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BMS 242 - Advanced Cell and Molecular > Gene Identification and Analysis > Flashcards

Gene Identification and Analysis Flashcards

1
Q

What 2 ways can we find genes in the nucleotide sequence?

How do these work?

A

1) Prediction software
- Scans the sequence for promoters, start + stop sequences and intron splice sites

2) Computer analysis to translate the DNA in the 6 reading frames (3 reading frames in each direction of DNA)
- THEN, search for similarities to known proteins, using BLAST software

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2
Q

What is the disadvantage of using prediction software to find genes in the nucleotide sequence?

A

Can not be sure if they are true

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3
Q

What is BLAST software?

What is the process using this?

A

Searches for similarities to KNOWN proteins:

1) Input amino acid sequence of the proposed protein (6 reading frames)
2) BLAST tries to align the unknown protein sequence to ALL known proteins in the database - searching for proteins with similar sequences
3) Produces a report of alignment of the proposed protein to known protein.

  • Shows how many amino acids are the same and in the same order
  • Show similar charges
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4
Q

What does it mean if there is similarity found between the proposed protein and a known protein?

A
  • Suggests that the proteins have evolved from the same common ancestor
  • Suggests they have similar molecular functions (eg. if known is a transcription factor, proposed may be too)
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5
Q

What is Vega and what is it used for?

A

Used to look at the genome online - to identify if there is any characteristics of a gene in that DNA sequence (prediction software)

(looks for promoters, intron splice sites etc.)

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6
Q

What are Ests and how are they expressed?

A

Expressed Sequence Tags
Short sequences at the end of cDNA
Expressed as RNA

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7
Q

When using the prediction software, can one piece of evidence (eg. intron splice site) be used as stand-alone evidence to say that there is a gene there?

A

NO, multiple parts of evidence must be used together

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8
Q

What are microarrays used for?

A

To identify genes

Allow to compare the TRANSCRIPTIOMES of different tissues

  • In different states (eg. normal and cancerous)
  • In different tissues (liver, spleen)
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9
Q

What are the advantages of microarrays?

A

High throughput - small scale, fast, automated

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10
Q

Describe the process of a microarray, to discover which genes are turned on in a specific tissue

A

1) Grid formed - each position in the grid contains ONE cDNA
2) Purify mRNA from a tissue and tag it with a fluorescent dye
3) Add the mRNA in solution, onto the array - allowing the mRNA to hybridise and rinse off the excess (removes any unbound DNA)
4) If the mRNA hybridises to any cDNA in the grid - shows that the genes are present
5) Use a sensitive camera to detect which genes are ‘ON’
6) Can compare to the microarray of a diseased tissue to discover which genes are up-regulated/ down-regulated

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11
Q

What carries out the microarray process?

A

A very precise robot - manufactures the array on a microscopic grid

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12
Q

What is cDNA?

What are its features?

A 
Complimentary DNA (complementary to mRNA)
Single stranded

Synthesised from a single strand of RNA
Contains only EXONS
Antisense DNA strand
Made by reverse transcriptase

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13
Q

When comparing microarrays between a normal tissue and one of a tumour, what POTENTIALLY are the genes that are LOST in the tumour?

A

POTENTIALLY tumour suppressor genes

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14
Q

When comparing microarrays between a normal tissue and one of a tumour, what POTENTIALLY are the genes that are GAINED in the tumour?

A

POTENTIALLY oncogenes

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15
Q

When comparing microarrays between a normal tissue and one of a tumour, what are the genes that are THE SAME in the tumour?

A

House-keeping genes and are specific to that tissue

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16
Q

From a microarray, how can the identity of the genes be found?

A

Using the grid coordinates

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17
Q

What 3 ways can genes be identified?

A

1) By making a cDNA library from mRNA (represents the transcriptome
2) By making a genomic library and then make PREDICTIONS based upon the genomic sequence using BLAST
3) Using microarrays

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18
Q

What are the different ways of analysing the function of the identified gene?

A

1) Reverse genetics in mice

2) Forward genetics

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19
Q

What must be done before forward/reverse genetics can be used to analyse the function of a gene?

A
  • Must have already made PREDICTIONS of the function (eg. using microarrays and BLAST)
  • Forward/reverse genetics BACKS up the prediction
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20
Q

What are the disadvantages of genetic engineering in mice?

A

Time consuming and expensive

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21
Q

What must you start with in order to genetically engineer mice?

A

A GENOMIC clone of the endogenous gene

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22
Q

What are the 2 approaches to genetic engineering in mice?

Describe them

A

1) Gene Replacement (Altering the gene)
- Make small changes to the endogenous gene

2) Gene Knock-out (Destroying the gene)

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23
Q

What does gene knock-out determine?

