Student Reader » MIMG 132

Insertion Sequence Elements

Levi Clancy — Sun, 05 Feb 2012 18:53:32 +0000

Bacterial IS elements (Insertion Sequence) and equivalent eukaryotic elements have a diagnostic structure: a central protein coding region containing genes for transposition enzymes; flanking Inverted Repeats that serve as recognition sequences for the transposase; and distal flanking short direct repeats. The Inverted Repeats are part of the transposon and are the same for all copies in a genome. Distal flanking repeats are derived from the host DNA and are different at each site of insertion.

For an inverted repeat the two copies of the repeated sequence are on opposite strands of the DNA. They are thus read 5’ – 3’ in inverted orientation. Conversely, for a direct repeat the two copies of the repeated sequence are on same strand of the DNA and are read 5’ – 3’ in the same direction.

Excision	In the first step of transposition, the IS element is excised from the DNA at the outside edges of the inverted repeats, and the target DNA undergoes a staggered cleavage.
Ligation	In the second step, the excised IS element is ligated to the exposed 5’ ends of the target DNA.
Filling In	Filling in of the gaps left by the staggered cleavage and ligation results in complete insertion into the target with a duplication of the sequence between the staggered cuts.

Cell Cycle

Levi Clancy — Tue, 18 Oct 2011 15:53:33 +0000

Mitosis	Mitosis is a eukaryotic process whereby a cell divides to produce two daughter cells identical to itself. Mitosis is the nuclear division that results in two daughter nuclei whose genetic material is identical with that of the original nucleus. In multicellular organisms, somatic cells undergo mitosis while germ cells undergo meiosis. Prokaryotic cells lack a nucleus and divide by binary fission. The mitotic phase is a relatively short action-packed period of the cell cycle. It alternates with the much longer interphase, where the cell prepares itself for division. Interphase is divided into three phases, G1 (first gap), S (synthesis), and G2 (second gap). During all three phases, the cell grows by producing proteins and cytoplasmic organelles. However, chromosomes are replicated only during the S phase. Thus, a cell grows (G1), continues to grow as it duplicates its chromosomes (S), grows more and prepares for mitosis (G2), and then finally enters mitosis.
Meiosis	Meiosis is the nuclear division by which a reproductive cell with two equivalent chromosome sets divides twice to produce four meiotic products, each of which has only one set of chromosomes.
Binary Fission

Adenovirus Promoter Experiment

Levi Clancy — Sun, 04 Sep 2011 04:20:58 +0000

Promoters in E. coli were found using bacteriophages like λ and T7, which act very strongly to encode massive quantities of viral proteins. This same idea was expanded to eukaryotes, using adenoviruses (which infect eukaryotic cells). Viral genes express massive amount of proteins and a great model for finding examples of eukaryotic promoter sequences.

Nuclear Run-On	A modified in-vitro nuclear run-on experiment was performed on cells that had been infected with an adenovirus; the hybridization step is below. Viral genes express massive amount of proteins and a great model for finding examples of eukaryotic promoter sequences.
Centrifugation	The RNAs were separated by size using rate-zonal centrifugation. These adenoviral RNAs were added to a tube containing a gradient of sucrose with ↓ at the top and ↑ at the bottom. Centrifugation allowed the RNAs to be separated by weight (and, accordingly, by length).
Hybridization	The RNA segments were hybridized to endonuclease-digested adenovirus DNA. Logically, longer RNA bound more DNA segments, while shorter RNA segments bound less. A loose adenovirus genome map was made by recording which DNA segments were bound by RNA segments of increasing length.
Filtration	Hybridized DNA was obviously from coding regions and remained bound to the filter. Any ssDNA was thus a putative control region, and was eliminated from the filter. Unhybridized RNA was eliminated by RNase A digestion (which targets ssRNA).
Repetition	At various stages of the adenovirus life cycle, if a certain DNA segment never binds to RNA then it must not encode any RNA. If they are not coding regions, then these DNA segments must be control regions instead. A single adenovirus promoter was found for all late phase genes.
Synthesis	Regulation of TF synthesis (transcription of the TF gene).
Activity	Regulation of transcription factor (TF) activity (activators and repressors). Regulation of TF activity by interaction with small molecules (ligands), and post-translational modifications, especially phosphorylation and dephosphorylation. Regulates Nuclear transport, import and export; DNA binding to cognate DNA site; Interactions with co-activators.
Degradation	Regulation of TF degradation.

Prokaryotic Transcription

Levi Clancy — Sun, 04 Sep 2011 04:11:59 +0000

RNAP (RNAP) recognizes and binds to promoter region on dsDNA, forming the closed complex. Around the initiation site (+1), the DNA is unwound & becomes single-stranded; the RNAP/ssDNA structure is the open complex. The RNAP transcribes the DNA, but produces about 10 abortive (short, non-productive) transcripts which are unable to leave the RNAP because the exit channel is blocked by the σ-factor. The σ-factor eventually dissociates from the holoenzyme, and elongation proceeds. Most transcripts originate utilizing adenosine-5′-triphosphate (ATP), GTP being used less often at the +1 site. UTP & CTP (pyrimidines) are disfavored.

	The σ-subunit binds to the promoter region, then the core polymerase binds to it (a holoenzyme is a core-polymerase-σ-subunit complex). The promoter refers to the start point of transcription. Since transcription starts at +1, the promoter is between -35 and -10. The start site is relative to the promoter region. There is no primer. Three stop codons: UGA, UAG, UAA
	RNAP does everything, including helicase activity by unwinding closed compled DNA. Then the RNAP starts unwinding it into an open complex. Elongation, you just keep adding nucleotides to your mRNA.

	The RNAP runs along the DNA, synthesizing the complementary RNA in the process. In prokaryotes, the nascent mRNA is translated co-transcriptionally by ribosomes. Some proofreading occurs during this process: Pyrophosphorolytic editing – RNAP immediately removes incorrect pairs reversing the reaction that put them together. Hydrolytic editing – RNAP backtracks one or more bases to remove an incorrect pair, stimulated by Gre factors. In prokaryotes, this occurs in the cytoplasm which is why translation occurs at the same time.
σ Factor Fate	It is not favorable to have σ-factor on holoenzyme since core polymerase does not want to associate it. The sigma factor falls away before elongation. Therefore, the core polymerase performs elongation (not the holoenzyme).

Termination has two mechanisms: intrinsic (ρ-independent) & ρ-dependent. In intrinsic termination, a terminator sequence within RNA signal RNAP to stop. Terminator sequence is palindromic, & forms a stem-loop hairpin structure leading to dissociation of RNAP from DNA template. In rho-dependent terminatio, ρ binds & runs along the mRNA toward the RNAP. When they collide, it causes RNAP to dissociate from DNA (terminating transcription). ρ stops RNA synthesis at specific sites.

