Background TOR, the  t arget  o f the antibiotic  r apamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. It acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner Raptor. In Arabidopsis, Raptor activity is essential for postembryonic growth. Though comparative studies suggest potential downstream effectors, no Raptor binding partners have been described in plants.
Background
TOR, the  t arget  o f the antibiotic  r apamycin in both yeast and mammalian cells, is a potent cell growth regulator in all eukaryotes. It acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner Raptor. In Arabidopsis, Raptor activity is essential for postembryonic growth. Though comparative studies suggest potential downstream effectors, no Raptor binding partners have been described in plants.Results AtRaptor1B, a plant Raptor homologue, binds the AML1 ( A rabidopsis  M ei2- l ike 1) protein in a yeast two-hybrid assay. This interaction is mediated by the N-terminal 219 residues of AML1, and marks AML1 as a candidate AtTOR kinase substrate in plants. The AML1 N-terminus additionally carries transcriptional activation domain activity. Plants homozygous for insertion alleles at the  AML1  locus, as well as plants homozygous for insertion alleles at all five loci in the  AML  gene family, bolt earlier than wild-type plants.
Results
AtRaptor1B, a plant Raptor homologue, binds the AML1 ( A rabidopsis  M ei2- l ike 1) protein in a yeast two-hybrid assay. This interaction is mediated by the N-terminal 219 residues of AML1, and marks AML1 as a candidate AtTOR kinase substrate in plants. The AML1 N-terminus additionally carries transcriptional activation domain activity. Plants homozygous for insertion alleles at the  AML1  locus, as well as plants homozygous for insertion alleles at all five loci in the  AML  gene family, bolt earlier than wild-type plants.AML1AMLConclusion AML1 interacts with AtRaptor1B, homologue of a protein that recruits substrates for phosphorylation by the major cell-growth regulator TOR. Identification of AML1 as a putative downstream effector of TOR gives valuable insights into the plant-specific mode of action of this critical growth regulator.
Conclusion
AML1 interacts with AtRaptor1B, homologue of a protein that recruits substrates for phosphorylation by the major cell-growth regulator TOR. Identification of AML1 as a putative downstream effector of TOR gives valuable insights into the plant-specific mode of action of this critical growth regulator.

Background TOR, the  t arget  o f the antibiotic  r apamycin in both yeast and mammalian cells, is a major regulator of cell growth and translation [ 1 ]. TOR is a large (over 2,400 residues) protein kinase [ 2 ] present in all eukaryotes analyzed. It is thought to act in a nutrient-sensitive complex TORC1 with Raptor ( r egulatory  a ssociated  p rotein of TOR) and another protein to regulate cell growth [ 3 - 7 ] – though there is some debate on the nutrient sensitivity of the complex [ 8 ]. Raptor, a protein with HEAT and WD-40 protein interaction domains, recruits substrates for phosphorylation by TOR in yeast and mammals [ 3 , 7 ]. TOR also acts in a second, nutrient-insensitive complex without Raptor to regulate the cytoskeleton [ 3 , 8 , 10 ]. Disruption of the Arabidopsis TOR homologue AtTOR is lethal early in plant embryonic development [ 11 ]. Disruption of AtRaptor (encoded by two paralogous loci in Arabidopsis) causes seedling developmental arrest but allows normal embryonic development [[ 12 ], manuscript in preparation], suggesting that AtTOR embryonic activity is independent of AtRaptor and that the TOR-Raptor complex has been adapted in the ancestor of the angiosperms to regulate post-embryonic growth. In support of this, AtTOR has been show to be expressed in dividing and expanding cells [ 11 ]. Thus, identifying downstream activators of TOR signaling may provide insights into the activity of AtTOR in post-embryonic growth. Mei2 is a putative TOR substrate and potent meiosis-signaling molecule identified in the fission yeast  Schizosaccharomyces pombe  [ 13 , 14 ]. Mei2 is bound by the Raptor homologue Mip1 [ 15 ], and is an inactive phosphoprotein under high nutrient conditions [ 16 , 17 ] – conditions which promote TOR kinase activity. The kinase governing two of the three Mei2 phosphorylation sites is known [ 16 ]; TOR is a strong candidate for the kinase governing the third. In diploids under low nutrient conditions, unphosphorylated Mei2 accumulates and localizes to the nucleus, where it binds to a noncoding, mRNA-like RNA molecule meiRNA in an interaction mediated by the third of its three RNA Recognition Motifs (RRMs)[ 14 , 16 , 18 ]. The Mei2-meiRNA interaction occurs as meiRNA is being transcribed, tethering Mei2 to the meiRNA locus [ 19 ]. Accumulation of Mei2 at this focused point immediately precedes meiosis. Mei2-like proteins are predicted in a wide range of organisms [ 20 , 21 ] including some fungi, alveolates, a diatom, and all land plants, but they are absent from metazoans and budding yeast. In land plants, predicted Mei2-like proteins form a small conserved gene family, many of whose members' transcripts accumulate in the shoot apical meristem specifically or in the shoot apical meristem in addition to a range of mature tissues [ 20 ]. A rabidopsis  M ei2- l ike 1 (AML1), the first member of this family to be described, was isolated in a screen for plant cDNAs whose expression could complement defects in the fission yeast meiosis signaling pathway [ 22 ]. Like Mei2, AML1 has three RRMs. Expression of a protein fragment containing only the third AML1 RRM was sufficient for restoration of meiosis signaling in fission yeast lines with defects upstream of Mei2, but not in lesions of the  mei2  locus itself.  T erminal  E ar 1 (te1), a more divergent member of the  mei2 -like family of genes, regulates leaf initiation in maize; the tassel of mutant plants is encased a whorl of leaves superficially resembling a maize ear [ 23 ]. Given the potential role of downstream AtTOR effectors in post-embryonic growth, the intriguing signaling activity of Mei2 in fission yeast, and the known interaction between Mei2 and the fission yeast Raptor protein, we asked if the Mei2-Raptor interaction was conserved between its Arabidopsis orthologues AML1 and AtRaptor1B.
