{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,4]],"date-time":"2026-10-04T23:41:33Z","timestamp":1791157293964,"version":"4.1.0"},"reference-count":34,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5400","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1999,1,15]]},"abstract":"<jats:p>\n            Phosphoinositide 3-kinase (PI3K) activation has been implicated in many cellular responses, including fibroblast growth, transformation, survival, and chemotaxis. Although PI3K is activated by several agents that stimulate T and B cells, the role of PI3K in lymphocyte function is not clear. The mouse gene encoding the PI3K adapter subunit p85\u03b1 and its splice variants p55\u03b1 and p50\u03b1 was disrupted. Most p85\u03b1-p55\u03b1-p50\u03b1\n            <jats:sup>\u2212\/\u2212<\/jats:sup>\n            mice die within days after birth. Lymphocyte development and function was studied with the use of the RAG2-deficient blastocyst complementation system. Chimeric mice had reduced numbers of peripheral mature B cells and decreased serum immunoglobulin. The B cells that developed had diminished proliferative responses to antibody to immunoglobulin M, antibody to CD40, and lipopolysaccharide stimulation and decreased survival after incubation with interleukin-4. In contrast, T cell development and proliferation was normal. This phenotype is similar to defects observed in mice lacking the tyrosine kinase Btk.\n          <\/jats:p>","DOI":"10.1126\/science.283.5400.393","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:50:55Z","timestamp":1027763455000},"page":"393-397","source":"Crossref","is-referenced-by-count":516,"title":["Impaired B Cell Development and Proliferation in Absence of Phosphoinositide 3-Kinase p85\u03b1"],"prefix":"10.1126","volume":"283","author":[{"given":"David A.","family":"Fruman","sequence":"first","affiliation":[{"name":"D. A. Fruman. C. M. Yballe, J. Y. Yu, L. C. Cantley, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215, and Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. S. B. Snapper, Department of Medicine, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115; Center for Blood Research, Boston, MA 02115; and Gastrointestinal Unit (Medical Services), Massachusetts General Hospital, Boston,..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott B.","family":"Snapper","sequence":"additional","affiliation":[{"name":"D. A. Fruman. C. M. Yballe, J. Y. Yu, L. C. Cantley, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215, and Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. S. B. Snapper, Department of Medicine, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115; Center for Blood Research, Boston, MA 02115; and Gastrointestinal Unit (Medical Services), Massachusetts General Hospital, Boston,..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudine M.","family":"Yballe","sequence":"additional","affiliation":[{"name":"D. A. Fruman. C. M. Yballe, J. Y. Yu, L. C. Cantley, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215, and Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. S. B. Snapper, Department of Medicine, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115; Center for Blood Research, Boston, MA 02115; and Gastrointestinal Unit (Medical Services), Massachusetts General Hospital, Boston,..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laurie","family":"Davidson","sequence":"additional","affiliation":[{"name":"D. A. Fruman. C. M. Yballe, J. Y. Yu, L. C. Cantley, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215, and Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. S. B. Snapper, Department of Medicine, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115; Center for Blood Research, Boston, MA 02115; and Gastrointestinal Unit (Medical Services), Massachusetts General Hospital, Boston,..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan Y.","family":"Yu","sequence":"additional","affiliation":[{"name":"D. A. Fruman. C. M. Yballe, J. Y. Yu, L. C. Cantley, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215, and Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. S. B. Snapper, Department of Medicine, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115; Center for Blood Research, Boston, MA 02115; and Gastrointestinal Unit (Medical Services), Massachusetts General Hospital, Boston,..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frederick W.","family":"Alt","sequence":"additional","affiliation":[{"name":"D. A. Fruman. C. M. Yballe, J. Y. Yu, L. C. Cantley, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215, and Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. S. B. Snapper, Department of Medicine, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115; Center for Blood Research, Boston, MA 02115; and Gastrointestinal Unit (Medical Services), Massachusetts General Hospital, Boston,..