Transformation-Griffith’s Expt
1928
DNA Mediates Transformation
Convert IIR
to IIIS
By DNA?
Avery MacLeod and McCarty Experiment
Circa 1943
Transforming Principle
DNAse activity
+ means that activity is present
All RNA gets degraded during enzyme preparation
Chapter 4
The Interrupted
Gene
3.12 Gene Knockouts and Transgenics
• transgenics – Organisms created by
introducing DNA prepared in test tubes into the
germline.
– The DNA may be inserted into the genome or exist in
an extrachromosomal structure.
Figure 03.27: Transfection can
introduce DNA directly into
the germline of animals.
Photo reproduced from P. Chambon, Sci. Am.
244 (1981): 60-71. Used with permission of
Pierre Chambon, Institute of Genetics and
Molecular and Cellular Biology, College of
France.
3.12 Gene Knockouts and Transgenics
• Embryonic stem (ES) cells that are injected into
a mouse blastocyst generate descendant cells
that become part of a chimeric adult mouse.
– When the ES cells contribute to the germline, the next
generation of mice may be derived from the ES cell.
– Genes can be added to the mouse germline by
transfecting them into ES cells before the cells are
added to the blastocyst.
3.12 Gene Knockouts and Transgenics
Figure 03.29: ES cells can be used to generate mouse chimeras.
3.12 Gene Knockouts and Transgenics
• An endogenous gene can be replaced by a transfected
gene using homologous recombination.
• The occurrence of successful homologous
recombination can be detected by using two selectable
markers, one of which is incorporated with the integrated
gene, the other of which is lost when recombination
occurs.
3.12 Gene Knockouts and Transgenics
• The Cre/lox system is widely used to make
inducible knockouts and knock-ins.
– knockout – A process in which a gene function is
eliminated, usually by replacing most of the coding
sequence with a selectable marker in vitro and
transferring the altered gene to the genome by
homologous recombination.
– knock-in – A process similar to a knockout, but more
subtle mutations are made.
3.12 Gene Knockouts and Transgenics
Figure 03.31: The Cre recombinase catalyzes a site-specific recombination between two
identical lox sites, releasing the DNA between them.
4.1 Introduction
• interrupted gene – A gene in which the coding
sequence is not continuous due to the presence of
introns.
• primary (RNA) transcript – The original unmodified
RNA product corresponding to a transcription unit.
• RNA splicing – The process of excising introns from
RNA and connecting the exons into a continuous mRNA.
4.1 Introduction
• intron – A segment of DNA that is transcribed, but later
removed from within the transcript by splicing together
the sequences (exons) on either side of it.
• mature transcript – A modified RNA transcript.
Modification may include the removal of intron
sequences and alterations to the 5′ and 3′ ends.
Figure 04.01: Interrupted genes are expressed via a precursor RNA.
What are Logo plots?
Logo for
a) Splice acceptor
b) Splice Donor
c) Initiator Met
AG/GT
exon 1
4321123456
CAG/NT
intron 1
exon 2
98765432112
The Shine-Dalgarno
Sequence
4.2 An Interrupted Gene Consists of Exons
and Introns
• Introns are removed by RNA splicing, which occurs in cis
in individual RNA molecules.
• Mutations in exons can affect polypeptide sequence;
mutations in introns can affect RNA processing and
hence may influence the sequence and/or production of
a polypeptide.
Figure 04.02: Exons remain in the
same order in mRNA as in DNA,
but distances along the gene do
not correspond.
4.3 Exon and Intron Base Compositions
Differ
• The four “rules” for DNA base composition are the first
and second parity rules, the cluster rule, and the GC
rule.
– Exons and introns can be distinguished on the basis of all rules
except the first.
• The second parity rule suggests an extrusion of
structured stem-loop segments from duplex DNA, which
would be greater in introns.
• The rules relate to genomic characteristics, or
“pressures,” that constitute the genome phenotype.
4.4 Organization of Interrupted Genes May
Be Conserved
• Introns can be detected when genes are compared with
their RNA transcription products by either restriction
mapping, electron microscopy, or sequencing.
• cDNA – A single-stranded DNA complementary to an
RNA, synthesized from it by reverse transcription in vitro.
Figure 04.03: Comparison of
the restriction maps of cDNA
and genomic DNA for mouse
β-globin.
4.4 Organization of Interrupted Genes May
Be Conserved
• The positions of introns are usually conserved
when homologous genes are compared
between different organisms.
– The lengths of the corresponding introns may vary
greatly.
• Introns usually do not encode proteins.
Figure 04.05: Mammalian genes for DHFR have the same relative organization of rather
short exons and very long introns.
4.5 Exon Sequences Under Negative
Selection Are Conserved but Introns Vary
• Comparisons of related genes in different species
show that the sequences of the corresponding exons
are usually conserved, but the sequences of the
introns are much less similar.
• Introns evolve much more rapidly than exons because
of the lack of selective pressure to produce a
polypeptide with a useful sequence.
Figure 04.06: The sequences of the mouse βmaj- and βmin-globin genes are closely related
in coding regions.