Rho dependent termination: rho uses it’s helicase activity to separate mRNA from DNA
Rho-independent termination:
- loop formed
- hairpin loop formed due to Gs and Cs wanting to bind together
- That association makes DNA-RNA hybrid unwind
- Due to weaknesses of DNA-RNA hybrids, and weak AU base pairs, hybrid falls apart

Eukaryotic Transcription

Levi Clancy — Sun, 04 Sep 2011 04:11:54 +0000

Eukaryotes have 3 RNAPs:

RNAPI makes rRNA
RNAPII makes mRNA
RNAPIII makes tRNA & small RNAs

Eukaryotes have 5 GTFs

Tata Binding Protein (TBP)
TFIIH
FT TFIID
TFIIB
TFIIH

Eukaryotic promoter has 4 parts

B Recognition Element
TATA box
Initiator
Downstream promoter element

	TBP recognizes and binds to the tatabox (-31 to -26). TBP is the σ of eukaryotes. Once bound, TBP recruits the other factors.
	TFIIH, a helicase, makes the DNA go from a closed to open complex. TFIIH phosphorylates the carboxy terminal domain of RNAP, causing a conformational change in RNAP which results in the factors being released (hydroxyl groups are phosphorylated; threonine, serine and tyrosine have 3 of them; serine and tyrosine are predominant on the carboxy terminal domain). TFIIH is a large General Transcription Factor (GTF) of 9 subunits, nearly as large as Pol II. Two of the subunits are homologous to DNA helicases, enzymes that use energy from ATP hydrolysis to separate the strands of a DNA double helix. One of the subunits is a kinase that phosphorylates Ser5 of the Pol II large subunit C-terminal domain (CTD) heptapeptide repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).

TBP recognizes and binds to the tatabox (-31 to -26). TBP is the σ of eukaryotes. Once bound, TBP recruits the other factors.

TFIIH, a helicase, makes the DNA go from a closed to open complex. TFIIH phosphorylates the carboxy terminal domain of RNAP, causing a conformational change in RNAP which results in the factors being released (hydroxyl groups are phosphorylated; threonine, serine and tyrosine have 3 of them; serine and tyrosine are predominant on the carboxy terminal domain). TFIIH is a large General Transcription Factor (GTF) of 9 subunits, nearly as large as Pol II. Two of the subunits are homologous to DNA helicases, enzymes that use energy from ATP hydrolysis to separate the strands of a DNA double helix. One of the subunits is a kinase that phosphorylates Ser5 of the Pol II large subunit C-terminal domain (CTD) heptapeptide repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).

The intial complex containing properly distorted DNA becomes the platform for assembly. TFIIB interacts specifically with TBP, and the promoter. Usually, RNAPII is preloaded with a variety of factors. As factors load onto this sort of open complex, a helicase protein is required to make this an open complex. This complex is tied to the promoter & keeps trying to commence transcription, but it is unable to go. It starts & makes short abortive-initiation transcripts about 10 base pairs long because it is ready to go but cannot. It has mousetail, the C-terminal domain tail is phosphorylated & the initiation factors are shed so that RNAPII remains on the RNA & can go beyond abortive initiation. Whether or not and how you phosphorylate this tail depends on the speed RNAP II CTD has YSPTSPS heptapeptide repeats. Each has sites for specific serine kinases (1 in TFIIH).

Phosphorylation enables the complex to shed initiation factors and proceed to elongation. In eukaryotes, transcription takes place in the nucleus (where the DNA is). This allows for the temporal regulation of gene expression via sequestration of RNA in nucleus, and allows for selective transport of RNAs to the cytoplasm, where the ribosomes reside. Further complexity is added by the multitude of transcripton factors and signaling pathways that may interact in combination to mediate cell-type and developmental transcriptional regulation. Primary (initial) mRNA transcripts in eukaryotic cells are synthesized as larger precursor RNAs that are processed by splicing out introns (non-coding sequences) and ligating exons (non-contiguous coding sequences) into the mRNA. Primary transcripts for some genes can be large.

RNA Polymerase

Levi Clancy — Thu, 01 Sep 2011 19:32:02 +0000

E. coli RNA Polymerase is α₂ββ’ω there are three eukaryotic RNA Polymerases. Binding of the RNA polymerase to the gene is highly regulated and requires lots of protein factors even for genes active in all cells. Transcription in eukaryotes requires the assembly of the RNA Polymerase into a very large initiation complex at the gene promoter. This is controlled by a large number of factors that effect both the packaging of the DNA and the binding of the polymerase.

Euk.	Prok.	Overview
Clamp		Binds DNA
Mg⁺ Ion		Important in catalysis of phosphodiester bond attack.
RPB 1	β’
RPB 2	β
α-like	α
Zipper
CTD		The carboxy terminal domain is very important for RNA processing. You have a protein and a peptide repeat and its serines can be phosphorylatd and then bind the RNA transcript and do RNA processing like cap, polyadenylation and more. CTD = (Tyr-Ser-Pro-Thr-Ser-Pro-Ser)n → n = ~26 in yeast, 52 in mammals Ser2 and Ser5 are phosphorylated and dephosphorylated.

Pol II requires the RNA polymerase II General Transcription Factors (Pol II GTFs) to transcribe virtually all eukaryotic protein coding genes. Purified RNA polymerase II cannot initiate transcription from a promoter without the assistance these proteins: (TFIIA); TFIIB; TFIID; TFIIE; TFIIF; TFIIH. These are required along with Pol II to transcribe virtually all eukaryotic protein coding genes.

Within TFIID is a TATA-box binding domain that binds the TATA-box; next, TFIIA binds upstream to TFIID and then TFIIB downstream to TFIID. This multi-protein complex binds RNA Pol II (forming the basal transcription complex) and transcription initiates. In genes without a TATA box, an initiator element binds TBP instead but without sequence specificity.

(TFIIA)
TFIIB
TFIID	Subunit. is TBP. TBP is associated with several other subunits in the general transcription factor TFIID. These are called TBP-Associated Factors, or TAFs. There is an anti-TBP that blocks initiation of transcription.
TFIIE
TFIIF
TFIIH	TFIIH is a large General Transcription Factor (GTF) of 9 subunits, nearly as large as Pol II. Two of the subunits are homologous to DNA helicases, enzymes that use energy from ATP hydrolysis to separate the strands of a DNA double helix. One of the subunits is a kinase that phosphorylates Ser5 of the Pol II large subunit C-terminal domain (CTD) heptapeptide repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).

Telomerase

Levi Clancy — Wed, 31 Aug 2011 23:57:03 +0000

What problem does the enzyme telomerase overcome? DNA replication requires an RNA primer to initiate synthesis, which is degraded after priming. The loss of these primers on the lagging strand of the chromosome ends will result in a loss of information with each round of replication. Telomerase is a special enzyme that uses its own RNA template to add telomeric repetitive DNA to chromosome ends.

