Molecular Tools for Studying Genes and Gene Activity P2

Question 1

(5.6 MAPPING AND QUANTIFYING TRANSCRIPTS)

locating their starting and stoppinig points

Answer 1

MAPPING TRANSCRIPTS -

Question 2

(5.6 MAPPING AND QUANTIFYING TRANSCRIPTS)

measuring how much of a transcripts exists at a certain time

Answer 2

QUANTIFYING TRANSCRIPTS -

Question 3

is a modification of the southern blot technique, it was designed to investigate RNA structure and quantity.

Answer 3

Northern Blot

Question 4

Most northern analyses were performed to investigate levels of gene expression (transcription from DNA) and stability, also it is used to investigate RNA structural abnormalities resulting from aberrations in synthesis or processing

Answer 4

Northern Blot

Question 5

Northern blot is similar to a Southern blot, but it contains __________ instead of DNAs. The RNAs on the blot can be detected by hybridizing them to a labeled probe. The intensities of the bands reveal the relative amounts of specific RNA in each.

Answer 5

electrophoretically separated RNAs

Question 6

used to locate 5’ - or 3’ - ends of RNA to quantify the amount of a given RNA in cells at a given time

Answer 6

S1 MAPPING

Question 7

S1 MAPPING principle: to label a ________ that can hybridize only to the transcript of interest

Answer 7

single – stranded DNA probe

Question 8

(S1 MAPPING THEME)

a variation on S1 mapping that uses an RNA probe and RNase instead of a DNA probe and S1 nuclease

Answer 8

RNase mapping (RNase protection assay)

Question 9

technique that is very popular because of the relative ease of preparing RNA probes by transcribing recombinant plasmids or phagemids in vitro with purified phage RNA polymerases

Answer 9

RIBOPROBES

Question 10

Can also give an estimate of the concentration of a given transcript

Answer 10

PRIMER EXTENSION

Question 11

(PRIMER EXTENSION)

one can locate the _______ of a transcript by hybridizing an oligonucleotide primer to the RNA of interest, extending the primer with __________ to the 5’-end of the transcript, and electrophoresing the reverse transcript to determine its size.

Answer 11

5’ -end ; reverse transcriptase

Question 12

The ___________ of transcript, the more molecules of labeled primer will hybridize and therefore the more labeled reverse transcripts will be made. The more __________, the darker the band on the autoradiograph of the electrophoretic gel.

Answer 12

higher the concentration ; labeled reverse transcripts

Question 13

a means of checking the efficiency and accuracy of in vitro transcription

Answer 13

RUN OFF TRANSCRPTION

Question 14

(RUN OFF TRANSCRIPTION)

A gene is truncated in the middle and transcribed in vitro in the presence of _________. The RNA polymerase runs off the end and releases an __________.

Answer 14

labeled nucleotides ; incomplete transcript

Question 15

Size of run off transcription: locates the _____________

Answer 15

transcription start site

Question 16

Amount of run off transcript: reflects the ____________

Answer 16

efficiency of transcription

Question 17

variation on the run-off theme of quantifying accurate transcription in vitro

Answer 17

G-LESS CASSETTE ASSAY

Question 18

(G-LESS CASSETTE ASSAY)

instead of cutting the gene, a ________, or _________ lacking guanines in the non-template strand, is inserted just downstream of the promoter

Answer 18

G-less cassette ; stretch of nucleotides

Question 19

(G-LESS CASSETTE ASSAY)

transcribed in vitro with CTP, ATP, and UTP, one of which is labeled, but no _____

Answer 19

GTP

Question 20

Transcription aborts at the end of the _______, yielding a predictable size band on _________.

Answer 20

cassette ; gel electrophoresis

Question 21

(5.7 MEASURING TRANSCRIPTION IN VIVO)

_______, ________, and _______ are useful for determining the concentrations of specific transcripts in a cell at a given time, but they do not necessarily tell us the rates of synthesis of the transcripts.

Answer 21

Primer Extension, S1 Mapping, Northern Blotting

Question 22

(5.7 MEASURING TRANSCRIPTION IN VIVO)

To measure transcription rates, we can employ other methods, including ___________ and ___________

Answer 22

nuclear run-on transcription and reporter gene expression

Question 23

a way of ascertaining which genes are active in a given cell by allowing transcription of these genes to continue in isolated nuclei.

