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Genetics 200A Course Concept List
Genetics – the study of genes; a set of gene-based techniques for studying biological problems.
Elements of Gene Structure & Regulation
Promoter is a region of DNA that binds RNA polymerase prior to initiation of transcription.
Enhancer element is a cis-acting regulatory DNA sequence that increases the utilization of a promoter
and can often function in either orientation and in any location (upstream or downstream) relative to
the promoter.
Operator is the site on DNA at which a repressor or an activator bind to prevent or enhance
transcription respectively from the adjacent promoter.
Repressor is a protein that binds to the operator to prevent transcription from the adjacent promoter.
Activator is a protein that binds to a cis-acting DNA site (an operator or an enhancer) to promote
transcription from the adjacent promoter.
Gene (cistron) is the segment of DNA that encodes a polypeptide chain or non-coding RNA molecule
and contains a regulatory sequence that makes the transcription possible.
Allele is a DNA sequence variant of a gene; one of the alternative forms of a gene.
Locus is the position on a chromosome at which a gene or a group of genes resides.
Operon is a bacterial transcriptional unit that can encode multiple polypeptides in a single RNA, and
includes the adjacent regulatory sequence.
Cistron is equivalent to gene in comprising a unit of DNA representing a protein. More technical
definition:
Cistron is the genetic unit defined by the cis/trans test.
cis/trans test assays the effect of relative configuration on expression of two mutations. In a
double heterozygote, two mutations in the same gene show mutant phenotype in trans
configuration, wild-type in cis configuration.
cis configuration describes two sites on the same molecule of DNA.
trans configuration decribes two sites on different molecules of DNA.
Polycistronic mRNA includes coding regions for multiple polypeptides.
Monocistronic mRNA codes for one polypeptide
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Open reading frame (ORF) is a sequence of DNA that begins with the start codon and ends with a
stop codon; sequence is (potentially) translatable into protein.
Exon is any segment of an interrupted gene that is represented in the mature RNA product.
Intron is a segment of DNA that is transcribed, but removed from within the transcript by splicing
together on the sequences (exons) on either side of it.
Genes can be regulated by proteins, RNAs, or other small molecules. Regulation can involve
accessibility of the transcription machinery to the DNA (chromatin structure, nuclear import of
transcription factors) or initiation, elongation and/or termination of transcription.
Chromosome Behavior in Mitosis
Mitosis is the process by which one cell divides to produce two cells that are genetically
indistinguishable from each other.
Prophase is the first stage of mitosis, during which the chromosomes condense and become visible,
the nuclear membrane breaks down (in plant and animal cells), and the spindle apparatus forms at
opposite poles of the cell.
Metaphase is the stage of mitosis or meiosis when chromosomes align in the equatorial plane of the
cell such that each sister chromatid (the products of DNA replication) is paired by cohesion
In anaphase sister cohesion is lost and each sister chromatid move toward the opposite pole of the cell.
In telophase, chromosomes uncoil, nuclei reform, a cleavage furrow forms and the cell divides.
Mitotic recombination or somatic crossing over occurs when a crossing over event generates a
diploid daughter cell with a combination of alleles different from that in the parental cell.
Mosaic screens typically utilize mitotic recombination to introduce homozygotic cells into an
organism. When this homozygosity results in a phenotype, it can produce a population of cells (within
the organism) that can be identified.
Chromosome Behavior in Meiosis
Prophase I the first stage of meiosis, during which the DNA replicates, and homologs undergo pairing
and crossing-over.
In metaphase I, kinetochores of the homologs are attached to opposite centrosomes via kinetochore
microtubules. Homologs are held together by cross-overs (also called chiasmata) and are aligned in
the equatorial plane of the cell.
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Anaphase I results from loss of cohesion along chromosome arms, with the retention of cohesion
around the centromeres.
In telophase I the cell divides.
Meiosis II is analogous to mitosis, where sister chromatids separate from each other.
Homologous recombination is the process by which homologous regions on chromosomes can be
exchanged or converted during meiosis.
Holliday junctions are physical structures of chromosomes that form during the crossover process of
homologous recombination.
Intragenic recombination is recombination that occurs within the same gene.
Intergenic recombination is recombination that occurs between different genes.
Nondisjunction is the failure of chromosome pairs to separate properly during meiosis or mitosis.
Note that when homologous chromosomes separate to opposite poles at meiosis I, they are said to
disjoin. The usual result of nondisjunction is gametes with altered chromosome content, and an
imbalance of chromosomes in the progeny, unless there is compensating loss in the gamete from the
other parent. When a chromosome is lost in progeny, it is called Monosomic. When a chromosome is
gained, it is called Trisomic.
Identifying Genes Using Mutants I - General Strategies
Mutation describes any change in the sequence of genomic DNA.
Spontaneous mutations are those that occur in the absence of any added reagent to increase the
mutation rate.
A Screen is a mutagenesis procedure in which mutagenized progeny are individually evaluated for a
desired phenotype.
A Selection is a screen in which only desired mutants survive.
Dominant screens are screens that look for phenotypes in the F1 generation.
Haploid screens are genetic screens in a haploid organism such that mating is not required to observe
phenotypes in generations after mutagenesis.