Background
TOR, the  t arget  o f the antibiotic  r apamycin in both yeast and mammalian cells, is a major regulator of cell growth and translation [ 1 ]. TOR is a large (over 2,400 residues) protein kinase [ 2 ] present in all eukaryotes analyzed. It is thought to act in a nutrient-sensitive complex TORC1 with Raptor ( r egulatory  a ssociated  p rotein of TOR) and another protein to regulate cell growth [ 3 - 7 ] – though there is some debate on the nutrient sensitivity of the complex [ 8 ]. Raptor, a protein with HEAT and WD-40 protein interaction domains, recruits substrates for phosphorylation by TOR in yeast and mammals [ 3 , 7 ]. TOR also acts in a second, nutrient-insensitive complex without Raptor to regulate the cytoskeleton [ 3 , 8 , 10 ].Disruption of the Arabidopsis TOR homologue AtTOR is lethal early in plant embryonic development [ 11 ]. Disruption of AtRaptor (encoded by two paralogous loci in Arabidopsis) causes seedling developmental arrest but allows normal embryonic development [[ 12 ], manuscript in preparation], suggesting that AtTOR embryonic activity is independent of AtRaptor and that the TOR-Raptor complex has been adapted in the ancestor of the angiosperms to regulate post-embryonic growth. In support of this, AtTOR has been show to be expressed in dividing and expanding cells [ 11 ]. Thus, identifying downstream activators of TOR signaling may provide insights into the activity of AtTOR in post-embryonic growth.Mei2 is a putative TOR substrate and potent meiosis-signaling molecule identified in the fission yeast  Schizosaccharomyces pombe  [ 13 , 14 ]. Mei2 is bound by the Raptor homologue Mip1 [ 15 ], and is an inactive phosphoprotein under high nutrient conditions [ 16 , 17 ] – conditions which promote TOR kinase activity. The kinase governing two of the three Mei2 phosphorylation sites is known [ 16 ]; TOR is a strong candidate for the kinase governing the third. In diploids under low nutrient conditions, unphosphorylated Mei2 accumulates and localizes to the nucleus, where it binds to a noncoding, mRNA-like RNA molecule meiRNA in an interaction mediated by the third of its three RNA Recognition Motifs (RRMs)[ 14 , 16 , 18 ]. The Mei2-meiRNA interaction occurs as meiRNA is being transcribed, tethering Mei2 to the meiRNA locus [ 19 ]. Accumulation of Mei2 at this focused point immediately precedes meiosis.Schizosaccharomyces pombeMei2-like proteins are predicted in a wide range of organisms [ 20 , 21 ] including some fungi, alveolates, a diatom, and all land plants, but they are absent from metazoans and budding yeast. In land plants, predicted Mei2-like proteins form a small conserved gene family, many of whose members' transcripts accumulate in the shoot apical meristem specifically or in the shoot apical meristem in addition to a range of mature tissues [ 20 ].A rabidopsis  M ei2- l ike 1 (AML1), the first member of this family to be described, was isolated in a screen for plant cDNAs whose expression could complement defects in the fission yeast meiosis signaling pathway [ 22 ]. Like Mei2, AML1 has three RRMs. Expression of a protein fragment containing only the third AML1 RRM was sufficient for restoration of meiosis signaling in fission yeast lines with defects upstream of Mei2, but not in lesions of the  mei2  locus itself.  T erminal  E ar 1 (te1), a more divergent member of the  mei2 -like family of genes, regulates leaf initiation in maize; the tassel of mutant plants is encased a whorl of leaves superficially resembling a maize ear [ 23 ].mei2mei2Given the potential role of downstream AtTOR effectors in post-embryonic growth, the intriguing signaling activity of Mei2 in fission yeast, and the known interaction between Mei2 and the fission yeast Raptor protein, we asked if the Mei2-Raptor interaction was conserved between its Arabidopsis orthologues AML1 and AtRaptor1B.Results AML1 and AtRaptor1B interact in a yeast two-hybrid assay To test for an interaction between the Arabidopsis proteins AML1 and AtRaptor1B, the open reading frames (ORFs) of each of the transcripts predicted to encode these proteins were amplified via PCR with primers carrying suitable restriction sites for cloning into the yeast two hybrid vectors pGADT7 and pGBKT7. pGADT7 encodes a transcriptional activation domain (AD) which can recruit the yeast transcription machinery. pGBKT7 encodes a DNA binding-domain (BD) which binds to the promoters of  ADE2  and  HIS3 . pGADT7 and pGBKT7 additionally carry the genes  LEU2  and  TRP1 . When transformed into the yeast line AH109, which is  leu2 - trp1 - ade2 - his3 - , co-transformed cells will grow on yeast medium lacking leucine and tryptophan. Cells co-transformed with two-hybrid constructs encoding proteins that interact will grow on media additionally lacking histidine and adenine (selective media). AH109 yeast cells co-transformed with clones encoding AML1 and AtRaptor1B in complementary two-hybrid vectors grew on selective media. Co-transformations of control pGBKT7 and pGADT7 empty vectors, or either control vector co-transformed with its complement harboring the AML1 or AtRaptor1B ORF, yielded cells able to grow on media lacking leucine and tryptophan but not on selective media. Evidence from other systems indicates that Raptor protein fragments lose the ability to bind substrates [ 4 ]. Therefore, only full length AtRaptor1B was tested in this assay. Mei2, the fission yeast AML1 homologue, is highly modular. It is divided into distinct N-terminal and C-terminal domains. The N-terminal half of the protein appears to play a regulatory role [ 24 ]. The C-terminal half of Mei2 is sufficient to complement lesions in the  mei2  locus [ 16 ]. Additionally, the AML1 C-terminal half, expressed in fission yeast meiosis signaling mutants, is able to complement meiosis signaling defects upstream of Mei2 [ 22 ]. Therefore we generated clones encoding fragments of AML1 and assayed them for interactions with full length AtRaptor1B. AML1 fragments N412 and N219, comprised of the first 412 or the first 219 residues of the 915 residue AML1 protein, restored growth on media lacking leucine, tryptophan, histidine and adenine when cloned into pGADT7 and co-transformed with pGBK-Raptor (Fig.  1 ). Neither N163, nor 155–219, (the fragments which together comprise N219), nor 155–412 (which with N163 comprises N412), could restore growth when cloned into pGADT7 and co-transformed with pGBK-Raptor. AML1 fragment 402C, comprised of residues 402 to the C-terminus of the protein, and all C-terminal fragments tested (695C, 402–704) failed to restore growth on selective medium. None of the AML1 fragments cloned into pGADT7, and cotransformed with empty pGBKT7 control vector, yielded transformants able to grow on selective medium. AML1 fragments N412 and N163 harbor activation domain activity in a yeast one-hybrid assay AML1 N412, cloned into pGBKT7, restored growth on selective medium to cells co-transformed with either pGAD:Raptor or pGADT7. To investigate this result further, we tested all AML1 fragments for native transcriptional activation domain activity in a yeast one-hybrid assay. AML1 fragments were cloned into pGBKT7, singly transformed into AH109 yeast cells, and assayed for growth on media lacking tryptophan (to confirm transformation) and media additionally lacking adenine. The DNA binding-domain of the pGBKT7 tethers any C-terminally fused fragments to the  ADE2  promoter. In the absence of a binding partner, BD-fusion chimeric proteins trigger transcription of  ADE2  only if the protein fused to the BD contains native transcriptional activation activity. AML1 N412 and AML1 N163, but not full length AML1, AML1 N219 or any C-terminal AML1 fragments, were able to restore growth on media lacking tryptophan and adenine (Fig.  2 ). AML1 N122 was similarly unable to restore growth. AML1 fragments 155–219 and 155–412, in pGBKT7, could not be stably transformed into yeast. These results were observed in multiple independent transformant lines for a given construct. Cotransformation of any of the pGBKT7-derived constructs with pGADT7 did not affect the growth of any of the transformed lines on media lacking tryptophan and adenine. Plants homozygous for insertion alleles of  AML1  and of all five  AML  family members show early flowering An interaction with AtRaptor1B points to AML1 as a downstream effector of TOR signaling in plants. Additionally, the dramatic phenotype  mei2  disruption and the intriguing mode of Mei2 action led us to ask what the consequences would be of disruption of the  AML1  locus and of all five  AML  gene family members. To obtain insertion alleles in  AML1  and other  AML  gene family members, we screened the insertion allele populations at the University of Wisconsin Arabidopsis Knockout facility [ 25 ], obtaining alleles harboring insertions in  AML1 ,  AML3 ,  AML4  and  AML5  (Fig.  3 ). An  AML2  insertion allele was obtained from the SIGnAL collection at the Salk Institute [ 26 ]. By RT-PCR using primers which anneal to the cDNA at sites spanning the insertion site of each insertion allele, we established that no wild-type transcripts accumulate in homozygous mutants (Fig.  4 ). By a series of crosses, we then generated higher-order insertion homozygotes, culminating in the quintuple insertion homozygotes Q6 and Q17. All lines were viable and fertile. AML insertion homozygotes bolted earlier than wild-type lines (Fig.  5A  and  5B ). This effect was independent of the number of insertion alleles carried by the mutants; AML 'quint' lines Q6 and Q17 were not qualitatively different than lower order insertion allele homozygotes. Additionally, AML insertion homozygote seedlings were assayed for a differential response to a range of signaling molecules. Seedlings were germinated on culture medium supplemented with the gibberellic acid GA 3 , paclobutrazol, the auxin 2,4-D, 1-amino-cyclopropane-1-carboxylic acid, 1% sucrose, 6% sucrose and kinetin, and in the dark. Quintuple insertion mutants responded slightly more than wild-type seedlings to GA 3  as measured by the change in seedling length in the presence vs. in the absence of the hormone. This effect was repeatable but weak, and no other differential hormonal response was observed (data not shown). Sequence downstream of the mutant allele insertion sites is transcribed Given the mild phenotype of the AML insertion homozygotes, we further investigated the extent of the effect of the insertions at each locus. RT-PCR, as previously stated, showed that no wild-type transcripts accumulate in lines Q6 and Q17. In fission yeast, however, the C-terminal half of the protein is sufficient to complement lesions of the  mei2  locus [ 16 ]. The insertion alleles of all but  AML4  are disrupted at or near the 5' ends of their predicted coding regions. We therefore designed primers that anneal to the region downstream of the insertion site in each allele and performed polymerase chain reactions to assay for accumulation of fragments capable of encoding the C-terminal half of any of the AML proteins (Fig.  4 ). To determine whether the amplified fragments corresponded to  AML  cDNAs, we performed restriction digests on the PCR products, which confirmed that the cDNAs originated from  AML  gene transcripts. Weak amplification of cDNA representing transcripts originating downstream of the insertion site was observed for AML1, AML2 and AML3; amplification of the AML5 3' region was indistinguishable from the amplification seen from of wild-type cDNA template. Transgenic lines overexpressing AML1:GFP or GFP:AML1 fusion proteins could not be recovered The  AML1  ORF was separately cloned into the pCambia1302 35S::GFP plant transformation vector both 5' and 3' of the  GFP  ORF, and the construct was transformed into Arabidopsis via  Agrobacterium -mediated floral dip [ 27 ]. Transformants, identified by resistance to the antibiotic hygromycin and confirmed through PCR, were recovered at a very low rate of less than .01%. No fluorescence was observed in any tissues of any transformants assayed, and  AML1:GFP  transcripts could not be detected via RT-PCR performed on cDNA transcribed from RNA extracted from bulk shoot tissue (data not shown).
