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Week 9 Lecture Content Flashcards

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1
Q

Random Insertional mutagenesis followed by gene-specific screening

A
  1. Create insertion library using transposons or T-DNA
  2. Isolate DNA
  3. PCR with primers g1, g2, t1, t2
    - If gene does not have insertion, only the combination of g1 + g2 results in a product
    - If a gene has an insertion, specific combination of g and t primers will yield products
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2
Q

Gene silencing by double-stranded RNA

A
  • RNAi silencing gene expression transcriptionally or post-transcriptionally
  • Impact: transcription, mRNA, stability, and/or translation
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3
Q

RNAi transcriptionally silencing gene expression

A
  1. exogenous dsRNA enters cell
  2. Dicer cuts RNA into 21-25bp fragments
  3. RISC complex binds and denatures RNA - passenger strand degraded
  4. Binds to mRNA by complementary case pairing
  5. Forming double-stranded RNA which is hen translationally blocked or destroyed
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4
Q

The evolution and applications of RNAi: Significance

A
  • Protect their genomes against mutational effects of transposable genetic elements
  • Protect against viral infection
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5
Q

Common use of RNAi

A

To ‘knock down’ expression of selected genes to determine the effect on the phenotype

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6
Q

RNAi silences gene expression post-transcriptionally

A
  1. exogenous dsRNA enters cell
  2. Dicer cuts RNA into 21-25bp fragments
  3. RISC complex binds and denatures RNA - passenger strand degraded
  4. transport into nucleus for bidirectional transcription
  5. Forms pre-siRNA that goes back to step 2
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7
Q

Applied RNAi steps

A
  1. Transfection or direct injection of dsRNA
  2. Cleavage of dsRNA into 21- to 24-base-long siRNA by dicer
  3. Cleavage of target mRNAs complementary to siRNAs by slicer activity of argonaute
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8
Q

CRISPR-Cas

A

-Clustered regularly interspaced short palindromic repeats
- Native function: defense against invading nucleic acids

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9
Q

How does CRIPR-Cas system work?

A
  • CRISPR sequences to produce crRNAs by cas-encoded RNases
  • TracrRNA at two regions - one binds to Cas DNA endonuclease and other to a crRNA
  • Cas endonuclease creates a double-strand break in the DNA homologous to the crRNA
  • Cas operon: DNA endonuclease and RNase
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10
Q

What is a reporter gene?

A

Gene whose product can be detected directly or produces a detectable substance

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11
Q

What forms can reporter genes come in?

A
  • Enzyme: can cleave a colorless substrate to produce a colored product
  • Fluorescent Protein: light emission
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12
Q

Beta-galactosidase as a reporter gene

A

lacZ
- can cleave colorless substrates ONPG and X-gal to produce yellow and blue products
- Cannot be used in plants as they have endogenous beta-galactosidase

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13
Q

Beta-glucronidase as a reporter gene

A

GUS
- Cleaves X-gluc into a blue product, can be used in plants

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14
Q

Luciferase as a reporter gene

A

Catalyzes a reaction between luciferin and ATP that results light - fireflies

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15
Q

Reporter genes: Transcriptional fusion

A

Regulatory sequences directing transcription of the gene of interest are fused with the reporter gene so as to direct transcription of the coding sequences of the reporter gene
- Gene will be transcribed in the pattern directed by the regulatory sequence to which it is fused

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16
Q

What can reporter genes help reveal

A
  1. Regulatory sequences
  2. Temporal patterns of gene regulation
  3. Spatial patterns of gene expression
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17
Q

Using reporter gene lacZ to identify enhancer of eve gene in flies

A
  1. Construct restriction map of 5’ upstream sequence
  2. Fuse the 5’ upstream sequence with lacZ
  3. Create various deletion mutations of the 5’ upstream sequence
  4. Transform the deletion constructs into fruit fly larvae
  5. Feed fly larvae with x-gal and observe color segments
  6. Confirmation of results
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18
Q

Recombinant DNA technology

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A

A set of techniques for amplifying, maintaining, and manipulating DNA sequences in vitro and in vivo
- divide genome into smaller segments
- assemble small DNA segments into larger ones

19
Q

What are the main recombinant DNA techniques?

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A
  1. Fragment DNA into smaller pieces and purify the pieces - restriction enzymes, sonication/nebulization, gel electrophoresis
  2. Create many copies of a DNA sequence - in vitro by PCR and om vivo plasmids and other vectors
  3. Determine the exact sequence of specific DNA molecules
  4. Combine DNA fragments
  5. Introduce specific DNA molecules into living organisms
20
Q

Restriction enzyme

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A

Recognizes a specific DNA sequence and cuts both strands of the sugar-phosphate backbone of DNA
- create sticky ends with single stranded segments at the ends of each fragment
- Prevalent in bacteria
- protect against phage infections

21
Q

Restriction-modification system

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A
  • DNA methylase: methylate the restriction recognition site of bacteria’s own genome for protection
  • Restriction enzyme: unable to cut own methylated DNA; however, invading viral DNA is not methylated and will be cut
22
Q

Reconstruction map

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A

A map of known restriction enzyme recognition sites along a sequence of DNA
- can be determined by analyzing fragment size when sliced

23
Q

How to make a recombinant DNA molecule?

