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| tRNA nucleotidyl transferase |
-in euks, CCA is added after transcription |
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mcs5109 Thu, 23 Oct 2008 21:11:10 GMT |
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| how to add CCA to tRNA? |
euks: tRNA nucleotidyl transferase adds CCA AFTER transcription
pros: CCA is encoded in the DNA |
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mcs5109 Thu, 23 Oct 2008 21:11:10 GMT |
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| pre-tRNA introns |
-very short (about 15 nts)
-no consensus sequences
-removed:
-Cleavage by an endonuclease
-Phosphodiesterase to open a cyclic intermediate and provide a 3’OH
-Activation of one end by a kinase (with ATP hydrolysis)
-Ligation of the ends (with ATP hydrolysis)
-Phosphatase to remove the extra phosphate on the 2’OH (remaining after phosphodiesterase ) |
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mcs5109 Thu, 23 Oct 2008 21:07:34 GMT |
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| RNase D |
Exonuclease trims 3’ to 5’, leaving the mature 3’ end |
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mcs5109 Thu, 23 Oct 2008 21:07:34 GMT |
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| RNase F |
Endonuclease cleaves 3 nucleotides past the mature 3’ end |
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mcs5109 Thu, 23 Oct 2008 21:07:34 GMT |
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| RNase P |
Endonuclease cleaves to generate the 5’ end |
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mcs5109 Thu, 23 Oct 2008 21:07:33 GMT |
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| RNase III |
-no apparent primary sequence specificity
-stems of stem-loops |
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mcs5109 Thu, 23 Oct 2008 21:03:09 GMT |
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| Cutting and Trimming RNA |
-endonucleases to cut at specific sites within a longer precursor RNA
-exonucleases cut at ends to make mature products
-in pro and euks for ALL RNA |
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mcs5109 Thu, 23 Oct 2008 21:03:09 GMT |
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| types of RNA processing |
A) Cutting and trimming
B) Covalent modification
C) Splicing |
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mcs5109 Thu, 23 Oct 2008 21:03:09 GMT |
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| nuclear pore complex |
-how mRNA gets out of nucleus
-proteins associated with the RNA carry nuclear export signals
-GTPase Ran hydrolizes GTP to get energy |
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mcs5109 Thu, 23 Oct 2008 21:03:09 GMT |
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| Guide RNA-directed uridine insertion or deletion |
-Part of the guide RNA is complementary to the mRNA in vicinity of editing (Fig. 13-26).
-Uses a guide RNA (in 20S RNP = editosome) that is encoded elsewhere in the genome |
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mcs5109 Thu, 23 Oct 2008 14:07:48 GMT |
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| Site-specific deamination |
-only in certain tissues
-regulated manner
-Cytidine deaminase converts the C to U
-ADAR (adenosine deaminase acting on RNA) converts Adenosine to Inosine. Inosine can base-pair with cytosine. |
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mcs5109 Thu, 23 Oct 2008 14:07:48 GMT |
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| Methods of RNA Editing |
1) Site-specific deamination
2) Guide RNA-directed uridine insertion or deletion |
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mcs5109 Thu, 23 Oct 2008 14:06:22 GMT |
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| RNA editing |
-changes RNA sequence after transcription
-as much as 55% of the nucleotide sequence added after transcription
-can add, delete or change nucleotides |
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mcs5109 Thu, 23 Oct 2008 14:06:22 GMT |
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| Exon shuffling advantage |
- exons usually encodes an independently folding unit of protein
-different proteins via exon duplication and divergence
-extra mileage: related exons are sometimes in unrelated genes
-Exons (~150 nt) while introns up to several hundred kb); rcombination is more likely to occur within the introns that within the exons |
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mcs5109 Thu, 23 Oct 2008 14:06:22 GMT |
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| The intron advantage |
- regulate gene expression via alternative splicing
-produce multiple products from a single gene.