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lewis C.","family":"Cantley","sequence":"additional","affiliation":[{"name":"D. A. Fruman. C. M. Yballe, J. Y. Yu, L. C. Cantley, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215, and Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. S. B. Snapper, Department of Medicine, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115; Center for Blood Research, Boston, MA 02115; and Gastrointestinal Unit (Medical Services), Massachusetts General Hospital, Boston,..."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/42648"},{"key":"e_1_3_1_2_3","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/S0968-0004(97)01061-X","volume":"22","author":"Vanhaesbroeck B.","year":"1997","unstructured":"Vanhaesbroeck B., Leevers S. J., Panayotou G., Waterfield M. D., Trends in Biochem. Sci. 22, 267 (1997).","journal-title":"Trends in Biochem. Sci."},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/S0014-5793(97)00617-0","volume":"410","author":"Domin J.","year":"1997","unstructured":"Domin J., Waterfield M. D., FEBS Lett. 410, 91 (1997).","journal-title":"FEBS Lett."},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1006\/geno.1996.0527","volume":"37","author":"Fruman D. A.","year":"1996","unstructured":"Fruman D. A., Cantley L. C., Carpenter C. L., Genomics 37, 113 (1996).","journal-title":"Genomics"},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"5317","DOI":"10.1074\/jbc.271.10.5317","volume":"271","author":"Inukai K.","year":"1996","unstructured":"Inukai K., et al., J. Biol. Chem. 271, 5317 (1996);","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_5_3","doi-asserted-by":"crossref","first-page":"2195","DOI":"10.1128\/MCB.16.5.2195","volume":"16","author":"Antonetti D. A.","year":"1996","unstructured":"Antonetti D. A., Algenstaedt P., Kahn C. R., Mol. Cell. Biol. 16, 2195 (1996).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.18.3.1379"},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","unstructured":"B. C. Duckworth and L. C. Cantley in Handbook of Lipid Research: Lipid Second Messengers R. M. Bell and J. H. Exton Eds. (Plenum New York 1996) vol. 8 pp. 125\u2013175.","DOI":"10.1007\/978-1-4899-1361-6_4"},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"911","DOI":"10.1016\/S0092-8674(00)81069-7","volume":"84","author":"Deng C.","year":"1996","unstructured":"Deng C., Wynshaw-Boris A., Zhou F., Kuo A., Leder P., Cell 84, 911 (1996).","journal-title":"Cell"},{"key":"e_1_3_1_9_2","unstructured":"A 4.5-kb genomic fragment encompassing exons 11 through 15 of the mouse p85\u03b1 gene (3) was replaced by a 2-kb pgk- neo cassette. TC-1 ES cells (2 \u00d7 10 7 ) were electroporated with 20 \u03bcg of linearized DNA and subjected to positive selection with G418 (0.4 mg\/ml) and negative selection with gancyclovir (1 \u03bcM). Two clones (designated 75 and 134) containing heterozygous disruptions of the gene were identified by Southern (DNA) blot analysis with probes flanking the 3\u2032 homology region and with probes internal to both the 5\u2032 and 3\u2032 homology regions. The presence of a single integration event was verified by hybridization with a Neo probe (10). Germ line transmission was obtained with clone 75. For genotyping of mice we subjected tail DNA to polymerase chain reaction analysis using two primer sets in the same reaction one set (5\u2032-TCCAAATGAAAAGAACGGCTATC-3\u2032 and 5\u2032-TGTACCAAAGTCACTGAAAAATCTGTT-3\u2032) detecting the endogenous allele and the other set (5'-AAGATAATATTGAAGCTGTAGGGAAAAA-3\u2032 and 5\u2032-TGTTAAGAAGGGTGAGAACAGAGTACC-3') detecting a DNA segment in the targeted allele. Homozygous mutant cell lines were isolated by selecting heterozygous cells (clone 75) in increased concentrations of G418 (4.8 mg\/ml). These lines and the parental clone 75 were subcloned before injection into RAG2-deficient blastocysts as described (11). Animal experiments were carried out in accordance with institutional guidelines."},{"key":"e_1_3_1_10_2","unstructured":"Southern blots were prepared with ZetaProbe GT membranes (Bio-Rad) and hybridized with 32 P-labeled DNA probes. Protein immunoblots were prepared with nitrocellulose membranes (Bio-Rad) probed with polyclonal or monoclonal antibodies and developed with enhanced chemilumiscence reagents (NEN). Monoclonal antibody to p85\u03b1 (anti-p85\u03b1) and polyclonal anti\u2013pan-p85 were purchased from Upstate Biotechnology Inc. polyclonal anti-p110\u03b1 from Santa Cruz and polyclonal anti-p85\u03b2 was generated by immunizing rabbits with a keyhole limpet hemacyanin (KLH)\u2013conjugated peptide derived from mouse p85\u03b2 TyrProPheArgArgGluArgProGluAspLeuGluLeu . Immunoprecipitations were performed with the same antibodies except that the monoclonal anti-p85\u03b1 was mixed with a second monoclonal anti-p85\u03b1 from Transduction Laboratories and the anti-p110\u03b1 was a gift of K. Auger."},{"key":"e_1_3_1_11_2","unstructured":"D. A. Fruman unpublished results."