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To identify splicing events in rice, orthologues of atRSp31 and atRSZ33 were searched using Arabidopsis protein sequences at The TIGR Rice Database ( ). Genomic sequences for retrieved proteins were used to get corresponding transcripts. Selected transcripts were aligned to genomic sequences using Geneseqer.
|
16936312_p6
|
16936312
|
Sequence retrieval and analysis
| 3.860395 |
biomedical
|
Study
|
[
0.9986951947212219,
0.00021514717082027346,
0.0010896481107920408
] |
[
0.9983097314834595,
0.001420330721884966,
0.00020460515224840492,
0.00006541669426951557
] |
en
| 0.999996 |
Splice variants of maize orthologues of atRSp31 were retrieved at Geneseqer using genomic sequences of zmRSp31A and zmRSp31B ( 19 ).
|
16936312_p7
|
16936312
|
Sequence retrieval and analysis
| 3.627262 |
biomedical
|
Study
|
[
0.9979439377784729,
0.00020993512589484453,
0.0018461451400071383
] |
[
0.9959906935691833,
0.0036543484311550856,
0.00025567508419044316,
0.0000992956556729041
] |
en
| 0.999997 |
To identify orthologues of either atRSp31 or atRSZ33 in the distant species Arabidopsis protein sequences were used to pre-screen translated EST databases at limited to Coniferophyta, Bryophyta or algae. A second screen was done using either atRSp31 or atRSZ33 protein sequences in a BLAST search limited to species detected in the pre-screen. EST sequences were assembled into contigs using the CAP2 program ( 20 ) at . Obtained contigs were analysed by blastx against Arabidopsis proteins. In addition, translated contigs were compared to Arabidopsis and rice SR proteins using ClustalW program ( 21 ) at . Shading of multiple alignment files was done with BOXSHADE at .
|
16936312_p8
|
16936312
|
Sequence retrieval and analysis
| 4.100044 |
biomedical
|
Study
|
[
0.9992595314979553,
0.0002035409997915849,
0.0005368926213122904
] |
[
0.9994606375694275,
0.00024877674877643585,
0.0002530756755732,
0.00003752812335733324
] |
en
| 0.999996 |
Contigs were used to retrieve genomic sequences at NCBI and species specific databases (PHYSCObase, ; ChlamyDB ). Genomic sequences were used in screen for additional ESTs representing splice variants. Gene structures and alternative splicing events were finally analysed using Geneseqer.
|
16936312_p9
|
16936312
|
Sequence retrieval and analysis
| 3.848561 |
biomedical
|
Study
|
[
0.9993491768836975,
0.00020289023814257234,
0.0004479348717723042
] |
[
0.9978440999984741,
0.0017916429787874222,
0.00026703739422373474,
0.00009719850640976802
] |
en
| 0.999996 |
Wild-type plants of A.thaliana (Col-0) and plants overexpressing atRSZ33 ( 16 ) were germinated and grown either on GM medium ( 22 ) or in soil under 16 h light/8 h dark photoperiod at 23°C.
|
16936312_p10
|
16936312
|
Plant growth and protoplast transformation
| 3.393352 |
biomedical
|
Study
|
[
0.9954540729522705,
0.00038222147850319743,
0.004163700621575117
] |
[
0.9806140661239624,
0.018614334985613823,
0.0005913822678849101,
0.00018016892136074603
] |
en
| 0.999996 |
Arabidopsis cell suspension growth conditions and protoplasts preparation and transformation were as described previously ( 23 ). Protoplasts were collected by centrifugation and frozen in liquid nitrogen for RNA isolation or resuspended in SDS–PAGE loading buffer for analysis by western blotting, 24 h after transformation.
|
16936312_p11
|
16936312
|
Plant growth and protoplast transformation
| 3.900413 |
biomedical
|
Study
|
[
0.9992038607597351,
0.00019250276091042906,
0.0006036602426320314
] |
[
0.9932987093925476,
0.005933226086199284,
0.000660440418869257,
0.00010762111196527258
] |
en
| 0.999997 |
Total RNA from Arabidopsis plants and transformed cell culture protoplasts was isolated using RNeasy Plant Mini Kit (Qiagen) and treated with DNase I (Promega).
|
16936312_p12
|
16936312
|
RNA isolation and analysis of alternative splicing forms
| 3.819227 |
biomedical
|
Study
|
[
0.9993429780006409,
0.00017181923612952232,
0.00048516306560486555
] |
[
0.9674747586250305,
0.031020915135741234,
0.001228547072969377,
0.0002758213959168643
] |
en
| 0.999998 |
cDNAs of mRNA isoforms of atRSp31 were obtained by RT–PCR ( 24 ).
|
16936312_p13
|
16936312
|
RNA isolation and analysis of alternative splicing forms
| 3.531588 |
biomedical
|
Study
|
[
0.9988070726394653,
0.00021981958707328886,
0.0009731379104778171
] |
[
0.9935057163238525,
0.005885004531592131,
0.00046044611372053623,
0.00014875023043714464
] |
en
| 0.999995 |
Primer 1, 5′-AAAT GAGCTC CATTATGAAGTTTCTACTG-3′, containing a SacI restriction site (underlined), was used to prime reverse transcriptase.
|
16936312_p14
|
16936312
|
RNA isolation and analysis of alternative splicing forms
| 3.507195 |
biomedical
|
Study
|
[
0.9980213642120361,
0.0005514880176633596,
0.0014271753607317805
] |
[
0.5918506383895874,
0.4062274396419525,
0.0008971059578470886,
0.0010247930185869336
] |
en
| 0.999996 |
Primer 2, 5′-AAACT GGATCC AGTCGTCGTCGTCGTCTAGGG-3′, and primer 3, 5′-ATATAG GGATCC CATAAGGTCTTCCTCTTGGGACTGGAG-3′, both of which contain an additional BamHI restriction site, were used for PCR. Splicing events in the second intron were investigated by PCR using primers from the adjacent exons: primer 4, 5′-AAT GAGCTC GAATTGCGAATTAAGATAAAG-3′, and primer 5, 5′-ATA GGATCC TTTGCCCATTCAACTGATAAC-3′, containing a SacI and BamHI restriction site, respectively.
|
16936312_p15
|
16936312
|
RNA isolation and analysis of alternative splicing forms
| 4.145454 |
biomedical
|
Study
|
[
0.9992727637290955,
0.000338678335538134,
0.00038861611392349005
] |
[
0.9822896122932434,
0.017056947574019432,
0.00037163199158385396,
0.0002817833737935871
] |
en
| 0.999999 |
To analyse regulation of the splicing profile in Arabidopsis cell suspension protoplasts transfected with either pHA-catRSp31 or pHA-gatRSp31 (see below), reverse transcription was carried out using primer 5′-CTTTGAGTAGCTTCAAGGG-3′. PCR was performed using the same reverse primer and a direct primer either to the HA tag 5′-GATCCTACCCATATGACGTTCCAGATTACGCTA-3′ (to detect transgenic atRSp31 ), or 5′-CGAATTAAGATAAAGATG↓AGGCCA-3′ (to detect endogenic atRSp31 ; the position of the HA tag insertion in the pHA-catRSp31 or pHA-gatRSp31 is indicated by an arrow).
|
16936312_p16
|
16936312
|
RNA isolation and analysis of alternative splicing forms
| 4.12466 |
biomedical
|
Study
|
[
0.9991424083709717,
0.00021133595146238804,
0.0006463183672167361
] |
[
0.9992563128471375,
0.000521598500199616,
0.00017722486518323421,
0.00004485778117668815
] |
en
| 0.999997 |
To control loading, RT–PCR of ubiquitin with following primers 5′-CTCCTTCTTTCTGGTAAACGT-3′ and 5′-CTCCTTCTTTCTGGTAAACGT-3′ ( 25 ) was used. All PCR products, except ubiquitin, were sequenced.
|
16936312_p17
|
16936312
|
RNA isolation and analysis of alternative splicing forms
| 3.522814 |
biomedical
|
Study
|
[
0.998695433139801,
0.000222198708797805,
0.0010823897318914533
] |
[
0.9818936586380005,
0.01727117970585823,
0.0005945865996181965,
0.00024059686984401196
] |
en
| 0.999996 |
A DNA template for in vitro transcription of the complete second intron was obtained by PCR amplification on the genomic clone using primers 4 and 5. The PCR product was subcloned into the SacI and BamHI sites of the vector pSP65 (Promega), linearized with BamHI and used for in vitro transcription as described previously ( 26 ). RNA was purified and stored in aliquots at −70°C. Splicing reactions were carried out using the HeLa cell RNA Splicing System (Promega). Products of splicing were analysed by RT–PCR using primers 4 and 5 and sequenced.
|
16936312_p18
|
16936312
|
In vitro transcription and splicing in HeLa splicing extracts
| 4.135886 |
biomedical
|
Study
|
[
0.9995371103286743,
0.0002341344952583313,
0.00022882029588799924
] |
[
0.9991063475608826,
0.0005423816037364304,
0.0002789180143736303,
0.00007235889643197879
] |
en
| 0.999997 |
The coding region of atRSp31 cDNA was amplified by PCR using primers 6, 5′-ATAT CCATG G GGCCAGTGTTCGTCGG-3′, and primer 3, containing NcoI and BamHI restriction sites, respectively. Following cleavage of the PCR product with these enzymes, it was subcloned into the pET-3d expression vector (Novagen). To obtain the NcoI restriction site, the fourth nucleotide of the coding region was changed to G (see above primer 6, boldface G). Thus, in the expressed protein the second amino acid is changed from an arginine to a glycine. Induction of recombinant protein expression in the Escherichia coli strain BL21(DE3) pLysS was done according to the Novagen protocol. atRSp31 was purified from inclusion bodies as described previously ( 12 ). The SR proteins from Arabidopsis , carrot and rabbit were purified using a two-step salt precipitation method as described in ( 26 ).
|
16936312_p19
|
16936312
|
Protein purifications
| 4.159624 |
biomedical
|
Study
|
[
0.9995348453521729,
0.00019207829609513283,
0.00027314413455314934
] |
[
0.9992645382881165,
0.0004081840452272445,
0.0002653142437338829,
0.00006204787496244535
] |
en
| 0.999997 |
The coding and genomic sequences of atRSp31 were amplified using primers 5′-CATG CCATGG CT TACCCATATGACGTTCCAGATTACGCT AGGCCAGTGTTCGTCG-3′(NcoI site, underlined; HA epitope tag, in boldface) and 5′-AAAA CTGCAG CATTGATCAAGGTCTTCCTC-3′ (PstI site is underlined). The resulting PCR fragments were cloned into NcoI and PstI sites of pDEDH-Nco ( 27 ). Next, translation initation sequence of pDEDH-Nco was replaced by the 5′-untranslated region (5′-UTR) of atRSp31 , which was amplified using primers 5′- CCCCGGGG AGTCGTCGTCGTCGTCTAG-3′ (XmaI site is underlined) and 5′-CATG CCATGG CTTTATCTTAATTCGCAATTCC-3′ (NcoI site is underlined). This yielded the pHA–catRSp31 and pHA–gatRSp31 constructs. The constructs were verified by sequencing.
|
16936312_p20
|
16936312
|
Preparation of constructs of HA-tagged atRSp31 for protoplast transformation
| 4.163937 |
biomedical
|
Study
|
[
0.9994346499443054,
0.0002686115331016481,
0.0002966226893477142
] |
[
0.9992189407348633,
0.0004919392522424459,
0.00021283353271428496,
0.00007619328971486539
] |
en
| 0.999998 |
Proteins were separated by SDS–PAGE on 15% gels and electroblotted on a Immobilon-P Transfer Membrane (Millipore). Antibodies used were rat monoclonal anti-HA 3F10 (Roche Diagnostics) and goat anti-rat IgG horseradish peroxidase-conjugated (1:10 000) (Sigma-Aldrich). Blots were developed using ECL western blotting detection reagents (Amersham Biosciences).
|
16936312_p21
|
16936312
|
Western blotting
| 3.806286 |
biomedical
|
Study
|
[
0.9992813467979431,
0.0002694562135729939,
0.00044920446816831827
] |
[
0.9195098876953125,
0.07846476137638092,
0.0015586443478241563,
0.0004667407483793795
] |
en
| 0.999994 |
The five introns of atRSp31 are arranged such that they predominantly separate protein domains . The first intron is situated in the 5′-UTR, whereas the much longer second intron divides the first RRM, separating the highly conserved RNP2 and RNP1 domains. The third and the fourth introns border the second RRM. Furthermore, the RS region is delimited by the fourth and fifth introns so that the last exon contains the remaining nine amino acids and the 3′-UTR. Previously, northern blot analysis of poly(A) + RNA from various tissues of A.thaliana plants revealed at least three transcripts of atRSp31 which were differentially expressed in roots, leaves, stems and flowers of wild-type plants ( 11 ). RT–PCR amplification using total RNA was performed, and the three main products of 1320, 1199 and 791 bp were subcloned and sequenced . The 791 bp form corresponds to the ‘correctly’ spliced cDNA (mRNA1) which was published earlier ( 11 ) and which gives rise to a full-length 31 kDa protein. The longest form arises from the usage of an alternative 3′ splice site in the long intron (mRNA3) and encodes a short protein of 71 amino acids (8.5 kDa) due to a new in frame stop codon in the included intron . The hypothetical protein contains the first 35 amino acids from the RRM1 domain comprising the conserved RNP2 and 36 amino acids from the included sequence of the second intron. In the 1199 bp product, the same alternative 3′ splice site is used as in mRNA3 but an additional alternative 5′ splice site is recognized in this intron resulting in a new alternative exon . However, this splicing event gives rise to the same potential protein product as the one encoded by mRNA3. Additionally, both mRNA2 and mRNA3 possess partial sequences of the third intron due to use of an alternative 3′ splice site.