-Create new genes by reshuffling exons |
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mcs5109 Thu, 23 Oct 2008 14:05:24 GMT |
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| introns late model |
-introns were inserted into genes by a transposon-like mechanism. |
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mcs5109 Thu, 23 Oct 2008 14:05:24 GMT |
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| Intron early model |
-introns were removed from pros because of a selective pressure to increase the rate of chromosome replication and cell division. |
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mcs5109 Thu, 23 Oct 2008 14:05:24 GMT |
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| Regulated alt splicing |
-repressor protein can overcome repressor site
-activator protein can splicing enhancer to not translate that region |
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mcs5109 Thu, 23 Oct 2008 14:04:13 GMT |
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| if intron is not removed from mRNA |
-no transport
-no protein synthesis |
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mcs5109 Thu, 23 Oct 2008 14:04:13 GMT |
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| if an exon contains a stop codon |
-truncated protein |
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mcs5109 Thu, 23 Oct 2008 14:04:13 GMT |
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| SR proteins |
-bind RNA using RNA-recognition motif
-recruits splicing machinery using RS domain |
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mcs5109 Thu, 23 Oct 2008 14:04:13 GMT |
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| Repressor |
exonic splicing silencers (ESS)
-mostly heterogeneous nuclear ribonucleoprotein (hnRNP)
-bind RNA but lack the RS domains
-can't recruit the splicing machinery |
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mcs5109 Thu, 23 Oct 2008 14:03:06 GMT |
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| Enhancer |
exonic splicing enhancers (ESE) |
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mcs5109 Thu, 23 Oct 2008 14:03:06 GMT |
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| Alternative splicing |
-switches gene expression |
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mcs5109 Thu, 23 Oct 2008 14:03:06 GMT |
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| SV40 T antigen |
-ratio of 2 proteins produced (T-ag vs. t-ag) depends on level of SF2/ASF (SR protein)
-if a lot of SF2/ASF, produces t-ag |
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mcs5109 Thu, 23 Oct 2008 14:03:06 GMT |
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| alt splicing occurs in... |
...all metazoa
...a lot in vertebrates
...60% of human structural genes are subject in alternative splicing |
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mcs5109 Thu, 23 Oct 2008 14:02:13 GMT |
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| products of alternative splicing |
-constitutive or regulated
-what spliced depends on cell type, conditions, tissue, etc. |
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mcs5109 Thu, 23 Oct 2008 06:58:23 GMT |
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| minor spliceosomes |
-in higher euks (mammals, plants)
-alternate spliceosome
-aka AT-AC
-recognizes rare introns that contain consensus sequence distinct of the most pre-mRNA introns.
-some components same, some different to recognize different introns
-same chemical pathway, though |
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mcs5109 Thu, 23 Oct 2008 06:58:22 GMT |
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| SR proteins recruit |
U2AF to 3' splice site
U1 to 5' splice site |
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mcs5109 Thu, 23 Oct 2008 06:58:22 GMT |
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| exon splicing enhancers |
-SR (Serine Argenine rich) proteins bind to exonic splicing enhancers (ESEs) with the exon
-SR proteins recruit spliceosomes to nearby splice site
-specially U2AF to the 3’ splice site and U1 snRNP to the 5’ site
-SR proteins essential for splicing |
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mcs5109 Thu, 23 Oct 2008 06:58:22 GMT |
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| Two ways to enhance splicing accuracy |
1. Coupling between RNA transcription and splicing
2. Use exonic splicing enhancers |
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mcs5109 Thu, 23 Oct 2008 06:46:59 GMT |
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| What genes lack introns in higher euks? |
histones and interferons |
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mcs5109 Thu, 23 Oct 2008 06:46:59 GMT |
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| Gene splicing |
-introns rare in pros, yeast
-introns more in higher euks
-introns constitute ~80% of typical vertebrate structural genes |
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mcs5109 Thu, 23 Oct 2008 06:46:59 GMT |
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| Why aren't Group II and I RNAs not enzymes? |
-b/c turnover= 1!
-to turn into enzyme, need free G and complementary IGS sequence
-can change IGS to cleave whatever RNA we want |
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mcs5109 Thu, 23 Oct 2008 06:46:59 GMT |
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| RNA evolution |
-Before, many catalytic functions in the pre-mRNA splicing may carried out by RNAs
-these functions now performed by proteins
-now, catalytic center is still formed solely by RNA
-but proteins still do their job |
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mcs5109 Thu, 23 Oct 2008 06:46:59 GMT |
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| Noble prize |
-Additional proteins are NOT needed for splicing of pre-rRNA!