A

The function of the gene (if remove the gene - no longer have the function)

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24
Q

What is the process of gene knock-out in mice? (of a single gene)

A

1) Insert 2 mammalian genes into the genomic clone of the gene
- NEO inserted directly into the exon of the genomic clone
- TK placed off to one side of the exon

2) Purify the DNA from the bacteria
3) Introduce the construct into mouse EMBRYONIC stem cells (ES) using cell culture techniques
4) Some of the DNA makes it into the nucleus of the mouse and into the MOUSE GENOME

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25
Q

In gene knock-out, what are ‘homologous arms’?

A

Gene sequences that flank NEO on either side in the GENOMIC CLONE

Have homology to the mouse genome

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26
Q

In gene knock-out, what is recombination of the construct into the genome of the mouse driven by and how?

Is this efficient?

A

The ENDOGENOUS DNA repair machinery

Identifies fragments of DNA swimming around the cell and tries to put things back together

NOT efficient

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27
Q

In gene-knock out, what 2 things can occur when the construct is integrated into the genome?

Which occurs more frequently?

A

1) HOMOLOGOUS recombination

2) NON-HOMOLOGOUS recombination
Occurs more frequently

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28
Q

Describe homologous recombination of the construct into the mouse genome

A
  • DNA repair mechanism recognises the HOMOLOGOUS ARMS flanking NEO
  • Inserts the construct into the homologous gene - forms a transgene
  • Endogenous gene is knocked out
  • TK is lost
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29
Q

Describe NON-homologous recombination of the construct into the mouse genome

A
  • Doesn’t involved the homologous arms - are not recognised
  • Occurs at ANY POINT in the genome (doesn’t target the gene of interest)
  • TK is taken in at the same time as NEO
30
Q

What is a transgene?

A

An organism whos genome has been altered by the transfer of genes from another species or breed

31
Q

How can you distinguish between homologous and non-homologous recombination into the mouse genome?

Which one is wanted?

A

Homologous - genome doesn’t contain TK

Non-homologous - genome contains TK

Homologous is wanted, as the gene becomes altered

32
Q

Describe the method used to identify which have homologous recombination

A

1) Grow cells on NEOMYCIN
- Cells which have integrates NEO will be able to grow in the media
- Cells which haven’t taken up the construct will die

2) Grow cells on GANC
- Cells which have taken up TK along with NEO will die (negative selection)

33
Q

What is reverse genetics?

A
  • Analysing the function of a KNOWN gene by analysing the phenotypic effects of specifically engineered sequences (knock-out)
34
Q

What is forward genetics?

A
  • Identifying genes of interest based upon a phenotype
  • RANDOMLY mutate the genome (in a non-specific way) - in many places, many genes and look for interesting phenotypes
  • Gene is NOT known
35
Q

What is the process of reverse genetics?

A

1) Insert the mouse Es cells which contain the knock-out gene into early mouse embyros

2) Insert the embryos into a recipient mouse
- This mouse will give birth to transgenic pups

3) First generation are mosaic and have mosaic gonads

4) Mosaic animals are bred to generate some non-mosaic carriers of the transgene (2nd generation)
- All cells carry the mutant gene

5) The carriers are interbred to create HOMOZYGOUS MUTANTS (3rd generation)

36
Q

What are ‘mosaic’ organisms?

A

Organisms which have a mixture of cells from the implanted stem-cell line and from the mother

Some cells are mutant and some cells aren’t

37
Q

What are the disadvantages of forward genetics?

A
  • Random mutagenesis - affects the WHOLE genome

- Need to analyse MANY mutagenised animals to find an interesting phenotype

38
Q

What are the advantages of forward genetics?

A

Inexpensive and easy to manage

39
Q

What is linkage analysis used as?

A

As a genetic map to find the mutation and identify the gene

40
Q

What is the process for forward genetics?

What traits are these usually done for?

A

Usually done for RECESSIVE mutations

1) Mutagenise MALE using EMS (a chemical mutagen)
2) Outcross males to WILDTYPE females to produce offspring
3) Incross this family - produce HOMOZYGOUS embryos
4) Keep doing this until find 2 separate (individual - start with DIFFERENT male fly) families with the SAME phenotype

41
Q

In forward genetics, why is it the male which becomes mutagenised?

A
  • Easier to produce new gametes, compared to females (already made)
42
Q

In forward genetics, what is the P0 generation?

A

Mutagenised males

43
Q

In forward genetics, what is the F1 generation?

A

The males which are outcrossed

44
Q

In forward genetics, what is the F2 generation?

A

The family which is incrossed

45
Q

In forward genetics, what is the F3 generation?

A

The offspring of the in-crossed family - to produce a phenotype

46
Q

When in-crossing the F2 generation in forward genetics, if there is a phenotype, what proportion of the offspring will show it?

A

1/4

47
Q

What organism is usually used for for forward genetics and why?