|
16936312_p22
|
16936312
|
Gene structure and alternative splicing forms of atRSp31
| 4.554905 |
biomedical
|
Study
|
[
0.9988597631454468,
0.0006597741739824414,
0.00048042935668490827
] |
[
0.99875807762146,
0.0005508481990545988,
0.0004908783594146371,
0.00020021703676320612
] |
en
| 0.999997 |
According to the RT–PCR analysis presented in the Figure 1 , all three splice forms of atRSp31 are expressed in Arabidopsis flowers. In contrast, in wild-type suspension protoplasts and wild-type seedlings , only mRNA1 encoding the full-length protein is produced. Interestingly, alternative mRNA2 and mRNA3 are the only transcripts of atRSp31 detected by northern blotting in the leaves and stems ( 11 ). Because expression of alternative splice forms is regulated in a tissue/organ-specific manner, it was interesting to check if a protein is produced from these alternatively spliced mRNAs. Arabidopsis cell culture protoplasts were transiently transfected with a genomic construct of atRSp31 under the control of a 35S CaMV promoter. A hemagglutinin (HA)-tag was introduced just after the start codon to allow monitoring the protein production. As shown in Figure 2A , lane 2, upper panel, both alternatively spliced mRNAs could be detected, but no 8.5 kDa protein was visible in a western blot analysis with HA-antibodies . This experiment shows that under the conditions employed no protein from mRNA2 and mRNA3 is detectable.
|
16936312_p23
|
16936312
|
Gene structure and alternative splicing forms of atRSp31
| 4.195856 |
biomedical
|
Study
|
[
0.9992251396179199,
0.00030628699460066855,
0.00046855187974870205
] |
[
0.9995589852333069,
0.000184726100997068,
0.0002029814204433933,
0.00005340409552445635
] |
en
| 0.999995 |
Experiments with another Arabidopsis SR protein, atRSZ33, had uncovered that this protein regulates splicing of its own pre-mRNA by changing alternative splicing in the similarly positioned long intron in the middle of the RRM ( 16 ). We therefore asked whether atRSp31 might also regulate its own splicing. Previously, we had shown that recombinant atRSp31 can stimulate splicing activity of a HeLa cell S100 extract ( 11 ). As no plant splicing extract is available, we used the HeLa cell system to test the ability of recombinant atRSp31 to influence splice site choice in its own pre-mRNA. A construct containing the long intron of atRSp31 with adjacent exon sequences was used for in vitro transcription . Obtained pre-mRNA was spliced in a HeLa cell nuclear extract, and the RNA products were characterized by RT–PCR using primers from the adjacent exon regions. A time course of the splicing reaction revealed the production of correctly spliced mRNA but not of alternatively spliced mRNAs. Neither the addition of recombinant atRSp31 nor of SR protein preparations from Arabidopsis (arabSR), carrot (carSR) or rabbit (rabSR) influenced this splicing pattern. These data show that the two splice sites of the long intron leading to the correct mRNA are strong splice sites in an animal splicing system. Therefore, if atRSp31 would indeed be able to influence splicing of its own pre-mRNA in vivo , then more plant-specific splicing factors might be needed for autoregulation. Moreover, these alternative splicing events might be controlled by cis -acting sequences not present on this short pre-mRNA construct.
|
16936312_p24
|
16936312
|
Regulation of alternative splicing in atRSp31
| 4.345582 |
biomedical
|
Study
|
[
0.9993200302124023,
0.0003684482944663614,
0.00031151605071499944
] |
[
0.9993033409118652,
0.00023693252296652645,
0.00036819136585108936,
0.00009148014214588329
] |
en
| 0.999998 |
To test these possibilities we transformed Arabidopsis cell culture protoplasts transiently with either a genomic or cDNA construct of atRSp31 containing an N-terminal HA-tag, and analysed splicing by RT–PCR. The RT reaction primer was from the end of the fourth exon, and the same reverse primer was used for the PCRs. To distinguish expression of transgenic or endogenous atRSp31 transcripts, the direct primer was either to the HA-tag or to the sequence which is disrupted in the transgene by the insertion of the HA-tag, respectively. The upper panel on Figure 2A shows expression of the transgenic cDNA construct (lane 1) and the genomic construct (lane 2) of atRSp31 . Lanes 3 and 4 are control transformations with empty vector or water, respectively. The middle panel shows the expression of endogenous atRSp31 in the same samples, and in this case only correctly spliced mRNA was detected. Western blot analysis with an HA-tagged antibody confirmed that a protein is produced from both constructs . The amount of protein is much lower in the case of the genomic construct which conforms to the lower level of mRNA1. As the mRNA patterns of the endogenous transcripts were unchanged by overexpression of atRSp31, these experiments show that atRSp31 does not influence splicing of its own pre-mRNA.
|
16936312_p25
|
16936312
|
Regulation of alternative splicing in atRSp31
| 4.245678 |
biomedical
|
Study
|
[
0.99921715259552,
0.000341063947416842,
0.0004417818272486329
] |
[
0.9994521737098694,
0.00024151602701749653,
0.0002475565124768764,
0.000058701833040686324
] |
en
| 0.999997 |
Because atRSZ33 regulates splicing of a similarly positioned intron of its own pre-mRNA ( 16 ), we decided to check whether atRSZ33 is capable to affect also splicing in the long intron of atRSp31 . Total RNA from 10 days old wild-type seedlings and seedlings overexpressing atRSZ33 was isolated and analysed by RT–PCR using primers to the second and fourth exons of atRSp31 . In wild-type seedlings, mRNA1 coding for the full-length protein is the only splice form detected . Overexpression of atRSZ33 drastically affected splicing in the long intron of atRSp31 as mRNA1 is decreased and the production of the alternative splice variants mRNA2 and mRNA3 is greatly enhanced , strongly resembling the splicing pattern of atRSp31 in wild-type flowers . The effect of atRSZ33 on alternative splicing of the long intron in atRSp31 is similar to the one in its own pre-mRNA, suggesting that atRSZ33 also regulates alternative splicing of atRSp31 .
|
16936312_p26
|
16936312
|
Regulation of alternative splicing in atRSp31
| 4.224637 |
biomedical
|
Study
|
[
0.9992709755897522,
0.0002894650970119983,
0.000439555908087641
] |
[
0.999474823474884,
0.00025900208856910467,
0.0002043888671323657,
0.00006190250860527158
] |
en
| 0.999996 |
atRSp31 is a member of the plant-specific RS subfamily, which includes also atRSp31a ( 6 ), atRSp40 and atRSp41 ( 11 ). atRSp31/atRSp31a and atRSp40/atRSp41 are two pairs of paralogues located at duplicated regions of the Arabidopsis genome ( 6 ). All four genes contain long introns at the same position between RNP2 and RNP1 of the first RRM. We were interested to analyse whether similar alternative splicing occurs in the other genes of this family as well. To identify potential alternative splicing events in atRSp31a , RT–PCR was performed with RNA from 6 days old seedlings, leaves, flowers and roots. Two main products were obtained (data not shown), one of them was the normally spliced mRNA and the other was a splice variant utilizing an alternative 5′ splice site in the long intron of atRSp31a with the sequence matching the alternative 5′ splice site in atRSp31 . We did not detect a splice form with the alternative 3′ splice site in the long intron of atRSp31a although this sequence is almost identical to atRSp31 . In silico search in the available cDNA and EST databases revealed also only the transcript using the alternative 5′ splice site (Supplementary Table S1). Because of the high sequence conservation of the 3′ alternative splice sites in both genes, we suppose that this splice site might be utilized in tissues and/or conditions different to those used in our analysis. However, we cannot exclude that certain cis -elements necessary for the utilization of the alternative 3′ splice site are absent in atRSp31a .
|
16936312_p27
|
16936312
|
Alternative splicing in the Arabidopsis plant-specific RS subfamily
| 4.278169 |
biomedical
|
Study
|
[
0.9991233944892883,
0.00034710048930719495,
0.0005295886658132076
] |
[
0.9994673132896423,
0.00022774303215555847,
0.00023140096163842827,
0.00007360870949923992
] |
en
| 0.999997 |
According to our previous data ( 11 ), only atRSp40 but not atRSp41 undergoes alternative splicing. In agreement with these data, database searches revealed alternative splice forms involving the long intron of atRSp40 only (Supplementary Table S1). Similar to atRSp31 , alternative 3′ and 5′ splicing occurs in the long intron of atRSp40 . However, comparison of the atRSp40 and atRSp31 / atRSp31a sequences did not reveal any sequence similarity at the regions of their alternative splice sites. There is no experimental indication for alternative splicing in the long intron of atRSp41 , and analysis of its intronic sequence did not reveal any potential splice sites similar to those in atRSp40 or in atRSp31 / atRSp31a.
|
16936312_p28
|
16936312
|
Alternative splicing in the Arabidopsis plant-specific RS subfamily
| 4.195743 |
biomedical
|
Study
|
[
0.9994020462036133,
0.0002507120370864868,
0.000347284076269716
] |
[
0.999467670917511,
0.0002753487788140774,
0.00018936560081783682,
0.00006762026168871671
] |
en
| 0.999998 |
Alternative splicing in most of the genes of the Arabidopsis RS subfamily prompted us to examine the splicing profile of their orthologues in other species. As rice genome sequencing is finished, we decided to analyse alternative splicing events in this species. A BLAST search showed that the rice genome encodes only two proteins which belong to the RS subfamily, Os02g03040 and Os04g02870. In a recent paper also these two proteins from this subfamily were identified, osRSp29 and osRSp33 ( 28 ). Both rice genes contain long introns at the identical positions in the first RRM . Moreover, analysis of available transcript data revealed alternative splicing events similar to those found in Arabidopsis . For osRSp29 , we were able to retrieve a single type of differential transcript utilizing alternative 3′ and 5′ splice sites at the same time. In osRSp33 , there are two types of splice variants, one which also utilizes alternative 3′ and 5′ splice sites simultaneously and one which uses only the alternative 3′ splice site . However, only osRSp29 revealed a surprisingly high conservation of the sequences adjacent to the alternative 3′ and 5′ splice sites in the long introns. In comparison, sequence conservation around any constitutive splice site is poor .
|
16936312_p29
|
16936312
|
Evolutionary conservation of alternative splicing in the RS subfamily
| 4.32057 |
biomedical
|
Study
|
[
0.9991586208343506,
0.00036135275149717927,
0.00048005778808146715
] |
[
0.9994046688079834,
0.00022854964481666684,
0.0002973544178530574,
0.00006942248728591949
] |
en
| 0.999998 |
Recently, orthologues of atRSp31 in maize were identified and termed zmRSp31A and zmRSp31B. It has been shown that they produce multiple alternatively spliced transcripts ( 19 ). We compared the genomic sequences of the maize genes with their orthologues in Arabidopsis and rice, and found again the conserved sequences around the alternative 3′ and 5′ splice sites in the long introns . Analysis performed using Geneseqer revealed the splice variants described by ( 19 ) as well as some new splice forms (Supplementary Table S1). Interestingly, different combinations of both conserved and non-conserved splice sites are utilized in the long introns of maize genes from RS subfamily .
|
16936312_p30
|
16936312
|
Evolutionary conservation of alternative splicing in the RS subfamily
| 4.161086 |
biomedical
|
Study
|
[
0.9993398785591125,
0.00022252069902606308,
0.0004375729477033019
] |
[
0.9995226860046387,
0.0002124907186953351,
0.00021331851894501597,
0.00005154589962330647
] |
en
| 0.999998 |
As both monocot species possessed the conserved sequences utilized for alternative splicing in the long intron we wanted to investigate how far this feature was conserved in more distant taxa. To find proteins from the RS subfamily we used the atRSp31 protein sequence in BLAST searches versus translated EST databases of gymnosperms ( P.taeda ), bryophytes ( P.patens ) and algae ( C.reinhardtii ) (Materials and Methods).
|
16936312_p31
|
16936312
|
Evolutionary conservation of alternative splicing in the RS subfamily
| 4.002929 |
biomedical
|
Study
|
[
0.9990498423576355,
0.00020868820138275623,
0.0007415285217575729
] |
[
0.9994692206382751,
0.00032008884591050446,
0.00016597947978880256,
0.0000447543898189906
] |
en
| 0.999997 |
We have identified five proteins that belong to the RS subfamily in P.taeda . In two of these genes, ptRSp34 and ptRSpNN , the intron position is conserved and the intron is alternatively spliced . Because we lack genomic sequences for P.taeda we do not know if the sequences surrounding these splice sites are still conserved.
|
16936312_p32
|
16936312
|
Evolutionary conservation of alternative splicing in the RS subfamily
| 3.055465 |
biomedical
|
Study
|
[
0.9936907291412354,
0.0003246263659093529,
0.005984693765640259
] |
[
0.9740059971809387,
0.025302257388830185,
0.0004143670084886253,
0.00027732294984161854
] |
en
| 0.999997 |
In Physcomitrella , a single protein from the RS subfamily was detected, which we named ppRSp27 . Comparison of the genomic sequence to ESTs revealed the presence of the long intron at the conserved position. However, no ESTs were found supporting alternative splicing in this gene. Alignment of the Physcomitrella and Arabidopsis /rice/maize genomic sequences also did not show any conserved alternative splice sites in the long intron.
|
16936312_p33
|
16936312
|
Evolutionary conservation of alternative splicing in the RS subfamily
| 4.130136 |
biomedical
|
Study
|
[
0.999171257019043,
0.00026012404123321176,
0.000568593037314713
] |
[
0.9989275336265564,
0.0008070154581218958,
0.00017810608551371843,
0.0000872900927788578
] |
en
| 0.999996 |
We have also detected a single protein for the RS subfamily in C.reinhardtii , crRSp35 . Interestingly, in crRSp35 , the number and positions of the majority of introns are not conserved. The only introns which have conserved positions and phases in Chlamydomonas are the one corresponding to the long intron and the next one separating the RRM1 and RRM2. In addition, most introns in the Chlamydomonas gene have symmetrical phases . Nevertheless, existing EST data support alternative 3′ splicing in the long intron of crRSp35 . However, comparison of the genomic sequence of crRSp35 to the ones from other species shows that sequence at this alternative splice site is not conserved, and no other putative conserved alternative splice sites were detected in the long intron.