Suppressor screens look for mutations that suppress an observed mutant phenotype, i.e. cause a
phenotype to change back towards wild-type.
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Temperature-sensitive mutation creates a gene product that is functional at low temperature but
inactive at higher temperature (the reverse relationship is usually called cold-sensitive).
Cold-sensitive mutant is defective at low temperature but functional at normal temperature.
Synthetic lethal screens look for mutations that although individually are viable, when combined in
the same organism result in lethality.
Sensitized screen – this is specialized way of screening for mutations that modify an existing
mutation. In such screens a particular function or pathway is partially compromised by mutation, or
dominant negative construct, or other means to a level that gives an easily scored visible phenotype. In
this background loss of function of even one of two copies of another gene that is required in the same
pathway will alter the severity of the phenotype (i.e. genes that usually have recessive phenotypes
behave as dominant modifiers of the sensitized phenotype). Note: it is important that an easily scored
quantitative trait is used for sensitization.
Identifying Genes Using Mutants II - Tools to Inactivate Genes
Mutagens are treatments that increase the rate of mutation.
ENU (N-ethyl-N-nitrosourea) is a chemical mutagen that typically alkylates thymines and results in
point mutations due to base mis-pairing during replication.
EMS (Ethyl methanesulfonate) is a chemical mutagen that alkylates guanine bases in DNA and
results in point mutations (and rarely, in small deletions) due to base mis-pairing during replication.
X-rays can produce radicals within the DNA that leads to cross-linking and breaks in the backbone.
Ultimately, this can result in large rearrangements and deletions in the chromosomes.
TILLING combines chemical mutagenesis with mutation screens of pooled PCR products, resulting in
the isolation of missense and nonsense mutant alleles of the targeted genes.
Insertional mutagenesis utilizes transposons or other methods to insert DNA segments into genes,
disrupting the gene. One advantage of this technique is that the gene that is disrupted can easily be
identified by PCR.
Transposons are mobile DNA elements that can move around the genome by 2 different mechanisms:
“conservative transposition” or “retrotransposition/non-conservative transposition”.
In one mechanism, the transposon codes for a transposase enzyme and contains transposase-recognized
sequences. Transposase removes the DNA element and reinserts it into other locations in the genome.
This behavior is called conservative transposition (only one transposon is in play).
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In a second mechanism, known as retrotransposition, small DNA elements are first transcribed into
RNA, then reverse transcribed back to DNA by reverse transcriptase (sometimes coded for by the
retrotransposon) and reinserted into the genome. This process is considered non-conservative, since
each round makes a new DNA element.
Excisions of transposons can be catalyzed by some transposases. These can be identified by loss of
an associated marker. Often excision is imprecise and results in deletion of flanking sequences and
hence useful mutations.
A Gene trap uses a construct that is only expressed when it is integrated into a gene. This construct
has no regulatory elements or transcription start codon, but codes for a reporter sequence.
Enhancer traps use a construct that is only expressed when it is integrated into a location that places it
under the control of an enhancer. These constructs contain minimal promoters and start codons and are
then subject to local enhancer regulation. They can be used to indicate patterns by which those
enhancers promote gene expression.
Promoter traps use a construct that can only be expressed when integrated to be under the control of a
promoter and/or enhancers. They contain a start codon and a reporter sequence.
RNAi (RNA interference) is a form of post-transcriptional gene silencing, in which very short RNA
molecules (siRNA or miRNA) target destruction of an endogenous RNA. siRNA is introduced
exogenously in the form of dsRNA which is processed through the action of several enzymes, while
miRNA is produced endogenously (miRNA) by the cell.
Morpholino knockdown induces inhibition of gene expression by base-pairing to endogenous RNA
molecules. Morpholinos are RNA-like molecules that can pair to complimentary RNA, but cannot be
degraded by RNAses.
Conditional mutants are any mutant organisms that display different phenotypes depending on the
environmental conditions.
Identifying Genes using Mutants III – types of mutations
Recessive allele is obscured in the phenotype of a heterozygote by the dominant allele, often due to
decreased activity and/or levels of the product of the recessive allele.
Recessive lethal is an allele that is lethal in the absence of the wild-type copy.
Essential gene is one whose deletion or lack of a functional product upon its expression is lethal to the
organism.
Sterile mutations result in the inability to reproduce. These are often sex specific: female sterile and
male sterile.
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Maternal effect mutation some mutations that appear as female sterile in cursory analysis are fertile,
however the embryos are defective and do not develop to adulthood. Typically, these represent
situations in which the maternal genome encodes gene products that are placed in the egg and required
for subsequent development.
Constitutive mutations cause genes that are usually regulated to be expressed without regulation.
Uninducible mutants cannot be induced to express under normal conditions.
Silent mutation is a DNA mutation in the coding sequence which does not change the amino acid
sequence of the protein; silent mutations occur as a result of the redundancy in the genetic code.
Gain of function – any mutation that results in the gene product to be over-expressed or to gain a
novel function. These are often, but not always, dominant mutations. They include:

Hypermorph – a mutant that produces increased activity or levels gene product.

Neomorph – a mutant with a novel gain of function phenotype.

Dominant-negative – a mutation which results in a gene product that neutralizes the wild-type
gene product. Antimorph is a mutant with a dominant-negative phenotype.
Loss of function – any mutation which results in the gene product having less activity or no function.
These are often, but not always, recessive mutations. Haplo-insufficiency is an exception, elimination
of a function of one allele results in a mutant phenotype; in which a loss of function condition causes a
dominant phenotype (see below). They include:

Null – a mutant where the function of a gene has been completely eliminated.

Hypomorph – a mutant with partial loss of function.
associated with missense mutations.
These are often, but not always,
Haplo-insufficiency - occurs when a diploid organism only has a single working copy of a wild-type
gene, and the single functional copy of the gene does not produce enough of a gene product to bring
about a wild-type condition.
Lethal – a mutation that results in death or the inability to reproduce
Ploidy refers to the number of copies of the chromosomes present in a cell.
 Haploid cells contain one copy of each chromosome; the haploid number n is characteristic of
gametes of diploid organisms. A single chromosome complement is defined as 1c.
 Diploid cells contain two copies of each chromosome, and are described as 2c.
 Polyploid cells have more than two chromosome complements, e.g. triploid (3c), tetraploid
(4c), pentaploid (5c), etc.
 Aneuploid chromosome constitution differs from the usual diploid (or haploid) constitution by
loss or duplication of individual chromosomes or chromosomal segments.
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
Euploid cells have the complete copy number of chromosomes. For instance, euploid human
cells have 46 total chromosomes.
Triplet repeat expansion – a type of mutation in which the copy number of a trinucleotide tandem
repeat increases. This expansion can be detrimental depending on the size of the expansion. Fragile X
Syndrome, Huntington’s disease, and myotonic dystrophy are all associated with triplet repeat
expansions.
Synthetic phenotype – the condition in which a combined double mutant shows an enhanced
phenotype that is stronger than either of the single mutant phenotypes alone.
Synthetic lethal – two mutations when combined in one organism are synthetically. A mutant
organism with either mutation individually is alive, but with both mutations is dead or incapable or
reproduction.
Penetrance – given an individual carries a mutation causing a genetic disorder, penetrance is the
probability that the individual will exhibit phenotypic signs of the disorder.
Expressivity – The degree to which a particular genotype is expressed in the phenotype.
Given an inherited genetic disorder, expressivity describes the range of possible phenotypic effects
among individuals manifesting the disorder. (i.e., if a person is penetrant for the mutation(s), how
severely is he/she affected?) Disorders with variable expressivity display phenotypic symptoms
ranging from mild to severe in different individuals. Disorders with in which expressivity is not very
variable have more homogeneous disease manifestations.
Quantitative Trait Locus (QTL) – a locus that quantitatively influences phenotype and acts with one
or more other loci.
Allelic series – A set of known alleles of one gene. A set of mutations in the same gene that display a
range of phenotypes (e.g. from weak to strong).
DNA Changes Associated With Mutations:
 Single base-pair changes
 Deletion
 Inversion
 Translocation
 Insertion
 Amplification (includes Duplication)
Identifying Genes using Mutants IV – methods to isolate genes based on
chromosomal position
Recombination frequency between two loci is the ratio of the number of individuals with a nonparental phenotype to the total number of individuals (among progeny of a cross). It is a measure of
how often crossing-over occurs between two loci and is used to construct linkage maps (1%
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recombination frequency = 1 centiMorgan). Two unlinked loci will have a recombination frequency of
50%.
Linkage describes the tendency of genes to be inherited together as a result of their location on the
same chromosome; measured by recombination frequency between loci.
Deletion mapping takes advantage of large deletions removing several or many genes to map
recessive mutations. When a mutation (or a vital part of gene affected by a mutation) falls within a
deletion, the deleted chromosome cannot provide the function. Hence, if a transherozygote of a
mutation and a deletion exhibits the mutant phenotype, the mutation is said to fall within the deletion.
