13. Analyzing Mixtures of DNA, RNA and protein

nucleic acid hybridization

means for detecting complementary in DNA or RNA samples

Southern blotting

DNA detection

northern blotting

RNA detectionn

protein-protein interaction

means detecting proteins with other proteins (antibodies)

western blotting

protein detection

what do we need in order to detect the specific gene or specific product of gene expression “target”

1. DNA/RNA need a labeled hybridization probe = 10 mer - 100’s mer of nucleotides complementary gene of interest (DNA; Southern) or mRNA (northern)

2. protein: need a labeled antibody

TARGET is unknown sequence or protein and PROBE is

known sequence or antibody

what does it mean to be labeled

something identifiable, detectable, measurable

Hybridization Probe or Antibody

• use radioactive atoms in nucleotides (typically “label” T for DNA, U for RNA probes)

• use covalently attached fluorescent molecules, variety of enzyme conjugates

• incorporated into the probe during synthesis of probe

• method for detection (measurement) of radioactivity, or light or enzymatic product

labeled probe will

hybridize with target and be detected

Source of our labeled probe DNA

1. whole or partial coding sequence for the gene of interest from same organism or

2. whole or partial coding sequence for the gene of interest from a different organism, or

3. sequence of the gene from the same gene family as our gene of interest

in general: we use labeled nucleotides to perform

de novo DNA or RNA sequences from one of the above DNA sources as a template

homologous probe

probe and target sequences are perfect match (100% complementarity)

heterologous probe

when probe and target are not 1—% complementary, some degree of mismatching of bases

Source of our labeled probe for protein

“target” protein in sufficient quantities for antibody production (monoclonal, polyclonal)

typically, labeling with enzymes or fluorescent tags can be added after antibody production and purification

how complementary is our probe with the target

it depends ont he template used to make the probe

• percentage of sequence identity (complementarity, homology) between target sequence and probe determines the hybridization conditions

higher homology

allows higher stringency

The conditions under which you hybridize (temperature, salt concentration) change

the “minimum” homology that will be required for hybridization to be successful between target and probe

• controlling these conditions is controlling stringency

stringency considerations are also affected by

probe size and the actual sequence used as a probe or targeted (e.g repeats in either)

the ultimate goal while performing nucleic acid hybridization (for both DNA and RNA detection)

is to bind the probe to the target in a controlled manner

depending on the source of the probe sequence the probe could have

more or fewer mismatches with the target nucleic acid when they hybridize

percentage of identity (similarity) between the target and the probe determines STRINGENCY which is

a measure of the tolerance for mismatches between the two

higher similarity - higher stringency

less changes for mistakes/mismatch

lower similarity - lower stringency

higher chance for mistakes/mismatch

homologous probe (100% similarity)

heterologous probe (<100%)

what do we need in order to detect the specific gene or specific product of gene expression “target”

labeled probe (nucleic acid or protein)

sour e of our labeled probe?

same organism, related not related

how complementary is our probe with the target

stringency and sequence similarity

southern blotting detects

specific DNA fragments (identify specific restriction fragments in a complex mixture of fragments)

southern blotting can be used for

1. estimating the # and position of gene copies in a genome

2. restriction mapping of genomic fragments

3. detection of

   1. cloned sequences

   2. transgenes

   3. homologous sequences in different genomes

   4. repetitive sequences

southern blotting steps

1. digest DNA with restriction endonucleases

2. perform agarose gel electrophoresis on the DNA fragments from different digests

3. DNA fragments fractionated by size (visible under UV light if gel is soaked in ethidium bromide)

4. soak gel in NaOH: neutralize

5. transfer (blot) gel to nitrocellulose or nylon membrane using Southern blot technique

6. DNA fragments are bound to the membrane in positions identical to those on the gel

7. hybridize membrane with radioactively labeled probe

8. expose membrane to X-ray film; resulting autoradiograph shows hybridized DNA fragments

DNA in each and every cell of the organism is the same; however,

the set of synthesized proteins is different in different tissue/cell types , and/or during the life cycle

Temporal control

genes that are expressed at a precise time during the life cycle of an organism, this is also called developmental regulation. (e.g ovalbumin, globin in haemoglobin, plant seed storage proteins)

spatial control

genes expressed in a specific tissue or cell type. also described as tissue-specific expression. (e.g different genes expressed in liver cells, muscle cells or root tip cells)

many genes are both temporally and spatially controlled such that they are expressed in

a specific tissue at a precise stage of development of the tissue (e.g developing seed or flower expresses certain genes specific to those organs at a precise stage of development)

induced gene expression

change in types or amount of gene expression in response to environmental signals, exposure to chemical subtance or physiological stress (e.g thermal stress - heat shock genes; gene expression controlled by steroid hormones; toxic substances such as heavy metals, antibiotics or anaesthetics)

northern blotting

1. mRNA is transcribed from the protein coding DNA - first in gene expression

2. total RNA (all three major groups) is isolated from cells and electrophoresed

Normal blotting detects specific mRNAs

northern blotting is similar procedure to southern blotting but

no denaturation (RNA is already ss)

Northern blotting method can be used to determine steady-state level of a specific transcript in a certain RNA mixture =

abundance of specific mRNA at certain time, under certain conditions

northern blotting depends on

both transcription and degradation rate for that specific mRNA

we could compare abundance of mRNA isolated from

1. different tissues of one organism (e.g brain and muscle)