Results
AML1 and AtRaptor1B interact in a yeast two-hybrid assay To test for an interaction between the Arabidopsis proteins AML1 and AtRaptor1B, the open reading frames (ORFs) of each of the transcripts predicted to encode these proteins were amplified via PCR with primers carrying suitable restriction sites for cloning into the yeast two hybrid vectors pGADT7 and pGBKT7. pGADT7 encodes a transcriptional activation domain (AD) which can recruit the yeast transcription machinery. pGBKT7 encodes a DNA binding-domain (BD) which binds to the promoters of  ADE2  and  HIS3 . pGADT7 and pGBKT7 additionally carry the genes  LEU2  and  TRP1 . When transformed into the yeast line AH109, which is  leu2 - trp1 - ade2 - his3 - , co-transformed cells will grow on yeast medium lacking leucine and tryptophan. Cells co-transformed with two-hybrid constructs encoding proteins that interact will grow on media additionally lacking histidine and adenine (selective media). AH109 yeast cells co-transformed with clones encoding AML1 and AtRaptor1B in complementary two-hybrid vectors grew on selective media. Co-transformations of control pGBKT7 and pGADT7 empty vectors, or either control vector co-transformed with its complement harboring the AML1 or AtRaptor1B ORF, yielded cells able to grow on media lacking leucine and tryptophan but not on selective media. Evidence from other systems indicates that Raptor protein fragments lose the ability to bind substrates [ 4 ]. Therefore, only full length AtRaptor1B was tested in this assay. Mei2, the fission yeast AML1 homologue, is highly modular. It is divided into distinct N-terminal and C-terminal domains. The N-terminal half of the protein appears to play a regulatory role [ 24 ]. The C-terminal half of Mei2 is sufficient to complement lesions in the  mei2  locus [ 16 ]. Additionally, the AML1 C-terminal half, expressed in fission yeast meiosis signaling mutants, is able to complement meiosis signaling defects upstream of Mei2 [ 22 ]. Therefore we generated clones encoding fragments of AML1 and assayed them for interactions with full length AtRaptor1B. AML1 fragments N412 and N219, comprised of the first 412 or the first 219 residues of the 915 residue AML1 protein, restored growth on media lacking leucine, tryptophan, histidine and adenine when cloned into pGADT7 and co-transformed with pGBK-Raptor (Fig.  1 ). Neither N163, nor 155–219, (the fragments which together comprise N219), nor 155–412 (which with N163 comprises N412), could restore growth when cloned into pGADT7 and co-transformed with pGBK-Raptor. AML1 fragment 402C, comprised of residues 402 to the C-terminus of the protein, and all C-terminal fragments tested (695C, 402–704) failed to restore growth on selective medium. None of the AML1 fragments cloned into pGADT7, and cotransformed with empty pGBKT7 control vector, yielded transformants able to grow on selective medium.
AML1 and AtRaptor1B interact in a yeast two-hybrid assay
To test for an interaction between the Arabidopsis proteins AML1 and AtRaptor1B, the open reading frames (ORFs) of each of the transcripts predicted to encode these proteins were amplified via PCR with primers carrying suitable restriction sites for cloning into the yeast two hybrid vectors pGADT7 and pGBKT7. pGADT7 encodes a transcriptional activation domain (AD) which can recruit the yeast transcription machinery. pGBKT7 encodes a DNA binding-domain (BD) which binds to the promoters of  ADE2  and  HIS3 . pGADT7 and pGBKT7 additionally carry the genes  LEU2  and  TRP1 . When transformed into the yeast line AH109, which is  leu2 - trp1 - ade2 - his3 - , co-transformed cells will grow on yeast medium lacking leucine and tryptophan. Cells co-transformed with two-hybrid constructs encoding proteins that interact will grow on media additionally lacking histidine and adenine (selective media).ADE2HIS3LEU2TRP1leu2trp1ade2his3AH109 yeast cells co-transformed with clones encoding AML1 and AtRaptor1B in complementary two-hybrid vectors grew on selective media. Co-transformations of control pGBKT7 and pGADT7 empty vectors, or either control vector co-transformed with its complement harboring the AML1 or AtRaptor1B ORF, yielded cells able to grow on media lacking leucine and tryptophan but not on selective media.Evidence from other systems indicates that Raptor protein fragments lose the ability to bind substrates [ 4 ]. Therefore, only full length AtRaptor1B was tested in this assay. Mei2, the fission yeast AML1 homologue, is highly modular. It is divided into distinct N-terminal and C-terminal domains. The N-terminal half of the protein appears to play a regulatory role [ 24 ]. The C-terminal half of Mei2 is sufficient to complement lesions in the  mei2  locus [ 16 ]. Additionally, the AML1 C-terminal half, expressed in fission yeast meiosis signaling mutants, is able to complement meiosis signaling defects upstream of Mei2 [ 22 ]. Therefore we generated clones encoding fragments of AML1 and assayed them for interactions with full length AtRaptor1B.mei2AML1 fragments N412 and N219, comprised of the first 412 or the first 219 residues of the 915 residue AML1 protein, restored growth on media lacking leucine, tryptophan, histidine and adenine when cloned into pGADT7 and co-transformed with pGBK-Raptor (Fig.  1 ). Neither N163, nor 155–219, (the fragments which together comprise N219), nor 155–412 (which with N163 comprises N412), could restore growth when cloned into pGADT7 and co-transformed with pGBK-Raptor. AML1 fragment 402C, comprised of residues 402 to the C-terminus of the protein, and all C-terminal fragments tested (695C, 402–704) failed to restore growth on selective medium. None of the AML1 fragments cloned into pGADT7, and cotransformed with empty pGBKT7 control vector, yielded transformants able to grow on selective medium.AML1 fragments N412 and N163 harbor activation domain activity in a yeast one-hybrid assay AML1 N412, cloned into pGBKT7, restored growth on selective medium to cells co-transformed with either pGAD:Raptor or pGADT7. To investigate this result further, we tested all AML1 fragments for native transcriptional activation domain activity in a yeast one-hybrid assay. AML1 fragments were cloned into pGBKT7, singly transformed into AH109 yeast cells, and assayed for growth on media lacking tryptophan (to confirm transformation) and media additionally lacking adenine. The DNA binding-domain of the pGBKT7 tethers any C-terminally fused fragments to the  ADE2  promoter. In the absence of a binding partner, BD-fusion chimeric proteins trigger transcription of  ADE2  only if the protein fused to the BD contains native transcriptional activation activity. AML1 N412 and AML1 N163, but not full length AML1, AML1 N219 or any C-terminal AML1 fragments, were able to restore growth on media lacking tryptophan and adenine (Fig.  2 ). AML1 N122 was similarly unable to restore growth. AML1 fragments 155–219 and 155–412, in pGBKT7, could not be stably transformed into yeast. These results were observed in multiple independent transformant lines for a given construct. Cotransformation of any of the pGBKT7-derived constructs with pGADT7 did not affect the growth of any of the transformed lines on media lacking tryptophan and adenine.