Synthesis (S) Phase

Levi Clancy — Wed, 11 May 2011 16:31:10 +0000

Eukaryotic chromosomes are replicated from multiple origins. Initiation of replication from these origins occurs throughout S phase. Some origins fire in early S, some in late S phase. However, no eukaryotic origin initiates more than once per S phase.

S phase continues until replication from multiple origins along the length of each chromosome results in complete replication of the entire chromosome. These two factors (SCF and APC/C) ensure that the correct gene copy number is maintained as cells proliferate.

How do the S-phase cyclin-CDKs activate DNA replication? And how is the process of DNA replication regulated so that each origin fires one time, and only one time during S-phase? In eukaryotes, DNA replication at an origin initiates only one time during S-phase. What mechanism accounts for this? In proliferating cells, how is this block to re-initiation at a replication origin overcome in preparation for replication during the next S-phase?

The Origin Recognition Complex binds the origin of replication and then recruits replication proteins. Once S-cyclin+CDK activates the complexes, replication can commence. The DNA replication proteins are deactivated and/or fall off the genome, or actively move along the DNA (for example, the DNA Polymerase). These DNA replication complexes are not expressed so the DNA replication complex cannot reform.

The problem of reassembling the pre-replication complex is resolved later via mitotic cyclins, which activate expression of DNA replication proteins but leave the DNA replication complexes inactive. Mitotic cyclins are quickly degraded during telophase and thus do DNA replication complexes are not expressed during S-phase.

What mechanism accounts for the sudden onset of DNA synthesis in S. cerevisiae? Active S-cyclin+CDK phosphorylates and activates proteins that initiate DNA synthesis at origins of replication. However, S-cylin+CDK is inhibited by Sic1 while S-phase cyclin and S-phase CDK is being produced.

The inhibitor is then precipitously degraded by Late-G₁-Cyclin+CDK. This de-repression unleashes a massive wave of active S-cyclin+CDK, as opposed to a slow rise in activity that would have occurred without the repressor. This permits the sudden activation of large numbers of DNA replication complexes and thus the sudden onset of DNA synthesis.

BK Channel

Levi Clancy — Thu, 11 Jun 2009 04:33:09 +0000

BK channels (aka Slo channels) are tuned via alternative splicing of α subunit exons, thereby controlling regulatory properties, conductance and voltage sensitivity of the channel. BK Channels are present in muscle tissue and in the cochlea

Nuclear Envelope

Levi Clancy — Thu, 11 Jun 2009 01:53:54 +0000

The nuclear envelope consists of a lipid bilayer.

Long and fibrous lamin proteins form a layer of structural support for the nuclear envelope. Lamin is phosphorylated in prometaphase, causing a conformational change and the loss of laminal structural properties. Without laminal support, the nuclear membrane breaks apart and absorbs into the smooth endoplasmic reticulum. The endoplasmic reticulum breaks apart, but is bound to the lamin via the inner nuclear membrane Lamin B Receptor; and the lamin binds to chromatin.

As anaphase ends, dephosphorylation of existing lamin begins. Once the genetic material has fully segregated at the completion of anaphase, production of new lamin is well underway. The new lamin drags tubes of smooth endoplasmic reticulum across the surface of the chromatin; these tubes flatten and merge, forming a solid nuclear membrane. The endoplasmic reticulum and lamin detach themselves from the chromatin. In the mature daughter cell, the lamina is a continous layer that is bound to the inner membrane of the nuclear envelope by emerin proteins.

Mitosis: Ubiquitin Protein Ligases

Levi Clancy — Wed, 10 Jun 2009 18:44:19 +0000

Polyubiquitination marks eukaryotic proteins for degradation by proteasomes. Three enzymes are required for the Ubiquitin to work: E1, the Ubiquitin-activating enzyme; E2, the Ubiquitin-conjugating enzyme; and E3, the Ubiquitin ligase. SCF and APC/C are ubiquitin protein ligase complexes that that control three major transitions in the cell cycle: onset of S-phase through degradation of Sic1 by SCF; initiation of anaphase via degradation of securin by APC-Cdc20; exit from mitosis via degradation of cyclin B’s by APC-Cdh2. APC has several substrates that must be degraded at different times in the cycle; thus, its activity is directed by specificity factors that bind it. SCF only degrades Sic1 and thus its activity is regulated only by phosphorylation of its substrate.

Complex

Overview

SCF

Degradation of phosphoryated Sic1 or p27 to activate S-phase cyclin. SCF is a ubiquitin protein ligase needed for polyubiquitination and proteasomal degradation of phosphorylated Sic1. In contrast to the APC/C, the SCF ubiquitin-protein ligase is not regulated by phosphorylation or other modifications of specificity factors, but rather by phosphorylation of its substrate, Sic1.

S.F.	Substrate	Overview
None	Sic1	SCF degrades phosphorylated Sic1 (inhibitor of S-cyclin+CDK) to initiate S-phase.

APC/C

The anaphase promoting complex/cyclosome (aka APC/C) is a E3 ubiquitin protein ligase that is bound by various specificity factors that direct it to degrade different substrates at different times in the cycle.

S.F.	Substrate	Overview
Cdc20	Securin	Cdc20 directs APC/C to degrade Securin, initiating anaphase. Induces partial degradation of cyclin Bs.
Cdh2	Cyclin Bs	APC/C-Cdh2 initiates telophase by degrading S-phase and mitotic cyclins, thus allowing prereplication complexes to form at DNA replication origins. Degrades geminin in metazoans. Inactivated by G₁ -cyclin+CDK.

Mitosis: Cell Cycle Activators

Levi Clancy — Wed, 10 Jun 2009 18:42:40 +0000

Human	Yeast	Activates	Overview
CAK Kinase		Cyclin-CDKs	CAK positive phosphorylates CDK1, CDK2 and CDK4. CAK is itself a member of the CDK family and is composed of CDK7, cyclin H and an assembly protein Mat1. Checkpoint controls, inactivate Cdc25C and Cdc25a phosphatases to induce cell-cycle arrest. (link)
Cdc25 phosphatase		Cyclin-CDKs	Involved in activating MPF.
Cdc25a phosphatase		S-phase Cyclin-CDK	Activates vertebrate S-phase cyclin-CDK
Cdc25c phosphatase		Mitotic Cyclin-CDK	Activates vertebrate mitotic cyclin-CDK
Cdc14 phosphatase		Cdh2	Activating Cdh2 thus inhibits mitotic cyclin-CDK
ATM/ATR Kinases		Chk1/Chk2	Checkpoint controls, activate Chk1/Chk2 kinases

