Sex Determination
SEX DETERMINATION.................................................................................................................................... 1
SPLICING..............................................................................................................................................................2
The Spliceosome:............................................................................................................................................2
Assembly of the spliceosome:...................................................................................................................................2
How do U1 and U2 know where the 3’ and 5’ ends of the introns are?....................................................................2
ALTERNATIVE SPLICING:..........................................................................................................................................2
5 modes of alternative splicing in eukaryotes:...............................................................................................3
ACTIVATION OF SPLICING:.......................................................................................................................................3
How do we study splicing?.............................................................................................................................3
SEX DETERMINATION IN DROSOPHILA.......................................................................................................... 4
X-CHROMOSOME SIGNAL ELEMENTS (XSES)...............................................................................................................4
THRESHOLD RESPONSE MODEL.................................................................................................................................4
SEX LETHAL (SXL) GENE...........................................................................................................................................4
Sxl and alternative splicing:............................................................................................................................5
Structure:........................................................................................................................................................5
Splicing of the late Sxl pre-mRNA in XX flies:..................................................................................................5
CURRENT SUMMARY:................................................................................................................................................5
Late Sxl protein...............................................................................................................................................5
How does late Sxl direct alternative splicing of tra?......................................................................................6
........................................................................................................................................................................6
Gel-Shift Assays..............................................................................................................................................6
EXPERIMENT 1&2................................................................................................................................................. 7
Transformer Protein.......................................................................................................................................8
Dsx (double sex) Transcription Factor............................................................................................................8
SUMMARY OF SEX DETERMINATION IN DROSOPHILA...............................................................................................9
SEX DETERMINATION IN OTHER INSECTS....................................................................................................... 9
MEDFLY...............................................................................................................................................................9
HONEYBEES........................................................................................................................................................11
CONCLUSIONS............................................................................................................................................. 12
, Sex Determination
Splicing
- Splicing = the removal of introns from primary RNA & joining together of exons
- Involves 2 trans-esterification reactions
o Nucleophilic attack at 3’ end of intron
o Nucleophilic attack at 5’ end of intron forms Lariat loop
o Lariat released
The Spliceosome:
- Massive macromolecular complex containing 5 small nuclear ribonucleoproteins
(snRNPs) and up to 300 other proteins
- The snRNPs are called U1, U2, U4, U5 & U6
- Each contains a single short RNA molecule (100-300 nucleotides in length)
- RNA mols in snRNPs play 2 major roles:
1. Play a role in recognizing intron-exon boundaries
2. Play a role in the catalysis of trans-esterification reactions
- Assembly of the spliceosome:
1. Splice site definition
a. U1 is snRNP responsible for defining 3’ splice site and U2 defines 5’ splice
site
2. Spliceosome assembly
a. Catalytically inactive at this point
3. Formation of catalytically active complex (Only becomes active when U1 and U4
are released) and splicing occurs.
How do U1 and U2 know where the 3’ and 5’ ends of the introns are?
- Recognition of intron-exon boundaries through specific nucleotide sequence motifs
- U1 recognizes dinucleotide sequence G-U at 5’ end of every intron
- Associated factor of U2 (U2AF35) recognizes dinucleotide sequence AG at 3’ end of
every intron.
- Associated factor of U2 (U2AF65) recognizes pyrimidine rich tract upstream of
U2AF35.
- Branch-Point site upstream of U2AF65 is recognition site for snRNP U2 itself.
- ALL 4 of these motifs have to be PRESENT and SPACED CORRECTLY for splice sites to
be defined.
o i.e. only introns with all 4 of these motifs in the right place will be spliced out
Alternative Splicing:
- Splicing of a primary RNA molecule into different mRNA variants that (usually)
encode different proteins.
- Different proteins formed might differ in function, stability, ability to interact with
other proteins in the cell etc.
SEX DETERMINATION.................................................................................................................................... 1
SPLICING..............................................................................................................................................................2
The Spliceosome:............................................................................................................................................2
Assembly of the spliceosome:...................................................................................................................................2
How do U1 and U2 know where the 3’ and 5’ ends of the introns are?....................................................................2
ALTERNATIVE SPLICING:..........................................................................................................................................2
5 modes of alternative splicing in eukaryotes:...............................................................................................3
ACTIVATION OF SPLICING:.......................................................................................................................................3
How do we study splicing?.............................................................................................................................3
SEX DETERMINATION IN DROSOPHILA.......................................................................................................... 4
X-CHROMOSOME SIGNAL ELEMENTS (XSES)...............................................................................................................4
THRESHOLD RESPONSE MODEL.................................................................................................................................4
SEX LETHAL (SXL) GENE...........................................................................................................................................4
Sxl and alternative splicing:............................................................................................................................5
Structure:........................................................................................................................................................5
Splicing of the late Sxl pre-mRNA in XX flies:..................................................................................................5
CURRENT SUMMARY:................................................................................................................................................5
Late Sxl protein...............................................................................................................................................5
How does late Sxl direct alternative splicing of tra?......................................................................................6
........................................................................................................................................................................6
Gel-Shift Assays..............................................................................................................................................6
EXPERIMENT 1&2................................................................................................................................................. 7
Transformer Protein.......................................................................................................................................8
Dsx (double sex) Transcription Factor............................................................................................................8
SUMMARY OF SEX DETERMINATION IN DROSOPHILA...............................................................................................9
SEX DETERMINATION IN OTHER INSECTS....................................................................................................... 9
MEDFLY...............................................................................................................................................................9
HONEYBEES........................................................................................................................................................11
CONCLUSIONS............................................................................................................................................. 12
, Sex Determination
Splicing
- Splicing = the removal of introns from primary RNA & joining together of exons
- Involves 2 trans-esterification reactions
o Nucleophilic attack at 3’ end of intron
o Nucleophilic attack at 5’ end of intron forms Lariat loop
o Lariat released
The Spliceosome:
- Massive macromolecular complex containing 5 small nuclear ribonucleoproteins
(snRNPs) and up to 300 other proteins
- The snRNPs are called U1, U2, U4, U5 & U6
- Each contains a single short RNA molecule (100-300 nucleotides in length)
- RNA mols in snRNPs play 2 major roles:
1. Play a role in recognizing intron-exon boundaries
2. Play a role in the catalysis of trans-esterification reactions
- Assembly of the spliceosome:
1. Splice site definition
a. U1 is snRNP responsible for defining 3’ splice site and U2 defines 5’ splice
site
2. Spliceosome assembly
a. Catalytically inactive at this point
3. Formation of catalytically active complex (Only becomes active when U1 and U4
are released) and splicing occurs.
How do U1 and U2 know where the 3’ and 5’ ends of the introns are?
- Recognition of intron-exon boundaries through specific nucleotide sequence motifs
- U1 recognizes dinucleotide sequence G-U at 5’ end of every intron
- Associated factor of U2 (U2AF35) recognizes dinucleotide sequence AG at 3’ end of
every intron.
- Associated factor of U2 (U2AF65) recognizes pyrimidine rich tract upstream of
U2AF35.
- Branch-Point site upstream of U2AF65 is recognition site for snRNP U2 itself.
- ALL 4 of these motifs have to be PRESENT and SPACED CORRECTLY for splice sites to
be defined.
o i.e. only introns with all 4 of these motifs in the right place will be spliced out
Alternative Splicing:
- Splicing of a primary RNA molecule into different mRNA variants that (usually)
encode different proteins.
- Different proteins formed might differ in function, stability, ability to interact with
other proteins in the cell etc.
