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Functional Genetics: FULL COURSE GUIDE

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This is a pack of notes designed and used by a student who obtained 93% for the course. It includes everything you need to succeed, including extensive and easy-to-understand lecture notes (re-written and combined with textbook and online sources) and the questions and answers to tutorials run in 2...

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  • February 10, 2022
  • 142
  • 2020/2021
  • Class notes
  • Rob ingle
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Functional Genetics
MCB2023S
Full Course Notes



Lecture notes
• Rob Ingle
◦ Sex Determination (pg 2-13)
◦ Dosage Compensation (pg 14-18)
◦ Genetic Mapping (pg 19-26)
• Suhail Rafudeen
◦ DNA Mutagenesis and Repair (pg 27-42)
◦ Lac Operon (pg 43-49)
◦ CRISPR/Cas (pg 50-55)
• Nicola Illing
◦ Cell Cycle Regulation (including cancer) (pg 56-87)
◦ Stem Cells (pg 88-92)
◦ Genetics of Axis Speci cation in Drosophila (pg 93- 105)


Tutorials (questions and answers)
• Sex determination and dosage compensation (pg 106-108)
• Genetic mapping 1 and 2, + past test Qs (pg 109-129)
• DNA mutagenesis (pg 130-132)
• Lac Operon and CRISPR (pg 133-135)
• Cell Cycle Regulation 1 and 2 (pg 136-142)

,SEX DETERMINATION

1. Alternative splicing as a mechanism for gene regulation

RNA splicing: a form of RNA processing in which nascent pre-mRNA is transformed
into mature mRNA by the removal of introns and the joining
together of exons (before it leaves the nucleus).
• Splice sites: short consensus sequences immediately
surrounding the exon-intron boundaries that allow for intron
recognition.
◦ The 5' splice site (at the 5' end of the intron) usually contains the sequence GU.
◦ The 3' splice site usually contains the sequence AG.
◦ The branch-point site (BPS) is 20-30 nucleotides upstream
of the 3' end of introns.
• Small nuclear ribonucleoproteins (snRNPs): a protein-RNA
complex defined by a core noncoding RNA molecule (100-300
nt) and associated proteins.
◦ They function in recognising the sequence motifs
(splice sites) by base pairing with their RNA
component. This only occurs if all motifs are present and
spaced correctly.
‣ U1 → 5'ss
‣ U2 → 3'ss, but more specifically, U2AF35 → 3'ss;
U2AF65 → pyrimidine-rich sequence just upstream
of 3'ss; U2 → BPS.
◦ They also play a role in catalysis of the trans-
esterification reactions.
◦ Note that while the full complement (U1, U2, U4, U5, U6) is
present in every cell and they are required for splicing, they
do not regulate the process. See splicing factors.
‣ Much like how general transcription factors are required
for transcription to occur, but its regulatory
transcription factors that determine whether or not it
occurs.
• Spliceosome: a very large macromolecular complex
consisting of five snRNPs and many other proteins, that
catalyses the splicing process.
◦ Sequence of assembly: (1) the recognition of splice sites by U1 and U2, (2) the
assembly of the other components, (3) the release of U1
and U4 to form a catalytically active complex, and (4)
splicing.
• Mechanism: two sequential trans-esterification reactions
◦ (1) The phosphodiester bond at the 5'ss is cleaved as a

, result of nucleophilic attack by the 2'-hydroxyl group of the branch-point
adenosine. This releases the upstream (5') exon and produces an intron lariat
intermediate (in which the 5' end of the intron is joined by a 2'-5' phosphodiester
◦ bond to the BPS-A).
◦ (2) The phosphodiester bond at the 3'ss is cleaved as
a result of nucleophilic attack by the free 3'-OH of the
liberated 5' exon. This releases the intron lariat and
simultaneously joins the exons by a 3'-5'
phosphodiester bond. The products are a mature
mRNA and the excised intron.