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A
  1. Digest fragments creating nonidentical, complementary sticky ends
  2. Insertion DNA combines with vector DNA
  3. DNA ligase catalyzes phosphodiester bond formation between 5’ phosphate and 3’ hydroxyl groups
24
Q

Strategies to prevent self-ligation

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A
  1. remove the 5’ phosphate group
  2. blunt end cloning
  3. double digest
25
What should an ideal cloning vector contain
- Origin of replication - Selectable marker - Multiple cloning sites - lacZ: Beta-galactosidase cleaves lactose analog X-gal and converts it to a blue by-product; an insert prevents cleavage of X-gal and the colonies are white
26
How to put recombinant DNA molecule into bacteria
Transformation - Divalent cations or electrical shock are often used to make bacterial cells competent by opening pores in their membranes - Plasmids are amplified by DNA replication transmitted to progeny by cell division
27
How to construct a cDNA library
1. Isolate mRNA 2. Add oligo dT primers 3. Synthesize first strand cDNA using reverse transcriptase 4. Partially degrade mRNA using RNase H 5. Synthesize second strand cDNA using DNA polymerase and remaining mRNA fragments as primers 6. Protect sites in cDNA from digestion through methylation 7. ligate linkers containing target gene 8. digest and clone into vectorM
28
Major differences between genomic and cDNA libraries
- Introns: none - Regulatory sequences: none - Copy numbers: based on level of expression - Tissue specificity present in cDNA
29
cDNA library
a collection of cloned DNA sequences that are complementary to the mRNA that was extracted from an organism or tissue
30
Things to consider when choosing appropriate cloning vectors and host cells to express specific transgenes
1. Regulatory elements 2. Codon optimization 3. Post-translational modification enzymes
31
How is insulin produced in humans
- Starts as preproinsulin with chain A, chain B, pre-amino acids and pro-amino acids - Cleavage of pre-amino acids produces proinsulin - disulfide bonds form between chain A and B - cleavage of pro-amino acids produces insulin
32
How is human insulin commercially produced?
1. Plasmid with lacZ and either sequence for chain A or B transform E. coli. 2. Under production of lactose gene, artificial A and B chains created 3. harvest culture and lyse cells to isolate fusion protein and cleave off beta-galactosidase peptide chain 4. Chains added together to form insulin
33
Production of insulin - creation of the B chain
1. AA sequence was determined by peptide sequencing 2. Nucleotide sequence was created by reverse translation of the amino acid sequence 3. Two successive stop codons were added following the open reading frame 4. Methionine codon inserted at the beginning 5. EcoRI and BamHI sites were added to the ends of the DNA to facilitate clonign into a vector
34
What are other commercial proteins produced by transgenic organisms
- Human growth hormones and erythropoietin - Proteases and lipases in laundry detergents - chymosin in cheese - Vaccines - PCR polymerases - Reverse transcriptase
35
What is Ti - plasmid and how does it work to transfer DNA from bacteria to plants
- Tumor inducing plasmid - Transfer DNA is the portion of the Ti-plasmid that is transfered from the bacterium into the nucleus of a plant cell - contains auxin and cytokinin biosynthetic genes and genes for amino acid biosynthesis - transferred into the plant cell and cause uncontrolled division of plant cells
36
How is Ti plasmid used to create transgenic plants
- Reengineering of Ti plasmid separates sequences responsible for transfer of T-DNA and T-DNA - Transformation vector contains T-region flanked by right and left border sequences - "Disarmed" plasmid contains genes required for virulence and conjugative transfer; lacking t-region, no longer able to induce crown gall disease - Genes on disarmed plasmid produce conjugative and virulence proteins that act in trans on T-DNA border sequences of transformation vector to effect transfer of T-DNA into plant cell - Infected plant cells are grown on selectable media containing herbicide and, after selection, regenerated into transgenic plants
37
Common traits engineered into transgenic crops
- herbicide-resistance - Insect toxins - faster growth, higher productivity, delayed maturation, firmer fruits - Enhanced nutrition
38
What are the concerns about transgenic plants
- Allergies to recombinant proteins - Development and spread of insecticide resistance in natural insect populations - Transfer of herbicide resistance to wild plants - Negative impacts on natural insect pollinators
39
Gene therapy
The use of genes to cure or alleviate disease symptoms
40
Gene therapy : sickle cell disease in mice
1. Harvesting adult cells 2. Reprogramming adult cells into induced pluripotent stem cells by activating four yamanka factors 3. Repairing the genetic defects using CRISPR and homologous recombination 4. Differentiating the iPS cells into hemopoietic precursors in vitro 5. transplanting the corrected cells into bone marrow of affected mice
41
How are microbes and plants clone
- All microorganisms are totipotent (capable of cloning by themselves easily - Many plants have the capacity for asexual propagation, which can produce clones that are all genetically identical
42
How to clone animals
- Most animals require sexual reproduction - Animals do not readily propagate clonally in nature - Most animals are not totipotent
43
Cloning animas by nuclear implantation - in goats
1. Remove egg from Scottish blackface 2. Remove cells form mammary gland in finn dorset 3. extract egg nucleus and inject mammary nucleus 4. electroshock to induce cell division and allow to develop until blastocyst stage 5. Implant blastocyst in surrogate mother's womb 6. Finn Dorst born

Genetics (18 decks)

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