When overlapping deletions are available along a chromosome this method provides rapid and reliable
mapping. Often the physical map of the deletion can be established cytologically or molecularly. This
defines the correspondence between the physical and genetic maps.
Segmental aneuploids a chromosome that has extra or missing copies of portions of chromosome as a
result of duplication or deletion.
Linkage disequilibrium describes a situation in which two genetic markers occur together more
frequently in the population than would be expected from their genetic distance from each other. It
implies that the markers have been inherited coordinately. It can result from a founder effect, in which
there has been insufficient time to reach equilibrium since one of the markers arose in the population.
Linkage group includes all loci that can be connected (directly or indirectly) by linkage relationships;
equivalent to a chromosome.
Linkage mapping is the process of constructing linkage groups or maps based on recombination
frequencies.
Three-point cross involves crossing an individual heterozygous for three mutations to a recessive
homozygote. The two most common phenotypes that result are the parental ones; the two least
common phenotypes come from a double crossover in gamete formation. By comparing the parental
and double-crossover phenotypes, the geneticist can determine which gene is located between the
others on the chromosome.
Tetrad analysis allows unambiguous identification of the four products of an individual meiosis. It is
a powerful tool of yeast genetics, which allows the genetic identification of centromeres.
Chi-square test is a statistical test to determine whether the outcome of an experimental cross is
consistent with an expected Mendelian ratio; for instance, such as 9:3:3:1. The formula is:
2 =  ((observed value-expected value)2/expected value)
The value obtained is compared to a Chi-Square Table (below) and if the value obtained is less than
the value in the table, we accept the hypothesis:
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Degrees of Freedom
1
2
3
4
5
Probability
0.9
0.5
0.02 0.46
0.21 1.39
0.58 2.37
1.06 3.36
1.61 4.35
0.1
2.71
4.61
6.25
7.78
9.24
0.05
3.84
5.99
7.82
9.49
11.07
0.01
6.64
9.21
11.35
13.28
15.09
Degrees of Freedom (df) = n-1, where n = number of classes of progeny.
For example: An AaBb x AaBb cross has four classes of progeny (A-B-, A-bb, aaB-, aabb) expected to
assort at a 9:3:3:1 ratio. So n = 4 and df = 3, so 2 < 7.82 is necessary to have 95% confidence (p =
0.05) that the alleles are assorting according to the expected Mendelian ratio (i.e. independently).
Sex linkage is the pattern of inheritance shown by genes carried on a sex chromosome (usually the X).
Cell hybrid is a somatic cell containing chromosomes derived from parental cells of different species
(e.g. a human-moue somatic cell hybrid), generated by fusing the cells to form a heterokaryon in which
the nuclei subsequently fused.
Balancer chromosomes are genetically altered chromosomes that cannot recombine with their wild
type homologs.
Balance chromosomes are used to maintain a stock of heterozygous flies carrying a recessive lethal
mutation. Consider the following cross:
mut/+ x mut/+  mut/+, mut/+, mut/mut (dead), and +/+
Because 100% of the progeny of the +/+ flies survive, compared to only 75% of the mut/+ flies, the
+/+ genotype will overtake the stock after a few generations and the mutation of interest will be lost.
Balancer chromosomes carry a second recessive lethal mutation, so that only heterozygotes can
survive:
mut/bal x mut/bal  mut/bal, mut/bal, mut/mut (dead), and bal/bal (dead)
Thus the stock can be maintained indefinitely. Balancers also carry a dominant, clearly visible marker
so that they can be easily followed in any cross, and typically have multiple inversions in order to
prevent recombination with the chromosome of interest.
Types of Genetic Markers
Genetic marker is any allele of interest in an experiment that can be detected by phenotyping or
genotyping.
Selectable marker is an allele that confers or prevents survival under particular growth conditions and
thus can be used in a selection.
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Phenotypic marker is an allele which confers a phenotype different from wild-type, usually an easily
(visually) observable one.
GFP tagging by insertion provides one way of producing a visible marker.
RFLP (restriction fragment length polymorphism) refers to inherited differences in sites for
restriction enzymes (for example, caused by base changes in the target site) that result in differences in
the lengths of the fragments produced by cleavage with the relevant restriction enzyme. RFLPs are
used for genetic mapping to link the genome directly to a conventional genetic marker.