A

Flies

Takes 3 generations to produce a phenotype, which is expensive to do with mice

48
Q

When mutagenise a male fly, why do each of the offspring (generation F1) have a unique mutation?

A

When mutagenise the male fly, the fly will carry many mutations throughout the genome

This results in each sperm carrying a DIFFERENT set of mutations

Forms offspring which have a unique set of mutation

49
Q

In F1, why are the mutagenised males outcrossed with WILD-TYPE females?

A

To produce many offspring, which all carry the same SET of mutations

50
Q

In F1, are the the sperm of the mutagenised males heterozygous or homozygous?

A

Heterozygous

51
Q

Why could 2 different mutations produce the SAME phenotype? (2 reasons)

A

1) The mutations could be in the SAME gene (but at different position)
2) The mutations could be in DIFFERENT genes, but they are involved in the SAME pathway, which give the same phenotype

52
Q

How can mutations occur in the same gene?

A

Genes are very large

53
Q

What are mutations in the same gene called?

A

Alleles - varient forms of a gene

54
Q

What is the process of the ‘complementation test’?

What does it identify?

A

Identifies if mutations with the same phenotype are in fact alleles of the SAME gene

Cross m1/+ and m2/+

If 1/4 MUTANT offspring:

  • Mutations FAIL to complement (they are alleles of the same gene) as produces a phenotype (genotype +/+)
  • Only have 1 gene of interest

If NO mutant offspring:

  • Mutations COMPLIMENT each other (they are mutations in DIFFERENT genes)
  • Genotype -/+
  • Non-allelic
  • 2 genes of interest
55
Q

What does complementation analysis allow?

A

Mutations to be sorted into distinct groups, which correspond to individual genes

56
Q

What 3 ways can mutations affect gene function?

How?

A

1) Changes in the regulatory sequence (which affect transcription - eg. enhancers)

  • TF cannot bind and activate the gene
  • Reduced expression of mRNA, BUT the mRNA produce would be functional

2) Changes in the non-coding sequence (may affect RNA splicing)

  • May splice out an intron
  • Truncated mRNA
  • Non functional

3) Changes in the coding sequence (may alter an important amino acid)

  • Affecting folding
  • May create a premature stop codon
57
Q

What are the 2 groups of mutations in the coding sequence?

What effects do these mutations cause?

A

1) Missense - amino acid substitution
- May not have an effect, depending upon the amino acid substituted (may be similar)
- Also, many codons code for one amino acid

2) Nonsense - early stop codon
- Truncation of the protein

58
Q

What is an amorphic mutation of a DNA binding domain?

What happens when +/- for this mutation?

+/+?

A

Non-functioning
Completely inactivates the binding domain

+/-: Haplosufficient

+/+: Strong phenotype - no transcriptional activation

59
Q

What does haplosufficient mean?

A

Single functioning allele of the gene is enough to restore function

60
Q

What is an hypomorphic mutation of a DNA binding domain?

What happens when +/- for this mutation?

+/+?

A

Weakening of the binding domain

+/-: Haplosufficient
Mutant can dimerise with WT and still activate transcription

+/+: Mild phenotype - complex often falls off the DNA

61
Q

What is an antimorphic mutation of a DNA binding domain?

What happens when +/- for this mutation?

+/+?

A

Destroys the dimerisation domain
Competitive inhibitors

+/-: Mutant poisons the WT and stops it from working (DOMINANT)
BUT, if 2 WT land together - transcription can take place

-/-: Completely amorphic (inactive)

62
Q

What is an hypermorphic mutation of a DNA binding domain?

What happens when +/- for this mutation?

+/+?

A

Activation is independant of dimerisation

+/-: Mutant form binds DNA - active ALL of the time
Constitutively active
Makes too much transcript
DOMINANT

+/+: Same phenotype

63
Q

What are loss-of-function mutations?

A

Amorphic

Hypomorphic

Antimorphic

64
Q

What are gain-of-function mutations?

A

Hypermorphic

65
Q

Which mutations are dominant?

A

Antimorphic (dominant negative)

Hypermorphic

66
Q

Which mutation is ‘dominant negative?’

A

Antimorphic

67
Q

Which mutations commonly affect one domain of the protein?

A

Antimorphic

68
Q

What 3 things cause an amorphic mutation?

A

1) Early nonsense mutation - null protein
2) Deletion of entire gene
3) Detrimental missense mutation

69
Q

What can cause an hypomorph mutation?

A

Missense mutations

OR

Mutations in the enhancer sequence

70
Q

What can cause an hypermorph mutation?

A

Mutation in a repressor sequence

71
Q

What happens during a antimorphic mutation?

A

The mutation form of the gene poisions the wildtype form

BMS 242 - Advanced Cell and Molecular (24 decks)

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