AML1 fragments N412 and N163 harbor activation domain activity in a yeast one-hybrid assay
AML1 N412, cloned into pGBKT7, restored growth on selective medium to cells co-transformed with either pGAD:Raptor or pGADT7. To investigate this result further, we tested all AML1 fragments for native transcriptional activation domain activity in a yeast one-hybrid assay. AML1 fragments were cloned into pGBKT7, singly transformed into AH109 yeast cells, and assayed for growth on media lacking tryptophan (to confirm transformation) and media additionally lacking adenine. The DNA binding-domain of the pGBKT7 tethers any C-terminally fused fragments to the  ADE2  promoter. In the absence of a binding partner, BD-fusion chimeric proteins trigger transcription of  ADE2  only if the protein fused to the BD contains native transcriptional activation activity.ADE2ADE2AML1 N412 and AML1 N163, but not full length AML1, AML1 N219 or any C-terminal AML1 fragments, were able to restore growth on media lacking tryptophan and adenine (Fig.  2 ). AML1 N122 was similarly unable to restore growth. AML1 fragments 155–219 and 155–412, in pGBKT7, could not be stably transformed into yeast. These results were observed in multiple independent transformant lines for a given construct. Cotransformation of any of the pGBKT7-derived constructs with pGADT7 did not affect the growth of any of the transformed lines on media lacking tryptophan and adenine.Plants homozygous for insertion alleles of  AML1  and of all five  AML  family members show early flowering An interaction with AtRaptor1B points to AML1 as a downstream effector of TOR signaling in plants. Additionally, the dramatic phenotype  mei2  disruption and the intriguing mode of Mei2 action led us to ask what the consequences would be of disruption of the  AML1  locus and of all five  AML  gene family members. To obtain insertion alleles in  AML1  and other  AML  gene family members, we screened the insertion allele populations at the University of Wisconsin Arabidopsis Knockout facility [ 25 ], obtaining alleles harboring insertions in  AML1 ,  AML3 ,  AML4  and  AML5  (Fig.  3 ). An  AML2  insertion allele was obtained from the SIGnAL collection at the Salk Institute [ 26 ]. By RT-PCR using primers which anneal to the cDNA at sites spanning the insertion site of each insertion allele, we established that no wild-type transcripts accumulate in homozygous mutants (Fig.  4 ). By a series of crosses, we then generated higher-order insertion homozygotes, culminating in the quintuple insertion homozygotes Q6 and Q17. All lines were viable and fertile. AML insertion homozygotes bolted earlier than wild-type lines (Fig.  5A  and  5B ). This effect was independent of the number of insertion alleles carried by the mutants; AML 'quint' lines Q6 and Q17 were not qualitatively different than lower order insertion allele homozygotes. Additionally, AML insertion homozygote seedlings were assayed for a differential response to a range of signaling molecules. Seedlings were germinated on culture medium supplemented with the gibberellic acid GA 3 , paclobutrazol, the auxin 2,4-D, 1-amino-cyclopropane-1-carboxylic acid, 1% sucrose, 6% sucrose and kinetin, and in the dark. Quintuple insertion mutants responded slightly more than wild-type seedlings to GA 3  as measured by the change in seedling length in the presence vs. in the absence of the hormone. This effect was repeatable but weak, and no other differential hormonal response was observed (data not shown).
Plants homozygous for insertion alleles of  AML1  and of all five  AML  family members show early flowering
AML1AMLAn interaction with AtRaptor1B points to AML1 as a downstream effector of TOR signaling in plants. Additionally, the dramatic phenotype  mei2  disruption and the intriguing mode of Mei2 action led us to ask what the consequences would be of disruption of the  AML1  locus and of all five  AML  gene family members. To obtain insertion alleles in  AML1  and other  AML  gene family members, we screened the insertion allele populations at the University of Wisconsin Arabidopsis Knockout facility [ 25 ], obtaining alleles harboring insertions in  AML1 ,  AML3 ,  AML4  and  AML5  (Fig.  3 ). An  AML2  insertion allele was obtained from the SIGnAL collection at the Salk Institute [ 26 ].mei2AML1AMLAML1AMLAML1AML3AML4AML5AML2By RT-PCR using primers which anneal to the cDNA at sites spanning the insertion site of each insertion allele, we established that no wild-type transcripts accumulate in homozygous mutants (Fig.  4 ). By a series of crosses, we then generated higher-order insertion homozygotes, culminating in the quintuple insertion homozygotes Q6 and Q17. All lines were viable and fertile.AML insertion homozygotes bolted earlier than wild-type lines (Fig.  5A  and  5B ). This effect was independent of the number of insertion alleles carried by the mutants; AML 'quint' lines Q6 and Q17 were not qualitatively different than lower order insertion allele homozygotes.Additionally, AML insertion homozygote seedlings were assayed for a differential response to a range of signaling molecules. Seedlings were germinated on culture medium supplemented with the gibberellic acid GA 3 , paclobutrazol, the auxin 2,4-D, 1-amino-cyclopropane-1-carboxylic acid, 1% sucrose, 6% sucrose and kinetin, and in the dark. Quintuple insertion mutants responded slightly more than wild-type seedlings to GA 3  as measured by the change in seedling length in the presence vs. in the absence of the hormone. This effect was repeatable but weak, and no other differential hormonal response was observed (data not shown).Sequence downstream of the mutant allele insertion sites is transcribed Given the mild phenotype of the AML insertion homozygotes, we further investigated the extent of the effect of the insertions at each locus. RT-PCR, as previously stated, showed that no wild-type transcripts accumulate in lines Q6 and Q17. In fission yeast, however, the C-terminal half of the protein is sufficient to complement lesions of the  mei2  locus [ 16 ]. The insertion alleles of all but  AML4  are disrupted at or near the 5' ends of their predicted coding regions. We therefore designed primers that anneal to the region downstream of the insertion site in each allele and performed polymerase chain reactions to assay for accumulation of fragments capable of encoding the C-terminal half of any of the AML proteins (Fig.  4 ). To determine whether the amplified fragments corresponded to  AML  cDNAs, we performed restriction digests on the PCR products, which confirmed that the cDNAs originated from  AML  gene transcripts. Weak amplification of cDNA representing transcripts originating downstream of the insertion site was observed for AML1, AML2 and AML3; amplification of the AML5 3' region was indistinguishable from the amplification seen from of wild-type cDNA template.
Sequence downstream of the mutant allele insertion sites is transcribed
Given the mild phenotype of the AML insertion homozygotes, we further investigated the extent of the effect of the insertions at each locus. RT-PCR, as previously stated, showed that no wild-type transcripts accumulate in lines Q6 and Q17. In fission yeast, however, the C-terminal half of the protein is sufficient to complement lesions of the  mei2  locus [ 16 ]. The insertion alleles of all but  AML4  are disrupted at or near the 5' ends of their predicted coding regions. We therefore designed primers that anneal to the region downstream of the insertion site in each allele and performed polymerase chain reactions to assay for accumulation of fragments capable of encoding the C-terminal half of any of the AML proteins (Fig.  4 ). To determine whether the amplified fragments corresponded to  AML  cDNAs, we performed restriction digests on the PCR products, which confirmed that the cDNAs originated from  AML  gene transcripts. Weak amplification of cDNA representing transcripts originating downstream of the insertion site was observed for AML1, AML2 and AML3; amplification of the AML5 3' region was indistinguishable from the amplification seen from of wild-type cDNA template.mei2AML4AMLAMLTransgenic lines overexpressing AML1:GFP or GFP:AML1 fusion proteins could not be recovered The  AML1  ORF was separately cloned into the pCambia1302 35S::GFP plant transformation vector both 5' and 3' of the  GFP  ORF, and the construct was transformed into Arabidopsis via  Agrobacterium -mediated floral dip [ 27 ]. Transformants, identified by resistance to the antibiotic hygromycin and confirmed through PCR, were recovered at a very low rate of less than .01%. No fluorescence was observed in any tissues of any transformants assayed, and  AML1:GFP  transcripts could not be detected via RT-PCR performed on cDNA transcribed from RNA extracted from bulk shoot tissue (data not shown).