-just need GMP, GDP, GTP or Guanosine |
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mcs5109 Thu, 23 Oct 2008 06:46:59 GMT |
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| enzyme RNAs |
Ribonuclease P (RNase P): Cutting and trimming to generate ends of rRNA, tRNA and mRNA
Group I introns
Group II introns
snRNAs in splisosome involved in splicing
Hammerhead ribozymes: cleavage
rRNA in ribosome: peptide bond formation |
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mcs5109 Thu, 23 Oct 2008 06:38:03 GMT |
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| internal guide sequence |
IGS determines 5' splice site |
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mcs5109 Thu, 23 Oct 2008 06:38:03 GMT |
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| Group I intron shape is... |
...linear
-ribose G nucleotide for RNA cleavage
-binds to G pocket
-Internal guide sequence (IGS)
-b/w 400-1,000 nts long and much of sequence important for folding/catalysis
-High [G] in vivo prevents reverse reaction |
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mcs5109 Thu, 23 Oct 2008 06:38:03 GMT |
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| self-splicing introns |
-intron folds upon itself into a specific conformation to catalyze own release
-Group II= lariat
-Group I= linear intron |
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mcs5109 Thu, 23 Oct 2008 06:38:03 GMT |
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| Group I introns |
-rare
-some organelles in euks, some pros
-branch site G
-RNA enzyme encoded by intron (self-splicing) |
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mcs5109 Thu, 23 Oct 2008 06:30:17 GMT |
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| Group II introns |
-rare
-mito, chloro (euk organelle introns)
-branch site A
-RNA enzyme encoded by intron (self-splicing) |
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mcs5109 Thu, 23 Oct 2008 06:30:17 GMT |
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| nuclear pre-mRNA introns |
-euks
-very common
-branch site A
-major/minor spliceosomes |
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mcs5109 Thu, 23 Oct 2008 06:30:17 GMT |
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| 3 types of introns |
nuclear pre-mRNA
Group II
Group I |
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mcs5109 Thu, 23 Oct 2008 06:30:17 GMT |
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| C complex |
U1, U4 leave
-triggers catalysis
-lariat half-formed |
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mcs5109 Thu, 23 Oct 2008 06:26:54 GMT |
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| B complex |
-all 5 U's bind |
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mcs5109 Thu, 23 Oct 2008 06:26:54 GMT |
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| A complex |
U1, U2 bind |
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mcs5109 Thu, 23 Oct 2008 06:26:54 GMT |
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| different spliceosome proteins can bind to same sequence |
ex: Same sequence is recognized by a protein (BBP) at one stage and displaced by snRNP U2 at another |
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mcs5109 Thu, 23 Oct 2008 06:26:54 GMT |
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| spliceosome assembly |
1. U1 snRNP binds (and base pairs) to the 5’ splice site.
2. BBP (branch-point binding protein) binds to the branch site
3. U2 snRNP binds (and base pairs) to the branch point, BBP dissociates
4. U4U5U6 snRNP binds
5. U1 snRNP dissociates
6. U4 snRNP dissociates
-all requires ATP hydrolysis |
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mcs5109 Thu, 23 Oct 2008 06:26:54 GMT |
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| snRNPs |
-U1, U2, U4, U5 and U6
-of spliceosome
-recognize the 5’ splice site and the branch site
-bring those sites together |
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mcs5109 Thu, 23 Oct 2008 06:17:13 GMT |
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| snRNAs |
-U1, U2, U4, U5 and U6
-in spliceosome
-100-300 nts long
-assembled w/ proteins (snRNPs) |
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mcs5109 Thu, 23 Oct 2008 06:16:23 GMT |
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| Spliceosome machinery |
-to splice exons!
-150 proteins and 5 RNAs, size is similar to a ribosome
-many functions expressed by RNA components |
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mcs5109 Thu, 23 Oct 2008 06:16:23 GMT |
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| trans-splicing |
-splice 2 different RNAs together
-Y shaped lariats
-in C. elegance and higher euks
-rare, though
-chemistry of trans and cis the same |
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mcs5109 Thu, 23 Oct 2008 06:16:23 GMT |
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| why does splicing go forward? |
1. increase in entropy
2. intron rapidly degraded (no 5' cap or polyA tail) |
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mcs5109 Thu, 23 Oct 2008 06:10:27 GMT |
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| spliceosome energy |
-needs ATP for assembly/use
-none for breaking/making bonds |
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mcs5109 Thu, 23 Oct 2008 06:10:27 GMT |
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| RNA splicing |
-no extra energy required
-2 phosphodiester bonds formed and broken
-only ATP needed to operate spliceosome |
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mcs5109 Thu, 23 Oct 2008 06:10:27 GMT |
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| to remove intron.... |
2 transesterification rxns:
1. 2’ OH of the conserved A at the branch site attacks phosphoryl group of the conserved G in the 5’ splice site. It forms three-way junction.
2. Newly formed 3’OH of 5’ exon attacks the phosphoryl group at the 3’ splice site
-joins the 5’ and 3’ exons
-release “lariat” |
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mcs5109 Thu, 23 Oct 2008 06:10:27 GMT |
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| AG |
-3' splice site |
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mcs5109 Thu, 23 Oct 2008 06:06:27 GMT |
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| GU |
-5' splice site |
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mcs5109 Thu, 23 Oct 2008 06:06:27 GMT |
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| Branch point site |
found entirely within the intron, usually close to its 3’ end, A followed by polypyrimidine track |
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mcs5109 Thu, 23 Oct 2008 06:06:27 GMT |
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| important splice sites |
-5’ splice site: 5’ end of the intron: GU
3’ splice site: 3’ end of the intron: AG
Branch point site |
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mcs5109 Thu, 23 Oct 2008 06:06:27 GMT |
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| # introns per gene |
-goes up higher on the animal chain |
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mcs5109 Thu, 23 Oct 2008 06:06:27 GMT |
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