Alternative splicing: the mechanism by which different
mRNA transcripts, and thus multiple protein isoforms, are
generated from the same primary RNA molecule.
• The particular combination of exons spliced together is
dependent on each cell's complement of splicing factors
(SF): proteins involved in regulating the removal of introns from strings of mRNA.
◦ SR proteins are splicing activators. They bind to exonic splicing enhancers
(ESE) or intronic splicing enhancers (ISE) to promote the binding of U1 and U2.
‣ 'SR' because they are Serine- and Arginine-rich.
◦ Heterologous nuclear RNPs (hnRNPs) are splicing
inhibitors. They bind to exonic or intronic splicing silencer
(ESS or ISS) to inhibit the binding of U1 and U2.
• The complement in a specific cell can change with time,
development, environmental factors and tissue type. For
example, the pre-mRNA of the human alpha-tropomyosin
gene is spliced in a tissue-specific manner; strained muscle,
smooth muscle, fibroblast, and brain all produce different proteins from the same
RNA (because they have different amounts of the regulatory proteins).
• Modes of alternative splicing in eukaryotes
◦ Exon skipping: an exon may be spliced or retained.
◦ Mutually-exclusive exons: one of two exons is retained, but not both.
◦ Alternative 5' donor and 3' acceptor sites: multiple binding sites for U1 and
U2, respectively (ie. multiple sites they will recognise).
◦ Intron retention: a sequence may be spliced out as an intron or retained, but in
either case it contains an open reading frame (it is not flanked by introns, as in
exon skipping).
• We can study how RNA is being spliced in a specific cell using RT-PCR and
electrophoresis.
◦ Using reverse transcriptase-PCR, spliced RNA transcripts are converted to
cDNA and amplified.
‣ Extract RNA from cells of interest and convert them to cDNA (easier to work
with) by adding a poly-T primer (which binds the poly-A tail of the mRNA) and
reverse transcriptase (which binds the primer and adds dNTPs to synthesize

, cDNA).
‣ Treat mixture with RNase to degrade the RNA.
‣ Design PCR primers that lie outside the region of the transcript that is
‣ alternatively spliced (in the flanking exons), and add them to the cDNA.
‣ Use the cDNA as a template in the PCR reaction; polymerase amplifies the
target region exponentially.
‣ The resulting RT-PCR products vary in size depending on how they were
spliced.
◦ Using electrophoresis, the products are separated according to size and the
splicing events that generated the detected RNA can be identified.
‣ For example, the MINK1 gene is alternatively spliced by exon skipping. In
some tissues, the predominant band is shifted up (greater size; the exon is
mostly being included), while in others the predominant band is shifted
down.
‣ Note: DNA is microscopic - each band contains many copies of the target
DNA region. Thus PCR is very necessary.


2. Sex Determination in Drosophila
This serves as an example of regulation of gene expression by alternative mRNA
splicing.

Sex determination is different in humans and flies
• In humans, femaleness is the default state, and maleness oc
curs due to the presence of the SRY gene on the Y chromosome.
• In flies, maleness is the default state, and
femaleness occurs due to the presence of two X
chromosomes and the subsequent central
female-determining event: activation of the Sxl
gene in early embryogenesis.

Sex-lethal (Sxl) is the master sex-determining gene in Drosophila.
• It acts as a binary genetic switch to regulate sex
determination and sexual dimorphism. In summary:
◦ XX produce Sxl and initiate a SF cascade
culminating in Dsx-F production and female somatic
differentiation. It also blocks male dosage
compensation.
◦ XY proceed on the default pathway of male somatic
differentiation, and male dosage compensation is activated.
• Once activated it controls its own expression by a self-sustaining
positive feedback splicing mechanism to maintain the initial sex fate
choice.
• The gene contains two promoters from which transcription can be

, driven.
◦ Establishment promoter (Pe) - XSE
transcription factors bind
◦ this region to activate transcription of sxl
prior to cellular blastoderm formation.
◦ Maintenance promoter (Pm) - transcription factors bind this region to activate
transcription after cellular blastoderm formation.

Early Drosophila development
• Multinucleate syncytium stage: after fertilisation, the zygotic nucleus undergoes 13
rapid mitotic divisions in a single syncytium.
• Syncytial blastoderm stage: the nuclei migrate to the perimeter of the egg.
• Cellular blastoderm stage: the plasma membrane folds inward between the nuclei,
eventually partitioning off each nucleus into a single cell. The cells surround the yolk
sac. This occurs during the 14th division cycle, about 3 hours after fertilisation.