Mitosis: Cell Cycle Inhibitors

Levi Clancy — Wed, 10 Jun 2009 18:42:04 +0000

Human	Yeast	Inhibits	Overview
Wee1 Kinase		Cyclin-CDKs	Inhibitory phosphorylation of CDK1 and CDK2. (link
INK4		Mid-G1 CDKs	INK4s include cyclin-dependent kinase 4 and 6, which have important tumor suppression activity by inhibiting the mid G1 CDKs, thus inhibiting passage through G1. Both genes encoding INK4a are mutated in many human tumors, lessening their ability to block passage into G1.
p21, p27, and p57	Sic1	S-Phase Cyclin CDKs	Binds and inhibits S-phase cyclin-CDKs (p21, p27, and p57 inhibit Cyclin E-CDK2). Activated by G1 cyclin-CDK (CDK=Cdc28 in budding yeast). Yeast Sic1 is phosphorylated by G1 cyclin-CDKs, and must be phosphorylated at at least six sites by G1 cyclin-CDKs before it is bound sufficiently well by SCF to be polyubiquitinated. Each of these sites are relatively poor substrates for the G1 cyclin-CDKs. Sic1 must be phosphorylated by G1 cyclin-CDKs at at least six sites by G1 cyclin-CDKs before it is bound sufficiently well by SCF to be polyubiquitinated. Each of these sites are relatively poor substrates for the G1 cyclin-CDKs.
Mad2	Mad2	Anaphase	Mad1 binds to the kinetochore. When Mad2 binds to that then it is activated. (One unattached kinetochore is sufficient to inhibit all Cdc20 in the cell.) But when microtubules attach, the tension at the kinetochore leads to Mad2 degradation. If the sister chromatids do not attach to opposite poles, the spindle checkpoint is triggered. Mad2, which is normally bound to Mad1 in a Mad1/Mad2 complex then binds to the anaphase-promoting complex (APC) to form an APC/Mad2 complex. Binding to the APC prevents the formation of the APC/Cdc20p complex which is necessary to begin anaphase. The binding of Mad2 proteins to the APC effectively prevents the cell from transitioning into the anaphase until all of the chromatids are properly attached to opposite spindle pole bodies.
Rb			See below.
	Sic1	S-phase cyclin-CDKs
Cdc14 Phosphatase			When MPF acts during the transition form prophase to anaphase, Cdc14 phosphatase reverse these effects during late anaphase, telophase and interphase.
Securin			An inhibitor related to anaphase triggering.
Chk1/Chk2 kinases		Cdc25C/Cdc25a	Checkpoint controls, inactivate Cdc25C and Cdc25a phosphatases to induce cell-cycle arrest.

Mitosis: Vertebrate Checkpoints

Levi Clancy — Sun, 07 Jun 2009 23:02:49 +0000

Checkpoint	Overview
DNA Damage A	Midway through G₁, ATM/R activates p53, which activates p21^CIP, which blocks Mid-G₁-Cyclin+CDK (Cyclin-D+CDK4 & CDK6) if DNA damage is detected.
DNA Damage B	At the start of S-phase, ATM/R activates: p53, which activates p21^CIP, which blocks the late G₁ cyclin (Cyclin E) and the S-Phase cyclin (Cyclin A) if DNA damage is detected; and Chk1/2, which blocks Cdc25A if DNA damage is detected. Cdc25A would otherwise activate the CDK2, which binds the late G₁ cyclin (Cyclin E) and the S-phase cyclin (Cyclin A). Cyclin-E+CDK2 and Cyclin-A+CDK2 are needed to initiate S-phase.
DNA Damage C	Midway through S-phase, ATM/R activates: p53, which activates p21^CIP, which blocks Cyclin A (S-Phase cyclin) if DNA damage is detected; and Chk1/2, which blocks Cdc25A if DNA damage is detected. Cdc25A would otherwise activate the CDK2, which binds Cyclin A. Cyclin-A+CDK2 is needed during S-phase.
Intra-S-Phase	At the cusp of G₂ and M phase, ATR activates Chk1, which inactivates Cdc25C. Cdc25C would otherwise activate mitotic cyclins (Cyclin A and Cyclin B).
DNA Damage D	At the cusp of G₂ and M phase, ATM/R activates p53, which activates p21^CIP, which inactivates mitotic cyclins (Cyclin A & Cyclin B) if DNA damage is detected. Arrest in G2 allows DNA double-stranded breaks to be repaired before mitosis.
Spindle Assembly	In the spindle assembly checkpoint (aka metaphase checkpoint), mitotic arrest deficient 2 (aka Mad2) blocks metaphase until every single kinetochore has properly attached to spindle microtubules. Mad2 exists in an open conformation (Mad2^O) and a closed conformation (Mad2^C). Mad1 and Mad2^C form a tetramer that binds unattached kinetochores via the Mad1 subunit. Mad2^C of the kinetochore-bound tetramer can transiently bind Free Mad2^O. This transient interaction causes Mad2^O to bind and inactivate Cdc20, and to convert from Mad2^O→Mad2^C. Mad2^C-Cdc20 transiently interacts with additional free Mad2^O. This causes Mad2^O to bind and inactivate additional Cdc20, forming a second Mad2^C-Cdc20. The cycle repeats and free Mad2^O is quickly converted to Mad2^C-Cdc20. Binding of microtubules to the kinetochore displaces Mad1-Mad2^C tetramers. Free tetrameric Mad2^C cannot bind Mad2^O. Instead, free tetrameric Mad2^C binds p31^comet. p31 then binds the Mad2^C of the Mad2^C-Cdc20 complexes, resulting in release of active Cdc20. Just a few Mad1-Mad2^C tetramers bound to kinetochores can generate enough Mad2^C-Cdc20 to overcome p31 activity. Once all kinetochores have attached to microtubules (thus releasing all Mad1-Mad2^C tetramers), p31 activity predominates and active Cdc20 is released from Mad2^C. The active Cdc20 binds APC/C, and the APC/C-Cdc20 degrades securin, the inhibitor of separase. Free separase digests the Kleisin subunit of cohesin, breaking open the Smc1-Smc3-Kleisin ring and allowing sister chromatids to separate. Cohesin is a Smc1/Smc3/Kleisin heterotrimer that holds together sister chromatids, with Kleisin acting like a clasp.
Spindle Position	The spindle position checkpoint (aka the chromosome segregation checkpoint) blocks the onset of telophase by inactivation of Cdc14. Cdc14 would otherwise activate Sic1 and the degradation of Cyclin B’s by APC/C-Cdh2.

Telophase

Levi Clancy — Fri, 05 Jun 2009 04:21:56 +0000

How is the dismantling of the nuclear lamina during prophase and its reassembly during telophase accomplished? Where does the nuclear envelope go during mitosis? Long and fibrous lamin proteins form a layer of structural support for the nuclear envelope. Lamin is phosphorylated in prometaphase, causing a conformational change and the loss of laminal structural properties. Without laminal support, the nuclear membrane breaks apart and absorbs into the smooth endoplasmic reticulum. The endoplasmic reticulum breaks apart, but is bound to the lamin via the inner nuclear membrane Lamin B Receptor; and the lamin binds to chromatin.

