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Chapter 5
Genetic Analysis in Cell Biology
(textbook: “Molecular Cell Biology” 6 ed, Lodish section: 5.1+5.4-5.5)
Understanding gene function:
relating function, location, and structure of gene products
Phenotype
DNA
Mutations: types and causes
a An organism’s genotype is its entire set of genes and may denote
whether an individual carries mutations in one or more genes
(genotype = basic genetic make-up of an individual organism)
a An organism’s phenotype is its function and physical
appearance, and depends on that individual’s genotype
a Normal (wild-type) organisms may develop changes termed
mutations in their DNA sequence thereby altering their genotype
and perhaps their phenotype
a Organisms that develop such mutations are termed “mutants”
a Mutations may be part of the genotype or occur as acquired
(somatic) mutations
Haploids and diploids
a A haploid organism has a single copy of each chromosome and its
phenotype is a consequence of that one copy
a A diploid organism has two copies of each chromosome and thus
two copies of each gene
a The two copies of each gene may be the same or the copies may
be different. Different forms of each gene are termed alleles (allele=
naturally occuring mutation)
a Diploids that carry identical alleles are termed homozygous
a Diploids that carry different alleles are termed heterozygous
Cross-over mainly occurs in meiosis
(gametogenesis)
Cross over allows exchange of
genetic information between
paired chromosomes
Genotype and phenotype in diploids
Alleles may be dominant or recessive
Segregation of a mutation
Segregation of Dominant mut
Dominant mutations usually lead to a
gain of function although occasionally
they may lead to a loss of function
Segregation of Recessive mut
Recessive mutations lead to loss of
function
Geno-type linked diseases
(non-cancerous)
An autosome is a non-sex chromosome. It is an ordinarily paired type of chromosome that is the same in both
sexes of a species. For example, in humans, there are 22 pairs of autosomes. The X and Y chromosomes are not
autosomal. Non-autosomal chromosomes are usually referred to as sex chromosomes, allosomes or heterosomes.
Segregation of geno-type linked diseases
Various types of mutations
NB: single base mutations leading to know functional coding-change are called sense mutations. Single base mutations
frequently occur in nature and are called “singel nucleotide polymorphisms” (SNP’s)
Spontaneous mutations may result from
errors in DNA replication
Spontaneous mutations may
also result from chemical
instability of purine and
pyrimidine bases, or
from exposure to UV light or
chemical carcinogens
-Or from cross-over
NB: chapter on repair
Cross-over can occur in somatic cells, but most
frequently is part of meiosis
Induction of point mutations by EMS
Scientists may induce mutations in experimental
organisms by treatment with chemical mutagens
(e.g, EMS) or exposure to ionizing radiation
EMS= ethylmethane sulfonate
Mutational agents are used to generate “libraries” of mutant organisms which can then be screened for specific
functional changes. Identification of mutated gene then gives information on which genes are involved in this
functionality.
Role of spontaneous somatic mutations in human
genetic diseases
Rb is a classical
Oncogene
Rb (retinoblastoma pocketprotein): normal function: prevents cells from entering S-phase (cell cycle). Inactivation by
phosphorylation (cyclins+Cdks, see previous lecture) allows progression through S-phase -> mitosis.
Mutated Rb = cronic inactivated -> continous cell-cycling (no growth arrest!) = cancer
Isolation and analysis of mutants
a The procedures used to identify and isolate mutants are
termed “genetic screens”
a The screen used depends on whether the organism is
haploid or diploid. If diploid, the screen used also depends
on whether the mutation is dominant or recessive.
a Screens with haploid organisms often involve temperaturesensitive mutants
a Screens for recessive lethal mutations utilize visible markers
Example: mutants in Drosophila
Mutations may cause only subtle
changes or may produce significant
changes in development, cellular
function, appearance and/or behavior
Drosophila melanogaster (fruitfly) is one of the most favored organisms for genetic studes. It has a short generation time
and phenotypes are often easily detected (visible)
Temperature-sensitive genetic screen used to identify cell cycle
mutants in yeast
Yeast (especially “bakers yeast; saccharomyces cerevisiae) is a favorite for cell cycle analysis. Eucaryotic single-cell
organism with: Fast propagation (generation time 2-4h), easy manipulation (DNA-transfection etc.) and a well
charachterized genome.