Transgenic lines overexpressing AML1:GFP or GFP:AML1 fusion proteins could not be recovered
The  AML1  ORF was separately cloned into the pCambia1302 35S::GFP plant transformation vector both 5' and 3' of the  GFP  ORF, and the construct was transformed into Arabidopsis via  Agrobacterium -mediated floral dip [ 27 ]. Transformants, identified by resistance to the antibiotic hygromycin and confirmed through PCR, were recovered at a very low rate of less than .01%. No fluorescence was observed in any tissues of any transformants assayed, and  AML1:GFP  transcripts could not be detected via RT-PCR performed on cDNA transcribed from RNA extracted from bulk shoot tissue (data not shown).AML1GFPAgrobacteriumAML1:GFPDiscussion Raptor proteins in yeast and mammals function by recruiting substrates for TOR, a central regulator of cell growth in response to nutrients [ 4 , 7 , 28 ]. An interaction with Raptor therefore strongly suggests that a given protein is a TOR substrate and downstream effector of TOR signaling. Plants homozygous for lesions at both  AtRaptor  loci show normal embryonic development but are unable to maintain shoot meristem activity [ 12 ]. TOR substrates, then, may play a role in regulating meristem-driven post-embryonic growth. The interaction between AML1 and AtRaptor1B implicates the AML family of proteins in TOR signaling. It points specifically to a role for the AML proteins in regulation of shoot meristem activity. An interaction between Mei2 and the fission yeast Raptor homologue Mip1 has been reported previously; indeed, Mip1 ( M ei2  i nteracting  p rotein 1) was the first Raptor homologue characterized in any eukaryote [ 15 ]. The conservation of this interaction from fission yeast to plants suggests that the well-characterized Mei2 signaling pathway may provide insight into the function of the AMLs. Mei2 is a potent meiosis-signaling molecule. It triggers pre-meiotic cell differentiation and meiosis in response to nutrient stress [ 13 , 14 ]. Meiosis signaling in fission yeast is a model for cell differentiation in response to external nutrient cues. Thus the AMLs may also play a role in cellular differentiation or in meiosis signaling. Aside from the effect of Mei2 in development, there is the intriguing issue of its mode of action. Mei2 sub-cellular localization is mediated by an interaction with a noncoding, mRNA-like molecule [ 14 , 19 ]. There is a fairly large population of mRNA-like transcripts conserved among land plants despite lacking large conserved open reading frames [ 29 ]. Of these, the conserved alfalfa transcript  ENOD40  has been shown to mediate the sub-cellular localization of an RNA-binding protein [ 30 ] and to mediate phytohormone responses [ 31 ]. AML1 may be a binding partner of one or more of these mRNA-like noncoding molecules in plants. The transcriptional activation activity of the AML1 N-terminus observed in the yeast one hybrid assay has not been ascribed to Mei2 and may represent a novel activity of plant Mei2-like proteins. This activation activity localizes to the N-terminal 163 residues, but is strongly influenced by the adjacent residues. Activity is lost in N219, regained on N412 and lost again in full length AML1. This suggests that the AML1 N-terminal half has multiple configurations, and that the accessibility of the activation domain varies among configurations. We should emphasize, however, that the AML1 N-terminal fragment transcriptional activation activity has yet to be shown  in planta , and that the activity may result from fusion to the DNA binding domain rather than being present in native AML1. AtRaptor1B binding to AML1 is also localized to the N-terminus, and appears to be mediated by multiple sites in this region. This suggests that the N-terminus may contain a TOR phosphorylation site, and that this site may influence the configuration of the N-terminus. The repeated failure to recover transgenic lines expressing AML1 suggests that its unregulated overexpression is lethal. Future efforts to characterize the AML proteins in transgenic plants may benefit from the use of inducible promoters driving transgene expression to circumvent the putative lethality of unregulated AML expression. Disruption of any of the AML loci causes early bolting in plants grown under long days. However, lines homozygous for insertions in all five AML loci did not differ dramatically from lower-order insertion homozygotes, despite the fact that RT-PCR performed with primers spanning the insertion sites show that the wild-type transcript does not accumulate. Transcripts originating downstream of the insertion sites but still capable of encoding the C-terminal half of the wild-type protein accumulate from all loci but  AML4 . This raises the possibility that the  AML  quintuple insertion homozygote lines do not represent total disruption of AML activity. Four of the five  AML  open reading frames in the insertion mutant are apparently truncated and all are divorced from their native promoters, but some promoter activity (perhaps from the 35S viral promoters harbored within the inserted DNA) remains and may be sufficient to cause transcription of AML coding region DNA downstream of the insertion site. Viewed in this light, the early flowering phenotype of AML insertion homozygotes may arise not from the total disruption of AML activity but from the accumulation of AML proteins which, due to the truncations in their N-termini caused by the insertions, are no longer bound by AtRaptor1B, no longer phosphorylated by AtTOR, or no longer able to activate transcription of floral repressors. Finally, these results provide a cautionary tale. RT-PCR performed using primers which span an insertion site may not be sufficient to conclude that all activity of a protein of interest is abolished.
Discussion
Raptor proteins in yeast and mammals function by recruiting substrates for TOR, a central regulator of cell growth in response to nutrients [ 4 , 7 , 28 ]. An interaction with Raptor therefore strongly suggests that a given protein is a TOR substrate and downstream effector of TOR signaling. Plants homozygous for lesions at both  AtRaptor  loci show normal embryonic development but are unable to maintain shoot meristem activity [ 12 ]. TOR substrates, then, may play a role in regulating meristem-driven post-embryonic growth. The interaction between AML1 and AtRaptor1B implicates the AML family of proteins in TOR signaling. It points specifically to a role for the AML proteins in regulation of shoot meristem activity.AtRaptorAn interaction between Mei2 and the fission yeast Raptor homologue Mip1 has been reported previously; indeed, Mip1 ( M ei2  i nteracting  p rotein 1) was the first Raptor homologue characterized in any eukaryote [ 15 ]. The conservation of this interaction from fission yeast to plants suggests that the well-characterized Mei2 signaling pathway may provide insight into the function of the AMLs.Mei2 is a potent meiosis-signaling molecule. It triggers pre-meiotic cell differentiation and meiosis in response to nutrient stress [ 13 , 14 ]. Meiosis signaling in fission yeast is a model for cell differentiation in response to external nutrient cues. Thus the AMLs may also play a role in cellular differentiation or in meiosis signaling.Aside from the effect of Mei2 in development, there is the intriguing issue of its mode of action. Mei2 sub-cellular localization is mediated by an interaction with a noncoding, mRNA-like molecule [ 14 , 19 ]. There is a fairly large population of mRNA-like transcripts conserved among land plants despite lacking large conserved open reading frames [ 29 ]. Of these, the conserved alfalfa transcript  ENOD40  has been shown to mediate the sub-cellular localization of an RNA-binding protein [ 30 ] and to mediate phytohormone responses [ 31 ]. AML1 may be a binding partner of one or more of these mRNA-like noncoding molecules in plants.ENOD40The transcriptional activation activity of the AML1 N-terminus observed in the yeast one hybrid assay has not been ascribed to Mei2 and may represent a novel activity of plant Mei2-like proteins. This activation activity localizes to the N-terminal 163 residues, but is strongly influenced by the adjacent residues. Activity is lost in N219, regained on N412 and lost again in full length AML1. This suggests that the AML1 N-terminal half has multiple configurations, and that the accessibility of the activation domain varies among configurations. We should emphasize, however, that the AML1 N-terminal fragment transcriptional activation activity has yet to be shown  in planta , and that the activity may result from fusion to the DNA binding domain rather than being present in native AML1.in plantaAtRaptor1B binding to AML1 is also localized to the N-terminus, and appears to be mediated by multiple sites in this region. This suggests that the N-terminus may contain a TOR phosphorylation site, and that this site may influence the configuration of the N-terminus.The repeated failure to recover transgenic lines expressing AML1 suggests that its unregulated overexpression is lethal. Future efforts to characterize the AML proteins in transgenic plants may benefit from the use of inducible promoters driving transgene expression to circumvent the putative lethality of unregulated AML expression.Disruption of any of the AML loci causes early bolting in plants grown under long days. However, lines homozygous for insertions in all five AML loci did not differ dramatically from lower-order insertion homozygotes, despite the fact that RT-PCR performed with primers spanning the insertion sites show that the wild-type transcript does not accumulate.Transcripts originating downstream of the insertion sites but still capable of encoding the C-terminal half of the wild-type protein accumulate from all loci but  AML4 . This raises the possibility that the  AML  quintuple insertion homozygote lines do not represent total disruption of AML activity. Four of the five  AML  open reading frames in the insertion mutant are apparently truncated and all are divorced from their native promoters, but some promoter activity (perhaps from the 35S viral promoters harbored within the inserted DNA) remains and may be sufficient to cause transcription of AML coding region DNA downstream of the insertion site.AML4AMLAMLViewed in this light, the early flowering phenotype of AML insertion homozygotes may arise not from the total disruption of AML activity but from the accumulation of AML proteins which, due to the truncations in their N-termini caused by the insertions, are no longer bound by AtRaptor1B, no longer phosphorylated by AtTOR, or no longer able to activate transcription of floral repressors.Finally, these results provide a cautionary tale. RT-PCR performed using primers which span an insertion site may not be sufficient to conclude that all activity of a protein of interest is abolished.Conclusion TOR is a major regulator of cell growth in eukaryotes, but little is known about its downstream effectors in plants. This work shows that AML1 binds AtRaptor1B, and suggests that the AML protein family may be phosphorylated by AtTOR in an AtRaptor1B-mediated interaction. The interaction with AtRaptor1B implicates AML1 as a downstream effector of AtTOR kinase signaling, and provides insight into the mode of action of this critical growth regulator.