Metaphase

Levi Clancy — Fri, 05 Jun 2009 04:21:42 +0000

How does the metaphase checkpoint prevent sister chromatid separation at the onset of anaphase until every kinetochore has become associated with spindle microtubules? In the spindle assembly checkpoint (aka metaphase checkpoint), mitotic arrest deficient 2 (aka Mad2) blocks metaphase until every single kinetochore has properly attached to spindle microtubules. Mad2 exists in an open conformation (Mad2^O) and a closed conformation (Mad2^C).

Mad1 and Mad2^C form a tetramer that binds unattached kinetochores via the Mad1 subunit.
Mad2^C of the kinetochore-bound tetramer can transiently bind Free Mad2^O.
This transient interaction causes Mad2^O to bind and inactivate Cdc20, and to convert from Mad2^O→Mad2^C.
Mad2^C-Cdc20 transiently interacts with additional free Mad2^O.
This causes Mad2^O to bind and inactivate additional Cdc20, forming a second Mad2^C-Cdc20.
The cycle repeats and free Mad2^O is quickly converted to Mad2^C-Cdc20.
Binding of microtubules to the kinetochore displaces Mad1-Mad2^C tetramers.
Free tetrameric Mad2^C cannot bind Mad2^O. Instead, free tetrameric Mad2^C binds p31^comet.
p31 then binds the Mad2^C of the Mad2^C-Cdc20 complexes, resulting in release of active Cdc20.

Just a few Mad1-Mad2^C tetramers bound to kinetochores can generate enough Mad2^C-Cdc20 to overcome p31 activity. Once all kinetochores have attached to microtubules (thus releasing all Mad1-Mad2^C tetramers), p31 activity predominates and active Cdc20 is released from Mad2^C. The active Cdc20 binds APC/C, and the APC/C-Cdc20 degrades securin, the inhibitor of separase. Free separase digests the Kleisin subunit of cohesin, breaking open the Smc1-Smc3-Kleisin ring and allowing sister chromatids to separate. Cohesin is a Smc1/Smc3/Kleisin heterotrimer that holds together sister chromatids, with Kleisin acting like a clasp.

Nonsense-Mediated Decay

Levi Clancy — Fri, 05 Jun 2009 04:19:00 +0000

Human disease mutations that create nonsense mutations do not always produce a truncated protein. Often they lead to rapid mRNA degradation via Nonsense-Mediated Decay. During splicing in the nucleus, the Exon Junction Complex, containing 4 proteins, is deposited ~20 nt upstream from each exon/exon junction in the final mRNA. This marks the junctions and stays on the RNA as it is transported to the cytoplasm and goes through the first round of translation. The EJC is thought to be displaced from the mRNA during translation.

If translation terminates >50 nt upstream of an EJC, it is not displaced and signals for that mRNA to be degraded. Nonsense mutations generally lead to an early stop codon, so this means that most exonic nonsense mutation will be degraded. The EJC recruits a protein called UPF1 to the mRNA. This in turn stimulates binding of decapping enzymes and deadenylation, leading to rapid degradation. Nonsense mediated decay is advantageous because it prevents the production of truncated and potentially dominant negative mutant proteins in the heterozygous state.

Note that the normal termination codon is almost always in the last exon of a gene. Nonsense mutations in this exon do not lead to nonsense mediated mRNA decay and produce slightly truncated proteins.

Mitosis Promoting Factor

Levi Clancy — Fri, 05 Jun 2009 04:36:31 +0000

Cycles in M-Phase Promoting Factor (MPF) activity control mitosis. As a protein kinase, MPF likely acts via phosphorylation of the major histone protein H1 and the major nuclear envelope protein lamin. This leads to the degradation of the nuclear envelope and condensation of chromatin into chromosomes in anticipation of mitosis. Found in all organisms, MPF is a heterodimer composed of cyclin-dependent kinase (cdk1) and cyclin B.

Cycles of MPF activity are based on synthesis and degradation of cyclin B. During the M phase (mitosis) the heterodimer is functional and drives cells into metaphase. Cyclin B is degraded, inactivating MPF once cells go into mitosis. During the S phase, newly synthesized Cyclin B from maternal mRNA leads to formation of a new functional MPF heterodimer.

Cytoplasm from mammalian cells arrested in metaphase of mitosis by treatment with drugs that inhibit the polymerization of microtubules (e.g. colchicine or nocodazole) had high oocyte maturation promoting factor (MPF).

When the ctoplasm from Xenopus eggs was injected into the cytoplasm of mammalian cells in G1, it caused the mammalian cells to undergo the events of early mitosis: chomosome condensation and nuclear envelope break down.

Also, when the cytoplasm from mammalian cells arrested in mitotic metaphase by treatment with micrtotubule inhibitors was injected into the cytoplasm of mammalian cells in G1, it caused the mammalian cells to undergo the events of early mitosis: chomosome condensation and nuclear envelope break down.

This is like the mitosis promoting activity first observed in the cell fusion experiments.

So, Xenopus ooctye MATURATION PROMOTING FACTOR = mammalian MITOSIS PROMOTING FACTOR.

(Fortunatley, “MPF” is the abbreviation for both Maturation Promoting Factor (revealed by injection into Xenopus oocytes) and Mitosis Promoting Factor (revealed by fusing an M-phase cell to a cell in G1).

Oocytes remain arrested in G2 until they mature and addition of a sperm nucleus (via fertilization when in vivo). This is a great model for figuring out what the egg must have synthesized beforehand for mitosis to occur.

Step	Overview
Preparation	G₂-arrested frog oocytes are arrested and suspended in buffer. They are passed through an electric field, which stimulates the oocytes to mature into eegs. The buffer is removed and the eggs are powerfully centrifuged, tearing them apart into three different layers resembling a parfait: lipid at top; cytoplasm in the middle; and yolk at the bottom. The layer of cytoplasm is then extracted from this egg parfait.
Sperm Nuclei	When sperm chromatin is added to an egg extract, a nuclear envelope forms around the sperm chromatin and the chromosomes decondense (30 min), then the chromosomes condense and the nuclear envelope breaks down (60 min), then the chromosomes decondense, a nuclear envelope forms around the chromosomes and the DNA is replicated (80 min).

Treatment	Overview
Untreated	When sperm nuclei were added to untreated egg extract, mitotic cycles ensued as expected.
RNase	When egg extract was first treated with RNase and sperm nuclei were added, chromosomes did not condense and no new proteins were synthesized. RNase degrades mRNA but leaves tRNA and rRNA intact (needed for transcription and translation).
RNase + cyclin B mRNA	When cyclin B mRNA was added to egg extracted treated with RNase, then mitosis ensued as expectd. Thus, cyclin B is the only protein that must be synthesized in the egg extract for cycles of mitosis to ensue after fertlization.
RNase + nondegradable cyclin B mRNA	(nondegradable cyclin B mRNA refers to mRNA encoding a cyclin B mutant that can’t be degraded). These results demonstrate that cyclin B must be degraded for cells to exit mitosis: chromosome decondensation and formation of a nuclear envelope.

