Supplementary InformationSupplementary Methods
EST librariesDigital Differential Display
We used the Digital Differential Display program (http://www.ncbi.nlm.nih.gov/UniGene/info_ddd.shtml) to analyze gene representation in cDNA libraries from ES cells and various tissues. EST libraries derived from ES cells were #10023, 500, 240, 1882 and 2512 274 and 220 (total 27705 39323 entries). Libraries used to represent various somatic tissues were #8901, 8902, 9741, 539, 598, 9978, 9980, 8621, 10030, 10402, 553, 519, 544, 7240, 10407, 5400, 12252, 5369, 9938, 1140, 9956, 2518, 2602, 2509, 2601, 5389, 4042, 9950, 2590, 2554, 9946, 4263, 2523, 12248, 509, 9974, 9959, 12249, 5497, 1771, 868, 5403, 497, 8907, 7222, 1870, 1783, 470, 2520, 5390, 2526, 2527, 1876, 5423, 5361, 9847, 1878, 2579, 9839, 5368, 1879, 3780, 5352, 5357, 12267, 12265, 12266, 7256, 12078, 12309 and 498 467, 318, 198, 408, 239, 400, 453, 109, 16, 523, 161, 483, 258, 264, 419, 529, 327, 411, 417, 418, 399, 196, 271, 255, 495, 101, 98, 351, 416, 321, 251, 412, 379, 549, 329, 265, 449, 328, 516, 320, 436, 427, 297, 366, 390, 315, 228, 277, 292, 284, 285 and 30 (total 1328835 625654 entries).
Primer sequences
45328-AS1 (5’- CCCATGTTACCACGTAACTT-3’)
45328-race11 (5’- GGTAACTTGGTCGGAAAGGGAAAT-3’)
ERas-gw-S (5’-AAAAAAGCAGGCTGGGGAATGGCTTTGCCTA-3’)
ERas-gw-AS (5’-AGAAAGCTGGGTCAAAGATCTTCAGGCTACAG-3’)
ERas-gw-AS2 (5’-AGAAAGCTGGGTCATTTCTGGTGTCGGGTC-3’)
PI3K-delta-gw-U (5’- AAAAAGCAGGCTCCATGCCCCCTGGGGTGGACTGCC-3’)
PI3K-delta-gw-L (5’-AGAAAGCTGGGTCTACTGTCGGTTATCCTTGGAC-3’).
hyg.upper (5’-AAAAAGCAGGCTACCATGAAAAAGCCTGAACTCACC-3’)
hyg.lower (5’-AGAAAGCTGGGTCTATTCCTTTGCCCTCGGACGAGTG-3’)
mutS (5’-CTTGCAGGATCGCCTAGGCTCCGGGCGTAT-3’)
mut AS (5’-ATACGCCCGGAGCCTAGGCGATCCTGCAAG-3’).
hygTopII.HpaI (5’-GTTAACCCAAAGATGAAAAAGCCTGAACTCACC-3’)
hygLast.XbaI (5’-TCTAGACTATTCCTTTGCCCTCGGACGAGT-3’)
ERAS-S118 (5’-ACTGCCCCTCATCAGACTGCTAC-3’)
ERAS-AS264 (5’-ACTGTGCCCAAGCCTCGTGACTTT-3’)
ERAS-U527 (5’-CTGGTGATGGTGTGCTGGGCGTCT-3’)
ERAS-S812 (5’-CGAATCAAGCAAGAAGACCCGACA-3’)
b-geo screening 1 (5’-AATGGGCTGACCGCTTCCTCGTGCTT-3’)
ERAS-screening1 (5’-GGGAGGGAGGGCAAGGGCAGAGGGCT-3’)
ERAS-TW1 (5’-CTCAAGAAAGTCCGCTTCCCGCTCAG-3’)
ERAS-TW2 (5’-GGAACGCCAGAGCCCTGCTTACCTGT-3’)
E-Ras3’probe-s (5’-AGCTGGAGCGTCCGGGTCATCGTC-3’)
E-Ras3’probe-as (5’-AGGCTGGGAATTAAAGGCGTGAAC-3’)
hHRAS2-S (5’-GATCAGCACACAATAGGCAT-3’)
hHRAS2-AS (5’-ACTCCCACCCACACAACACT-3’)
Ha-ras2-gw-s (5'-CACCATGGAGCTGCCAACAAAGCC-3')
Ha-ras2-gw-as (5'-TCAGGCCACAGAGCAGCCACAGTG-3')
B-Raf-gw-s (5'-CACCTATAAGATGGCGGCGCTGAGC-3')
B-Raf-gw-as (5'-TCAGTGGACAGGAAACGCACCATATC-3')
Construction of entry vectors for Gateway cloning system
EcoRI/BamHI fragments of pCMV-Ras, pCMV-RasV12 and pCMV-RasN17 (Clontech) were inserted into the same site of pENTR-1A (Invitrogen) to produce construct pENTR-H-Ras, pENTR-H-RasV12 and pENTR-H-RasN17. Coding region of mouse E-Ras was amplified by RT-PCR from ES-cell derived total RNA with primers E-Ras-gw-S and E-Ras-gw-AS. Coding region of mouse ERas lacking the CAAX motif was amplified by RT-PCR from ES-cell derived total RNA with primers ERas-gw-S and ERas-gw-AS2. PCR product was inserted into pDONR201 (Invtirogen) with BP recombinase (Invitrogen) to produce pDONR-E-Ras. Coding region of mouse PI3K∂kinse p110d was amplified from an EST clone (#4192906) with primers PI3K-delta-gw-U and PI3K-delta-gw-L. After adding the attB1 and attB2 recombination sites at both ends by additional PCR with primers attB1 and attB2, we inserted these PCR products into pDONR201 (Invitrogen) with BP recombinase to construct pDONR-mERas, pDONR-mERasDC and pDONR-PI3K. Coding regions of human BRaf and hERas were amplified with primers BRaf-gw-s/as and Ha-ras2-gw-s/as from human testis cDNA and pCR2.1-hERas. PCR products were inserted into pENTR/D-TOPO (Invitrogen) by TA cloning to construct pENTR-BRaf and pENTR-hERas.