Conclusion
TOR is a major regulator of cell growth in eukaryotes, but little is known about its downstream effectors in plants. This work shows that AML1 binds AtRaptor1B, and suggests that the AML protein family may be phosphorylated by AtTOR in an AtRaptor1B-mediated interaction. The interaction with AtRaptor1B implicates AML1 as a downstream effector of AtTOR kinase signaling, and provides insight into the mode of action of this critical growth regulator.Methods Generating the two-hybrid constructs AML1  was cloned via RT-PCR. The cDNA template was reverse-transcribed using Omniscript (Qiagen) from RNA extracted from bulk shoot tissue using Trizol Reagent (Invitrogen). Restriction sites  Nco I and  Xma I/ Sma I were added to the 5' and 3' ends of the ORF and of all smaller AML1 fragments via PCR using ExTaq high-fidelity polymerase (Takara). An EST clone (RZL03b06) tagging  AtRaptor1B  was obtained from Kazusa DNA institute and sequenced. Restriction sites  Nco I and  Eco RI were added at the 5' and 3' ends of the ORF via PCR. pGBKT7 and pGADT7 are distributed by BDBiosciences. Yeast two-hybrid assay AH109 cells ( leu2 - trp1 - ade2 - his3 - ) were grown in YEPDA liquid plates or on YEPDA plates with 17 g/L Agar-Y (Bio101 Systems). Cells were transformed using the Yeastmaker Yeast Transformation System2 (BD Biosciences) and plated on medium lacking the appropriate macronutrients (Bio101 Systems). Colonies were observed 3–7 days after transformation. Genotyping of insertion alleles DNA from lines harboring insertion alleles was extracted using the alkaline boiling method [ 32 ]. Provisional homozygotes were confirmed via a second extraction using the C-TAB DNA extraction protocol. PCR to assay for wild-type and insertion alleles was performed in 20 uL volumes using ExTaq polymerase and buffers and the following cycling parameters: 94°C, 15 seconds; 61°C, 30 seconds; 72°C, 2 minutes; 35 cycles. Genotyping primers were as follows: AML1-5sm 5'atagaaggaaacaaaaaggaaaggaggaa3'; AML1-3sm 5'tagcatatcacttccctgtagccgcactg3'; AML2-5sm 5'attgctctgtctctgatgatgttttgtcg3'; AML2-3sm 5'gcagcaatatttctaaagcatcgggttca3'; AML3-5sm 5'ctttagttccctctttcctctgctgtgat3'; AML3-3sm 5'ctgccaagaacgggaaaacaaacataaa3'; AML4-5sm 5'ttgcaagcggtagtccatataaatcctc3'; AML4-3sm 5'atgctaccgggagaacctaagtgaaatc3'; AML5-5sm 5'tctttagccacatcaatcattctcatcct3'; AML5-3sm 5'atcagcgtcaagttccattcctcctccac3'; JL-202 5'cattttataataacgctgcggacatctac3'; JL-270 5'tttctccatattgaccatcatactcattg3'; pROC-737 5'gggaattcactggccgtcgttttacaa3'. The wild-type loci were assayed with the above pairs. The insert was assayed using the following pairs: AML1-5sm or AML1-3sm with JL-270 or JL-202, AML2-5sm with pROC-737, AML3-5sm with JL-270 or JL-202, AML4-3sm with JL-270 or JL-202, AML5-5sm with JL-202. Insertions in the  AML1  and  AML5  loci were obtained from the University of Wisconsin alpha collection using their described protocol and are in the Wassiljewskia (Ws) ecotype background. The AML1 insertion was found in pool CSJ8-46-H35. The AML5 insertion was found in pool CSJ1091-H45. Insertions in the  AML3  and  AML4  loci were obtained from the University of Wisconsin Basta collection and are in the Ws background. The  AML3  insertion was found in pool 67-6-F. The  AML4  insertion was found in pool 18-2-H. The insertion in  AML2  was obtained from the line 029713 from the Salk collection and is in the Columbia (Col-0) ecotype background. Aside from regions genetically linked to any of the insertion loci, the  AML  quintuple insertion allele homozygote lines are in the genomic background of a Col-0 × WS F 3  individual once backcrossed to the WS background. RNA extraction, RT-PCR RNA extraction was performed using TRIzol™ Reagent (Invitrogen) essentially according to manufacturer's instructions. Total RNA was treated with DNA- free ™ DNase (Ambion) and reverse transcribed using Omniscript reverse transcriptase (Qiagen) with an oligo-dT primer. Primer pairs spanning the insertion site for RT-PCR on all five lines are as follows: AML1 117–138 5'gtgatggatgcgattggataga3', AML1 556-534rc 5'attgtggcttcagctggtaactt3'; AML2 86–109 5'tttgcttctccgattctcttcctt3', AML2 456-435rc 5'agcatcgggttcaacatcttcc3'; AML3 548–568 5'gtagcggaggaggtcttgaat3', AML3 1060-1039rc 5'tctccttgatctcgccataaac3'; AML4 1932–1955 5'aagcggtagtccatataaatcctc3', AML4 2944-2927rc 5'tcccctgaatccgaccat3'; AML5 104–124 5'cgtgatcatcgtcggtgttgg3', AML5 1047-1024rc 5'ctcgacgaatttgtgatgcctctta3'. Reactions were performed using Takara ExTaq and 35 cycles of 94°C for 30 sec, 58°C annealing ( AML1,2,4 ) or 60°C annealing ( AML3,5 ) for 30 sec, and 72°C for one minute. Primer pairs amplifying a region 3' of the insertion site for RT-PCR on  AML1 ,  AML2 ,  AML3  and  AML5  insertion homozygotes are as follows: AML1 +1635 5'aggctctcgccgccctatta3', AML1 -2466 5'cgttgccaccttctcgctatt3'; AML2 +1779 5'accggggaacagtagtgaac3', AML2 -2107 5'ctgtcggcaagcatagaaag3'; AML3 +1756 5'tctggcctgctgctacaatgg3', AML3 -2326 5'cgccgacaagaagatgagaaaac3'; AML5 +1268 5'gcaacggcttccaacagtca3', AML5 -1869 5'acgaggcctaccattttcatacaa3'. All reactions were performed with a 59°C annealing temperature.
Methods
Generating the two-hybrid constructs AML1  was cloned via RT-PCR. The cDNA template was reverse-transcribed using Omniscript (Qiagen) from RNA extracted from bulk shoot tissue using Trizol Reagent (Invitrogen). Restriction sites  Nco I and  Xma I/ Sma I were added to the 5' and 3' ends of the ORF and of all smaller AML1 fragments via PCR using ExTaq high-fidelity polymerase (Takara). An EST clone (RZL03b06) tagging  AtRaptor1B  was obtained from Kazusa DNA institute and sequenced. Restriction sites  Nco I and  Eco RI were added at the 5' and 3' ends of the ORF via PCR. pGBKT7 and pGADT7 are distributed by BDBiosciences.
