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Molecular Genetics > Week 4 > Flashcards

Week 4 Flashcards

Non-coding RNA and cloning, genetic libraries

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1
Q

What is non-coding RNA (ncRNA)?

A

RNA that is not translated

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2
Q

What does the term proteome mean?

A

The entire set of proteins that are or can be expressed by a genome at a certain time.

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3
Q

What happens to the proportion of ncDNA when the overally proportion of protein coding DNA decreases?

A

the proportion of ncDNA increases

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4
Q

What are the main roles of ncRNA?

A

RNA is involved in many cellular processes via ribonucleoprotein (RNP) complexes including: - DNA replication - RNA processing - Protein synthesis - Protein targeting - Catalysing chemical reactions in living cells - Gene regulation

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5
Q

What are siRNA?

A

siRNA: Small interfering RNA

  • Involved in gene regulation, transposon control and viral defense
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6
Q

What are miRNA?

A

miRNA: Micro RNA

  • help in post transcriptional gene regulation
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7
Q

What are piRNA:

A

piwi interacting RNA (Bound by piwi proteins)

  • Involved in silencing germline transposable elements
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8
Q

What are lncRNA?

A

Long Regulatory non-coding RNA

- greater than 200nc long

- Makes up about 1% of genome

  • Largest portion of mammalian nc-transcriptome
  • Involved in x-inactivation and imprinting

- Epigenetic regulation, sequence-specific tethers for protein complexes and specify subcellular localisation.

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9
Q

What are snRNA?

A

Small nuclear RNA:

  • Assist in pre-mRNA splicing

- Components of the spliceosome

  • Form snRNP’s (‘snerps”)
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10
Q

What are snoRNA?

A

Small Nucleolar RNA:

  • Involved in the rRNA processing process and chemical modification of rRNA
  • Form snoRNP’s (“snorps”)
  • Bind to specific sites in precursor rRNA to modify bases
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11
Q

Where are snoRNA’s usually found?

A

encoded in introns and released via splicing of pre-mRNA

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12
Q

What makes up a spliceosome?

A

5 snRNA proteins(snRNP’s): U1, U2, U4, U5, and U6

+

shitloads of protiens (just shy of 300)

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13
Q

What is imprinting phenomenon?

A

The inheritance of only one paternal working copy of a specific gene due to epigenetic silencing. (this can be from either mum or dad)

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14
Q

What is the Prader-Willi syndrome?

A

The absence of snoRNA HBII-52 causing alternative splicing of the serotonin receptor precursor RNA, resulting in a protein that has a greatly reduced function.

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15
Q

What does the term epigenetics mean?

A

the study of how genes are “turned on and off”

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16
Q

What are transposon/transposable genes?

A

A DNA sequence/gene that can change its location within a genome

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17
Q

What are transcriptomes?

A

The transcriptome is the set of all RNA molecules in one cell or a population of cells. (can be either refering to the nc-RNA or mRNA)

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18
Q

What is the link between lncRNA and Alzheimer’s Disease?

A

lncRNA BACE1-AS (anti-sense strand), is upregulated contributing to an increase in the ammount of BACE1 protien produced resulting in an increase of ß-Amyloid which then leads to cognitive dysfunction etc.

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19
Q

What is RNA interference?

A

RNA challenging the Dogma of DNA -> RNA -> Protein

  • happens when the mRNA is destroyed before it can be transated into protein
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20
Q

What can RNAi (RNA interferance) be used for?

A
  • Defence against viral infection
  • Control of transposons
  • Regulation of gene expression
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21
Q

What was Fire and Mello’s Experiment?

A

They injected RNA corresponding to a gene important for muscle function in the a worm. Single stranded RNA had no effect but the double stranded RNA caused the offspring to twitch in a similar way to worms that lack a functional gene for the muscle protein.

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22
Q

What did Fire and Mello’s experiment conclude?

A
  • Gene silencing was triggered efficiently by dsRNA but not ssRNA
  • Silencing was specific for an mRNA matching the dsRNA
  • Neither intron or promotor sequence triggered a response as the effect could only take place after transcription.
  • target mRNA dissapeared indication degradation
  • Only minimal dsRNA were needed to accomplish full silencing indicating that dsRNA was amplified and/or acted catalytically rather than stoichiometrically.
  • dsRNA effect could spread between tissues and even to progeny (offspring)
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23
Q

What are the differences between origin of miRNA and siRNA?

