays

51
Q

define genetic defect

A

mutations that do not occur naturally and therefore cannot be fixed

52
Q

define somatic mutations and in what kind of organisms they occur in

A
  • passed on during mitosis but not to the following generations
  • occurs in multicellular organisms
53
Q

define germline mutations and in what kind of organisms they occur in

A
  • occur in multicellular organisms in gametes and are passed over to the next generation
54
Q

define gene expression

A
  • when gene is transcribed and translated to produce the corresponding protein
55
Q

why do genes need to be regulated?

A
  • gene’s don’t need to be transcribed all of the time
56
Q

what are the levels of controlling gene expression?

A
  1. DNA -> RNA -> mRNA -> polypeptide -> protein
  2. transcription -> post-transcription -> translation -> post translation
57
Q

what are the proteins involved in controlling gene expression? Define them

A
  • transcription factors: turns genes on
  • repressors: turns genes off
  • enhancers: increases transcription
  • inducers: allow transcription to occur
58
Q

what is an operon?

A
  • cluster of genes transcribed together that are cut up and translated seperately with one promoter region
59
Q

what is an operator?

A
  • regulatory region upstream of opeon
60
Q

which enzyme catalyzes the reaction of the breakdown of lactose in e coli?

A

betagalactosidase

61
Q

explain the lac operon structure

A
  • cluster of genes that make the enzymes that help with the metabolism of lactose
  • contains regulator gene (lac 1 that codes for repressor)
  • promoter
  • operator (repressor binds here)
  • Lac Z gene (makes betagalactosidase)
  • Lac Y gene (membrane protein pumping lactose into cell)
  • Lac A
62
Q

what happens to the lac operon when there is no lactose present?

A

repressor binds to operator and partially covers promoter so RNA polymerase cannot bind to it thus precenting transcription of Lac Z, Y, and A genes

63
Q

what happens to the lac operon when there is lactose present?

A

allolactase binds to the repressor to change its shape so that it does not bind to the operator, so RNA polymerase can continue with transcription

64
Q

what is allolactase considered?

A

inducer

65
Q

what allows allolactase to let go of the operator?

A

lactose breaks down and levels of it are reduced in cells so allolactase lets go of repressor so it can bind to operator

66
Q

what is tryptophan?

A
  • amino acid that e coli uses to make protein which can be obtained from the environment or synthesized
67
Q

what happens to the tryp operon when tryptophan is absent?

A

regulator gene encodes for repressor that remains inactive and does not attach to the operator so the genes to produce tryptophan

68
Q

what happens to the tryp operon when tryptophan is present?

A

corepressor or effector molecule activates repressor so no transcription occurs

69
Q

what is the corepressor for the tryp operon

A

tryptophan

70
Q

what are restriction enzymes?

A

cut DNA at specific points in a base sequence, occurring naturally in prokaryotes and specific to a given recognition site

71
Q

explain in what ways restriction enzymes can cut up a gene

A
  • sticky end method (palindrome)
  • blunt ends method (straight)
  • DNA ligase is added to join DNA and selected gene into a plasmid
72
Q

what kind of ligase is used for blunt ends?

A

T4 DNA ligase

73
Q

what are plasmids?

A
  • natural circle of DNA in bacteria and can also be engineered
  • have restriction enzyme recognition sites
74
Q

what are selective markers?

A

features that allow one to differentiate between regular plasmids and recombinant

75
Q

what is a recombinant plasmid?

A
  • a plasmid that took up the added target gene
76
Q

name the two types of selective markers

A
  • ampicilin resistant gene
  • lac Z gene
77
Q

explainthe selection for plasmid uptake

A
  • ampicilin resistance becomes the selecting agent and only the bacteria with the plasmid will grow in an antibiotic ampicilin plate because it is resistant, which allows for the successful uptake of marker to determine the presence of plasmid
78
Q

explain transformation in genetic technologies

A
  • process where plasmid carrying gene is TRANSFORMED or put into a cell or bacteria
79
Q

what is a recombinant bacteria?

A
  • when the plasmid enters the bacteria and contains the desired gene
80
Q

define blue white screening

A
  • process that quickly detects whether transformation is successful to eliminate plasmids that did not have the new DNA,detecting whether some DNA got added to the plasmid
81
Q

explain the process of blue white screening

A
  • the gene of Lac Z is inserted into the plasmid
  • interacts with the growth medium to produce blue betagalactosidase
  • one part comes from the plasmid that attaches to another protein in the bacteria to produce betagalactoisidase that cleaves the molecule x-gal turning the bacteria blue
  • when the gene of interest is spliced into Lac Z, the latter cannot make betagalactosidase, therefore we know that the plasmid became recombinant as the bacteria remained white
82
Q

explain gel electrophoresis

A
  • method that separates large molecules
  • restriction enzymes cleave DNA and pieces are put into wells filled with gel that floats in a buffer solution
  • electrical current passes through the gel and DNA moves towards the + charge because DNA is -
  • the smaller pieces move more quickly than the larger ones
  • several bands form that are visible under UV light and is used to compare genetic similarities to determine parent or sibling
83
Q

Explain the Sanger method

A
  • used to determine actual sequence of DNA based on replication and the use of dideoxynucleotides to prevent the bonding to the next nucleotide to terminate the growing chain
  • by adding 4 of the dd-nucleotides to 4 test tubes with four bases and radioactive primers, complementary DNA strands can be synthesized in vitro
  • then the complementary strand is assessed and assembled using the dd-nucleotides to determine the sequence
84
Q

what are the advancements to sequencing?

A
  • instead of using manual separate lane method fluorescent tagging in one tube with different colors that are lit up by a laser and recorded by a computer after being interpreted by a CCD detector
85
Q

What does PCR stand for

A

polymerase chain reaction

86
Q

explain PCR

A
  • method that generates copies of DNA fragments from small quantities without having to be inserted into a plasmid
  • 30 cycles in 3 hours
  • temperature varies:
    1. heat DNA to approx 95 degrees C to separate
    2. cool to about 55 degrees C to let DNA primers anneal
    3. heat to approx about 72 degrees C to extend complementary DNA strand using taq polymerase

SBIU4 (4 decks)

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