Generating the two-hybrid constructs
AML1  was cloned via RT-PCR. The cDNA template was reverse-transcribed using Omniscript (Qiagen) from RNA extracted from bulk shoot tissue using Trizol Reagent (Invitrogen). Restriction sites  Nco I and  Xma I/ Sma I were added to the 5' and 3' ends of the ORF and of all smaller AML1 fragments via PCR using ExTaq high-fidelity polymerase (Takara). An EST clone (RZL03b06) tagging  AtRaptor1B  was obtained from Kazusa DNA institute and sequenced. Restriction sites  Nco I and  Eco RI were added at the 5' and 3' ends of the ORF via PCR. pGBKT7 and pGADT7 are distributed by BDBiosciences.AML1NcoXmaSmaAtRaptor1BNcoEcoYeast two-hybrid assay AH109 cells ( leu2 - trp1 - ade2 - his3 - ) were grown in YEPDA liquid plates or on YEPDA plates with 17 g/L Agar-Y (Bio101 Systems). Cells were transformed using the Yeastmaker Yeast Transformation System2 (BD Biosciences) and plated on medium lacking the appropriate macronutrients (Bio101 Systems). Colonies were observed 3–7 days after transformation.
Yeast two-hybrid assay
AH109 cells ( leu2 - trp1 - ade2 - his3 - ) were grown in YEPDA liquid plates or on YEPDA plates with 17 g/L Agar-Y (Bio101 Systems). Cells were transformed using the Yeastmaker Yeast Transformation System2 (BD Biosciences) and plated on medium lacking the appropriate macronutrients (Bio101 Systems). Colonies were observed 3–7 days after transformation.leu2trp1ade2his3Genotyping of insertion alleles DNA from lines harboring insertion alleles was extracted using the alkaline boiling method [ 32 ]. Provisional homozygotes were confirmed via a second extraction using the C-TAB DNA extraction protocol. PCR to assay for wild-type and insertion alleles was performed in 20 uL volumes using ExTaq polymerase and buffers and the following cycling parameters: 94°C, 15 seconds; 61°C, 30 seconds; 72°C, 2 minutes; 35 cycles. Genotyping primers were as follows: AML1-5sm 5'atagaaggaaacaaaaaggaaaggaggaa3'; AML1-3sm 5'tagcatatcacttccctgtagccgcactg3'; AML2-5sm 5'attgctctgtctctgatgatgttttgtcg3'; AML2-3sm 5'gcagcaatatttctaaagcatcgggttca3'; AML3-5sm 5'ctttagttccctctttcctctgctgtgat3'; AML3-3sm 5'ctgccaagaacgggaaaacaaacataaa3'; AML4-5sm 5'ttgcaagcggtagtccatataaatcctc3'; AML4-3sm 5'atgctaccgggagaacctaagtgaaatc3'; AML5-5sm 5'tctttagccacatcaatcattctcatcct3'; AML5-3sm 5'atcagcgtcaagttccattcctcctccac3'; JL-202 5'cattttataataacgctgcggacatctac3'; JL-270 5'tttctccatattgaccatcatactcattg3'; pROC-737 5'gggaattcactggccgtcgttttacaa3'. The wild-type loci were assayed with the above pairs. The insert was assayed using the following pairs: AML1-5sm or AML1-3sm with JL-270 or JL-202, AML2-5sm with pROC-737, AML3-5sm with JL-270 or JL-202, AML4-3sm with JL-270 or JL-202, AML5-5sm with JL-202. Insertions in the  AML1  and  AML5  loci were obtained from the University of Wisconsin alpha collection using their described protocol and are in the Wassiljewskia (Ws) ecotype background. The AML1 insertion was found in pool CSJ8-46-H35. The AML5 insertion was found in pool CSJ1091-H45. Insertions in the  AML3  and  AML4  loci were obtained from the University of Wisconsin Basta collection and are in the Ws background. The  AML3  insertion was found in pool 67-6-F. The  AML4  insertion was found in pool 18-2-H. The insertion in  AML2  was obtained from the line 029713 from the Salk collection and is in the Columbia (Col-0) ecotype background. Aside from regions genetically linked to any of the insertion loci, the  AML  quintuple insertion allele homozygote lines are in the genomic background of a Col-0 × WS F 3  individual once backcrossed to the WS background.
Genotyping of insertion alleles
DNA from lines harboring insertion alleles was extracted using the alkaline boiling method [ 32 ]. Provisional homozygotes were confirmed via a second extraction using the C-TAB DNA extraction protocol. PCR to assay for wild-type and insertion alleles was performed in 20 uL volumes using ExTaq polymerase and buffers and the following cycling parameters: 94°C, 15 seconds; 61°C, 30 seconds; 72°C, 2 minutes; 35 cycles. Genotyping primers were as follows: AML1-5sm 5'atagaaggaaacaaaaaggaaaggaggaa3'; AML1-3sm 5'tagcatatcacttccctgtagccgcactg3'; AML2-5sm 5'attgctctgtctctgatgatgttttgtcg3'; AML2-3sm 5'gcagcaatatttctaaagcatcgggttca3'; AML3-5sm 5'ctttagttccctctttcctctgctgtgat3'; AML3-3sm 5'ctgccaagaacgggaaaacaaacataaa3'; AML4-5sm 5'ttgcaagcggtagtccatataaatcctc3'; AML4-3sm 5'atgctaccgggagaacctaagtgaaatc3'; AML5-5sm 5'tctttagccacatcaatcattctcatcct3'; AML5-3sm 5'atcagcgtcaagttccattcctcctccac3'; JL-202 5'cattttataataacgctgcggacatctac3'; JL-270 5'tttctccatattgaccatcatactcattg3'; pROC-737 5'gggaattcactggccgtcgttttacaa3'. The wild-type loci were assayed with the above pairs. The insert was assayed using the following pairs: AML1-5sm or AML1-3sm with JL-270 or JL-202, AML2-5sm with pROC-737, AML3-5sm with JL-270 or JL-202, AML4-3sm with JL-270 or JL-202, AML5-5sm with JL-202.Insertions in the  AML1  and  AML5  loci were obtained from the University of Wisconsin alpha collection using their described protocol and are in the Wassiljewskia (Ws) ecotype background. The AML1 insertion was found in pool CSJ8-46-H35. The AML5 insertion was found in pool CSJ1091-H45. Insertions in the  AML3  and  AML4  loci were obtained from the University of Wisconsin Basta collection and are in the Ws background. The  AML3  insertion was found in pool 67-6-F. The  AML4  insertion was found in pool 18-2-H. The insertion in  AML2  was obtained from the line 029713 from the Salk collection and is in the Columbia (Col-0) ecotype background. Aside from regions genetically linked to any of the insertion loci, the  AML  quintuple insertion allele homozygote lines are in the genomic background of a Col-0 × WS F 3  individual once backcrossed to the WS background.AML1AML5AML3AML4AML3AML4AML2AMLRNA extraction, RT-PCR RNA extraction was performed using TRIzol™ Reagent (Invitrogen) essentially according to manufacturer's instructions. Total RNA was treated with DNA- free ™ DNase (Ambion) and reverse transcribed using Omniscript reverse transcriptase (Qiagen) with an oligo-dT primer. Primer pairs spanning the insertion site for RT-PCR on all five lines are as follows: AML1 117–138 5'gtgatggatgcgattggataga3', AML1 556-534rc 5'attgtggcttcagctggtaactt3'; AML2 86–109 5'tttgcttctccgattctcttcctt3', AML2 456-435rc 5'agcatcgggttcaacatcttcc3'; AML3 548–568 5'gtagcggaggaggtcttgaat3', AML3 1060-1039rc 5'tctccttgatctcgccataaac3'; AML4 1932–1955 5'aagcggtagtccatataaatcctc3', AML4 2944-2927rc 5'tcccctgaatccgaccat3'; AML5 104–124 5'cgtgatcatcgtcggtgttgg3', AML5 1047-1024rc 5'ctcgacgaatttgtgatgcctctta3'. Reactions were performed using Takara ExTaq and 35 cycles of 94°C for 30 sec, 58°C annealing ( AML1,2,4 ) or 60°C annealing ( AML3,5 ) for 30 sec, and 72°C for one minute. Primer pairs amplifying a region 3' of the insertion site for RT-PCR on  AML1 ,  AML2 ,  AML3  and  AML5  insertion homozygotes are as follows: AML1 +1635 5'aggctctcgccgccctatta3', AML1 -2466 5'cgttgccaccttctcgctatt3'; AML2 +1779 5'accggggaacagtagtgaac3', AML2 -2107 5'ctgtcggcaagcatagaaag3'; AML3 +1756 5'tctggcctgctgctacaatgg3', AML3 -2326 5'cgccgacaagaagatgagaaaac3'; AML5 +1268 5'gcaacggcttccaacagtca3', AML5 -1869 5'acgaggcctaccattttcatacaa3'. All reactions were performed with a 59°C annealing temperature.