Answer 23

NUCLEAR RUN - ON TRANSCRIPTION

Question 24

(NUCLEAR RUN - ON TRANSCRIPTION)

idea of this assay is to isolate _____ from cells, then allow them to extend _______ the transcripts they had already started _____

Answer 24

nuclei ; in vitro ; in vivo

Question 25

(NUCLEAR RUN - ON TRANSCRIPTION)

__________: continuing transcription in isolated nuclei

Answer 25

run on transcription

Question 26

(NUCLEAR RUN - ON TRANSCRIPTION)

Specific transcripts can be identified by their _______ to known DNAs on dot blots. The run-on assay can also be used to determine the effects of assay conditions on ________.

Answer 26

hybridization ; nuclear transcription

Question 27

Place a surrogate reporter gene under control of a specific promoter, and then measure the accumulation of the product of this reporter gene.

Answer 27

REPORTER GENE TRANSCRIPTION

Question 28

(REPORTER GENE TRANSCRIPTION)

Reporter genes have been carefully chosen to have products that are very convenient to assay—more convenient than ______ or ________

Answer 28

S1 mapping or primer extension.

Question 29

lacZ, whose product, b-galactosidase, can be measured using chromogenic substrates such as X-gal, which turns blue on cleavage.

Answer 29

REPORTER GENES

Question 30

Bacterial gene

encoding the enzyme chloramphenicol acetyl transferase (CAT)

Answer 30

cat

Question 31

from firefly lanterns.

mixed with ATP and luciferin, converts the luciferin to a chemiluminescent compound that emits light

Answer 31

luciferase

Question 32

(MEASURING PROTEIN ACCUMULATION IN VIVO)

_________ can also be measured by monitoring the accumulation of the ultimate products of genes – proteins

Answer 32

GENE ACTIVITY:

Question 33

(MEASURING PROTEIN ACCUMULATION IN VIVO)

Done in two ways:

Answer 33

1. immunoblotting (western blotting)
2. Immunoprecipitation

Question 34

begins with labeling proteins in a cell by growing the cells with a
labeled amino acid, typically [35S] methionine.

Answer 34

IMMUNOPRECIPITATION

Question 35

(IMMUNOPRECIPITATION)

typically uses an ______ that will bind specifically to the protein of interest followed with a secondary antibody complexed to protein A on resin beads using a _______- centrifuge

Answer 35

antibody ; low – speed

Question 36

(IMMUNOPRECIPITATION)

the labeled cells are _______ and a particular labeled _____ is bound to a specific antibody or antiserum directed against that protein

Answer 36

homogenized ; protein

Question 37

(IMMUNOPRECIPITATION)

the antibody-protein complex is precipitated with a secondary antibody or protein A coupled to resin beads that can be sedimented in a _________, or coupled to magnetic beads that can be isolated ________

Answer 37

low-speed centrifuge ; magnetically

Question 38

(IMMUNOPRECIPITATION)

the _________ is released from the antibody, electrophoresed, and
detected by __________-

Answer 38

precipitated protein ; autoradiography.

Question 39

(5.8 ASSAYING DNA PROTEIN INTERACTIONS : FILTER BINDING)

Have been used for decades to filter – sterilize solutions

Answer 39

Nitrocellulose membrane filters

Question 40

(5.8 ASSAYING DNA PROTEIN INTERACTIONS : FILTER BINDING)

Nitrocellulose membrane filters:

• Can bind _____ but only under certain conditions
• Single – stranded DNA binds readily to ______, but double stranded DNA by itself does not
• _____does bind
• _____ bound to double stranded DNA — protein – DNA complex will bind

Answer 40

• DNA
• nitrocellulose
• Proteins
• Protein

Question 41

An assay that detects interaction between a protein and DNA by the reduction of the electrophoretic motility of a small DNA that occurs on binding to a protein

Answer 41

GEL MOBILITY SHIFT

Question 42

GEL MOBILITY SHIFT IS ALSO KNOWN AS

Answer 42

electrophoretic mobility shift assay (EMSA)

Question 43

(GEL MOBILITY SHIFT)

____________: the mobility is reduced still further because of the greater mass of protein clinging to the DNA

Answer 43

SUPERSHIFT

Question 44

(DNase footprinting)

________: A method for detect protein – DNA interactions that can tell where target site lies on the DNA and even which bases are involved in protein binding

Answer 44

Footprint

Question 45

(DNase footprinting)

relies on the fact that a protein, by binding to DNA, covers the binding site and so protects it from attack by _____. It leaves its “________” on the DNA