|
16936312_p34
|
16936312
|
Evolutionary conservation of alternative splicing in the RS subfamily
| 4.220802 |
biomedical
|
Study
|
[
0.9992231130599976,
0.00025481038028374314,
0.000522100948728621
] |
[
0.9993801116943359,
0.00037960615009069443,
0.0001723190798657015,
0.00006793441571062431
] |
en
| 0.999996 |
These analyses demonstrate that, in the RS subfamily, the presence of an alternatively spliced long intron at the position between RNP2 and RNP1 of the first RRM is highly conserved from green algae to angiosperms. Furthermore, the sequences of particular alternative splice sites in this intron are highly conserved between monocots and dicots (and possibly in gymnosperms) arguing for the evolution of a conserved alternative splicing mechanism for these genes in higher plant species.
|
16936312_p35
|
16936312
|
Evolutionary conservation of alternative splicing in the RS subfamily
| 4.229679 |
biomedical
|
Study
|
[
0.9993708729743958,
0.00023959760437719524,
0.00038959947414696217
] |
[
0.9990542531013489,
0.0004598693922162056,
0.00042110192589461803,
0.00006478194700321183
] |
en
| 0.999996 |
The fact that atRSZ33 could equally modulate the conserved alternative splicing in the long intron of atRSp31 as well as in its own pre-mRNA prompted us to investigate the evolutionary conservation of the long introns in the RRM of the plant specific RS2Z subfamily. Proteins of this subfamily are characterized by a single N-terminal RRM, two zinc knuckles, followed by an RS- and SP-rich domain ( 13 ). Arabidopsis has a paralogous pair of genes, atRSZ33 / atRSZ32 , which are located on duplicated regions of the Arabidopsis genome ( 6 ). We have published previously that alternative 3′ splicing occurs in the long intron of atRSZ33 situated in the RRM similar to the one in atRSp31 ( 13 , 16 ). Analysing the second RS2Z gene in Arabidopsis , atRSZ32 , we found that it is also alternatively spliced and produces similar splice variants as atRSZ33 . One of them originates from the usage of the 3′ alternative splice site in the long intron [ Figure 5A and Supplementary Table S1 ]. Another splice variant found in both Arabidopsis genes is created by the same event in the long intron and the retention of the following intron [ Figure 5A and Supplementary Table S1 ].
|
16936312_p36
|
16936312
|
Evolutionary analysis of alternative splicing in the plant specific RS2Z subfamily
| 4.353241 |
biomedical
|
Study
|
[
0.9992160797119141,
0.000369626737665385,
0.00041424884693697095
] |
[
0.9994040727615356,
0.0002515944361221045,
0.0002556591061875224,
0.00008866057760315016
] |
en
| 0.999998 |
The BLAST search for proteins of this subfamily in rice revealed four genes: osRSZ36 , osRSZ37a , osRSZ37b and osRSZ39 ( 28 ). All four rice genes contain long introns at the conserved position and two of them, osRSZ37a and osRSZ37b , show alternative splicing in the long intron .
|
16936312_p37
|
16936312
|
Evolutionary analysis of alternative splicing in the plant specific RS2Z subfamily
| 4.027382 |
biomedical
|
Study
|
[
0.9985734224319458,
0.00015712999447714537,
0.0012694031465798616
] |
[
0.996258020401001,
0.0034019809681922197,
0.0002515814849175513,
0.00008852437167661265
] |
en
| 0.999998 |
Again the alignment of the genomic sequences of the Arabidopsis and rice genes within the RS2Z subfamily revealed that sequences around the alternative splice sites are highly conserved , even more than those within the RS subfamily . The degree of conservation is much higher than around constitutive splice sites in any of the respective genes .
|
16936312_p38
|
16936312
|
Evolutionary analysis of alternative splicing in the plant specific RS2Z subfamily
| 4.047682 |
biomedical
|
Study
|
[
0.9990737438201904,
0.00019940876518376172,
0.0007268759072758257
] |
[
0.9980506896972656,
0.0014381840592250228,
0.0004447939863894135,
0.00006643157394137233
] |
en
| 0.999997 |
Further, we retrieved sequences of RS2Z proteins in gymnosperms, bryophytes and algae in a similar way as for the RS subfamily. The alignment of proteins which belong to RS2Z subfamily is shown in Figure 6 . Intron positions and phases are conserved in all detected genes.
|
16936312_p39
|
16936312
|
Evolutionary analysis of alternative splicing in the plant specific RS2Z subfamily
| 3.251804 |
biomedical
|
Study
|
[
0.9910774230957031,
0.00047547469148412347,
0.008447233587503433
] |
[
0.9970152378082275,
0.0026115060318261385,
0.000263767724391073,
0.00010946675320155919
] |
en
| 0.999998 |
In P.taeda , we have found a protein, ptRSZ35, which belongs to this subfamily . Comparison of the ESTs revealed alternative splicing in the conserved position between RNP2 and RNP1 . We conclude that the position and alternative splicing of this intron in RS2Z subfamily is conserved in angiosperms and gymnosperms; however, as genomic sequences are not available in P.taeda we cannot draw any conclusions about the conservation of the sequences preceding the alternative 3′ splice site.
|
16936312_p40
|
16936312
|
Evolutionary analysis of alternative splicing in the plant specific RS2Z subfamily
| 3.990231 |
biomedical
|
Study
|
[
0.9978296160697937,
0.00024760354426689446,
0.0019227055599913
] |
[
0.9988221526145935,
0.0009269999573007226,
0.00018357366207055748,
0.00006722050602547824
] |
en
| 0.999997 |
In Physcomitrella , at least one protein with all the domain features as well as exon–intron structure of the RS2Z subfamily is present, which we named ppRSZ38 . This protein contains an unusual glycine-rich stretch in front of the RNP2. ppRSZ38 contains a long intron at the conserved position. Alignment of genomic sequences revealed highly conserved alternative 3′ splice site in the long intron of ppRSZ38 almost identical to the corresponding Arabidopsis /rice sequences . However, no EST was found in the available databases which would support alternative splicing in this intron. Whether this splice site is utilized in Physcomitrella needs to be proven.
|
16936312_p41
|
16936312
|
Evolutionary analysis of alternative splicing in the plant specific RS2Z subfamily
| 4.2466 |
biomedical
|
Study
|
[
0.9990659356117249,
0.00025918963365256786,
0.0006749159656465054
] |
[
0.9985620379447937,
0.0011317962780594826,
0.0002047716552624479,
0.00010148249566555023
] |
en
| 0.999996 |
Interestingly, in C.reinhardtii we found a single protein with an N-terminal RRM, an SP and RS regions, but no zinc knuckles (data not shown). This protein might therefore not be considered a true member of the RS2Z subfamily.
|
16936312_p42
|
16936312
|
Evolutionary analysis of alternative splicing in the plant specific RS2Z subfamily
| 3.383824 |
biomedical
|
Study
|
[
0.9964975714683533,
0.0002861843095161021,
0.0032163190189749002
] |
[
0.9828835129737854,
0.016423406079411507,
0.0004685464664362371,
0.0002244756615255028
] |
en
| 0.999997 |
In summary, the RS2Z subfamily possesses a long intron in a conserved position in the RRM which contains intron sequences highly conserved from mosses to angiosperms. These conserved sequences are used for alternative splicing in monocots and dicots and most likely also in gymnosperms and mosses.
|
16936312_p43
|
16936312
|
Evolutionary analysis of alternative splicing in the plant specific RS2Z subfamily
| 3.995141 |
biomedical
|
Study
|
[
0.9972782731056213,
0.0001895975146908313,
0.002532107289880514
] |
[
0.955411434173584,
0.041651830077171326,
0.0026936938520520926,
0.00024305596889462322
] |
en
| 0.999999 |
The SR protein family from Arabidopsis includes three plant-specific subfamilies (RS, RS2Z and SCL) characterized by the presence of an alternatively spliced relatively long intron in their N-terminal RRM ( 6 ). Analysis of the alternative splice forms in all three subfamilies shows that they encode truncated putative proteins which would contain only a part of the RRM due to premature termination codons (PTC) generated by the inclusion of intronic sequences ( 13 , 14 ). If produced, these proteins should have no influence on splicing activity as they lack both RNA and protein interaction domains. To investigate the significance of these alternative splicing events we have analysed the regulation of alternative splicing of atRSp31 from the RS subfamily. We found that its alternative splicing is regulated by atRSZ33, a member of the RS2Z subfamily. As this regulation is reminiscent of the autoregulation of the similar long intron in atRSZ33 ( 16 ) we were interested to follow the evolutionary conservation of these splicing events in both plant-specific subfamilies in different lineages.
|
16936312_p44
|
16936312
|
DISCUSSION
| 4.313758 |
biomedical
|
Study
|
[
0.999045193195343,
0.00039580094744451344,
0.0005590285873040557
] |
[
0.9992859959602356,
0.0002638787846080959,
0.00037464304477907717,
0.00007552132592536509
] |
en
| 0.999996 |
Our analysis shows that positions and phases of introns are conserved in the majority of the orthologous genes in the RS and RS2Z subfamilies. In addition, introns are arranged such that they predominantly separate protein domains. This is in agreement with the hypothesis of a modular assembly of functional or structural domains in the evolution of complex genes ( 29 ). The only exception is a Chlamydomonas member of RS subfamily, crRSp35 . Beside two introns at conserved positions in the first RRM, this gene has additional introns, most of them are in symmetrical phases. At present it is not clear whether Chlamydomonas has gained the additional introns or if the exon–intron structure of the Chlamydomonas gene represents an ancestral arrangement and the other orthologues of the RS subfamily went through loss of introns.
|
16936312_p45
|
16936312
|
Evolutionary conservation of splicing profiles
| 4.22507 |
biomedical
|
Study
|
[
0.9992890357971191,
0.00033057728433050215,
0.0003803259751293808
] |
[
0.9993904829025269,
0.00024207918613683432,
0.000301601889077574,
0.00006574318831553683
] |
en
| 0.999997 |
One of the most conserved intron positions in both subfamilies is occupied by the relatively long intron separating RNP2 and RNP1. In addition to its particular length and conserved position, we have observed alternative splicing of these introns in some members of the plant-specific SR subfamilies. The functional significance of alternative transcripts can be best assessed by comparison of splicing profiles of orthologous genes in different species. Interestingly, in both subfamilies alternative splicing was found to be highly conserved in evolution in all species tested from angiosperms to green algae for the RS subfamily, and most likely down to the mosses for the RS2Z subfamily. In addition, a subset of alternative splicing events involves conserved 3′ and/or 5′ alternative splice sites, which are specific for each subfamily. These alternative splice sites conform to the splice site consensus; however, the surrounding sequences are much more conserved than in any of the constitutive splice sites in the corresponding genes. In the RS subfamily, the conserved intronic sequences are found both in monocots and dicots. In gymnosperms, the lack of genomic sequences does not allow to prove whether the observed alternative splicing events utilize the same conserved sequences. In contrast, the conserved intron sequences are present in the RS2Z family from angiosperms to mosses, although supporting experimental data are still missing for the latter class. Taken together the evolutionary preservation of these particular alternative splicing events suggests a functional, yet unknown significance. During preparation of this manuscript, an in silico analysis of alternative splicing events in plant SR proteins revealed that also the SCL subfamily possess conserved alternative splicing events although no hint for conserved intron sequences was presented ( 30 ).
|
16936312_p46
|
16936312
|
Evolutionary conservation of splicing profiles
| 4.51417 |
biomedical
|
Study
|
[
0.9990440011024475,
0.00045106708421371877,
0.0005049096653237939
] |
[
0.9976897239685059,
0.00033572432585060596,
0.001828103675507009,
0.00014638023276347667
] |
en
| 0.999998 |
It is worth noting that not all members of the subfamily in a species possess this particular alternative splicing event. For example, in the Arabidopsis RS subfamily, two genes, atRSp31 and atRSp31a , utilize these conserved alternative splicing signals, but not atRSp40 and atRSp41 . Similarly, both rice orthologues, osRSp29 and osRSp33 , are alternatively spliced, but only in osRSp29 alternative splicing involves the conserved sequences. In maize, both orthologues, zmRSp31A and zmRSp31B , produce multiple splice variants [( 19 ) and this study], and some of them are generated by the simultaneous use of both conserved and non-conserved alternative splice signals. Similarly, among the four rice genes in the RS2Z subfamily, two have the conserved alternative splicing event. It therefore seems that in a given species selective pressure has preserved conserved alternative splicing events at least in one member of the subfamily. These findings corroborate the significance of these highly conserved alternative splicing events. They might be also a good example for the notion that gene function is more often correlated with splicing profile similarity between orthologues in different species than with sequence similarity of paralogues in the same species ( 31 ).
|
16936312_p47
|
16936312
|
Evolutionary conservation of splicing profiles
| 4.361651 |
biomedical
|
Study
|
[
0.9991362690925598,
0.0003609675040934235,
0.0005027365405112505
] |
[
0.9985989928245544,
0.0002952681970782578,
0.0010141577804461122,
0.00009163885988527909
] |
en
| 0.999997 |
Alternative splicing is an important mechanism for generation of proteome diversity and regulation of gene expression at the post-transcriptional level. In both RS and RS2Z subfamilies, all conserved alternative splicing transcripts potentially encode extremely truncated proteins containing only a part of the RRM and some sequences from the included introns due to the generation of PTC. Our current data from the analysis of atRSp31 suggest that no truncated protein is made from these variant transcripts. Similarly, no truncated protein could be detected for atGRP7 which also contains an alternatively spliced long intron in the RRM domain ( 32 ). For maize orthologues of the RS subfamily, it has been shown that PTC-containing alternatively spliced transcripts associate with polysomes, but there is no evidence of their translation ( 19 ). Even if the alternative splice forms are translated, these short proteins will certainly have no splicing activity as they lack a complete RRM and RS domains. These alternative splicing events are different from those leading to the production of diverse protein isoforms. For example, alternative splicing events in the Arabidopsis homologues of human ASF/SF2 occur in long introns located in the 3′ ends of genes. Here, splice variants encode protein isoforms with a shortened RS domains ( 12 , 15 ), which might affect the phosphorylation status of the protein and/or its ability to interact with other proteins.