Construction of destination vectors for Gateway systems
We constructed pCAG-IRES-neo by replacing the NdeI/EcoRI fragment of pIRES-neo (Clontech), which contained the CMV promoter, with the NdeI/EcoRI fragment of pCX-EGFP, which contained the CAG promoter. Gateway refA Cassette (Invitrogen) was blunt-ligated into the EcoRI site of pCAG-IRES-neo to construct pCAG-gw-IRES-neo. Gateway refA Cassette was blunt-ligated into the XhoI site of pCAG-IP to construct pCAG-IP-gw. Two oligonucleotides , oligo-HA-s and oligo-HA-as were annealed and inserted into the EcoRI/BamHI site of pCAG-IRES-neo to construct pCAG-HA-IRES-neo. Gateway refB Cassette was blunt-ligated into the BamHI site of pCAG-HA-IRES-neo to construct pCAG-HA-gw-IRES-neo. An SpeI/XhoI fragment of pCAG-HA-gw-IRES-neo was inserted into the SpeI/XhoI site of pCAG-IP to construct pCAG-IP-HA-gw. pCAG-IP-myc-gw was constructed as described elsewhere (Y. Tokuzawa et al., Mol. Cell. Bio., submittedin press).
pCAG-gw-IRES-hygIH was constructed as follows. : cDNA of hygromycin resistance gene was amplified from pHPCAG-IP with primers hyg.upper and hyg.lower. Additional PCR with primers attB1 and attB2 was performed and PCR product was inserted into pDONR201 to construct pDONR-hygro. The SacII site in the hygromycin resistance gene was mutated without changing encoded amino acid (CCGCGG to CCTAGG) by Quick Change Site Site-Directed Mutagenesis Kit (Stratagene) to construct pDONR-hygromut. Primers used were mutS and mut AS. cDNA of the mutated hygromycin resistant gene was amplified from pDONR-hygromut with primers hygTopII.HpaI and hygLast.XbaI and ligated into pCR2.1. An HpaI/XbaI fragment of this plasmid was ligated into the SmaI/XbaI site of pIRES-neo (Clontech) to construct pIRES-hygIH. An SpeI fragment of pIRES-hygIH was replaced with an SpeI fragment of pCAG-gw-IRES-neo containing the CAG promoter and Gateway cassette to construct pCAG-gw-IRES-hygIH.
Construction of retroviral vectors
An SalI/EcoRI fragment of pCX-EGFP was blunt-ligated into the MluI/EcoRV site of pIRESpuro (Clontech) to construct pCAG-IRES-puroIP. A NotI/BamHI fragment of pCR2.1-E-Ras was introduced into the NotI/BamHI site of pCAG-IRES-puro to construct pCAG-E-Ras-IRES-puro. An EcoRI/BamHI fragment of pCMV-H-RasV12 was introduced into the EcoRI/BamHI site of pCAG-IRES-puroIP to construct pCAG-H-RasV12-IRES-puroIP. An EcoRV/XbaI fragment of pIRES-puro was blunt-ligated into the BamHI/SalI site of pMX to construct pMX-IRES-puro. An EcoRV/XbaI fragment of pCAG-E-Ras-IRES-puro was blunt-ligated into the BamHI/EcoRI site of pMX to construct pMX-E-Ras-IRES-puro. An EcoRI/XbaI fragment of pCAG-HRasV12-IRES-puroIP was blunt-ligated into the BamHI/EcoRI site of pMX to construct pMX-H-RasV12-IRES-puroIP. An EcoRV/XbaI fragment of pIRES-puroIP was blunt-ligated into the BamHI/SalI site of pMX to construct pMX-IRES-puroIP. Gateway refB Cassette was blunt-ligated into the BamHI site of pMX-IRES-puroIP to construct pMX-gw-IRES-puroIP. pMX-mERas-IRES-puroIP and pMX-hERas-IRES-puroIP were constructed by the Gateway technology. .
