BIO340 Mangone Exam 4 Prep Slides

what is a mutation

alteration of DNA sequence

T or F: mutations can only occur in germ cells

F they can occur in germ and somatic

Are somatic mutations heritable?

nooo

what are autosomal mutations?

mutations that occur within a chromosome that is not a sex chromosome

what is another type of chromosomal mutation?

X or Y linked mutations

define spontaneous mutation

happen randomly and naturally; linked to normal bio processes

define induced mutation

result from influence of exraneous factor

• UV

• radiation

• chemicals

What did the Luria-Delbruck fluctuation test show

mutations are not adaptive; they occur randomly

• arise in absence of selection rather than response to selection

what are the 5 phenotypic effects of mutations

1. loss of function

2. gain of function

3. visible

4. nutritional (biochemical)

5. behavioural

6. regulatory

What are the 7 types of mutations

1. missense

2. nonsense

3. frameshift

4. deletion

5. insertion

6. duplication

7. expanding

Where can neutral mutations occur and what significance does this have on gene expression?

most mutations occur in portions of genome with no genes; no effect on gene expression

what type of inheritance pattern does sickle cell have

autosomal recessive

what diseases can trinucleotide repeats cause

• fragile X

• huntingtons disease

what is inheritance patter for HD

autosomal dominant

what is significance of inducible enzymes in bacteria?

they are only produced when certain substrates are present

describe positive and negative control regarding transcription

positive - transcription occurs only if regulator stimulates RNA production

negative - expression occurs unless it is shut off by regulator

define operon

functioning unit of DNA containing cluster of genes under control of 1 regulatory signal/promoter

what was 1st operon discovered

lac operon

what is general structure of an operon?

• promoter

• repressor binding site (operator)

• structural genes

what enzyme recognizes the promoter on an operon?

RNA polymerase

Where are the genes in an operon located?

all clustered/located in same region

what is the inducer of the lac operon?

lactose

what does cis-acting mean?

regulatory elements that affect genes on the same molecule

define trans-acting

regulatory elements that affect genes on dif molecules

what are the 3 structural genes of lac operon? what do each code for

lacZ - B-galactosidase

lacY - permease

lacA - transacetylase

what is polycistronic mrna in context of lac operon?

one mrna codes for multiple cistrons/areas that code for a single gene

(1 mrna → multiple genes)

lac operon is optimally expressed in___

presence of lactose, absence of glucose

T or F: control of lac operon is only negative

F; positive and negative

What happens when glucose is present and lactose is absent?

lacI (repressor) binds to operator region to stop transcription

what is the lactose deriviative that is the inducer of the lac operon?

allolactose; binds to the repressor so that it can release the operator

what happens when glucose and lactose levels are high

since the inducer (allolactose) is present, transcription will occur but slowly

what is E coli’s preferred energy source?

glucose; lactose is secondary source

what is the relationship btwn cAMP and glucose concentration?

inversely proportional (if glucose is up cAMP is down and vice versa)

what does cAMP actually do?

activates expression of lac operon by recruiting CRP to bind to promoter which recruits RNA polymerase

how does the repressor actually stop transcription

repressor binds to operator and crates a repression loop; prevents RNA Pol from promoter

list the components of the lac operon from L to R

lacI, lacP, lacO, lacZ, lacY, lacA

if the operator region of lac operon mutated what would happen

constitutive expression of operon whether or not lactose is present bc repressor cant bind

what does trp operon encode for

synthesis of tryptophan

what are the 3 differences in the trp operon compared to lac?

1. gene for repressor is not adjacent to promoter but in another part of E coli genome

2. operator is entirely within the promoter

3. repressible operon is usually TURNED ON (not inducible; negative regulation)

how many genes code for enzymes that make trp

5

what is role of tryptophan in trp operon

it is a corepressor; its presence helps with repression of operon

what is attenuator region

seuqences in transcribed RNA of operon that control transcription after RNA pol starts synthesis of protein (premature termination)

name elements of trp operon in order from L to R

trpR (somewhere else on genome)

promoter (operator within)

leader (attenuator within)

5 genes (E,D,C,B,A)

is gene regulation in euks the same as in proks

NO ofc its more complex

what are the kinds of gene regulation in euks

pre, co, and post transcriptional

do euks have operons?