|
16936312_p48
|
16936312
|
Functional significance and regulation of the alternative splicing events
| 4.571728 |
biomedical
|
Study
|
[
0.9990867376327515,
0.0005074399523437023,
0.0004057483747601509
] |
[
0.9981570839881897,
0.0003879709402099252,
0.0012830966152250767,
0.00017183268209919333
] |
en
| 0.999996 |
Generally, alternative splicing events preserving the reading frame are more conserved than those leading to frame-shifts. It has been shown that only 5% of cassette alternative exons are both conserved and have the potential to introduce PTC ( 33 ). It is therefore very unlikely that the evolutionary conserved alternative splicing events seen in the plant SR protein subfamilies have occurred just by chance as aberrant splicing events, as they are preserved in different subfamilies of genes and across distant taxa.
|
16936312_p49
|
16936312
|
Functional significance and regulation of the alternative splicing events
| 4.161816 |
biomedical
|
Study
|
[
0.9994845390319824,
0.000170210434589535,
0.0003452280070632696
] |
[
0.99772709608078,
0.0010413705604150891,
0.0011498973472043872,
0.00008166775660356507
] |
en
| 0.999997 |
Given that no or only inactive proteins are made from the alternative transcripts, what functional significance could these conserved alternative splicing events in the long introns of the plant specific SR proteins have? Either the alternative splicing event is crucial for modulating the level of the splicing factor or the alternative transcript itself encodes an yet unknown activity. Although there is no evidence for the latter case, it has been shown previously that the presence of PTC in splice isoforms can promote nonsense-mediated decay (NMD) of such transcripts, and some splicing factors are subjected to such regulation ( 34 , 35 ). However, it has been shown recently by quantitative microarray profiling that NMD does not significantly influence the overall steady-state levels of transcripts with PTC generated by alternative splicing events ( 36 ). The alternative transcripts of atRSp31 are readily detectable by northern blotting and in some tissues they represent the only type of transcripts ( 26 ). It is therefore unlikely that the variant transcripts of atRSp31 are controlled by NMD, however, experimental proof is required.
|
16936312_p50
|
16936312
|
Functional significance and regulation of the alternative splicing events
| 4.319319 |
biomedical
|
Study
|
[
0.9993880987167358,
0.0002526252355892211,
0.00035932022728957236
] |
[
0.9982317090034485,
0.0004974156036041677,
0.001175746670924127,
0.00009520796447759494
] |
en
| 0.999998 |
Tight control of the atRSZ33 protein levels by autoregulation of alternative splicing has been demonstrated previously ( 16 ). Autoregulation seems to be conserved in the RS2Z family as the rice homologue, osRSZ36, has also been shown to cause changes in its splicing pattern ( 28 ). Autoregulatory circuits have been shown for several non-plant splicing factors, such as SXL ( 37 ), TRA-2 ( 38 ), SWAP ( 39 ) and SRp20 ( 40 ). On the other hand, we have demonstrated here that overexpression of atRSp31, either in vitro or in vivo , does not stimulate usage of the alternative splice sites. Interestingly, also in rice none of the two homologues of atRSp31, osRSp29 and osRSp33, can change the splicing pattern of their pre-mRNA ( 28 ). Together with this, we show that atRSZ33 is involved in the regulation of alternative splicing in the long intron of atRSp31 . However, atRSZ33 does not interact with atRSp31 in pull-down assays ( 13 ) which would imply that regulation of splicing in the long intron of atRSp31 by atRSZ33 occurs without direct interaction of these two proteins.
|
16936312_p51
|
16936312
|
Functional significance and regulation of the alternative splicing events
| 4.428573 |
biomedical
|
Study
|
[
0.9990732669830322,
0.0004267505428288132,
0.0005000227829441428
] |
[
0.999213457107544,
0.0002776699257083237,
0.00040786314639262855,
0.0001009961633826606
] |
en
| 0.999998 |
How and why are these alternative splicing events regulated? It has been noted that similar to animals, many of the alternatively spliced genes in Arabidopsis encode proteins with regulatory functions and many stress related genes are alternatively spliced ( 41 ). A genome wide analysis in A.thaliana uncovered many alternative splicing events in splicing factors and several of them were strongly influenced by cold stress ( 42 ). This is especially interesting as these alternatively spliced splicing regulators might coordinate alternative splicing of a particular set of stress response genes. Our unpublished data indicate that expression of atRSp31 is tightly regulated both transcriptionally and post-transcriptionally during plant development and in response to hormones, sugars and different light conditions. It would be extremely interesting to find out whether the highly conserved alternative transcripts of plant-specific SR genes have a specific role in development and/or environmental responses. Preservation of alternative splicing events that have often been attributed to accidents or erroneous action of splicing machinery argues for basic plant cell specific regulatory circuits established early in the plant evolution.
|
16936312_p52
|
16936312
|
Functional significance and regulation of the alternative splicing events
| 4.282925 |
biomedical
|
Study
|
[
0.9994964599609375,
0.00021414209913928062,
0.00028937499155290425
] |
[
0.9975858926773071,
0.0008357393089681864,
0.0014765524538233876,
0.00010182853293372318
] |
en
| 0.999997 |
Supplementary Data are available at NAR Online.
|
16936312_p53
|
16936312
|
SUPPLEMENTARY DATA
| 0.985075 |
biomedical
|
Other
|
[
0.7697952389717102,
0.0054101841524243355,
0.22479452192783356
] |
[
0.01419480424374342,
0.982682466506958,
0.0018953380640596151,
0.00122743786778301
] |
en
| 0.999993 |
The use of genetic knockout mice has greatly facilitated the study of gene function in a wide range of biological processes and has aided in the discovery of new therapeutics. Indeed, a retrospective investigation of knockouts of top-selling drug targets revealed a correlation between phenotypes, mechanism of action and therapeutic effectiveness ( 1 ). However, there are drawbacks to the use of knockout technology. These include the time required to generate the founder knockout animal and to breed a population of sufficient size for the acquisition of statistically significant data, the difficulties in generating knockout animals for embryonic lethal genes and the fact that knockout technology is practically limited to a few strains of mice and not readily applicable for use in other species.
|
16945951_p0
|
16945951
|
INTRODUCTION
| 4.029798 |
biomedical
|
Study
|
[
0.9994189739227295,
0.0002652648836374283,
0.00031583980307914317
] |
[
0.6892492771148682,
0.006270002573728561,
0.3040115237236023,
0.00046923503396101296
] |
en
| 0.999997 |
The discovery that small interfering RNA (siRNA) could be used in mammalian cells to elicit RNA interference (RNAi) has opened another avenue for studying gene function in mammals ( 2 ). The use of siRNA could circumvent some limitations of knockout technology, allowing rapid generation of relatively large numbers of knockdown mice of practically any strain of interest. Indeed, there have been several recent reports of the use of RNAi in mice [reviewed in ( 3 )]. However, the paucity of data detailing the molecular and phenotypic effects of delivering siRNA and of the subsequent knockdown of target gene expression in these studies has made it difficult to definitively discern the effectiveness and specificity of the RNAi approach. Here, we performed a molecular and phenotypic comparison of mice treated with siRNA targeting peroxisomal proliferator activated receptor alpha ( Ppara ) and Ppara knockout mice.
|
16945951_p1
|
16945951
|
INTRODUCTION
| 4.161241 |
biomedical
|
Study
|
[
0.9995983242988586,
0.00022319084382615983,
0.0001785480708349496
] |
[
0.99810791015625,
0.00023241806775331497,
0.0015793798957020044,
0.00008027643343666568
] |
en
| 0.999995 |
Ppara is a member of the nuclear hormone receptor superfamily and is involved in regulating fatty acid metabolism. In the liver, Ppara is expressed exclusively in hepatocytes ( 4 ). During an overnight or prolonged fast, fatty acids released from adipose tissue are transported to the liver, robustly inducing PPARα activity ( 5 ). Upon binding fatty acid ligand, PPARα stimulates transcription of genes containing PPARα response elements in their enhancers, most notably genes involved in lipid metabolism and energy homeostasis ( 6 ). Drugs belonging to the fibrate class act as synthetic PPARα ligands and can be used to treat patients with hypertriglyceridemia. Consistent with the proposed role for PPARα, these drugs improve plasma lipid profiles by promoting fatty acid β-oxidation and reducing hepatic triglyceride production.
|
16945951_p2
|
16945951
|
INTRODUCTION
| 4.467471 |
biomedical
|
Study
|
[
0.999419093132019,
0.0003292675828561187,
0.0002515818923711777
] |
[
0.8546960353851318,
0.00498338183388114,
0.13972458243370056,
0.0005959466216154397
] |
en
| 0.999996 |
Analyses of Ppara −/− mice have provided valuable insight into the role of PPARα in regulating metabolism. Interestingly, young adult Ppara −/− mice have slightly elevated levels of cholesterol but do not display an obvious phenotype under normal dietary conditions ( 7 , 8 ). However, under fasting conditions or when fed a high fat diet, these mice suffer from hypoglycemia and dyslipidemia, and accumulate massive amounts of lipid in their livers ( 5 , 7 , 9 ). These phenotypes can be partially explained by an inability of the Ppara −/− mice to meet energy demands and a rate of fatty acid uptake by the liver that exceeds the capacity of the liver to secrete triacylglycerols. In contrast to young adult Ppara −/− mice, aged Ppara −/− mice are obese and have increased serum triglycerides in the absence of fasting ( 10 ). These phenotypes were found to be sexually dimorphic, being more pronounced in females than in males.
|
16945951_p3
|
16945951
|
INTRODUCTION
| 4.270088 |
biomedical
|
Study
|
[
0.9996106028556824,
0.00021815995569340885,
0.00017117822426371276
] |
[
0.9928652048110962,
0.0003755892103072256,
0.006624323315918446,
0.00013493037840817124
] |
en
| 0.999996 |
In this study, we used hydrodynamic tail vein injection to deliver siRNA targeting Ppara and compared resulting genome-wide transcriptional profiles and phenotypes to those of Ppara −/− mice. We found that knockdown of Ppara using RNAi results in a transcript profile in the liver that is highly comparable, both in magnitude and direction, to that observed in Ppara −/− mice. Phenotypic analyses revealed that siRNA-treated mice displayed hypoglycemia and hypertriglyceridemia, phenotypes observed in similarly aged Ppara −/− mice. Together, these results indicate that hydrodynamic delivery of siRNA can be effectively used to study gene function in the liver of mice.
|
16945951_p4
|
16945951
|
INTRODUCTION
| 4.132113 |
biomedical
|
Study
|
[
0.9995670914649963,
0.00026951346080750227,
0.0001633382198633626
] |
[
0.9992988109588623,
0.00022438145242631435,
0.000402328121708706,
0.00007453203579643741
] |
en
| 0.999996 |
The siRNAs used in this study were obtained from Dharmacon and consisted of 21 nt sense and antisense oligonucleotides each containing fluoro substitutions at the 2′ position of pyrimidine nulceotides, a 5′-PO 4 and a two deoxynucleotide overhang at the 3′ end. The 2′ACE protected oligonucleotides were purified by HPLC and then deprotected according to the manufacturer's instructions. Control siRNA targeting the secreted alkaline phosphatase reporter gene : sense 5′-pAGGGcAAcuuccAGAccAudTdT-3′, antisense 5′-pAuGGucuGGAAGuuGcccudTdT-3′; Ppara siRNA#1: sense 5′-pGAucGGAGcuGcAAGAuucdAdT-3′, antisense 5′-pGGAucuuGcAGcuccGAucdAdT-3′; Ppara siRNA#2: sense 5′-pucAcGGAGcucAcAGAAuudCdT-3′, antisense 5′-pAAuucuGuGAGcuccGuGAdCdT-3′; Ppara siRNA#3: sense 5′-pGAAGuucAAuGccuuAGAAdAdT-3′, antisense 5′-puucuAAGGcAuuGGAcuucdAdT-3′. Nucleotides containing 2′-fluoro substitutions are lower-case; d, deoxynucleotides; p, 5′ PO 4 . Sense and antisense oligonucleotides for each target sequence were annealed by mixing equimolar amounts of each and heating to 94°C for 5 min, cooling to 90°C for 3 min, then decreasing the temperature in 0.3°C steps 250 times, holding at each step for 3 s.
|
16945951_p5
|
16945951
|
siRNAs
| 4.126397 |
biomedical
|
Study
|
[
0.9995655417442322,
0.00024897255934774876,
0.00018554388952907175
] |
[
0.9973580241203308,
0.0021729720756411552,
0.0003684747207444161,
0.00010056664905278012
] |
en
| 0.999997 |
Primary hepatocytes were harvested from adult mice (strain C57BL/6) using the two-step collagenase perfusion procedure as described previously ( 11 ). Hepatocyte viability was 85–90% as determined by Trypan blue exclusion. Hepatocytes were plated at a density of 1.5 × 10 5 cells per well in collagen coated 12-well plates. Cells in triplicate wells were transfected with siRNA at a final concentration of 100 nM using TransIT ® -siQuest (Mirus Bio Corporation) according to the manufacturer's protocol, 24 h post plating. Hepatocytes were harvested 24 h after transfection and total RNA was isolated with Tri Reagent (MRC, Inc.).