Results	A cyclin is periodically synthesized and degraded in the egg extract. When MPF activity is assayed (here by the simple histone H1 kinase assay), MPF activity peaks when the cyclin concentration peaks. Nuclear envelope breakdown and chromosome condensation occur when MPF activity peaks.

Eukaryotic Cell Cycle

Levi Clancy — Fri, 05 Jun 2009 04:35:07 +0000

Step	Overview
Growth 1	In a diploid eukaryotic cell, there are two versions of each chromosome, one from the mother and another from the father. The two corresponding chromosomes are called homologous chromosomes. Homologous chromosomes need not be genetically identical. During growth 1 (G1), an interphase, phase is the normal growth phase. Chromosomes are highly decondensed in most regions, allowing access of regulatory proteins to the DNA. Within the nucleus, individual chromosomes are found within diffuse but non-overlapping domains.
S Phase	Synthesis (S) DNA synthesis occurs. Results in 4n chromosomes in diploid organisms (like vertebrates); 2n chromosomes in haploid organisms (like yeast). n = number of distinct types of chromosomes. During the S phase of the cell cycle chromosomes are replicated to produce two complete copies of each. DNA replication results in an identical copy of each chromosome. These copies are called sister chromatids. Together, these chromatids are considered one chromosome. When separated, though, each sister chromatid is a chromosome. The 2 copies of the original chromosomes are called sister chromosomes.
Growth 2	During Growth 2 (G2), another interphase, the cell doubles in size. Centromeres (Microtubule Organizing Centers–MTOCs) form.
Mitosis	When conditions are good, the cell will replicate.

Centrosome

Levi Clancy — Fri, 05 Jun 2009 04:28:28 +0000

Microtubule organizing centers (aka centrosomes) are composed of asters at each end, with centrosomes spanning between them. The tubules that connect to the chromosome kinetochore are called kinetochore microtubules, while the tubules which interact with each other are polar microtubules (aka non-kinetochore microtubules). Microtubules are composed of α- and β-tubulin monomers polymerized to form hollow tubes. Depolymerization shortens the microtubules, while the chromosomes simultaneously migrate toward the asters, driving apart the spindle poles.

Mitosis: Biochemical Pathways

Levi Clancy — Fri, 05 Jun 2009 02:45:23 +0000

An obvious advantage of proteolysis for controlling passage through these critical points in the cell cycle is that protein degradation is an irreversible process, ensuring that cells proceed irreversibly in one direction through the cycle.

Spindle Assembly Checkpoint
Step	Initiation	Overview
Early G1		DNA prepreplication complexes assemble at origins. However, they are not activated. Mitotic cyclin-CDKs activate early steps in mitosis.
G₀	Most cells pause midway at a so-called G₀ interphase to carry out their functions for most or all of their existence.
Mid G1	G1 cyclin-CDK inactivates human Cdh2 and human inhibitors of cyclin-dependent kinase 4 and 6 (INK4s), important tumor suppressors that inhibit passage through G1 by inhibiting the mid G1 cyclin-CDKs. Both genes encoding INK4a are mutated in many human tumors, as they are less able to inhibit passage into G1.	E2F is a critical transcription factor that is bound by its inhibitor, Rb. Mid G1 Cyclin-CDKs phosphorylate Rb, thus releasing it from E2F. This activated E2F stimulates expression of late G1 cyclin and CDK, S phase cyclin, and factors needed for DNA synthesis.
Late G1	E2F stimulates its own transcription, and the Late-G1-cyclin+CDK can phosphorylate Rb; this forms a positive feedback loop whereby mid-G1 cyclin-CDKs are no longer needed to enter S-phase.	Late-G₁-cyclin+CDK activates expression of S-cyclin+CDK subunits. However, S-cyclin+CDK is promptly bound by its inhibitor Sic1.
S Phase	Late-G₁-cyclin+CDK phosphorylates the S-cyclin+CDK inhibitor (Sic1). This is a checkpoint: once the inhibitor is phosphorylated, G₁-cyclin+CDKs are no longer needed for entry into S-phase. SCF degrades phosphorylated Sic1.	After SCF degrades Sic1 and thereby de-represses S-cyclin+CDK, S-cyclin+CDK is free to activate pre-replication complexes at the DNA origins. Active S-cyclin+CDK phosphorylates and activates proteins that initiate DNA synthesis at origins of replication. However, S-cylin+CDK is inhibited by Sic1 while S-phase cyclin and S-phase is being produced. The inhibitor is then precipitously degraded by Late-G₁-Cyclin+CDK. S-cyclin+CDK is suddenly active en masse as a sudden event, rather than a slow rise in activity that would have occurred without the repressor. This permits the sudden activation of large numbers of DNA replication complexes. During the S phase, newly synthesized Cyclin B from maternal mRNA leads to formation of a new functional MPF heterodimer.
G2		Cdc25 phosphatase activates mitotic-cyclin+CDK (aka MPF).
Prophase	MPF activates early mitotic events.	Synthesis of mitotic cyclin leads to high MPF activity.
Metaphase	MPF drives cells into metaphase.	There are high levels of mitotic cyclin during metaphase, which results in high levels of MPF (mitotic-cyclin+CDK) activity.
Chromsome Segregation Checkpoint
Anaphase	APC-Cdc20 degrades phosphorylated securin, an inhibitor bound to separase. Separase then digests cohesin’s Kleisin subunit, allowing sister chromatids to separate.
Telophase	APC-Cdh2 degrades mitotic cyclins	When sister chromosomes have moved apart sufficiently to ensure their complete separation into the two daughter cells, Cdc14 phosphatase activates CdcA phosphatase, which activates APC/C-Cdh2 to degrade mitotic cyclins. Thus, APC and Cdc14 phosphatase induce late steps in mitosis, telophase and cytokinesis. This results in low levels of mitotic cyclin and thus low MPF activity.
Cytokinesis

Mitosis: Other Mitotic Factors

Levi Clancy — Fri, 05 Jun 2009 02:45:22 +0000

Factor	Overview
E2F	E2F is a transcription factor that by itself activates transcription.
Rb	Rb binds to E2F and represses its activation function. Rb is deactivated upon phosphorylation by mid G1 cyclin-CDKs (and, eventually, late G1 cyclin-CDKs).
Cohesin	Cohesin is a heterotrimeric complex of Smc1, Smc3 and Kleisin (Scc1). Smc1 and Smc3 form a circle that is clasped together by Kleisin. This ring fits over the chromatin near the centromere, remaining snugly over the sister chromatids after DNA replication.