Construction of targeting vector
A bacterial artificial chromosome (BAC) clone containing the mouse E-Ras gene was identified by colony hybridization with the full-length E-Ras cDNA as a probe. A DNA fragment from containing intron 1 was amplified from the BAC clone by PCR with primers ERAS-S118 and ERAS-AS264. An amplified fragment (3.3 kbp) was used as the 5’-homologous region of a targeting vector that replaces exon 2 with a b-geo cassette. To make the 3’-homologous region, we obtained 3’-flanking region with TOPO Walker Kit (Invitrogen). Primers used were ERAS-U527 for primer extension and ERAS-S812 for PCR. A 1.84-kbp fragment obtained from a PstI-digested DNA digested withpool PstI was used as the 3’- homologous region. The resulting targeting vector was linearized with SacII and introduced into RF8 ES cells by electroporation.
PCR screening for clones with correct homologous recombination
G418-resistant colonies were screened by PCR with primers b-geo screening 1 and ERAS-screening1. PCR was performed with Expand Long Template PCR System (Roche). The PCR Program consisted of an initial denature at 94 °C for 10 sec., 36 cycles of 94 °C for 5 sec., 55 °C for 5 sec. and 68 °C for 3 min. and an additional extension at 68 °C for 20 min. Specific amplification with these primers yielded a 2.6-kbp product from correctly targeted clones.
Preparation of probes for Southern hybridization.
To makeproduce the 5'-¢'-flanking probe for hybridization, we obtained generated a DNA fragment from the 5'¢'-flanking region with TOPO Walker Kit. Primers used were ERAS-TW1 for primer extension and ERAS-TW2 for PCR. A 4-kbp fragment obtained from DNA amplified from an NsiI-digested DNA pool was inserted into pCR2.1. A 0.7-kbp EcoRI fragment of this plasmid was used as the 5'-¢ probe. The 3'-¢ probe was amplified from the BAC clone with primers E-Ras3'Ras 3¢probe-s and E-Ras3’Ras 3¢probe-as.
Construction of the expression vector for activated PI3 kinase
An MluI/EcoRV fragment of pIRES-hygIH was replaced with an MluI/EcoRI fragment of pCAG-IRES-neo containing the CAG promoter to construct pCAG-IRES-hygIH. A HindIII/EcoRV fragment of pUSEamp-myr-p110 (Upstate Biology) was treated with Taq polymerase and subcloned into pCR2.1 by TA cloning. A BamHI fragment of this plasmid was subcloned into the same site of pCAG-IRES-hygIH to produce pCAG-myr-p110-IRES-hygIH
AP1 enhancer reporter assays.
pCAG-IP, pCAG-IP-ERas, pCAG-IP-HRasV12, or pCAG-IP-HRasN17 was introduced into MG1.19 ES cells along with pAP1-TAL (Clontech), in which a firefly luciferase (FL) is driven by a minimum thymidine kinase promoter and AP1 enhancer. To monitor transfection efficiency, pRL-TK (Promega), in which Renilla luciferase (RL) is driven by a thymidine kinase promoter was co-transfected. After a 24-hour culture in medium devoid of FBS, cell lysates were collected and luciferase activities were measured with Dual Luciferase Reporter assay System (Promega).
Legend for Supplementary figures
Figure S1
a. pMX-IP vector. LTR, long terminal repeat; y packaging signal; MCS, multiple cloning site; IVS, intron; IRES, internal ribosome entry site; puro, puromycin resistant gene
b. Flowcytometry analysis of cells transfected with pMX-IP or pMX-EGFP-IP.
Figure S2
a. Interaction of with BRaf. MG1.19 cells were transfected with myc-hERas (1), myc-mERas (2), myc-HRas (3), myc-HRasV12 (4), myc-HRasN17 (5), or EGFP (6), along with HA-BRaf. Twenty-four-hours later, cell lysates were collected and blotted with a -myc antibody (bottom) and a-HA antibody (middle). Lysates were then purified with agarose-conjugated a-myc antibody and precipitates were blotted with a-HA antibody (upper).
b. Effect of ERas and HRas on the AP1 enhancer reporter construct. Shown are averages and standard deviations of normalized luciferase activity from four independent experiments. *, P < 0.05; **, P < 0.01, compared to mock (pCAG-IP).
c. Interaction with endogenous ERas and PI3K p85. RF8 ES cell lysates were precipitated with a-p85 antibody (#06-195, Upstate Biotechnology) or normal rabbit IgG. Precipitates were separated by SDS-PAGE, transferred to a membrane, and blotted with a-ERas antiserum.
d. Akt phosphorylation in ERas-null ES cells. ES cells described in fig. 3f were grown on gelatin-coated plates in the medium supplemented with LIF. Lysates from log-phase growing cells were analyzed by Western blotting with a-phospho-Akt (Ser 473) or a-Akt antibody (#9270, New England Biolabs).
e. Inhibition of growth promoting effect of ERas by LY294002. NIH 3T3 Cells transfected with parent pMX vector (mock), pMX-hERas, -mERas, myrP110, or HRasV12 were plated at 5000 cells per well in 24-well plates and cultured with DMSO (vehicle), LY294002 (10 M) or PD098059 (50 M). Cell numbers were determined after 10 days. **, P < 0.01, compared to mock (n=4).