|
16945951_p6
|
16945951
|
Primary hepatocyte isolation and transfection
| 4.107477 |
biomedical
|
Study
|
[
0.999606192111969,
0.0002190055383834988,
0.0001748331997077912
] |
[
0.9991486072540283,
0.00045218682498671114,
0.0003367151948623359,
0.00006251796003198251
] |
en
| 0.999997 |
All animal studies were conducted at Mirus Bio Corporation with approval from Mirus' Institutional Animal Care and Use Committee. Six- to eight-week-old mice (strain C57BL/6, 19–21 g) were obtained from Harlan Sprague Dawley Inc. and housed at least 10 days on a 12 h light/dark cycle before injection. Mice had free access to food and water throughout the course of the experiments (Harlan Teklad Rodent Diet). For siRNA delivery, mice were injected in the tail vein using the hydrodynamic technique without anaesthesia as described previously ( 12 ). Briefly, siRNA (40 µg) was diluted in a volume of Ringer's solution (147 mM NaCl, 4 mM KCl and 1.13 mM CaCl 2 ) equal to 10% of the animal's body weight. The entire volume was injected into the tail vein in 5–7 s using a 3 ml syringe fitted with a 27 gauge needle.
|
16945951_p7
|
16945951
|
Mice and injection procedures
| 4.093315 |
biomedical
|
Study
|
[
0.9995630383491516,
0.0002942777646239847,
0.0001426418893970549
] |
[
0.99822598695755,
0.0013109149876981974,
0.00035560355172492564,
0.00010756011761259288
] |
en
| 0.999998 |
Mice had free access to food throughout the duration of the experiments and were sacrificed within 1 h of each other. Total RNA was isolated from mouse liver using the RNeasy Midi Kit (Qiagen) according to the manufacturer's protocol with minor modification. Briefly, mouse liver was harvested and immediately placed in 30 ml of RLT buffer and homogenized for 45 s using a PRO200 homogenizer (PRO Scientific). Homogenates were spun at 4000 r.p.m. for 20 min in a RT7 Plus centrifuge (Sorvall). Supernatant (2 ml) was transferred to a new tube and 1 vol of 50% ethanol was added before loading the RNeasy column. The manufacturer's protocol was followed for the rest of the procedure.
|
16945951_p8
|
16945951
|
Liver harvest and RNA isolation
| 4.134563 |
biomedical
|
Study
|
[
0.9995700716972351,
0.00024457622203044593,
0.00018523515609558672
] |
[
0.9934524297714233,
0.005677323788404465,
0.0006936631980352104,
0.00017664371989667416
] |
en
| 0.999997 |
Total RNA (500 ng) was reverse transcribed using SuperScript III (Invitrogen) and oligo-dT primers according to the manufacturer's protocol. Quantification of gene-specific mRNA levels was performed by RT-qPCR using an iCycler iQ system (Bio-Rad). Relative levels of Ppara and GAPDH mRNA were measured in biplex reactions performed in triplicate using TaqMan ® Universal PCR Master Mix and the TaqMan ® Gene Expression Assay for Ppara (Applied Biosystems) as per the manufacturer's protocol. The GAPDH primers and probe (IDT) were as follows: GAPDH-forward, 5′-AAATGGTGAAGGTCGGTGTG-3′; GAPDH-reverse, 5′-CATGTAGTTGAGGTCAATGAAGG-3′; and GAPDH-probe, 5′-Hex/CGTGCCGCCTGGAGAAACCTGCC/BHQ-3′.
|
16945951_p9
|
16945951
|
Quantitative PCR Assays
| 4.138335 |
biomedical
|
Study
|
[
0.9995986819267273,
0.0002517211833037436,
0.00014961753913667053
] |
[
0.9970604777336121,
0.002341188956052065,
0.00046742180711589754,
0.00013079539348836988
] |
en
| 0.999998 |
Expression profiling was carried out using custom arrays consisting of ∼23 000 60mer oligonucleotides (plus control sequences) representing mouse genes as described previously ( 13 ). All hybridizations were performed in duplicate, with fluor reversal (Cy3 or Cy5) in the second hybridization. For knockdown mice, each experiment consisted of three groups injected either with Ppara siRNA, control siRNA or injection buffer alone (Ringer's). RNAs from individual siRNA-treated animals and buffer-treated animals were hybridized against a pool of RNA from time-matched buffer-treated animals. Ppara −/− mice originated from the colony established at the National Cancer Institute ( 8 ). For fenofibrate treatment (200 mg/kg/day) wild-type (C57BL/6) and Ppara −/− mice (6 animals per group) were treated for 1 and 7 days. RNA from individual animals were paired according to their genetic background and hybridized against a pool of RNA (6 animals) from time-matched wild-type or Ppara −/− mice, respectively. The transcriptional response for Ppara −/− mice was determined by hybridization of RNA against a wild-type pool.
|
16945951_p10
|
16945951
|
Microarray procedures
| 4.110878 |
biomedical
|
Study
|
[
0.9995198249816895,
0.00024454708909615874,
0.00023571080237161368
] |
[
0.9994462132453918,
0.00023757076996844262,
0.0002588620700407773,
0.00005725008304580115
] |
en
| 0.999998 |
Significance between groups was determined using a two-tailed t -test of either unequal [e.g. Experiments A and B, treatments ( n > 15) versus control siRNA ( n = 4)] or equal variance [e.g. Experiment C ( n = 4)]. The significance of Pearson product-moment correlation coefficients ( r ) was determined using t -distribution tables for a two-tailed test and the formula: t = ( r ) N − 2 1 − r 2 df = N − 2 , where N = number of pairs.
|
16945951_p11
|
16945951
|
Statistical tests
| 4.047081 |
biomedical
|
Study
|
[
0.9995443224906921,
0.00017983492580242455,
0.0002757880138233304
] |
[
0.9986032843589783,
0.0009978011948987842,
0.0003400282876100391,
0.00005898921881453134
] |
en
| 0.999996 |
We first screened five siRNAs against Ppara for activity using mouse primary hepatocytes. In anticipation of in vivo studies, the siRNAs were synthesized with 2′F substitutions on pyrimidines and 2′-H substitutions in the 2 nt at each 3′ end to increase nuclease resistance. We found that three of the five siRNAs ( Ppara siRNA#1, #2 and #3) were highly active in this in vitro system .
|
16945951_p12
|
16945951
|
Delivery of Ppara siRNA to mice by hydrodynamic injection results in knockdown of Ppara expression in liver
| 4.068467 |
biomedical
|
Study
|
[
0.9995761513710022,
0.0001775831333361566,
0.0002462901175022125
] |
[
0.999477207660675,
0.00027840115944854915,
0.0001940930524142459,
0.00005033082197769545
] |
en
| 0.999996 |
Our initial studies in mice were composed of two independent experiments, A and B. In both experiments, Ppara siRNA#1 (40 µg) was delivered to 6- to 8-week-old C57Bl/6 female mice using hydrodynamic tail vein injection. Control groups included mice injected with a non-targeting siRNA (control siRNA) or Ringer's buffer alone. Livers were harvested 24 h after injection. A pairwise comparison of the Ppara siRNA-treated groups to the groups treated with control siRNA revealed a significant ( P ≤ 0.01) average reduction in Ppara mRNA as determined by quantitative PCR (RT-qPCR) . The Ppara RT-qPCR data across all individuals in our studies correlated significantly ( r = 0.81, P < 0.001) with Ppara expression in individuals for which data were obtained from high-density microarrays . The data indicated that the amount of Ppara knockdown was variable between mice, ranging from ∼80% knockdown to little or no apparent knockdown. This is likely due to variable siRNA delivery efficiency.
|
16945951_p13
|
16945951
|
Delivery of Ppara siRNA to mice by hydrodynamic injection results in knockdown of Ppara expression in liver
| 4.08894 |
biomedical
|
Study
|
[
0.9994995594024658,
0.0003020936856046319,
0.00019841927860397846
] |
[
0.9995383024215698,
0.0001814263960113749,
0.0002189378283219412,
0.00006135887088021263
] |
en
| 0.999997 |
We utilized high-density microarrays to compare the genome-wide transcriptional response in liver of mice injected with Ppara siRNA#1 to that of the well-characterized Ppara −/− mouse ( 8 ). Knockdown mice from Experiment A were used for these initial analyses. Using the ROAST ® correlation tool in the Resolver ® v5.0 gene expression data analysis system, we identified 622 genes that correlated ( r > 0.7, P < 1 × 10 −8 ) with Ppara expression levels. The transcriptional response for these 622 genes when projected onto the microarray responses of Ppara −/− mice revealed high concordance, both in direction and magnitude, with that observed with Ppara siRNA#1-treated animals . Transcriptional concordance was also maintained with larger gene sets representing lower Ppara correlation thresholds ( r > 0.5, P < 1 × 10 −5 , data not shown). Accordingly, the majority of mice treated with Ppara siRNA#1 grouped with Ppara −/− mice. However, three mice, Ppara siRNA#1 animals 15, 17 and 19, grouped in the clade containing the mice injected with control siRNA or Ringer's buffer alone . These mice showed little to no Ppara knockdown by RT-qPCR and may represent animals with suboptimal siRNA delivery . Upon visual inspection, the transcript profiles of these three animals does reveal some resemblance to those within the clade containing the majority of the Ppara siRNA#1-treated and the Ppara −/− mice. The magnitude of the response in these three mice was very low and is likely to be the reason why these animals group in the clade dominated by the control animals. There were other Ppara siRNA#1-treated mice displaying only apparently slight Ppara knockdown that did group in the clade containing Ppara −/− mice. It is possible that it is the change in Ppara levels that is important for perturbing expression of genes modulated by Ppara rather than the absolute levels of Ppara . If the pre-injection levels of Ppara expression in these animals were higher than average, then this change would be reflected in the overall transcriptional response to Ppara knockdown as observed here, and not necessarily in the absolute level of Ppara expression.
|
16945951_p14
|
16945951
|
Genome-wide transcript profiles of Ppara siRNA-treated mice and Ppara −/− mice are concordant
| 4.223444 |
biomedical
|
Study
|
[
0.9994322657585144,
0.00034106223029084504,
0.00022660545073449612
] |
[
0.9994274377822876,
0.00020329290418885648,
0.0002966445463243872,
0.00007259293488459662
] |
en
| 0.999998 |
In the following analyses, the transcript profiles of Ppara knockdown and Ppara −/− mice were compared to those of wild-type mice treated with the PPARα agonist fenofibrate in order to identify candidate genes that are proximal to PPARα regulation as well as to further confirm the specificity of the siRNA response ( 14 ). Although PPARα agonism is not the precise opposite of Ppara knockdown, we expected that genes proximal to PPARα regulation would be oppositely regulated in the two scenarios. Profiles of knockdown mice from Experiments A and B were included in this analysis as well as mice from a third experiment, Experiment C, in which Ppara siRNA#1, #2 or #3 were injected and livers harvested at 24, 48 or 96 h. The use of three different siRNAs targeting Ppara in Experiment C would help to ensure the profiles obtained were specific for Ppara knockdown and not due to off-target effects of any individual siRNA. A timecourse would allow us to determine the duration of the siRNA effect.
|
16945951_p15
|
16945951
|
Identification of proximal transcriptional responses to Ppara perturbation
| 4.085555 |
biomedical
|
Study
|
[
0.9995009899139404,
0.0002540842106100172,
0.0002449353050906211
] |
[
0.9995226860046387,
0.00016785356274340302,
0.0002589045907370746,
0.00005054958091932349
] |
en
| 0.999997 |
k -means clustering was used to separate the 622 genes modulated by Ppara siRNA treatment shown in Figure 2 into eight sets . Two of the sets, sets 1 and 7, were composed of 71 genes that were oppositely regulated between fenofibrate-treated mice, and Ppara siRNA-treated and Ppara −/− mice. When compared to the Gene Ontology (GO) gene sets for Biological Process annotation, these gene sets had significant correlation to metabolic pathways pertaining to oxidative phosphorylation ( P = 1.16 × 10 −6 ) and fatty acid β oxidation ( P = 5.50 × 10 −3 ), respectively ( Table 1 ). These two pathways have in common the fatty acid metabolic intermediate acetyl-CoA and are known to be directly regulated by PPARα ( 5 ). Thus, gene sets 1 and 7 are likely enriched for genes that are proximal to Ppara regulation. In addition, the fact that the genes in sets 1 and 7 show decreased expression in Ppara knockdown and Ppara −/− mice and increased expression upon fenofibrate treatment is evidence for the on-target effects of the Ppara siRNAs used. Two of the remaining gene sets, sets 5 and 8, were also identified to have significant ( P < 0.01) overlap with other GO gene sets (Table 1). Gene set 8 correlated with components of the ubiquitin-proteosome system and gene set 5 with those involved in mRNA processing. It has been shown that PPARα activity is controlled by regulating stability at the protein level and that Ppara mRNA levels are increased during fasting ( 5 , 15 ). Given the important role of PPARα in regulating fatty acid and glucose metabolism, modulation of these systems may be indicative of compensatory mechanisms utilized to maintain metabolic homeostasis when expression or activity of Ppara is perturbed. Together, these observations are consistent with the known proximal on-target effects of Ppara deficiency (gene sets 1 and 7) and putative distal responses to Ppara perturbation (gene sets 2–6 and 8).