NO they j have promoter and gene

is the E coli lac operon negatively inducible?

yas

whats a simple example of euk gene reg

GAL system in yeast; makes genes to transport galactose

is GAL systen inducible or repressible

inducible

what happens in presence and absence of galactose

absence - Gal80p binds to Gal4p which covers Gal4p’s activation domain to prevent transc

presence - galactose interacts with Gal3p and goes thru conformational change that lets it bind to Gal80/Gal4 complex

what is a cool example of post transcriptional reg

RNAi

in what organism was RNAi discovered

petunia; cosuppression of pigment

whats an important model organism

C elegans

why is C elegans a good model organism

• fast life cycle

• defined cell lineage

• hermaphrodite (male and female repro organs)

• transparent

• annotated genome

what C elegans gene was analyzed

unc-22 (uncoordinated twitching)

explain unc-22 experiment

injected sense/antisense/both into wild type C elegans

• sense → normal

• antisese → normal

• double stranded RNA → twitching

in which process does RNAi interfere

translation

what are the 2 steps of RNAi

initiation - generatinon of mature siRNA

execution - siRNA silences target gene and degrades or inhibits translation

what do DICER and RISC have in common

both cleave dsRNA

Explain what RISC (RNA-induced silencing complex) does

cleaves dsRNA and binds short antisense RNA strans that bind complementary strands execute RNAi

what RNA molecule is key to the RNAi process

siRNA (small interfering RNA)

what does siRNA do in the RNAi process

siRNA-RISC complex look for perfect complementarity in mRNA and cleave in that location; mRNA is degraded and translation is repressed

what is the purpose of RNAi silencing

primitive immunity; protect genome from exogenous nucleic acid invasion (RNA viruses)

where do miRNAs (micro RNA) originate from

piRNA (PIWI RNA)

what do miRNAs do

bind to 3’ UTR of mRNAs with partial complementarity and triggers deadenylation and decapping of target

when does mRNA regulate genes

posttranscriptionally

what is the difference between miRNAs and their target and siRNAs and their target

miRNAs - imperfect complementarity to target mRNA; inhibit translation

siRNAs - perfect duplex w target mRNA; trigger degredation

where do miRNAs and siRNAs come from

miRNA is genomically encoded (endogenous)

siRNA is produced exogenously

define recombinant DNA

joining of DNA from dif sources not found tgthr in nature

what 2 tools founded rDNA

• restriction enzymes

• DNA cloning vectors (plasmids)

what is an important example of the uses of rDNA?

insulin; first protein to be chemically synthesized by rDNA

what type of symmetry do rDNA recognition sequences exhibit

palindromic symmetry (ex: RADAR, TACOCAT)

what is an example of an important vector?

plasmid

what are 2 important aspects of vectors?

• can replicate independently

• carry a selectable gene marker to distinguish host cells that do not take up plasmid

what is a common marker on vectors/plasmids?

antibiotic resistance genes

how are plasmids introduced to the host?

transformation; done via heat shock and electroporation so the cell is forced to take up plasmid

T or F: expression vectors are engineered to make small quantities of encoded protein

FALSE; make large quantities

what is a powerful technique for copying dna?

PCR; amplify target DNA sequences present in small quantities

• eliminates need for host cell for cloning!

what is an important enzyme in PCR?

Taq polymerase

why is Taq polymerase so useful in PCR?

it is heat stable

how many primers does PCR need?

2; forward and reverse

what are the 3 steps of PCR?

denaturation

annealing

extension

what are some limitations to PCR?

• some info abt sequence must already be known

• minor contamination is bad

• cant amplify segments that are too long

define restriciton endonuclease

enzyme that recognizes and cleaves DNA in sequence specific manner

why are restriction endonucleases important?

allowed production of rDNA in vitro

how are DNA sequences replicated in vivo?

dna inserted into cloning vectors and amplified by replication after being put in host cell via transformation

T or F: PCR amplifies DNA in vitro

TRUE

what is gel electrophoresis?

separation of DNA fragments according to size

why is GFP important in vectors?

used to monitor gene expression; cells that take up gene will fluoresce

what is a DNA/genomic library?

contains at least one copy of all sequences in genome of interest

how are genomic libraries constructed?

cut genomic DNA w restriction enzyme + ligating fragments into vectors

what is difference between cDNA library and genomic library?

cDNA library only contains coding region

what is a more specific definition of cDNA?

(complementary dna) genes transcriptionally active @ time cells are collected for mrna isolation

how is cDNA made?

reverse transcriptase PCR (generate cDNA from mRNA)

what is cell potency?

developmental potential/range of dif cell types the cell can become

explain the phrase ‘the zygote is totipotent’

cell shave potential to develop into complete organism

is totipotency common in both plants and animals?

uncommon in animals after 2-4 cell stage

explain the concept of a cell fate

progressive restrictions put on cell developmental potential

what comes first, differentiation or determination?

determination

are there many genes and mechanisms that control the developmental process?

no! nature is lazy; fraction of genes control dvlpmt and the same mechanisms are used