Naming genes, mutations and complementation

Question 1

how are bacterias named

Answer 1

genus with capital
species lower case
all in italics

Question 2

how are genotype and phenotype linked

Answer 2

genotype directly affects the phenotype, if have a mutation may lose or gain a characteristic

Question 3

what is the genotype

Answer 3

genetic material of an individual

Question 4

what is the phenotype

Answer 4

the observable characteristics

Question 5

what affects phenotype

Answer 5

environment

Question 6

what are the 3 essential features in bacterial gene

Answer 6

a promoter
transcribed region
terminator region

Question 7

what is the promoter for in bacterial gene structure

Answer 7

All the sequences in the DNA required for expression and regulation of a gene
These sequences are NOT all included in the mRNA

Question 8

what is the transcribed region in bacterial gene structure for

Answer 8

makes mRNA
Can include more than one ORF (operon)
These sequences ARE included in the mRNA

Question 9

what terminator sequence in bacterial gene structure for

Answer 9

determines where mRNA finishes
Defines end of gene or operon
Often a hair-pin loop

Question 10

how is mRNA transcribed from prokaryotic gene structure

Answer 10

mRNA formed 5’ to 3’

Question 11

what is included in mRNA structure

Answer 11

5’ UTR
translated region
terminator sequence

Question 12

where is the 5’ UTR

Answer 12

before the AUG start codon

Question 13

where is the translated region

Answer 13

Each ORF (open reading frame, must have a RBS) is preceded by a ribosome binding site (RBS)

Question 14

what is the terminator sequence for

Answer 14

Normally a hair-pin loop

Gives mRNA stability

Question 15

how are genes identified

Answer 15

First studies defined genes in terms of observable properties
i.e. by PHENOTYPE

Question 16

how are phenotypes written

Answer 16

as non-italics and often

with a superscript letter to indicate further details

Question 17

how is something that is resistant to ampicillin antibiotic written

Answer 17

Amp^r

Question 18

how is something that is sensitive to ampicillin antibiotic written

Answer 18

Amp^s

Question 19

what does His ^- mean

Answer 19

strain can’t make histidine (would need histidine in media for growth)

Question 20

what does Lac- mean

Answer 20

can’t break down lactose (not necessarily mutant, may have never done it in the first place, so would not be a mutant varies in different strains

Question 21

how were genes responsible for phenotypes identified

Answer 21

Mostly by finding a loss-of-function mutant strain

Question 22

what is a mutant

Answer 22

strain containing a mutation in its genome

Question 23

how are genotypes written

Answer 23

3 letter codes, in lower case and italics. An additional capital letter indicates a specific gene

Question 24

what indicates gene defined by a mutation, first one found

Answer 24

xyz-1

Question 25

what indicates gene linked to a phenotype, first gene = A

Answer 25

xyzA

Question 26

what indicates gene defined as first mutation in gene A

Answer 26

xyzA1

Question 27

what is a lacZ gene

Answer 27

gene that encodes the enzyme ß-galactosidase

Question 28

what is a lacY gene

Answer 28

gene that encodes the enzyme lactose permease

Question 29

what does the number mean lacZ19

Answer 29

No other lac mutation will have the number 19 (not just lacZ mutations)

Question 30

what is needed for tryptophan biosynthesis

Answer 30

several genes

Question 31

how may a Trp^- phenotype be formed

Answer 31

trp mutant may have a mutation in one of a number of genes to give the Trp- phenotype

Question 32

how would trp mutants be named as they are found

Answer 32

named trp-1, trp-2 etc

Question 33

what happens to the trp name if analysis shows which gene is mutated

Answer 33

a letter can replace the number: trpA or trpB etc.

Question 34

if particular mutations identified what happens to trp name

Answer 34

numbers are added: trpA1 or trpB2

Question 35

what is Δ

Answer 35

deletion

Question 36

what is ::

Answer 36

insertion

Question 37

what is :

Answer 37

gene fusion

Question 38

what is xyz'

Answer 38

xyz is the gene | 3' end of gene missing

Question 39

what is 'xyz

Answer 39

xyz is the gene | 5' end of gene missing

Question 40

what is xyz^-

Answer 40

undefined mutation with gene function lost

Question 41

what is (pXY123)

Answer 41

plasmid XY123 inside cell

Question 42

what can cause mutations

Answer 42

chemical physical biological

Question 43

what can cause chemical mutations

Answer 43

Ethyl Methyl Sulphonate (EMS)

Question 44

what can cause physical mutations

Answer 44

UV

Question 45

what can cause biological mutations

Answer 45

transposons

Question 46

what conditions are ideal in selection

Answer 46

Ideally we use Selective conditions | i.e. when either the mutant or ‘wild-type’ will not grow

Question 47

what does the selection method depend on

Answer 47

nature of the mutation and its effect on the bacterium

Question 48

what is an auxotrophic mutant

Answer 48

require something for growth that the wild-type (prototrophic) bacteria don’t

Question 49

examples of auxotrophic mutants

Answer 49

- wild-type will grow on minimal medium of inorganic salts and glucose - an E. coli trp mutant won’t grow on this medium unless tryptophan is also added

Question 50

how are trp mutants found

Answer 50

we need to use replica plating colonies on to media +/- tryptophan

Question 51

what happens in selection by replica plating

Answer 51

Put all these colonies on and agar plate, hope some have the trp mutant Want to know which ones are – need to characterize these further Press agar plate onto velveteen ,get an imprint Press onto two different plates: one with minimal medium, other with complete that contains trp, (cant make trp if it is mutant for it) Workout which grow on complete but not minimal

Question 52

what are the replica plating problems

Answer 52

In practice using velvet replica plating has a number of drawbacks It’s easy to smear colonies together often not all colonies are lifted transferred colonies can be confused with contaminating bacteria

Question 53

what is a better method than replica plating

Answer 53

often cleaner/clearer to use sterilised wooden cocktail sticks to duplicate colonies in a grid on plates

Question 54

which are the easiest mutants to select

Answer 54

those which gain a new phenotype as the mutation introduces a marker gene e.g. resistance to antibiotics (or other biocides) The only things that grow on a plate containing the biocide or gaining a new trait Cells that grow have a visible phenotype

Question 55

what is used for selection by new traits

Answer 55

green fluorescent protein bioluminescence genes (lux) lacZ - blue/white colony screening

Question 56

what does GFP do

Answer 56

Good marker on plasmids –if they're green, taken up plasmid and expressing GFP marker

Question 57

what causes bacteria to glow (selection by new traits)

Answer 57

Plasmid contains many lux genes put into bacteria e.g. e.coli will glow Use as selection markers

Question 58

what does lacZ show

Answer 58

Determine if bacteria has taken up a particular plasmid

Question 59

what is complementation

Answer 59

ability of a cloned DNA fragment to ‘complement’ a mutation | i.e. the conversion of the mutant phenotype back to wild type

Question 60

what happens in plasmid complementation - lacZ or lac^- produced

Answer 60

beta-galactosidase mutant produces lacZ or Lac^- is white colonies

Question 61

what happens in plasmid complementation lacZ^- (placZ^+) or Lac^+

Answer 61

beta-galactosidase insert lacZ gene on a plasmid forms complemented mutant lacZ^- (placZ^+) or Lac^+ blue colonies