|
16945951_p16
|
16945951
|
Identification of proximal transcriptional responses to Ppara perturbation
| 4.405888 |
biomedical
|
Study
|
[
0.9993914365768433,
0.00039610525709576905,
0.00021238747285678983
] |
[
0.9987603425979614,
0.00028524789377115667,
0.0008311544661410153,
0.00012326540309004486
] |
en
| 0.999996 |
Unsupervised agglomerative clustering of all mice using gene sets 1 and 7 resulted in two major experimental clades . Clade 1 consists of Ppara siRNA#1, #2 and #3-treated mice at 24, 48 and 96 h post administration (with two exceptions) together with the Ppara −/− mice. Thus, the signatures of mice treated with three different Ppara siRNAs are similar to that of Ppara −/− mice, with the effect of the siRNAs persisting to at least 96 h post administration. The fact that a similar signature was obtained using three different Ppara siRNAs is evidence that any potential off-target effects of individual siRNAs did not significantly affect the outcome. Clade 2 consists primarily of mice receiving control treatments (highlighted in red). A few Ppara siRNA-treated animals are also found in Clade 2. These mice had only a weak signature for genes in sets 1 and 7 used for the cluster analysis, and their presence in Clade 2 likely due to suboptimal siRNA delivery. Conversely, 2 of 56 control mice had signatures of sufficient similarity to Ppara knockdown and knockout mice that they were grouped into Clade 1. It is known that Ppara expression levels are modulated by dietary intake ( 5 , 9 ). Even though all mice had free access to food for the duration of the experiments, minor variations in Ppara expression due to different feeding behaviours among individual mice were not unexpected. These minor differences appear to be reflected at least to some extent in the transcript profiles of two of the control mice.
|
16945951_p17
|
16945951
|
Identification of proximal transcriptional responses to Ppara perturbation
| 4.145292 |
biomedical
|
Study
|
[
0.9993996620178223,
0.0003378169785719365,
0.0002625088964123279
] |
[
0.9994661211967468,
0.00016640388639643788,
0.0003099319583270699,
0.00005758617407991551
] |
en
| 0.999997 |
We also note that for six of the animals treated with Ppara siRNAs in Experiment C a markedly different transcription signature was observed (data not shown). We were unable to reproduce this response in independent experiments, and as the signature was sufficiently high to obscure our analyses these mice were excluded. We did not detect evidence of an interferon-like transcriptional response in siRNA-treated animals in any of our experiments, consistent with previous reports using hydrodynamic delivery of naked siRNA ( 16 ).
|
16945951_p18
|
16945951
|
Identification of proximal transcriptional responses to Ppara perturbation
| 3.970393 |
biomedical
|
Study
|
[
0.9993889331817627,
0.00024010676133912057,
0.0003709751763381064
] |
[
0.999527096748352,
0.00023380080529022962,
0.00018738718063104898,
0.00005170002259546891
] |
en
| 0.999997 |
As a final test of the specificity of the transcriptional response to Ppara knockdown, we identified nine genes present on our microarray and reported in the literature to have functional (i.e. demonstrated by transfection and/or DNA binding assays) peroxisome proliferator response elements (PPREs) in their promoters ( 17 – 25 ). Unsupervised agglomerative clustering of all 125 transcription signatures revealed that genes with functional PPREs were predominantly down-regulated in Ppara siRNA-treated animals . In addition, all nine genes investigated were up-regulated with the PPARα agonist fenofibrate, lending support to this rationale for identifying proximal Ppara regulated candidate genes as described in the previous section.
|
16945951_p19
|
16945951
|
Treatment with Ppara siRNA alters expression of known PPARα targets
| 4.113082 |
biomedical
|
Study
|
[
0.9995026588439941,
0.00023612116638105363,
0.00026118941605091095
] |
[
0.9995110034942627,
0.00017222385213244706,
0.0002647216897457838,
0.00005205674096941948
] |
en
| 0.999998 |
The phenotype of Ppara −/− mice is readily apparent after fasting and includes decreased blood glucose levels and increased triglycerides ( 5 , 9 , 26 ). To determine the phenotypic effect of Ppara knockdown, we collected blood from mice prior to delivery of Ppara siRNA#1, and then at 24 h (Experiments A and C), 48 h (Experiment C) and 96 h (Experiment C) after siRNA delivery and performed assays for the appropriate physiological markers. In these analyses, mice had free access to food and were not purposely fasted before, during or after siRNA delivery.
|
16945951_p20
|
16945951
|
Mice treated with Ppara siRNA display a similar but distinct phenotype to Ppara −/− mice
| 4.061911 |
biomedical
|
Study
|
[
0.9995373487472534,
0.00023893329489510506,
0.00022373427054844797
] |
[
0.9995080232620239,
0.00018511840607970953,
0.00024877788382582366,
0.000058059369621332735
] |
en
| 0.999995 |
As expected, we observed an increase in ALT and AST levels in the serum at 24 h due to the effects of the hydrodynamic injection method in the liver. However, these returned to near normal levels by 96 h, and were not significantly different between treated and control groups. In contrast, a group comparison of the Ppara siRNA#1-treated animals to those treated with control siRNA in Experiment A revealed a significant decrease in plasma glucose ( P < 0.05) and increase in triglyceride ( P < 0.01) concentrations 24 h after siRNA delivery . Ppara knockdown as determined by microarrays relative to all Experiment A animals correlated significantly with modulations in glucose ( r = 0.59, P < 0.01) and triglyceride ( r = −0.60, P < 0.001) concentrations . The fact that these phenotypes were observed in the absence of fasting is contradictory to the situation in similarly aged Ppara −/− mice, where fasting is required. These data indicate that knockdown of Ppara expression using siRNA yields a phenotype consistent with the known function of Ppara , but one that does not fully recapitulate that of the knockout.
|
16945951_p21
|
16945951
|
Mice treated with Ppara siRNA display a similar but distinct phenotype to Ppara −/− mice
| 4.186973 |
biomedical
|
Study
|
[
0.9995513558387756,
0.0002877462829928845,
0.00016085935931187123
] |
[
0.9992569088935852,
0.00022740711574442685,
0.0004381122125778347,
0.0000775178341427818
] |
en
| 0.999996 |
In Experiment C, the treatment related trend for both plasma glucose and triglyceride concentrations was consistent with findings in Experiment A. However, the decrease in blood glucose observed in mice treated with Ppara siRNA#1 compared to control mice was not above a statistically significant threshold in Experiment C , despite the fact that Ppara knockdown by siRNA#1 in Experiment C at 24 h was on average equivalent to that observed in Experiment A. The increase in blood triglyceride concentration was statistically significant at 48 h ( P = 0.02), but increased below a statistically significant threshold at 24 h ( P = 0.20) and 96 h ( P = 0.41) . One possible explanation for the less pronounced phenotype observed in Experiment C versus Experiment A mice may lie in the fact that the Experiment C mice were male, whereas Experiment A mice were female. Sexually dimorphic responses have been reported previously in Ppara −/− mice ( 10 , 27 ).
|
16945951_p22
|
16945951
|
Mice treated with Ppara siRNA display a similar but distinct phenotype to Ppara −/− mice
| 4.099379 |
biomedical
|
Study
|
[
0.9995009899139404,
0.00027847522869706154,
0.0002205287164542824
] |
[
0.9995090961456299,
0.00017858629871625453,
0.0002563913876656443,
0.000055870041251182556
] |
en
| 0.999996 |
The main objective of this study was to determine if delivery of naked siRNA using hydrodynamic tail vein injection would result in functional inhibition of target gene expression in mouse liver. We chose to target Ppara , a well-characterized gene that is critically important in fatty acid metabolism, for which a genetic knockout exists, and whose protein product is the target of therapeutically relevant drugs. We obtained several lines of evidence indicating that delivery of Ppara siRNA induced target-specific inhibition. First, quantification of Ppara mRNA levels by RT-qPCR or microarrays indicated a significant reduction in mice receiving Ppara siRNA compared to those receiving a control. Second, we observed high-transcriptional concordance in both magnitude and direction between Ppara siRNA-treated mice and in Ppara −/− mice using genome-wide transcriptional profiling. The transcriptional changes were maintained for at least 96 h and were evident with three different siRNAs. Third, sets of genes were identified that were oppositely regulated in siRNA-treated mice compared to mice treated with the PPARα agonist fenofibrate. These gene sets were highly correlated with GO gene sets pertaining to oxidative phosphorylation and fatty acid β oxidation, processes shown previously to be directly regulated by PPARα ( 5 ). Fourth, genes known to contain functional PPARα-binding sites in their enhancers were expressed at lower levels in mice receiving Ppara siRNA than in control mice. Finally, mice treated with Ppara siRNA displayed phenotypes similar to those observed in Ppara −/− mice, namely hypoglycemia and hypertriglyceridemia. Thus, both molecular and phenotypic data indicate that functional delivery of naked siRNA to the liver was achieved.
|
16945951_p23
|
16945951
|
DISCUSSION
| 4.315389 |
biomedical
|
Study
|
[
0.9993801116943359,
0.0004377781879156828,
0.00018208161054644734
] |
[
0.999035120010376,
0.0003072350227739662,
0.0005397570785135031,
0.00011787281255237758
] |
en
| 0.999997 |
Although the phenotypes of animals treated with Ppara siRNA were similar to those reported for Ppara −/− mice, we also noted important differences. The most significant difference was the appearance of hypoglycemia and hypertriglyceridemia in Ppara siRNA-treated animals in the absence of fasting. This suggests that Ppara functions to maintain lipid and glucose homeostasis regardless of the fed state of the animal. This was an unexpected finding given the requirement for fasting to uncover the phenotypes in Ppara −/− mice. It is possible that in Ppara −/− mice, which are devoid of Ppara function throughout development, compensatory mechanisms are induced during growth and development that are sufficient to maintain homeostasis in the fed state, but insufficient in the fasted state. Fasting places a greater reliance on fatty acid oxidation in the liver, which is required to generate ketone bodies needed to supply the energy needs of tissues such as muscle and brain ( 28 ). In animals treated with Ppara siRNA, we speculate that Ppara expression is inhibited before the putative compensatory mechanisms can be fully induced. Alternatively, it is possible that the lack of complete knockdown impacted the phenotypes we observed in the knockdown mice versus those of the Ppara −/− mice. We cannot differentiate these possibilities based on the data presented in this report.
|
16945951_p24
|
16945951
|
DISCUSSION
| 4.207662 |
biomedical
|
Study
|
[
0.9995788931846619,
0.0002629109949339181,
0.0001582620170665905
] |
[
0.9992271661758423,
0.0002626215573400259,
0.00043120502959936857,
0.00007895465387264267
] |
en
| 0.999997 |
How an animal responds to decreased Ppara function may also depend on its gender. In our study, we observed more dramatic phenotypes in female versus male mice treated with siRNA. Gender-related differences in the phenotypes of Ppara −/− mice have also been noted. Djouadi et al . ( 27 ) found that inhibition of fatty acid oxidation in Ppara −/− mice by administration of an irreversible inhibitor of carnitine palmitoyltransferase I resulted in more severe phenotypes in males than in females. These phenotypes could be rescued by pre-treatment with estradiol. In aged Ppara −/− mice, Costet et al . ( 10 ) reported sexually dimorphic phenotypes including obesity and increased serum triglyceride levels in females, and steatosis and increased hepatic triglyceride levels in males. Together, these studies and the present one indicate that the gender of the animal affects how it responds to perturbations in Ppara expression.
|
16945951_p25
|
16945951
|
DISCUSSION
| 4.104947 |
biomedical
|
Study
|
[
0.9996198415756226,
0.00019272879580967128,
0.0001874919980764389
] |
[
0.9989705085754395,
0.0001940568326972425,
0.0007771902601234615,
0.0000582899920118507
] |
en
| 0.999997 |
We have shown here that a single hydrodynamic tail vein injection of a relatively small dose of naked siRNA leads to inhibition of Ppara expression in mouse liver to biological effect. These results suggest that this method can be used as a means to uncover gene function in vivo . However, there are caveats to the use of siRNA including the potential of off-target effects, incomplete knockdown and non-targeting of splice variants by the selected siRNA sequence. The use of multiple siRNAs aids in determining if the observed results are due to inactivation of the target gene itself and not due to other effects. Incomplete knockdown of target gene function as well as the transient nature of knockdown using siRNA may also impact the severity and specifics of the phenotype observed. The use of traditional gene knockout technology would be preferable to gene knockdown technology in this sense. However, the cost and time required to generate knockout mice is substantial. In light of this, one potential application of the siRNA approach would be as a screening method to gain insight on the phenotypes of large numbers of genes quickly. The function of the genes identified in the RNAi screen could then be verified or analyzed in more detail by creating knockout strains. The data presented in this report indicate that this could be a viable strategy for gaining an understanding of gene function in liver.
|
16945951_p26
|
16945951
|
DISCUSSION
| 4.253499 |
biomedical
|
Study
|
[
0.9995429515838623,
0.0003147563838865608,
0.0001422998757334426
] |
[
0.9981834292411804,
0.00041705917101353407,
0.0012892904924228787,
0.00011018503573723137
] |
en
| 0.999997 |
Comparative genomics is a fundamental and quickly developing evolutionary approach ( 1 , 2 ). While an increasing diversity of genomes are currently being sequenced ( 3 ) and promising new technologies could greatly reduce the cost and speed up the process of whole genomic sequencing ( 4 , 5 ), the vast majority of Earth's biodiversity will not have its genome sequenced in the near future. Given the current pace of large-scale environmental change, particularly inthe tropics ( 6 – 8 ) where biodiversity is greatest ( 9 ), novel techniques are required for biologists to rapidly develop the genomic resources for understudied organisms. Additionally, many of these tropical organisms, like rainforest trees, possess radically different life history strategies and evolutionary dynamics than current model organisms, which are short-lived with simple genomes, implying a rather limited amount of knowledge gained from current approaches will be transferable. The ability to understand historical patterns of genomic diversity created over geological and glacial time scales is essential for the future management of natural populations. Human activities are erasing the traces of these patterns before we can define them. This ignorance will lead to an extinction of the historical past, which we desperately need to properly interpret the present and plan for the future. While a certain amount of information can be leveraged out of available whole genome sequences ( 2 ), a fast and direct method for gathering genomic samples of genetic variation from previously unstudied organisms is needed.