SNP (single nucleotide polymorphism) refers to an inherited difference of a single nucleotide (point
mutation) in an otherwise shared sequence. SNPs are used for genetic mapping. RFLP is a variation of
SNP.
Tandem repeats are multiple copies of the same sequence lying in series. SSLP (single sequence
length polymorphism) are typically short (di-, tri-, tetra-, and penta-) nucleotide repeats.
VNTR (variable number tandem repeats) are repeats of larger size, typically 50-100 of base pairs in
length, that are repeated tandemly n number of times (i.e. (50-100)n = total length of the VNTR).
They can cause RFLPs and are sometimes assayed that way.
Modes of Non-Mendelian Inheritance
Maternal inheritance describes the preferential inheritance of genetic markers and often phenotypic
traits provided by the mother.
Cytoplasmic inheritance describes the inheritance of non-nuclear genetic elements, including DNA
from mitochondria and chloroplasts, endogenous viruses, dsRNA, and prions.
Prion is a proteinaceous infectious agent, which behaves as a heritable trait, although it contains no
nucleic acid. Examples are PrPSc, the agent of scrapie in sheep and bovine spongiform encephalopathy,
and Psi, a prion-like element in yeast. In both cases, protein amyloids are inherited.
Chromosomal Inheritance not Involving Changes in DNA Sequence
Epigenetic changes influence the phenotype without altering the genotype. They consist of changes in
the properties of a cell that are inherited but that do not represent a change in genetic information.
Examples are the lysogenic state of λ virus, DNA methylation, and possibly certain histone
modifications.
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Methods for Detecting DNA Sequences
Southern blotting is a technique for transferring denatured DNA from an agarose gel to a
nitrocellulose filter where it can be hybridized to a complementary nucleic acid. (Named for its
inventor, Ed Southern)
Northern blotting is a technique for transferring RNA from an agarose gel to a nitrocellulose filter on
which it can be hybridized to a complementary nucleic acid. (Named because someone thought it
would be funny :D.
In situ hybridization is performed by hybridizing a labeled probe to permeabilized cells. The
technique can be used to label DNA or RNA.
FISH (Fluorescent In Situ Hybridization) is like in situ hybridization except the probe is
fluorescently, rather than radioactively, labeled and hybridization is followed by microscopy.
Chromosome painting is a variation of FISH in which different chromosome-specific probes carry
different fluorophores.
PCR (Polymerase Chain Reaction) describes a technique in which cycles of denaturation, annealing
with primer, and extension with DNA polymerase, are used to amplify the number of copies of a target
DNA sequence by >106 times.
QPCR (a.k.a. real time PCR) is an assay that monitors the accumulation of product during a PCR
reaction (rather than simply the end product) in order to quantitate the initial number of copies of a
target DNA sequence. It is often used in combination with reverse transcription reaction to determine
relative expression levels of RNA.
Methods of Cloning DNA Molecules
Plasmid is an autonomous self-replicating extrachromosomal circular DNA.
Cosmids are plasmids into which phage lambda cos sites have been inserted; as a result, the plasmid
DNA can be packaged in vitro in the phage particle.
cDNA clone is a duplex DNA sequence representing an mRNA, carried in a cloning vector. Prior to its
cloning into a vector, it was reverse transcribed from mRNA using reverse transcriptase.
cDNA library is a collection of cDNA clones representing all the RNA expressed by a particular
group of cells under a particular condition.
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BAC (Bacterial Artificial Chromosome) & YAC (Yeast Artificial Chromosome) are DNA
constructs that contain all the sequences necessary to be duplicated during the cell cycle in bacteria or
yeast. They typically carry an insert with a gene or fragment of interest.
Methods for Altering the Genome
Transformation of bacteria or fungi describes the acquisition of new genetic markers by incorporation
of added DNA.
Transfection of higher eukaryotic cells is the acquisition of new genetic markers by incorporation of
added DNA.
Transduction refers to the transfer of a bacterial gene or genes from one bacterium to another by a
phage; a phage carrying host as well as its own genes is called a transducing phage. It also describes
the acquisition and transfer of eukaryotic cellular sequences by retroviruses.
Conjugation describes “mating” between two bacterial or yeast cells, when (part of) the chromosome
is transferred from one to the other.
Transposition refers to the movement of a transposon to a new site in the genome:

Nonreplicative transposition describes the movement of a transposon that leaves a donor site
(usually generating a double-strand break) and moves to a new site.

Replicative transposition describes the movement of a transposon by mechanisms in which
first it is replicated, and the one copy is transferred to a new site.
Conservative (Site-Specific) Recombination involves breakage and reunion of pre-existing strands of
DNA without any synthesis of new stretches of DNA. It often occurs between two specific (often, but
not necessarily, homologous) sequences. Examples include phage integration/excision, the resolution
of conintegrate structures during transposition, and the CRE/Lox or FLP/FRT system.
Homologous Recombination – involves the exchange of DNA sequences between two homologous
genetic elements. The process involves pairing of single-stranded DNA intermediates, which requires
that highly homologous sequences of substantial length be aligned for the exchange. Unlike
conservative site-specific recombination, limited DNA synthesis and mismatch repair is involved.
Illegitimate recombination – an exchange of genetic material, which is not homologous
recombination, DNA transposition, or conservative site-specific recombination
Reporter gene refers to any sequence construct that reads out desired aspects of a gene; associated
with tagging (see below).
Tagging – the insertion of an assayable marker to a gene, which can be used for a variety of purposes,
including the following:
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1. Assaying Promoter Activity – fusion of reporter genes to promoter sequences can be used to
assay patterns of promoter activation, regulation, and inactivation. Usable reporter genes
include:

GFP – green fluorescent protein, fluoresces green (509 nm) when exposed to blue
light (395 nm). eGFP is enhanced GFP, which can be excited at 490 nm and
fluoresces at 509 nm. The other major fluorescent proteins include CFP, YFP and
RFP for cyan, yellow, and red, respectively.

LacZ – or -galactosidase converts lactose into glucose and galactose (also may act
on X-gal to give a blue by-product).

Expression reporters – are specialized vectors used to identify expression driven
by particular promoter elements.
2. Determining Intracellular Protein Localization – fusion of reporter gene to coding sequences,
such that the reporter is directly incorporated into the final protein product, can be used to
identify the cellular location of the protein. GFP and LacZ are common reporters used for this
purpose.
3. Determining Expression Patterns of mRNA – incorporation of reporter genes like GFP or
LacZ into coding sequences can be used to detect expression of mRNA. Often, an IRES must
be used to allow expression of a reporter that has been internally inserted into an mRNA.

IRES (Internal Ribosome Entry Site) – a nucleotide sequence that allows for
translation initiation in the middle of an mRNA message; it is used to aid in the
expression of a reporter.
4. Protein Detection – fusion of epitope tags to a coding sequence allows the translated protein to
be bound to specific antibodies conjugated to easily identifiable molecules.