2. same tissue from different organism (brain from frog and brain from bird)

3. different treatments or conditions (time studies, normal versus transformed/treated)

remember probe is designed to detect certain mRNA, the one which is

transcribed from our gene of interest which codes for our protein of interest

in situ hybridization: probe binds to

complementary nucleic acids within cell or tissue (proves are the same as for southern and northern)

in situ hybridization: similar to southern and northern blotting BUT identifies

1. genes directly in chromosomes (FISH - Fluorescence In Situ Hybridization shown)

2. transcripts (mRNA) directly in cell or tissue for developmental expression studies, following treatments or environmental changes

expression studies of more htan one gene at the same time

• many genomes have been sequenced - info on various gene sequences easily obtainable

• pattern of genes expressed in a cell is characteristic of its present state

• all or most differences in a cell state are correlated with changes in mRNA levels of genes

• even expression patterns of uncharacterized genes may provide clues to their function

traditional methods are “one gene in one experiment” (obtained by northern hybridization” do not

show the whole picture of total gene expression in a cell and interaction of gene products

yeast genome microarray

1. target (unknown sequences) are getting labeled during experiment (= labeled cDNAs; made from isolated mRNAs during experiment)

2. Probe is known (made by/for us); its fixed and unlabeled

DNA microarray summary Grow cells in two conditions (e.g., glucose vs. ethanol).

1. Isolate mRNA → reverse transcribe to cDNA with different fluorescent dyes (green vs. red).

2. Mix cDNAs → hybridize to microarray (each spot = 1 gene).

3. Scan colors:

   • Green → higher in condition 1

   • Red → higher in condition 2

   • Yellow → equal in both

4. Principle: Complementary base pairing to measure relative gene expression.

measuring gene expression by measuring translation

SDS sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western Blotting

SDS - negatively charged detergent; binds to hydrophobic protein regions

helps protein unfolding

• protein bind lots of SDS in constant ratio to their mass

• gives them a negative charge proportional to their mass (equal charge density per unit length)

  • protein intrinsic charge is masked

electrophoresis under denaturing conditions:

proteins migrate towards the positive electrode when voltage is appleid to a gel

• separate on the basis of molecular weight rather than intrinsic charge

• can add a 2nd dimension (isolectric point) = 2D gel

• proteins detected in the gel by different stains: varying sensitivity and specificity

western blotting

1. SDS PAGE - proteins are denatured by heat and detergent (SDS) and electrophoresed, separating by size

2. the proteins are transferred to a membrane (here using “electroblotting”) - western blotting

3. the membrane is incubated with an antibody specific for one of the proteins

4. bound antibody is detected by a secondary antibody that is conjugated to an enzyme or tagged (radioactive or fluorescent tags) = visualization

mapping transcriptional start sites

DNA regulatory elements (promoter elements) that control transcription are often located near the start site of transcription of the gene

• in order to define the promotor region for a gene it is necessary to know the start of transcription

• also: important regulatory elements in the 5’ UTR of mRNA

methods used to locate transcriptional start sites

1. S1 nuclease protection (S1 mapping)

2. primer extension

S1 nuclease protection (S1 mapping)

collect RNA from study organism, hybridize to labelled antisense probe, digest remaining single stranded nucleic acids (unhybridized organism RNA and probe). run on gels for analysis

primer extension

hybridize selected mRNA with radiolabeled 20-50 mer complimentary to a region close to 3’ end. reverse transcript to produce cDNA then compare to original DNA sequence for transcript start site

gene expression

process in which information carried by a gene is converted into observable product

transcription

first step in gene expression - process where one strand of a DNA molecule is used as a template for synthesis of a complementary RNA, mRNA which carries information for a specific protien

one-cell system (bacteria)

• has to survive and reproduce

• gene expression regulation - to adjust to changes in its nutritional environment to enable cell growth and cell division

multi-cellular organism: has to survive and reproduce but also to grow and develop; different parts of the body - different function

gene expression regulation during:

1. development (time)

2. tissue differentiation (space and '‘space/time combo”)

3. stress (as a response to environmental stress - induction)

regulation of gene expression: prokaryotes

transcription initiation is controlled

• transcription and translation occur in the same compartment

• mRNA is polycistronic, without introns and has a short half-life

regulation of gene expression: eukaryotes

1. eukaryotic cell is “compartmentalized” - regulation in each compartment

2. gene expression can be regulated at various levels in eukaryotes

3. transcription initiation is most important level fo regulation

transcription (general) similar process to DNA synthesis

• enzyme - multi-subunit complex produces nucleotide strand in 5’ to 3’

• mg2+ is a co-factor (necessary to add to buffers for in vitro transcription)

• both DNA strands could be templates for RNA synthesis

enzyme is RNA polymerase (RNAP)

RMAP does not require a primer

Promoter sequences (RNAP binding sites) are

asymmetrical RNAP is positioned so it can only transcribe one strand from one promoter

DNA is unwound locally only

ATP is not required (DNA and RNAP undergo spontaneous reversible structural changes - energetically favourable state)

Product is single stranded RNA

released from the template immediately (DNA helix re-forms) many copies from same gene

precursors use ribose -

ribonucleotide triphosphates (rNTPs)

RNAP is less

efficient in proofreading (mistake every 10^4 nucleotides)