|
17000641_p0
|
17000641
|
INTRODUCTION
| 4.126414 |
biomedical
|
Study
|
[
0.9990160465240479,
0.0003626782854553312,
0.0006212607258930802
] |
[
0.7610318660736084,
0.014103305526077747,
0.22428575158119202,
0.000579098064918071
] |
en
| 0.999997 |
Here, we present an anonymous DNA microarray capable of capturing genomic signatures of DNA sequence variation from any organism using only a few hybridizations. The microarray probe sequences are generated using a pre-determined set of selection criteria, which could be modified and refined with increasing knowledge and specificity of application. In brief, the probe sequences are SHyPs: short (25 bp and less), hyperdispersed in sequence probability space, *anonymous* because they are generated without knowledge of the target genome and primers as they should fit optimality criteria for a PCR primer. A theoretical framework of such an approach, aimed at expressed sequences, has been described ( 10 ) and other microarray based approaches have targeted specific groups of organisms ( 11 ). The criteria used for the selection of the SHyP probes should harvest presence/absence information for a large and complex set of DNA sequences scattered throughout the entire genome. Major changes in copy number of these sequences, due to genomic re-organization or proliferation of certain elements, should be detectable ( 12 ). After construction of a genomic signature database, virtual comparisons between any subset of genomes would be possible and simple queries would generate optimal sets of oligonucleotide sequences to distinguish among target genomes. These informative markers could be used in larger-scale and cheaper downstream macroarray or PCR-based screening techniques. The results from comparative analyses of two genomes will also offer an access point directly related to the interesting and different regions of the targets.
|
17000641_p1
|
17000641
|
INTRODUCTION
| 4.274148 |
biomedical
|
Study
|
[
0.9994449019432068,
0.00030808564042672515,
0.00024693741579540074
] |
[
0.9984992742538452,
0.0006971170078031719,
0.0007109002908691764,
0.00009272926399717107
] |
en
| 0.999997 |
To prove the basic elements of the concept, we hybridized three known genomes (human, mouse and rat) and the previously undescribed genome of a tropical tree species (‘ramin’: Thymelaeaceae: Gonystylus bancanus ) to a custom 44 K feature microarrays fabricated by Agilent's SurePrint technology. The use of whole genomic DNA has been shown to be reliable in these types of hybridizations ( 13 ). ‘Ramin’ is an endangered species found only in peat-swamp forests along the inner margins of the South China Sea ( 14 ) and is vulnerable to extinction ( 15 ). Two ramin population samples, one from the east coast of Sumatra and one from the northwestern coast of Borneo, each composed of five individuals, were compared to discover genetic markers related to their geographic origin. The study design will allow us to testthe sensitivity of the approach, in this first iteration, to a broad range of genomic relatedness and the reliability and transitivity of hybridization signal across hybridization experiments. A simple and direct analysis is also presented to take advantage of the replication of hybridization experiments to identify the oligonucleotide probe sequences that provide results with relatively little variance across genomes and experiments. A BLAST study of a subset of probe sequences was performed to examine the distribution of these sequences in the known genomes and its correlation with observed hybridization intensities. We also discuss potential improvements in the probe sequence, microarray design and experimental procedures.
|
17000641_p2
|
17000641
|
INTRODUCTION
| 4.143468 |
biomedical
|
Study
|
[
0.9993370175361633,
0.0002684654900804162,
0.00039438146632164717
] |
[
0.9995006322860718,
0.00016352266538888216,
0.0002762653457466513,
0.00005953816798864864
] |
en
| 0.999999 |
Our strategy was to construct oligonucleotide probe sequence of 25 bp using a random sequence generator and several filtering criteria. These criteria included:
|
17000641_p3
|
17000641
|
Generation of SHyP microarray probes
| 2.857281 |
biomedical
|
Study
|
[
0.9961916208267212,
0.00044278238783590496,
0.00336557743139565
] |
[
0.9471978545188904,
0.05176442116498947,
0.0005386049160733819,
0.0004991262685507536
] |
en
| 0.999994 |
Numerous iterations were performed to generate 44 000 anonymous probe sequences fitting these criteria. Measures discussed below of ‘maximum sequence identity’ refer to the maximum amount of identical sequence between two oligonucleotide probes, within any 17 bp window of comparison.
|
17000641_p4
|
17000641
|
Generation of SHyP microarray probes
| 3.489928 |
biomedical
|
Study
|
[
0.9979536533355713,
0.0002799203502945602,
0.0017664418555796146
] |
[
0.9862246513366699,
0.013243142515420914,
0.0003365399606991559,
0.00019558427447918802
] |
en
| 0.999996 |
Samples of G.bancanus (ramin) were obtained through the cooperation of the Forest Research Center in Kuching, Malaysia and P.T. Diamond Raya Timber, Pekanbaru, Sumatra, Indonesia, and whole genomic DNA was extracted using a standard CTAB protocol. ‘Population samples’ for ramin were created by mixing equal amounts of genomic DNA of five different individuals from each population (Sarawak individuals = ‘RaminSK5’; Sumatra individuals = ‘RaminSU5’). To assess the impact of false positives, a self-self hybridization experiment was also performed using the rat genomic DNA .
|
17000641_p5
|
17000641
|
Preparation and hybridization of genomic DNA
| 3.688976 |
biomedical
|
Study
|
[
0.990993082523346,
0.00026082212571054697,
0.008746066130697727
] |
[
0.9981546998023987,
0.0016545905964449048,
0.00013686303282156587,
0.00005384465475799516
] |
en
| 0.999995 |
The complexity of mouse (Promega), human (Promega), rat (EMD Biosciences) and ramin genomic DNA was reduced by restriction endonuclease digestion. Recognizing that the active site for any single enzyme could involve a fairly large percentage of the SHyP probe sequences, e.g. both AluI and RsaI would affect 17.2% of the probes, we used a combination of three restriction endonucleases (MboI, AluI and RsaI: NEB) and performed separate digestions with each of the enzymes and then pooling these DNAs together prior to enzymatic labeling, the restriction reactions affect only 4 probes (0.009%) on the microarray. Purified digestion products were assessed on an Agilent Bioanalyzer to assess the distribution of fragment sizes before proceeding to labeling reactions. Four micrograms of pooled genomic DNA was labeled using either Cy 3 or Cy 5 following the BioPrime Array CGH Genomic Labeling System (Invitrogen). Purified labeling reaction products were quantified on a Nanodrop-1000 Spectrophotometer to determine the amount of product prior to hybridization. Labeled products were hybridized to a SHyP array for 40 h at 65°C before the microarray was washed and scanned using an Agilent DNA Microarray Scanner BA. The resulting image was then processed through Agilent's Feature Extraction software (version 7.5.1) in order to obtain intensity measurements and determine statistical difference in intensity level.
|
17000641_p6
|
17000641
|
Preparation and hybridization of genomic DNA
| 4.182453 |
biomedical
|
Study
|
[
0.9995613694190979,
0.00024583260528743267,
0.00019273828365840018
] |
[
0.9989591836929321,
0.0006401018472388387,
0.00032062927493825555,
0.00008010253077372909
] |
en
| 0.999997 |
Each comparison between genomes was performed twice, using each of the labeling dyes . Subsequent analyses across hybridizations were performed on the average intensity from these two fluor-flipped hybridizations to minimize any labeling bias or experimental effects. Therefore, five genomic comparisons were performed using ten separate microarrays: (i) mouse><rat; (ii) mouse><human; (iii) rat><human; (iv) rat><rat and (v) raminSK5><raminSU5.
|
17000641_p7
|
17000641
|
Preparation and hybridization of genomic DNA
| 3.972183 |
biomedical
|
Study
|
[
0.9994701743125916,
0.00016489952395204455,
0.0003649468708317727
] |
[
0.9990066885948181,
0.0006691264570690691,
0.0002658517623785883,
0.00005839174991706386
] |
en
| 0.999997 |
‘Genome-indicator’ probes were determined by (i) discarding all probes with a hybridization intensity value below the mode intensity value for each hybridization and (ii) choosing probes that were significantly ‘up-regulated’ in relation to the other genomes, at a P < 1 × 10 −8 confidence limit, as determined by image analysis using Agilent's gene expression software. This significance level is highly stringent and removed most of the false positives, as determined in the rat–rat hybridization. A subset of high intensity sequences in all mammal genomes was mapped onto the known chromosome database at NCBI, using the basic BLAST search tool, to examine the genomic distribution of these sequences.
|
17000641_p8
|
17000641
|
Standard microarray analysis
| 4.133555 |
biomedical
|
Study
|
[
0.9994996786117554,
0.00020970510377082974,
0.0002906115842051804
] |
[
0.9994091987609863,
0.00028777093393728137,
0.0002542991715017706,
0.00004877196624875069
] |
en
| 0.999998 |
Because the statistical analysis used in standard gene expression software is concerned with the up or down regulation of expressed sequences known to be present in the target genomes, low to no hybridization signal in one or both channels is usually interpreted as failure of the probe. This pattern, where hybridization fails in one genome but is reliably detected in the other, is ideal to capture informative genomic signatures. We have found in both downstream PCR work and in silico BLAST studies that probes with high signal values but ‘upregulated’ in one channel, are actually frequent in both genomes. While changes in copy number across genomes is important, the current approach is focused on detecting the presence and absence of evolutionarily labile DNA sequences across genomes.
|
17000641_p9
|
17000641
|
Low variance analysis
| 4.143479 |
biomedical
|
Study
|
[
0.9995282888412476,
0.00016505368694197387,
0.00030674715526401997
] |
[
0.9989902377128601,
0.0005873037152923644,
0.0003639078058768064,
0.000058569650718709454
] |
en
| 0.999996 |
In order to adopt the analysis of the resulting array image to this purpose, a simple protocol for establishing intensity thresholds and stability across hybridizations was developed. First, a threshold was determined for ‘detectable’ signal by simply examining the overall pooled hybridization intensities for each genomic comparison and throwing out all data that fell below the mode of the distribution. Distributions of signal are strongly skewed towards ‘empty’ values because most probes are not present in any one genome. A second threshold was determined by examining the variance of the hybridization signals near the initial threshold value. A higher threshold where detectable signals were stable was established for each hybridization. Probes with an average signal in all genomes below this second threshold were discarded. Signal levels were then compared across hybridizations for each genome and only those probes that varied <40% of the average were then considered ‘low variance’ probes. In this context, average signal above the second threshold was determined as ‘presence’ while average signal strength below was considered ‘absence’ in the target genome. With repeated experiments (only the rat is present in more than two hybridization), this standardization process will become more powerful and reliable. This analysis should be considered very stringent and not mutually exclusive to the gene expression analysis. The results of both will be included in future publications and databases. The Mathematica notebook to perform this analysis is available for download on the lead author's website.
|
17000641_p10
|
17000641
|
Low variance analysis
| 4.146209 |
biomedical
|
Study
|
[
0.9996422529220581,
0.00016504513041581959,
0.00019270517805125564
] |
[
0.9977637529373169,
0.0014958757674321532,
0.0006470076041296124,
0.00009326256986241788
] |
en
| 0.999996 |
To test for the effects of our selection criteria on the SHyP oligonucleotide sequences, we compared a subset of our probes to 3000 sequences generated in a completely random fashion. The SHyP probes were only slightly more similar to one another than random expectations (9.19 versus 9.13 maximum sequence identity within any 17 bp window). The standard deviation (1.4 bp) of the average maximum sequence identity was the same between the random and SHyP probes. Additionally, the bias towards GC enrichment slightly raises the average content to 52% for the SHyP probes. Each of the eight possible heteromeric dinucleotide combinations were present in repeats of two in roughly 14% of the probes and the proportion followed a Poisson distribution with repeats of four dinucleotides found in <0.1% of the probes (data not shown). The probe set can be downloaded from the lead author's webpage ( ).
|
17000641_p11
|
17000641
|
Probe characteristics
| 4.085374 |
biomedical
|
Study
|
[
0.9994896650314331,
0.00021130942332092673,
0.0002990257344208658
] |
[
0.9994487166404724,
0.0002799539070110768,
0.00021709217980969697,
0.00005421784953796305
] |
en
| 0.999996 |
Over half of the probes produced no detectable hybridization signal with any genome ( 23 531 out of 42 033 SHyP probes). The mouse genome had the greatest affinity for the SHyP array with 11 611 probes producing detectable signal, while the ramin genome had roughly an order of magnitude less affinity . The three mammalian genomes had an observable affinity for the same 15% of the probes . The average maximum sequence identity among these detectable mammal probes was 9.18 bp but the standard deviation (2.9) was much greater than the entire set. Among the probes with detectable signal for the ramin genomes, maximum sequence identity and GC enrichment were both high (9.54 bp and 54%, respectively). A large fraction of these detectable probes were discarded in the following more stringent analyses. Overall, these numbers indicate that the majority of the array did not hybridize with any of the study genomes and the resolving power of the array for future genomic comparisons is far from being saturated.
|
17000641_p12
|
17000641
|
Probe characteristics
| 4.143919 |
biomedical
|
Study
|
[
0.9994162321090698,
0.00024295275215990841,
0.00034090373083017766
] |
[
0.9994295239448547,
0.0002381872764090076,
0.00027459883131086826,
0.000057587752962717786
] |
en
| 0.999998 |
The mouse genome consistently hybridized to the greatest number of ‘indicator’ probes, at any level of statistical significance ( Table 1 ). Across the range of significance levels ( P < 0.01 to P < 1 × 10 −11 ), the number of ‘indicator’ probes for the three mammal genomes drops by roughly a factor of four while the rate of decline is sharper for the tree genome. The rate of false positives declines very rapidly, by well over two orders of magnitude. In comparison to the overall set of SHyP probes, most genomes demonstrate an increased degree of maximum sequence identity within each set of ‘indicator’ sequences, at any significance level, and this maximum sequence identity increased at higher levels of statistical stringency. ‘Indicator’ probes for rat and rodent genomes have higher levels of maximum sequence identity than the other genomes. Hybridization intensity for no genome had any appreciable relationship with simple GC content.