Answer 45

DNase ; footprint

Question 46

(DNase footprinting)

end-label the DNA. Either strand can be labeled, but only ________ per experiment

Answer 46

one strand

Question 47

(DNase footprinting)

________ represents the region of DNA protected by the protein, and therefore tells where the protein binds.

Answer 47

footprint

Question 48

(DNase footprinting)

3 popular methods:

Answer 48

DNase
Dimethylsulfate (DMS)
Hydroxyl radical footprinting

Question 49

(DMS FOOT PRINTING and OTHER FOOTPRINTING METHOD)

gives a good idea of the location of the binding site for the protein

Answer 49

DNASE FOOT PRINTING:

Question 50

(DMS FOOT PRINTING and OTHER FOOTPRINTING METHOD)

_________ – a macromolecule and is therefore a rather blunt instrument
for probing the fine details of the binding site

Answer 50

DNase

Question 51

(DMS FOOT PRINTING and OTHER FOOTPRINTING METHOD)

Starts in the same way as DNase foot printing, with end – labeling the DNA and binding the protein

Answer 51

DMS foot printing

Question 52

(DMS FOOT PRINTING and OTHER FOOTPRINTING METHOD)

DMS foot printing:

______ – protein complex
- Methylated with _____, using a mild treatment so that on average only one methylation event occurs per DNA molecule

Answer 52

DNA ; DMS

Question 53

(DMS FOOT PRINTING and OTHER FOOTPRINTING METHOD)

___________: other reagents are commonly used to foot print protein –DNA complexes by breaking DNA except where it is protected by bound
proteins

Answer 53

DNase and DMS

Question 54

(DMS FOOT PRINTING and OTHER FOOTPRINTING METHOD)

Uses copper – or iron – containing organometallic complexes to generate
hydroxyl radicals that break DNA strands

Answer 54

Hydroxyl radical footprinting

Question 55

Is way of discovering whether a given protein is bound to a given
gene in chromatin—the DNA–protein complex that is the natural state of DNA in a living cell

Answer 55

CHROMATIN IMMUNOPRECIPITATION (CHIP)

Question 56

(CHROMATIN IMMUNOPRECIPITATION (CHIP))

Detects a specific protein–DNA interaction in chromatin ______

Answer 56

in vivo.

Question 57

(CHROMATIN IMMUNOPRECIPITATION (CHIP))

It uses an ________ to precipitate a particular protein in complex
with ______, ______ to determine whether the protein binds near a
particular gene.

Answer 57

antibody ; DNA, and PCR

Question 58

(5.9 ASSAYING PROTEIN - PROTEIN INTERACTIONS)

Also extremely important in molecular biology

Answer 58

PROTEIN – PROTEIN INTERACTIONS

Question 59

(5.9 ASSAYING PROTEIN - PROTEIN INTERACTIONS)

IN PROTEIN – PROTEIN INTERACTIONS: There are number of ways to assay them:

Answer 59

Immunoprecipitation
Yeast two – hybrid assay

Question 60

takes advantage of two facts:
1. that transcription activators typically have a DNA-binding
domain and a transcription activating domain;
2. these two domains have self-contained activities.

Answer 60

yeast two - hybrid assay

Question 61

(5.10 FINDING RNA SEQUENCES THAT INTERACT WITH OTHER MOLECULES)

method that was originally developed To discover short RNA
sequences (aptamers) that bind to particular molecules.

Answer 61

SELEX (systematic evolution of ligands by exponential enrichment)

Question 62

(5.10 FINDING RNA SEQUENCES THAT INTERACT WITH OTHER MOLECULES)

• similar to classical SELEX in that it finds a few “needles” (RNA
  sequences) in a “haystack” of starting sequence
• But instead of finding aptamers that bind to other molecules, it
  finds RNA sequences that carry out, or make possible, some
  function

Answer 62

FUNCTIONAL SELEX

Question 63

(5.10 FINDING RNA SEQUENCES THAT INTERACT WITH OTHER MOLECULES)

detected a ribozyme (an RNA with enzymatic activity), and this ribozyme activity altered the RNA itself to allow it to be amplified.

Answer 63

First functional SELEX procedures

Question 64

(5.11 KNOCKOUTS AND TRANSGENICS)

Answer 64

KNOCKOUT MICE
TRANSFENIC MICE