Epitope Tag – most include a short stretch of amino acids that bind to an easily
detected antibody (biotin and DIG are exceptions, which are molecules added
covalently after translation).

c-myc is a 10 amino acid tag from the human protooncogene myc

HA triple tag is an amino acid sequence derived from the human influenza
hemaglutinin protein
5. Protein Purification – in addition to protein detection, particular tags can be employed to
easily purify large amounts of protein from cellular extracts.
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
TAP tag – a “tag” containing an IgG-binding unit of protein A from Staphylococcus
aureus (ProtA) and the Calmodulin Binding Peptide (CBP) can be genetically added
to proteins. This tag allows proteins to be isolated by Tandem Affinity Purification
(TAP), in which fusion proteins are recovered based on the tag’s affinity on an IgGmatrix, followed by incubation on calmodulin-coated beads in the presence of
calcium.
Gene Activation and Control Strategies – investigators employ a variety of methods to activate or
deactivate the expression of genes during the course of their experiments. These methods include
using:

Regulated promoter – allows you to turn a gene on or off when desired; the promoter is
activated by transcription factors that are controlled by the external addition of various
compounds. Common inducible systems include Gal4 (galactose), rTet, (tetracycline), and
pMT (Cu).

Overexpression – you can use either a constitutively active promoter or an inducible promoter
to express your gene of interest at above-normal levels.

Cre/Lox – Cre recombinase recombines the sequence surrounding LoxP sites. It is commonly
used to inducibly knock-out genes in specific tissues or at key developmental time points, when
a completely null mutant would be dead.

FLP/FRT – FLP recombines the sequence surrounding FRT sites. It is commonly used to
inducibly knock-out genes in specific tissues or at key developmental time points, when a
completely null mutant would be dead (similar to Cre/Lox)
Embryonic Stem (ES) cells – pluripotent cells that can be isolated from early embryos (first isolated
by Gail Martin at UCSF).
Nuclear transfer – transfer of a nucleus (with target DNA) to an enucleated egg to clone your favorite
organism.
Using Genome-Wide Methods for Studying Genes and Genomes
Cytogenetic methods provided the original way of examining the whole genome and these methods
still provide the method of choice for defining some types of genetic aberrations.
Karyotype is the complete set of all chromosomes of a cell of any living organism. The
chromosomes are arranged and displayed (often on a photo) in a standard format: in pairs,
ordered by size and position of centromere for chromosomes of the same size which is known
as an idiogram. Karyotypes are examined in searches for chromosomal aberrations, and may be
used to determine other macroscopically visible aspects of an individual's genotype, such as sex
(XX vs. XY pair). Typically used mammalian genetics.
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Polytene banding – multiple rounds of DNA replication greatly amplify the chromosomes in
Drosophila larvae and the precise alignment of all the copies yields a banded structure that is
stereotyped in wild-type. A naming system designates each band. Genetic rearrangements are
characterized by the alterations (deletion, translocation etc) the bands, and gene expression
could be assessed directly by puffing of particular loci.
Two Color Transcriptional Profiling is a procedure where the pattern of gene expression in a cell is
measured on a whole-genome scale. A microarray chip is made by printing complementary DNA that
covers every gene in a genome onta a slide. mRNA from two sample populations is reverse transcribed
into cDNA and labeled with either green or red fluorophores. The labeled cDNA samples are
competitively hybridized to the complementary DNA on the microarray. Ratios of fluorescence on
each gene spot are then assayed to determine how transcription of that gene varies between the two
samples.
Phenotypic Profiling is generally defined as using genome-wide knockout collection or RNAi library
to systematically screen for previously uncharacterized genes involved in a particular cellular pathway.
A researcher sets up experimental conditions that will cause an observable phenotype if a mutant strain
contains a defective gene involved in the pathway of interest. Using high throughput methods, a large
set (or possibly all) of the mutant strains in a collection will undergo testing under the appropriate
conditions for identifying a phenotype.
ChIP-chip refers to the procedure for using ChIP-microarray methods to locate where in the genome
proteins physically bind to DNA. Chromatin Immunoprecipitation (ChIP) is a procedure for
determining if a protein binds to DNA in vivo. Cells are treated with formaldehyde, which to crosslinks DNA binding proteins to chromatin. Cell extract is mixed with antibody for a particular DNA
binding protein. The antibody binds to the protein, removing it from the extract, along with any bound
cross-linked DNA segments. This DNA that was “pulled down” in the ChIP is amplified and labeled
with fluorophores. The fluorescently labeled DNA is then hybridized to a microarray (the “ChIP-chip”
namesake) containing the genomic regions of interest. If the protein was bound to a particular
genomic region, that DNA sequence will be enriched, and show a fluorescence shift on the microarray.