A

siRNAs are from dsRNA made up of two separate strands of perfectly basepaired RNA from outside or inside the cell whereas, miRNAs are from single, long transcripts from inside the cell that form imperfectly base-paired hairpin structures .

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24
Q

How does siRNA work to trigger gene silencing?

A

IN THE CYTOPLASM:

  1. dsRNA with 3’ overhangs bind to Dicer
  2. Dicer cleaves dsRNA into smaller fragments
  3. One of the strands (the guide strand) is loaded into the RNA induced silencing complex (RISC)
  4. RISC links the complex to mRNA through base pairing
  5. mRNA is cleaved and degraded
  6. Protein cannot be synthesised therefor resulting in gene silencing
25
Q

Is the RNA-Induced silencing complex specific or non-specific?

A

non-specific: It can bind to different ncRNA to help silence different genes

26
Q

How does miRNA work?

A
  1. In nucleus: long strand of pri-miRNA is transcribed with RNA pol II.
  2. pri-miRNA is folded into a stem loop structure imperfectly with some non binded nucleotides
  3. pri-miRNA > pre-miRNA and is moved out of nucleus to cytoplasm where it binds to dicer.
  4. Dicer catalyses a second cleaving resulting in a guide strand and passenger strand that both have 3’ overhangs.
  5. Guide strand moves into RISC (passenger strand is discarded)
  6. RISC binds non-specifically to target sequence on mRNA and essentially inhibits translation and therefor results in mRNA degredation.
27
Q

What is the difference in action between miRNA and siRNA?

A

miRNA: Inhibits translation of mRNA

siRNA: Cleaves mRNA

28
Q

In more detail what happens during the dicing step for siRNA and miRNA?

A

Dicer cleaves dsRNA at 21-25bp intervals beginning at one end of the dsRNA

Long dsRNA is processed to siRNA or miRNA molecules in the cytoplasm by Dicer.

29
Q

In more detail what happens during the RISC assembly?

A

Dicer combines with other dsRNA binding proteins TRBP and AGO2 to direct the siRNA or miRNA to the other RISC components:

TRBP recruits AGO2 (Argonaut)

AGO2 contains PIWI (endonucleases) and PAZ (docking region) domains

The passenger strand is removed upon activation of the RISC. Only the guide strand remains within the RISC.

30
Q

What happens during the effector step in miRNA and siRNA silencing?

A
  • Argonaut component of RISC cleaves mRNA that is complementary to its guide strand:

miRNA: Partial base pairing results in traslational repression

siRNA: complete complementary base pairing results in cleaving of the mRNA.

Once mRNA target is cleaved, RISC dissociates from the cleaved mRNA to repeat the cleavage cycle with other targets (Same or different gene)

31
Q

How can RNAi protect against viral infections?

A

Cells can process injected dsRNA and eliminate homologous ssRNA.

32
Q

What is the significance of RNAi in regards to transposons?

A

RNAi maintains stability by keeping transposons silent.

33
Q

In a nut shell, why would we use RNAi experimentally?

A

We can use RNAi to supress specific genes and look for the resulting phenotypic effect. We can essentially study the functionality of any gene in an organism. We can do this in vivo or in vitro to cells from almost all organisms.

34
Q

What is DNA cloning?

A

The isolation of specifc DNA sequences and the reproduction of those sequences seperate from the DNA that normally surrounds them. Each cell makes up to thousands of copies of the inserted DNA but it is not guarenteed that the cell will necessarily express the genetic information encoded on the peice of DNA. (it may take a few attempts or may not work at all).

35
Q

What are plasmids?

A

A plasmid is a small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria.

36
Q

Explain the Natural DNA Transfer Processes of Conjugation?

A

Conjugation is the process by which one bacterium transfers genetic material to another through direct contact. During conjugation, one bacterium serves as the donor of the genetic material, and the other serves as the recipient.

37
Q

Explain the Natural DNA Transfer Processes of Transformation?

A

alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane.

  • Occurs naturally with low frequency but can be made to be done more efficiently by chimically treating the cells with CaCl2 or by electric shock
38
Q

Explain the Natural DNA Transfer Processes of Transfection?

A

Using a bacteriophage (Lambda) as a vector to inject the DNA from inside the bacteriophage into the Bacteria (usually E. coli) as circular DNA and then it can replicate.

39
Q

What is meant by the term recombinant DNA?

A

Recombinant DNA molecules are DNA molecules formed by laboratory methods of genetic recombination to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

40
Q

What must be present in order for DNA sequences to be able to join together through a phosphodiester bond?