Mitosis, Part III: Detail of Mitotic Events

Levi Clancy — Fri, 05 Jun 2009 02:45:20 +0000

	Mitotic Events

Process	Genomic	Cellular
Prophase	The genome condenses. Visible chromosomes form. Each chromosome has 2 sister chromatids bound at the centromere by cohesin.	Spindle fibers (aka spindle poles) emanate from the centromere. The two centrosomes form G₂ sprout microtubules by polymerizing free-floating tubulin. The microtubules repel each other, pushing the centrosomes to opposite ends of the cell. This microtubular network is the start of the mitotic spindle. The nuclear double-membrane begins to reabsorb into the endoplasmic reticulum; when this finishes in prometaphase, the cell no longer has a nucleus.
Prometaphase	Each sister chromatid forms a kinetochore at its centromere as a place for microtubules to latch onto the chromosome (thus, there are two kinetochores per centromere). With the nuclear envelope gone, microtubules invade the nuclear space and (once the centromere’s spindles are sufficiently long) clasp onto the kinetochore. The kinetochore has an ATP-dependent motor that is activated by the microtubules. Kinetochore motors push each chromosome along the microtubules toward the metaphase plate. The metaphase plate is an imaginary plane that is perpendicular to the plane between the two centrosomes. Microtubules not bound to a kinetochore find and bind other “free” microtubules from the opposite centrosome; this forms the mitotic spindle.
Metaphase	The chromosomes have aligned such that their centromeres convene on metaphase plate. This precise alignment is due to opposing kinetochores functioning like equally strong people in a tug of war. Some species’ chromosomes do not align, but instead move randomly back and forth between the poles before roughly lining up at the metaphase plate. The mitotic spindle checkpoint refers to an arresting signal emitted by any kinetchores still unattached to microtubules. This is important because proper chromosomes separation in anaphase requires attachment of every kinetochore to many microtubules. Anaphase commences only once every kinetochore has attached to a cluster of microtubules, with every chromosome thus lined up along the metaphase plate.
Anaphase	Sister chromatids separate, resulting in two distinct populations of genetic material (one at each centrosome) that are identical. This occurs in two steps sometimes labeled early anaphase and late anaphase. Also, cytokinesis begins in anaphase and ends in telophase. Cohesin is cleaved, and sister chromatids thus become sister chromosomes. Sister chromosomes are pulled apart toward their attached centrosome via shortening of kinetochore microtubules. The non-kinetochore microtubules elongate, pushing the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell.
Telophase	The nonkinetochore microtubules continue to lengthen, elongating the cell even more. Sister chromosomes attach to opposite ends of the cell. Fragments of the parental nuclear envelope attach around each set of separated sister chromatids. Each set of nuclei, now surrounded by a new nuclear envelope, unfold back into chromatin. The cleavage furrow forms.
Cytokinesis	Often, (mistakenly) thought to be the same process as telophase cytokinesis, if slated to occur, is usually well under way by this time. In animal cells, a cleavage furrow develops where the metaphase plate used to be, pinching off the separated nuclei. In plant cells, vesicles derived from the Golgi apparatus move along microtubules to the middle of the cell, coalescing into a cell plate that develops into a cell wall, separating the two nuclei. Each daughter cell has a complete copy of the genome of its parent cell. Mitosis is complete.
Growth 1	Each chromosome is copied during S phase, forming two identical sister chromatids that then separate into the future daughter cells during anaphase. Thus, each generation has exactly as much DNA as its predecessor.	Upon completion of cytokinesis, the two daughter cells are identical to their predecessor and enter G1, an interphase. Most cells spend the majority of the cell cycle in G1; some animal cells never undergo mitosis.

This is called open mitosis, and it occurs in most multicellular organisms. Some protists, such as algae, undergo a variation called closed mitosis where the microtubules are able to penetrate an intact nuclear envelope.

Temperature Sensitive Mutant Experiment

Levi Clancy — Thu, 04 Jun 2009 21:32:17 +0000

Mutations often render a protein unstable, meaning that it can function at lower temperatures but will quickly fall apart at higher temperatures. This feature can be manipulated to identify genes important in different processes; at a lower temperature, the mutant will be wild-type; at a higher temperature, the mutant will reveal its phenotype. Experiments that make use of this are temperature-sensitive mutant experiments. The follow temperature-sensitive mutant experiment identified genes important in identifying genes relevant to cellular division in S. cerevisiae (baker’s yeast). Temperature sensitive mutants are often denoted with a ^ts superscript.

Step

Overview

Mutagen

Add a mutagen to a liquid culture of yeast, then distribute the culture into smaller aliquots. Incubate at 23°C for 5 hours. Next, plate each aliquot onto an agar plate and incubate overnight at 23°C. This provides colonies which will later be screened for temperatures sensitivity.

Screen

Make two replica-plates of each plate prepared earlier: one will be incubated at 23°C; the other will be incubated at 36°C. Colonies which form at 23°C (the permissive temperature) but not at 36°C (the non-permissive temperature) represent temperature-sensitive mutants.

Assay

Colonies are microscopically examined to find microbes that are alive, but arrested at some point in the cell cycle. Some colonies may show no signs of division, while other colonies may be stuck midway through division.

Complement

Complementation tests determine whether different recessive mutations are in the same gene. It was accomplished by mating together temperature-sensitive haploid yeasts, then plating the diploid offspring and examining for growth at 23°C and 36°C.

Mating of Colony 1 and Colony 2		Mating of Colony 2 and Colony 3
This formed wild-type progeny that grew at both 23°C and 36°C. Being wild-type, the diploid progeny had at least one healthy allele for every gene. Thus, the mutations in Colony 1 and Colony 2 were on different genes.		This formed mutant progeny that only grew at 23°C. Being mutant, the diploid progeny had a gene with two non-functioning alleles. Thus, the mutations in Colony 2 and Colony 3 were on the same gene.

Transformation

Wild-type yeast DNA was used to form a plasmid library, which was then used to transform the temperature-sensitive cell cycle mutants isolated earlier. These transformed cells were plated and grown at 35°C — if growth was observed, then the mutant must have been transformed with a functional version of its nonfunctional gene. The plasmids were then extracted and sequenced so that the genes involved in cell cycle mutations could be compiled.

Results

First, this experiment identifies any temperature-sensitive mutation that inhibits colony formation. However, these mutations are not just those which directly stop cell division; they could also be lethal mutations. Thus, temperature-sensitive mutants were examined microscopically to ensure that they were indeed alive but unable to divide. Some mutants were even arrested midway through division. Next, these temperature-sensitive mutants of cell division were consolidated via complementation. This allowed mutants to be organized based on whether they contain a mutation in the same gene. However, the mutated gene was still unknown. Lastly, transformation with a plasmid library allowed identification of the precise genes that had blocked the cell cycle.