Why is it an advantage to use single cells in culture particulary for cell-cycle studies ? ;-)
Some chromosome abnormalities can be mapped by
banding analysis
Molecular cloning of genes defined by
mutations
a The use of recombinant DNA techniques to isolate
mutation-defined genes is referred to as positional
cloning
a In higher organisms, the cloning of a mutation-defined
gene usually involves four steps
Positional cloning:
Step 1: identification of a cloned segment of DNA
near gene of interest
Linkage disequilibrium study
In situ hybridization
Positional cloning:
Step 2, and 3
Step 2:
isolation of a contiguous
stretch of DNA and
construction of a physical
map in that region
Step 3:
correlation of the physical
map with the genetic map to
locate the position of the
gene of interest
YAC= yeast artificial chromosome,
BAC= bacterial artificial chromosome
Function like plasmid vectors but can
harbor very large foreign DNA
fragments (= you can clone smaller
DNA fragments in plasmid vectors, but
large fragments in BAC’s and very
large fragments in YAC vectors)
Therefore very good for generating
tiling libraries
Step 4:
detection of alterations in expression of transcripts or changes in
DNA sequence between the mutant and wild-type
Detection of point mutations:
Gene replacement and transgenic animals
a Some genes are identified through means other than mutant analysis
a To determine the function of these genes, it is possible to replace an
organism’s wild type gene with an inactive gene to create a “gene knockout”
a It is also possible to introduce additional genes (transgenes) to create a
transgenic organism
a Gene knockout and transgenic techniques can involve mutagenesis of cloned
genes prior to transfer into the organism (= in vitro mutagenesis)
Creation of mice embryonic stemm cells (ES cells) carrying
a knockout mutation
Specific targeting of gene of interest (GOI)
Gene knockout in mice
Cell-type-specific gene knockouts in mice
Advantage:
Effect on specific
tissue / process can
be studied
Embryonal lethal
knock-outs can be
studied in tissues
where the knock out
is not lethal
Gene-silencing with small mRNA-interfering RNAs
(siRNA)
Dicer is an endogenous
enzyme which cleaves
specific RNA structures
(part of the miRNA
system)
siRNAs are designed to basepair
with specific mRNA, which will
then not be translated
An alternative to Knock-out. Gene-silencing addtionally allows controlled silencing through regulatable promoter
upstream of hairpin construct coding region.
Endogenous= naturally ocurring within cells
Production of transgenic mice
The foreign DNA contains a
promoter+GOI
If the promoter is a
constitutive active promoter
the GOI will be expressed in
all cells
If the promoter is regulated
by proteins only present in
certain cells, a tissuespecific expression can be
achieved
Example: transgenic mouse (GH+)
Promoter function seen in transgenic mice. (A) Recombinant plasmid containing rat growth hormone structural gene, mouse
metallothionein regulatory region, and bacterial plasmid pBR322. The plasmid, pMGH, was injected into the mouse oocytes.
The dark boxes on the injected plasmid correspond to the exons of the GH gene. The direction of transcription is indicated by
an arrow. (B) A mouse derived from the eggs injected with pMGH (left) and a normal littermate (right). (From Palmiter et al.
1982, photograph courtesy of R. L. Brinster.)
A plasmid containing this fused gene was grown in bacteria (see Chapter 2), the Mt-1/rGH piece was isolated, and about 600
copies of this fragment were injected into pronuclei of recently fertilized mouse eggs. DNA hybridization showed that many of
these newborn mice had incorporated numerous copies of the rat growth hormone gene into their chromosomes. These
transgenic mice were then fed a diet supplemented with zinc. The zinc induced large amounts of rat growth hormone to be
secreted by the livers of these mice. (The liver is where metallothionein is usually made. Growth hormone is usually secreted
from the pituitary gland.) The amount of growth hormone secreted correlated with the size of these mice. The transgenic mice
became enormous, up to 80 percent larger than their normal littermates
After completion of this lecture you should be able to:
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Define the terms: genotype, phenotype, mutants, wild-type, somatic mutation, haploid, diploid, allele, homozygous,
heterozygous, dominant allele, recessive allele
Know that cross-over allows exchange of genetic information between paired chromosomes, and mainly happens in
meiosis
Explain the genetic reasons underlying potential differences between diploid genotype and diploid phenotype
Describe and schematically draw the segregation of dominant and recessive mutations
Know the difference between X-linked and autosomal gene-linked diseases and how they segregate
Briefly define the following concepts of mutations: missense, nonsense, frameshift, insertion, deletion, translocation,
inversion, single nucleotide polymorphism (SNP)
Know that induction of pointmutations by chemical stimuli is a valuable tool for genetic analysis. –and that
pointmutations lead to either missense, nonsense or non-affected (sense) mutants
Understand that somatic mutation of cell-cycle regulating genes is the prevalent cause of cancer.
Know what a “genetic screen” is and explain why different methods are applyed for investigating recessive and
dominant mutations
Know why yeast is an excellent organism for studying cell cycle regulator genes
Briefly explain the 4 steps involved in Positional cloning of a mutation-defined gene from a higher organism
(phenotype Æ gene)
Know the term artificial chromosome (BAC and YAC) and that AC’s are vectors that can harbor very large foreign DNA
fragments
Know that in addition to genetic screens for mutants, specific genes may be investigated through generation of geneknock out and transgene organisms (gene Æ phenotype)
Briefly describe the classical strategy for generating knock-out mice
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Formation of ES cells carrying a knock out mutation
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Generation of knock-out vector carying disrupted (non-functional) form of GOI and selection markers (ie. antibiotic resistance genes)
Facilitated homologous recombiantion
Selection of ES cells with targeted disruption of GOI
Injection of the ES cells into mouse blastocyst. Insertion of blastocyst into pseudopregnant mouse
Selection of full knock out mice which arise from progeny carrying the mutation in their germline cells
Know that it is possible to generate not only full body knock out mice but also cell type specific knock outs applying
the Cre-Lox system.
Know the term siRNA, and that it is natures own tool for temporary gene silencing, which biotechnologically can be
applied as an alternative to the classical knockout
Know that transgene mice can be created by “simple” injection of DNA vector carrying the GOI into fertilized mice
eggs.