Data provided by Philip Leder, Harvard Medical School
4.6 Exon Sequences Under Positive
Selection Vary but Introns Are Conserved
• Under positive selection an individual with an
advantageous survives (i.e., is able to produce more
fertile progeny) relative to others without the mutation.
• Due to intrinsic genomic pressures, such as that which
conserves the potential to extrude stem-loops from
duplex DNA, introns evolve more slowly than exons that
are under positive selection pressure.
Figure 04.07: The sequences of snake venom phospholipase genes differ in coding regions,
but are closely related in introns and flanking regions.
Modified from D. R. Forsdyke, Conservation of Stem-Loop Potential in
Introns of Snake Venom Phospholipase A2 Genes: An Application of FORS-D
Analysis, Mol. Biol. Evol., vol. 12 (6), pp. 1157-1165, by permission of
Oxford University Press.
4.7 Genes Show a Wide Distribution of Sizes Due
Primarily to Intron Size and Number Variation
• Most genes are uninterrupted in S. cerevisiae but are
interrupted in multicellular eukaryotes.
Figure 04.08: Most genes are uninterrupted in yeast, but most genes are interrupted in flies
and mammals.
4.7 Genes Show a Wide Distribution of Sizes Due
Primarily to Intron Size and Number Variation
• Exons are usually short,
typically encoding fewer
than 100 amino acids.
Figure 04.10: Exons encoding for
polypeptides are usually short.
4.7 Genes Show a Wide Distribution of Sizes Due
Primarily to Intron Size and Number Variation
• Introns are short in
unicellular/oligocellular
eukaryotes but can be
many kb in multicellular
eukaryotes.
• The overall length of a
gene is determined largely
by its introns.
Figure 04.11: Introns range from
very short to very long.
4.8 Some DNA Sequences Encode More
Than One Polypeptide
• The use of alternative translation initiation or termination
codons allows multiple variants of a polypeptide chain.
• overlapping gene – A gene in which part of the
sequence is found within part of the sequence of another
gene.
Figure 04.12: Two proteins can be
generated from a single gene by
starting (or terminating) expression at
different points.
4.8 Some DNA Sequences Encode More
Than One Polypeptide
• Different polypeptides can be produced from the same
sequence of DNA when the mRNA is read in different
reading frames (as two overlapping genes).
Figure 04.13: Two genes may overlap by reading the same DNA sequence in different
frames.
4.8 Some DNA Sequences Encode More
Than One Polypeptide
• Otherwise identical polypeptides, differing by the
presence or absence of certain regions, can be
generated by differential (alternative) splicing
when certain exons are included or excluded.
– This may take the form of including or excluding
individual exons, or of choosing between alternative
exons.
Figure 04.15: Alternative splicing uses the same pre-mRNA to generate mRNAs that have
different combinations of exons.
4.9 Some Exons Can Be Equated with
Protein Functional Domains
• Proteins can consist of independent functional modules
the boundaries of which, in some cases, can be equated
with those of exons.
Figure 04.16: Immunoglobulin light and heavy chains are encoded by genes whose
structures correspond to the distinct domains in the protein.
4.9 Some Exons Can Be Equated with
Protein Functional Domains
• The exons of some
genes appear
homologous to the
exons of others,
suggesting a common
exon ancestry.
Figure 04.17: The LDL receptor gene consists of
18 exons. Triangles mark the positions of
introns.
4.10 Members of a Gene Family Have a
Common Organization
• gene family – A set of genes within a genome
that encodes related or identical proteins or
RNAs.
– The members were derived by duplication of an
ancestral gene followed by accumulation of changes
in sequence between the copies.
– Most often the members are related but not identical.
4.10 Members of a Gene Family Have a
Common Organization
• superfamily – A set of genes all related by presumed
descent from a common ancestor, but now showing
considerable variation.
• A set of homologous genes (homologs) should share
common features that preceded their evolutionary
separation.
Figure 04.19: The rat insulin gene with one
intron evolved by loss of an intron from an
ancestor with two introns.
4.10 Members of a Gene Family Have a
Common Organization
• All globin genes have a common form of organization
with three exons and two introns, suggesting that they
descended from a single ancestral gene.
Figure 04.18: The exon structure of
globin genes corresponds to protein
function, but leghemoglobin has an
extra intron in the central domain.
4.10 Members of a Gene Family Have a
Common Organization
• Intron positions in the actin gene family are highly
variable, which suggests that introns do not separate
functional domains.
Figure 04.20: Actin genes vary widely in their organization.
4.11 There Are Many Forms of Information
in DNA
• Genetic information includes not only that related to
characters corresponding to the conventional phenotype,
but also that related to characters (pressures)
corresponding to the genome “phenotype.”
• In certain contexts, the definition of the gene can be
seen as reversed from “one gene-one protein” to “one
protein-one gene.”
• Positional information may be important in development.
4.11 There Are Many Forms of Information in
DNA
• Sequences transferred “horizontally” from other
species to the germline could land in introns or
intergenic DNA and thence transfer “vertically”
through the generations.
– Some of these may be involved in intracellular
nonself-recognition.