Drosophila Sex-Lethal (Sxl) Protein

Levi Clancy — Mon, 25 May 2009 09:02:09 +0000

Sex-lethal is a sequence-specific RNA binding protein that recognizes a specific UGUUUUUUU element in its target RNAs. It has a Β1,2,3 & 4 domains as well as RNA recognition motifs RRM1 and RRM2. The presence or absence of Sxl in an early embryo will determine whether it develops as a male or a female.

Early Female Embryo

There is transcription from the Sxl P_E promoter. An mRNA is encoded starting at the E1 exon. A functional Sxl protein is expressed.

Late Female Embryo

The P_L promoter is activated. Its ORF is different than P_E and L3 now has a premature stop codon. However, Sxl^early binds near L3 to block U2Af from binding the 3′ splice site. Thus L3 (and its stop codon) is skipped in females. Functional Sxl is expressed.

Early Male Embryo

No transcription from P_E. No Sxl expressed.

Late Male Embryo

The P_L promoter is activated. There is no Sxl^early, so the stop codon in L3 leads to expression of a truncated and inactive Sxl.

Sxl and Tra

The female produced Sex-lethal protein also regulates splicing of Transformer (Tra), the next gene downstream in the Sexual Differentiation Pathway.

An exon in Tra pre-mRNA has two 3’ splice sites. U2AF has higher affinity for the primary upstream 3’ splice site, so splicing occurs there. However, this polypyrimidine tract also contains a high affinity Sxl binding element allowing Sxl to bind if it is present.

Cochlear Hair Cells

Levi Clancy — Mon, 25 May 2009 03:40:56 +0000

Cochlear Hair Cells are tuned to respond to different sound frequencies. These cells are arrayed in a tonotopic gradient, with low frequency responders at the apical end of the cochlea and high frequency responders at the basal end. Birds and reptiles uses alternative splicing of BK Channels as one facet of tuning these hair cells to transduce different sound frequencies. Isolation of cochlear cell mRNA has revealed that each cell expresses a different subset of BK Channel mRNA.

Alternative Splicing

Levi Clancy — Mon, 25 May 2009 01:15:50 +0000

Humans	3×109 base pairs	~25,000 genes
Fruit fly	1.2×108 base pairs	~13,600 genes
Round Worm	9.7×107 base pairs	~19,100 genes
Bakers Yeast	1.2×107 base pairs	~6000 genes

Does gene number correlate with complexity? As shown to the left, it obviously does not. Rather, alternative splicing correlates with complexity. 95% of Human genes are known to exhibit alternative splicing. Complex Transcription Units use alternative splicing to produce more than one type of mRNA. Patterns of RNA spliciing can combine to produce a dizzying array of alternatively spliced isoforms.

The Slo or Bk channel has over 500 isoforms.the neurexin protein has 2,346 isoforms; the para-sodium channel has 1,536 isoforms; the Drosophila DSCAM Receptor can potentially be made in 38,016 different spliced isoforms. Somatic sexual development in Drosophila is controlled by a cascade of splicing factors each regulating the splicing of genes downstream in the pathway. And cochlear hair cells are tuned to respond to different frequencies via alternative RNA splicing.

Alternative RNA splicing is an important aspect of gene control in metazoans (multicellular animals). Two general molecular mechanisms can regulate the use of alternative splice sites in complex transcription units: repress exon inclusion (as Sxl does); and promote exon inclusion (as Tra does). Sxl sits on L3 and blocks assembly of spliceosome components, thus causing the entire exon to get skipped. Tra stabilizes the exon recognition complex by binding SR proteins, thus causing an exon to get included that otherwise would not have been.

Sxl sits on L3, forming a functional L1-L2-L4 Sxl protein. This Sxl sits on Tra’s 2^nd exon and a function Tra protein is expressed composed of Exons 1 and 3. Rbp1 and Tra2 sit at the 5′ end of the 4^th Dsx exon, promoting splicing at that region and thus inclusion of Exon 4 (to encode a functional Dsx protein).

There are many examples of regulation in mammals where important changes in gene activity are regulated by alternative splicing. These systems use pre-mRNA binding proteins to enhance or repress particular splicing choices. These proteins have different types, numbers, and arrangements of RNA binding domains, and other domains that can be involved in protein/protein interactions.

The RNA binding domains target the proteins to specific short sequence elements adjacent to sites of regulation. Alternative splicing is especially common in the mammalian nervous system, where it is used to diversify many proteins important for neuronal development and function. Small cassette exons create short peptide inserts that determine precise changes in ligand binding, electrophysiology, or subcellular targeting.

BK Channels	Cochlear cells are tuned use alternative splicing of α subunit exons.
Sxl Protein	Drosophila use alternative splicing for sex differentiation via Sxl protein.

Spinal Muscular Atrophy

Levi Clancy — Sun, 24 May 2009 23:47:08 +0000

Spinal Muscular Atrophy is the most common genetic cause of infant death, occurring in 1 in 6,000-10,000 births. This is an autosomal recessive disorder caused by mutations in the SMN1 gene, encoding a protein involved in snRNP maturation. Humans have undergone an SMN gene duplication generating the SMN2 gene. Even though it encodes an identical protein, the presence of SMN2 cannot compensate for the loss of SMN1 in spinal muscular atrophy, because its splicing is abnormal and it does not produce functional protein.

Splicing targeted therapies are being tested for Spinal Muscular Atrophy. A single C to T mutation in an exonic splicing enhancer of SMN2 exon 7 causes exon skipping and production of a truncated protein from 80% of the SMN2 mRNA. If drugs or other therapies could be found that increase exon 7 splicing of SMN2, one could potentially treat the disease even without correcting the SMN1 defect.

mRNA Splicing Aberrations

Levi Clancy — Sun, 24 May 2009 22:54:36 +0000

Nearly all disease genes have mutant alleles which affect splicing of the mRNA, rather than protein coding. Exon skipping can arise via mutations in the 5′ splice site, the 3′ splice site, and/or exonic or intronic splicing enhancers. Activation of cryptic splice sites (thus shortening the exon) can arise via mutated exonic splicing enhancers. Examples of diseases due to mRNA splicing errors include Familial Isolated Growth Hormone Deficiency (mutations in the GH-1 gene), Cystic Fibrosis (mutations in the CFTR gene), Spinal Muscular Atrophy and some thalassemias (mutations in the β-globin gene that activate a cryptic 3′ splice site).

Self-Splicing Introns

Levi Clancy — Wed, 20 May 2009 04:31:21 +0000

Some mitochondrial, protist and archaeal genes have autocatalytic self-splicing introns (as opposed to nuclear spliceosome-dependent introns). These introns fold into specific secondary and tertiary structures that catalyze their own excision, without proteins. There are two families of these introns — Group I and and Group II — and Group II autocatalytic introns are thought to operate with a similar mechanism to the spliceosome catalytic center.