Sex determination is the culmination of the following cascade of
gene regulation.
(1) In early embryogenesis, females (and not males) produce small
amounts of early Sxl protein.
• a) Presence of two X-chromosomes activates the Sxl establishment promoter.
◦ SxlPe is activated by sufficiently high (threshold) concentrations of XSE
transcriptional activator proteins: Scute, Runt, Sisterless, and Unpaired.
◦ The genes that encode these transcription factors are X-linked and collectively
make up the X-chromosome signal elements (XSE, signal to the embryo how
many X-chromosomes it has).
‣ Three (scute, sisterliness, runt) directly encode transcription factors.
‣ One (unpaired) encodes a ligand that activates a maternally-supplied
transcription factor.
◦ This threshold response model predicts XX:AA as female (the XSE threshold is
reached/exceeded), XY:AA and X0:AA as male. BUT X:A haploids (rare) are
female because the formation of the cellular blastoderm is delayed by a single
division cycle to 15; they have sufficient extra time to accumulate XSE protein.
◦ The window of opportunity for SxlPe activation ends at cellular blastoderm stage
when rapid degradation of XSE mRNAs and proteins occurs.
• b) The activated promoter stimulates the transcription of the Sxl gene to produce
early Sxl transcripts and early Sxl protein. This small amount of protein synthesized
very early in development will go on to regulate post-transcriptional
splicing of the late Sxl transcript; it establishes a positive feedback
or auto-regulatory loop that ensure more synthesis later in female
development.

(2) In later embryogenesis, females (and not males) produce late Sxl
protein.

, • (a) Both females and males activate Sxl transcription from
the maintenance
• promoter.
◦ SxlPm is activated by other transcription factors that
are produced equally in XX and XY (unrelated to XSE
or the number of chromosomes)
• b) The activated promoter stimulates the transcription of the Sxl gene, producing
late Sxl transcripts. However, due to sex-specific splicing only females produce a
functional late protein.
◦ LSxl transcripts contain a premature stop codon (UAG) in exon 3.
◦ Intron sequences flanking exon 3 act as binding sites for ESxl protein (splicing
repressor). REALLY GOOD PICS ONLINE
◦ In females, the early Sxl protein acts as a splicing repressor; by binding these
sites it prevents the binding of U2 (to 3'ss of intron 2) and of U1 (to the 5'ss of
intron 3). This results in skipping of exon 3 and the production of a full-length,
functional Sxl protein.
◦ In males, exon 3 is included and the resulting protein is truncated with no
biological activity.
◦ This sex-specific splicing can be visualised in a RT-PCR experiment (see above;
using RNA from both XY and XX individuals). The
primers bind to exon 2 and exon 4 (either side of the
spliced exon 3). In XY, the band is shifted up which
represents a higher molecular weight and thus the
exon has been included. See pic.

(3) Females (and not males) produce functional transformer protein.
• a) Both females and males activate tra transcription and produce tra primary
transcripts. However, due to sex-specific splicing, only
females produce a functional tra protein.
◦ Transformer transcripts contain a premature UAG stop
codon in exon 2.
◦ In females, the late Sxl protein acts as a splicing
repressor; by binding the proximal 3'ss of intron 1, it forces U2AF to bind the
distal 3'ss instead ("alternative 3' acceptor site"), causing the 5' end of exon 2 to
be excluded from the mRNA. The result is productively spliced mRNA
translatable to a full-length, functional protein.
‣ Without any competition from Sxl, U2AF binds the proximal 3' splice site.
***See note below on how late Sxl drives alternative splicing of tra.
◦ In males, the entirety of exon 2 is included in the mature mRNA, which translates
into a nonfunctional protein.

(4) Females and males produce different versions of double-sex protein.
• a) Both females and males transcribe the dsx gene into dsx pre-mRNA. Sex-specific
splicing of this transcript produces two functional, but different (in terms of the

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