A

Ligase

5’ must be PO4 group

this must join a

3’ end that contains a OH group

and ATP must be present for the 3’ and 5’ to be able to join.

41
Q

What is Genetic engineering?

A

That DNA could:

  • be extracted as high molecular weight material
  • Be cut (aka restricted, digested)
  • Spliced (aka joined, ligated)
  • Returned to the bacterial cell
  • Alter the cells genetic and metabolic capabilities
42
Q

List an overview of the basic steps in DNA cloning?

A
  1. Obtain the disired DNA fragment
  2. Prepare a suitable vector
  3. Ligation of fragment (insert) to vector
  4. Transformation of cells with the recombinant vector
  5. Screen the transformed population for the presence of the desired sequence
43
Q

Explain step 1 in DNA cloning: Obtain desired Fragment?

A

Thee desired fragment (<10Kb) can be generated by:

  • Restriction endonuclease digestion
  • Physical fragmentation, e.g sonication, plus end-filling, to make the ends blut.
  • Making a complementary copy of RNA using reverse transcriptase
  • Amplifying specific regions of DNA by PCR
44
Q

How does reverse transcriptase work?

A

RT is an enzyme that is used to generae complementary DNA (cDNA) from an RNA template.

45
Q

Explain step 2 in DNA cloning: Preparing a suitable Vector?

A
  • A vector is the molecule into which a fragment of DNA can be inserted. (Plasmid or Phage)
  • Vector is generally digested with a restriction enzyme that leaves the ends complemetary to those of the DNA that is to cloned.
  • Optional to dephophorylate the termini of the cevtor using a phosphotase enzyme, to prevent it from being re-circularized without an insert during ligation.
  • Vector should be purfied after processing to avoid reagents interfering with the ligase reaction.
46
Q

Explain step 3 of DNA cloning: Ligation of fragment to vector?

A

DNA ligase will join two blunt ended peices of DNA or two peices with complementary overhangs.

47
Q

Explain step 4 of DNA cloning: Transformation of cells with the recombinant vector?

A
  • Cells are made competent (ready to uptake foreign DNA)
  • Newly created “recombinant” vector is inserted into the competent cells
  • Cells are allowed to recover from the transformation process by growth in rich media
48
Q

Explain step 5 of DNA cloning: Screen for the desired fragments?

A
  • Transformed cells are plated onto selcetive media (antibiotics e.g. ampicillin)
  • Cells containing the vector will be able to grow (whether they contain inserted fragments or not)
  • An additional selection process is used (i.e. Coloured and Lethal genes)
49
Q

What is A DNA library?

A

A collection of clones that represent the genome of an organism:

  • A portion of the genome or
  • the whole genome
50
Q

What are the two main types of DNA libraries?

A

Genomic and cDNA

51
Q

Why are DNA libraries useful?

A
  • Good for finding genes and markers
  • Allows PCR of unknown sequence
  • usually contains entire genome of an organism
52
Q

How do you make a gDNA (genomic) library?

A
  1. Make small peices of the genome
  2. Clone into appropriate vector
  3. Insert recombinant vector into bacterial cells
  4. Seperate individual cells each containing a piece of the genome.
53
Q

what are cDNA libraries?

A
  • mRNA
  • Reverse transcription into cDNA
  • More difficult than gDNA library
  • smaller subset of genome (expressed genes only)
  • allows comparisons (e.g. cancerous vs non-cancerous tissue)
54
Q

What is a screening library?

A

A library that allows you to “find the bit that you want”

  • Plasmid library (4Kb insert)
  • Cosmid library (40Kb insert)
  • BAC library (200Kb insert)
55
Q

How do we make a screening library?

A
  1. Prepare a library for screening by immobilizing the clones.
  2. Selecting the probe (Short ssDNA generated in vitro or by PCR)
  3. Hybridisation of probe to library
  4. Detecting the hybridised probe
56
Q

What is a hybridization probe?

A

A hybridization probe is a fragment of DNA or RNA of variable length (usually 100–1000 bases long) which can be radioactively labeled. It can then be used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe.

57
Q

What are the 4 different ways we can label probes?

A
  • Radioactively
  • by Colour
  • by light
  • through flourescence
58
Q

What is NGS?

A

Next Generation Sequencing:

  • Extremely thorough sequencing that can be used to sequence DNA and RNA of an entire genome.

Molecular Genetics (4 decks)

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