RNA extraction, RT-PCR
RNA extraction was performed using TRIzol™ Reagent (Invitrogen) essentially according to manufacturer's instructions. Total RNA was treated with DNA- free ™ DNase (Ambion) and reverse transcribed using Omniscript reverse transcriptase (Qiagen) with an oligo-dT primer. Primer pairs spanning the insertion site for RT-PCR on all five lines are as follows: AML1 117–138 5'gtgatggatgcgattggataga3', AML1 556-534rc 5'attgtggcttcagctggtaactt3'; AML2 86–109 5'tttgcttctccgattctcttcctt3', AML2 456-435rc 5'agcatcgggttcaacatcttcc3'; AML3 548–568 5'gtagcggaggaggtcttgaat3', AML3 1060-1039rc 5'tctccttgatctcgccataaac3'; AML4 1932–1955 5'aagcggtagtccatataaatcctc3', AML4 2944-2927rc 5'tcccctgaatccgaccat3'; AML5 104–124 5'cgtgatcatcgtcggtgttgg3', AML5 1047-1024rc 5'ctcgacgaatttgtgatgcctctta3'. Reactions were performed using Takara ExTaq and 35 cycles of 94°C for 30 sec, 58°C annealing ( AML1,2,4 ) or 60°C annealing ( AML3,5 ) for 30 sec, and 72°C for one minute.freeAML1,2,4AML3,5Primer pairs amplifying a region 3' of the insertion site for RT-PCR on  AML1 ,  AML2 ,  AML3  and  AML5  insertion homozygotes are as follows: AML1 +1635 5'aggctctcgccgccctatta3', AML1 -2466 5'cgttgccaccttctcgctatt3'; AML2 +1779 5'accggggaacagtagtgaac3', AML2 -2107 5'ctgtcggcaagcatagaaag3'; AML3 +1756 5'tctggcctgctgctacaatgg3', AML3 -2326 5'cgccgacaagaagatgagaaaac3'; AML5 +1268 5'gcaacggcttccaacagtca3', AML5 -1869 5'acgaggcctaccattttcatacaa3'. All reactions were performed with a 59°C annealing temperature.AML1AML2AML3AML5Authors' contributions GHA conceived of the study and performed all experimental manipulations. MRH provided guidance and arranged funding for the project. GHA and MRH drafted the manuscript, and both authors approve the final manuscript.
Authors' contributions
GHA conceived of the study and performed all experimental manipulations. MRH provided guidance and arranged funding for the project. GHA and MRH drafted the manuscript, and both authors approve the final manuscript.


A leading cause of death and disability, diabetes affects some 16 million Americans and up to 135 million people worldwide. While environmental factors such as diet and lifestyle are known to influence an individual's risk of getting adult-onset, or Type 2, diabetes, there is also a substantial inherited component, though many of the genetic pathways involved remain unidentified.The challenge of defining these genetic pathways lies in the fact that diabetes is what is known as a “complex trait”: not only is it likely that variations in many different genes or some combination of genes contribute to an increased risk, but there are probably different genes associated with diabetes in different populations. Tackling the monumental task of deciphering this genetic puzzle in the largest known study of its kind, Inês Barroso and colleagues confirm the genetic complexity of the disease and clearly demonstrate that untangling the genetics will require even larger studies.In diabetes, defects in both the secretion and function of insulin—which is produced by the pancreas—impair the body's ability to metabolize glucose. Based on what is known about the biology of pancreatic function and diabetes, the researchers chose 71 potential suspect genes that could reasonably be expected to contribute to the disease if they malfunctioned. Some of these genes are involved in the function of pancreatic beta-cells, which secrete insulin; a second group influences the function of insulin and glucose metabolism; and a third plays a broader role in energy metabolism. The results show that none of the genes on their own had a large effect, though a number of gene variations increased risk slightly. They also suggest the existence of variations in several genes that influence the risk of Type 2 diabetes.The dataset will be valuable for future studies of diabetes and supports the view that variation in genes affecting insulin production as well as insulin action can influence the risk of Type 2 diabetes. However, the genetic complexity of the disease—with a number of genes conveying a slightly increased risk— demands additional studies of larger populations to reliably identify the genes involved and the genetic variations that, alone or in combination, increase or lower an individual's risk of developing the disease.


What makes us human? From a philosophical perspective, the answer may lie in part in our apparently unique need—and self-awareness—to ask the question in the first place. From a biological perspective, the answer lies in part in the sequence of our DNA. While fossil evidence has provided a rough draft of the story of human evolution, much more remains to be learned about the path our genes followed, a path that diverged millions of years ago from our closest living hominid relatives, the chimp and bonobo. Charting differences between human genomes and those of our evolutionary relatives—both near and distant—has become a powerful tool for filling in the gaps in the human fossil record.Comparing the human genome to the genomes of other great apes can provide a window into the molecular changes that may ultimately spell the difference between human and nonhuman primates. That task was recently aided by the release of the draft sequence of the chimpanzee genome. Comparing the protein-coding sequences of human and chimp has identified molecular dissimilarities between us, which is to be expected. Though many differences between species can be explained at the molecular level by differences in protein structure, where and when a given protein is produced can be just as or even more important. Differences in protein expression arise from sequences in non-coding DNA that influence the timing and regulation of protein production and action.In a new study, Peter Keightley and colleagues conduct parallel comparative genomics studies—comparing regulatory regions in the chimp and human genome with those of mouse and rat—and make a startling discovery. The hominid lineages show a surprising lack of selective constraint—deleterious mutations have apparently accumulated—compared to the rodents, racking up an estimated additional 140,000 harmful mutations fixed, or retained, in the human and chimp lineages since they diverged. Such mutations have been selectively eliminated in mouse and rat.The authors focused on DNA sequences making up the bulk of gene-regulating elements—regions immediately preceding or following protein-coding sequences, as well as the first intron of each gene (an intron is a non-coding DNA sequence squeezed between two adjacent coding fragments). The degree of conservation in these areas was weighed against the conservation in other nearby non-coding sequences, which were assumed to be free of selective constraints. Keightley and colleagues found marked conservation in the regulatory regions between mice and rats, but nearly none between humans and chimps. This result suggests that the gene-regulating elements of hominids are subject to nearly unfettered mutation accumulation, likely due to an absence of natural selection forces strong enough to stabilize the ancestral sequences common to both human and chimpanzee.How can one explain these puzzling results? Keightley and colleagues propose that selection is ineffective against mildly unfavorable mutations in the gene-regulating regions because of the small effective population size in the evolutionary history of hominids.What do these results suggest for the future of human evolution? It's unlikely that the regulatory gatekeepers of our genome will allow mutations to spin out of control. Even if the number of unwanted mutations were to increase, stronger natural selection against them is likely to develop in parallel, Keightley and colleagues explain, protecting our fitness from a downward spiral. The authors' results support the notion that population size exerts a powerful influence on evolutionary changes at the molecular level and that many changes in gene control regions are under weak selection. With each new sequenced genome added to the comparative genomics lexicon, scientists are becoming increasingly conversant in the grammar and syntax of gene sequences—and filling in more and more gaps in the human story, letter by letter.