|
17000641_p13
|
17000641
|
Standard microarray analysis
| 4.177581 |
biomedical
|
Study
|
[
0.9992920160293579,
0.00027285338728688657,
0.00043512642150744796
] |
[
0.9995527863502502,
0.00017505094001535326,
0.00022298155818134546,
0.000049256068450631574
] |
en
| 0.999997 |
Among the known genomes, intensity values were highly correlated across hybridization experiments ( Table 2 ) and the ‘indicator’ probes were clearly distinguished in each pair-wise comparison . The two rodent genomes, individually and as a clade, had a much larger ‘signature’ on the SHyP array than the human genome . This result matches the reported accelerated rate of genomic evolution in the rodents, in comparison to humans ( 16 ). A very low rate of false positives was detected . While the overlapping ‘indicator’ probes for the rodents largely indicate shared descent, the overlapping probes between human:rat and human:mouse are clearly homoplasious. The degree of homoplasy seems to be substantially higher between the rat and the human.
|
17000641_p14
|
17000641
|
Standard microarray analysis
| 4.156511 |
biomedical
|
Study
|
[
0.9994470477104187,
0.00027361782849766314,
0.00027928518829867244
] |
[
0.9994480013847351,
0.0001840076147345826,
0.00031169920112006366,
0.00005636050627799705
] |
en
| 0.999998 |
The single plant genome, G.bancanus , used in these experiments was not directly compared with the three known genomes but instead a population level comparison was performed by mixing five individuals, each population found on different landmasses in Southeast Asia (Sumatra, Indonesia and Sarawak, Malaysia). In general, the genomes appeared to have less affinity for the SHyP array than the three mammalian genomes but this might have been due to the unknown nature of the genome and lower absolute amounts of whole genomic DNA. While no direct comparison was made, the hybridization signal can be compared across experiments. The ramin genome only produced 122 ‘indicator’ probes, when compared to all three mammals, but individual comparisons with mouse and human revealed 959 and 1137 ramin ‘indicator’ probes, respectively. The correlation of hybridization intensities between these two populations was quite high similar but over 107 probes were significantly different between the two populations.
|
17000641_p15
|
17000641
|
Standard microarray analysis
| 4.107746 |
biomedical
|
Study
|
[
0.999300479888916,
0.00021378467499744147,
0.00048566836630925536
] |
[
0.9995468258857727,
0.00024282559752464294,
0.00016247067833319306,
0.00004785205237567425
] |
en
| 0.999997 |
Less than half of the detectable probes (10 935) produced enough signal to be included in the low variance analysis and three quarters of these ‘active’ probes were rejected because of high variance between hybridizations, leaving only 3453 ‘low variance’ probes. The low variance analysis applied here should be considered highly stringent. By definition, no false positives are allowable in the self-self comparison. Correlation values generally improved when only the low variance probes were considered ( Table 2 ), except for the human genome where correlation values actually went down slightly. For inclusion in the low variance analysis, signal variance for each probe was averaged across all genomes, so the results should be expected to be biased towards the two rodent genomes, where intensity signals were highly correlated . As the taxonomic balance of the genomic signature database improves, this type of bias should disappear. The overall correlation among genomes roughly followed phylogenetic relatedness, although the signal for the human genome was not as highly correlated with the other genomes and was substantially less correlated with signal for the ramin genome. Low variance probes also produce higher correlation values among genomic comparisons than when all probes are considered, except in the comparison between humans and ramin.
|
17000641_p16
|
17000641
|
Low variance analysis
| 4.178118 |
biomedical
|
Study
|
[
0.9994157552719116,
0.0002527828037273139,
0.000331470335368067
] |
[
0.9994142055511475,
0.0001974566257558763,
0.0003327081212773919,
0.000055643013183726
] |
en
| 0.999998 |
The phylogenetic signal becomes even more obvious when the low variance probes are broken down into classes based upon their presence or absence in each genome ( Table 3 ). ‘Rodent’ probes (729) were slightly more frequent than ‘Mammal’ probes (682) while ‘Mouse’ probes (644) were the most common probes private to any one genome. Relatively few probes (224) were present in all four genomes. Several examples of homoplasious relationships among sets of genomes were observed: 146 probes unite rat:humans while 42 unite mice:humans. These probes could occur either through the loss or gain of the sequence within one member of the mammal clade or convergent evolution. Probes homoplasious between plants and mammals were quite rare but all possible combinations were present. The two rodent species demonstrated these homoplasious patterns in reverse order to their frequency of private probes. In both cases of human and ramin, the rat was more highly homoplasious than the mouse.
|
17000641_p17
|
17000641
|
Low variance analysis
| 4.16427 |
biomedical
|
Study
|
[
0.9992945194244385,
0.00025982639635913074,
0.00044564425479620695
] |
[
0.9994578957557678,
0.00018636073218658566,
0.0003072035906370729,
0.00004863954018219374
] |
en
| 0.999998 |
The hybridization signal of each genome within each class of ‘low variance’ probe was correlated to the phylogenetic relatedness of these genomes as well . A general trend in decreasing peak and average signal can be seen as the classes become less and less inclusive. The greatest signal was generally observed among ‘all’ and ‘mammal’ probes. This increased signal intensity is related to the repetitive nature of many of these probe sequences. Most of the ‘all’ probes have strings of dinucleotide and trinucleotide repeats, which are common in all tested genomes. These probes are particularly enriched for ‘CA’ repeats but not for other heteromeric repeats, which were equally frequent on the array. Strings of monomeric repeats, such as ‘TA’, were generally excluded given the selection criteria. The mammal class is particularly enriched with ‘TC’ and ‘TCC’ repetitive motifs, while the rodent class is enriched for ‘GC’ and ‘GCC’ repetitive motifs, although not as strongly. The human class is strongly enriched for ‘TGG’ repeats, which occur twice in several of the most common 11 letter words. While the rat, human and ramin probe classes are generally more similar to one another than would be expected, the mouse class is actually quite close to the overall similarity of probe sequence across the entire array.
|
17000641_p18
|
17000641
|
Low variance analysis
| 4.256719 |
biomedical
|
Study
|
[
0.999214768409729,
0.00027116434648633003,
0.0005141248111613095
] |
[
0.9993496537208557,
0.00021113015827722847,
0.0003848427440971136,
0.00005444381167762913
] |
en
| 0.999998 |
Because the two ramin genomes were not identical but were obtained from two populations on different landmasses, some fraction of the probes informative to the intraspecific comparison will have been excluded in the low variance analysis. Despite this bias, the proportion of probes, which were present in the ramin genome versus the other three was greater than would be expected, given the overall number of low variance probes, which had substantial signal for ramin. This result indicates that this class seems to be enriched in terms of indicator probes, in comparison to the other three much more closely related genomes. Additionally, a large proportion of the ramin probes appear to be also significantly ‘up-regulated’ in one population versus the other ( Table 1 : numbers in bold). The first ramin individual in this comparison was from a relatively small population from the Malaysian state of Sarawak while the second individual was collected along the northeastern coast of Indonesian Sumatra, where very large populations of this species can be found. The relative proportion of private probes in each population is probably a result of this difference in overall historical population size.
|
17000641_p19
|
17000641
|
Population level comparisons
| 4.104495 |
biomedical
|
Study
|
[
0.9965493679046631,
0.0003584563091862947,
0.003092213300988078
] |
[
0.999625563621521,
0.0001980850356630981,
0.0001401900517521426,
0.00003614111483329907
] |
en
| 0.999997 |
The probe sequences are evenly distributed through the known chromosome structure of the target genomes , with an obvious positive correlation in the length of the chromosome and the number of BLAST hits . The accelerated rate of accumulation of probe sequences in the two rodent genomes is quite clear, as the human chromosomes consistently contain fewer probes per chromosome length (Mb). Looking in greater detail at the human genome, the distribution of these BLAST hits are evenly distributed across all of the chromosomes . Occasional ‘hotspots’, where probe sequence abundance was high relative to chromosome length, were evident . Likewise, occasional ‘deserts’, where probe sequences were completely absent, were also evident .
|
17000641_p20
|
17000641
|
Genomic distribution of high intensity probe sequences
| 4.209574 |
biomedical
|
Study
|
[
0.9994603991508484,
0.00019290509226266295,
0.00034675790811888874
] |
[
0.9988391995429993,
0.0007434725412167609,
0.0003536541189532727,
0.0000636867989669554
] |
en
| 0.999997 |
In using three well-studied genomes as the initial step in these ‘proof of concept’ experiments, the results of this novel approach can be fully explored and verified against this rich body of knowledge. One comparative genomic aspect of our results, which agrees well with previous studies ( 2 ), is the greater number of ‘indicator’ probes in the two rodent genomes, both individually and as a clade. This indicates that the SHyP hybridization captured both phylogenetic signal and the generally increased rate of neutral DNA sequence evolution observed previously in rodents. If rodent genomes evolve roughly three times as fast as the human genome, they should be expected to accumulate more ‘anonymous’ probe sequences in their genomes. Differential measures of homoplasious evolution were also generated by our analysis, indicating a higher level of overall homoplasy in the rat genome, relative to the mouse and human genomes. The effects of homoplasy would also explain the slightly smaller number of rat versus mouse genome-indicator probes , particularly if the rat is somehow more constrained in its genomic evolution and basically re-evolves the same sequences. Ongoing BLAST studies will be used to examine the distribution of SHyP probes across all known genomes, available through the NCBI database. Predictive analyses for these other known genomes could be used to further explore the reliability and sensitivity of the array to genomic variation and in the design of the probe sequences.
|
17000641_p21
|
17000641
|
DISCUSSION
| 4.220104 |
biomedical
|
Study
|
[
0.9994595646858215,
0.00032462197123095393,
0.00021574039419647306
] |
[
0.9993231296539307,
0.00021007774921599776,
0.00039237304008565843,
0.00007441572961397469
] |
en
| 0.999996 |
The use of the array for screening plant genomes needs further exploration. The overall signal for the ramin population samples was substantially lower than any of the mammal genomes. Because these samples were mixtures of five individuals, contaminants or inhibitors may have been present in these genomic DNA extracts. These plants are poorly studied and little previous work has been performed on their DNA ( 17 , 18 ). In comparison to mammal tissue samples, plant tissues often contain a wide variety of potential compounds, which may act as inhibitors in downstream DNA protocols and extraction conditions frequently have to be optimized to improve overall results. The success of these experiments, in their initial attempt, is a testament to the robustness and ability of the technique to screen completely unknown samples. Given the overall lower intensity of the ramin hybridizations, the number of informative probe sequences detected, both between plants and animals and between populations, was substantially higher than would be expected.
|
17000641_p22
|
17000641
|
DISCUSSION
| 4.103095 |
biomedical
|
Study
|
[
0.9994376301765442,
0.00017678264703135937,
0.0003854933602269739
] |
[
0.9993964433670044,
0.00028266303706914186,
0.0002737008035182953,
0.000047228630137396976
] |
en
| 0.999997 |
While this study demonstrates the feasibility and power of the SHyP approach against an information rich background, further refinement of the technique is necessary and possible. In terms of probe sequence design, the ongoing BLAST studies indicate that none of the 25 bp SHyP probe sequences have perfect maximum sequence identity with any known genome sequence. The vast majority of matches are between 17–19 bp in length. By shortening the length of the oligonucleotide probe sequences and the hybridization/wash conditions, the specificity of these interactions can be increased. The current length probably loses some discriminatory power. Also, in the future, hybridizations will be performed after careful screening via flow cytometry to carefully measure C-content ( 19 ), particularly important for examining polyploidy plant genomes. These C -values would allow researchers to control for the amount of genomic DNA used in comparative hybridizations between organisms with different ploidy numbers in order to better interpret the resulting dye intensities. Higher density DNA microarray platforms will also allow a greater diversity of probe sequence representation. These higher densities will not only increase the overall sensitivity and power of the analysis but will also facilitate the inclusion of specific classes of genomic probes ( 20 ), such as the AT-rich chloroplast genomes ( 21 ). With increasing knowledge, the use of consistently present ‘universal’ probe sequences across the array could provide an important positive control for the array design.
|
17000641_p23
|
17000641
|
DISCUSSION
| 4.191392 |
biomedical
|
Study
|
[
0.9992609620094299,
0.0002984826860483736,
0.0004405461368151009
] |
[
0.9993540644645691,
0.00018450831703376025,
0.00041228230111300945,
0.00004915904355584644
] |
en
| 0.999997 |
The construction of a genomic signature database, using quickly developing, increasingly flexible DNA microarray technologies and the SHyP-CGH protocol described here, would allow biologists from a wide range of fields and interests to examine genomic scale data. While DNA microarray studies are cost-intensive, the approach described here would use ‘type’ specimens, formed by pooling genomes from several individuals of each target evolutionary unit, such as a ‘species’ or ‘population’. These types would be incorporated into the genomic signature database after a minimum number of comparative hybridizations, against both comparative targets and standards. Theoretically, only three or four hybridizations would be necessary for each type. After these initial set of hybridizations, no further DNA microarray experiments would be necessary for that target genome. Because the results are transitive across experiments and the hybridizations are performed against a standard platform, virtual comparisons would be possible against any other genome already present in the database. The results would be easily transferable to cheap downstream screening protocols designed for large numbers of individuals, such a macroarray or PCR-based techniques ( 22 ). A simple query of the genomic signature database would produce informative sets of probe sequences, based upon the target genomes. These informative probes would each represent an independently segregating locus sampled from across all genomic compartments. Given the large number of informative sites produced in this simple proof of concept experiment, this approach should be very cost-effective, using the DNA microarray based experiments only as a way to generate the database while all further assays use proven population-based techniques with a smaller subset of informative markers.
|
17000641_p24
|
17000641
|
DISCUSSION
| 4.299409 |
biomedical
|
Study
|
[
0.9994993209838867,
0.00029851376893930137,
0.00020214599499013275
] |
[
0.9978275895118713,
0.0010732036316767335,
0.0009838319383561611,
0.00011532293137861416
] |
en
| 0.999995 |
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