},{"key":"e_1_3_1_12_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.90.10.4528"},{"key":"e_1_3_1_13_2","unstructured":"Single-cell suspensions were prepared from lymphoid organs and red blood cells lysed in hypotonic buffer. Cells were stained with fluorescein isothiocyanate (FITC)\u2013 phycoerythrin- and CyChrome-labeled antibodies (Pharmingen and Southern Biotech) and analyzed on a FACScalibur with CellQuest software (Becton-Dickinson) with the use of a live cell gate."},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","first-page":"1961","DOI":"10.1093\/emboj\/17.7.1961","volume":"17","author":"Scharenberg A. M.","year":"1998","unstructured":"Scharenberg A. M., et al., EMBO J. 17, 1961 (1998).","journal-title":"EMBO J."},{"key":"e_1_3_1_15_2","first-page":"6241","volume":"15","author":"Salim K.","year":"1996","unstructured":"Salim K., et al., ibid. 15, 6241 (1996);","journal-title":"ibid."},{"key":"e_1_3_1_15_3","doi-asserted-by":"crossref","first-page":"22059","DOI":"10.1074\/jbc.272.35.22059","volume":"272","author":"Rameh L. E.","year":"1997","unstructured":"Rameh L. E., et al., J. Biol. Chem. 272, 22059 (1997);","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_15_4","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1016\/S1074-7613(00)80555-5","volume":"8","author":"Bolland S.","year":"1998","unstructured":"Bolland S., Pearse R. N., Kurosaki T., Ravetch J. V., Immunity 8, 509 (1998).","journal-title":"Immunity"},{"key":"e_1_3_1_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(93)90667-F"},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1038\/361226a0"},{"key":"e_1_3_1_18_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.8332900"},{"key":"e_1_3_1_18_3","doi-asserted-by":"crossref","unstructured":"; D. J. Rawlings et al. ibid. p. 358; J. D. Kerner et al. Immunity 3 301 (1995).","DOI":"10.1016\/1074-7613(95)90115-9"},{"key":"e_1_3_1_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/1074-7613(95)90114-0"},{"key":"e_1_3_1_20_2","unstructured":"We measured Ig's in serial dilutions of serum using microtiter plates (Dynatech) and antibody pairs specific for different mouse Ig isotypes (Pharmingen). P -Nitrophenylphosphate was used as a substrate for the alkaline phosphatase\u2013conjugated secondary antibodies and the absorbance at 405 nm was measured in a microplate reader (Molecular Devices)."},{"key":"e_1_3_1_21_2","unstructured":"T cells were purified from lymph node by negative selection with anti\u2013class II magnetic beads (Miltenyi Biotech) yielding greater than 90% T lymphocytes (Thy1 + ). B cells were purified from spleen and lymph node by negative selection with a combination of magnetic beads specific for CD43 CD11b and CD11c (Miltenyi) yielding greater than 95% B lymphocytes (B220 + ) except in the case of p85\u03b1-deficient cells which were 60 to 85% pure. T or B cells (5 \u00d7 10 4 ) were cultured in triplicate in 96-well dishes (Falcon) in a total volume of 100 \u03bcl of medium [RPMI containing 10% heat-inactivated fetal calf serum (FCS) 10 mM Hepes pH 7.4 penicillin (100 units\/ml) streptomycin (100 \u03bcg\/ml) 2 mM l -glutamine and 50 \u03bcM 2-mercaptoethanol]. For plate-bound antibody stimulation plates were precoated with anti-CD3\u03b5 (Pharmingen) at various concentrations or anti-CD28 (Pharmingen) at 10 \u03bcg\/ml (or both) in 1\u00d7 phosphate-buffered saline (PBS) for 1.5 hours at 37\u00b0C and washed in 1\u00d7 PBS containing 5% FCS before cell addition. Soluble agonists were included at the following final concentrations: phorbol 12-myristate 13-acetate (PMA) (20 ng\/ml Calbiochem) ionomycin (1 \u03bcM Calbiochem) anti-IgM [F(ab\u2032) 2 10 \u03bcg\/ml Jackson ImmunoResearch] LPS (10 \u03bcg\/ml Sigma) anti-CD40 (1 \u03bcg\/ml Pharmingen) recombinant murine IL-2 (100 U\/ml Genzyme) and IL-4 (2 ng\/ml Pharmingen). Approximately 5 \u00d7 10 4 cells in 50 \u03bcl were added to wells containing 50 \u03bcl of agonists at 2\u00d7 final concentration. Where indicated cells were pretreated for 15 min at 37\u00b0C with 10 \u03bcM Ly294002 (Calbiochem) before they were added to wells. Forty-eight hours later 50 \u03bcl of medium containing [ 3 H]thymidine (20 \u03bcCi\/ml NEN) was added to each well. Cells were harvested 12 hours later with a TomTec plate harvester and counted in a BetaPlate scintillation counter (Wallac)."},{"key":"e_1_3_1_22_2","doi-asserted-by":"crossref","first-page":"4071","DOI":"10.4049\/jimmunol.156.11.4071","volume":"156","author":"Hutchcroft J. E.","year":"1996","unstructured":"Hutchcroft J. E., Bierer B. E., J. Immunol. 156, 4071 (1996);","journal-title":"J. Immunol."},{"key":"e_1_3_1_22_3","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1042\/bj3180361","volume":"318","author":"Ward S. G.","year":"1996","unstructured":"Ward S. G., Biochem. J. 318, 361 (1996).","journal-title":"Biochem. J."},{"key":"e_1_3_1_23_2","doi-asserted-by":"crossref","first-page":"4702","DOI":"10.4049\/jimmunol.155.10.4702","volume":"155","author":"Truitt K. E.","year":"1995","unstructured":"Truitt K. E., et al., J. Immunol. 155, 4702 (1995).","journal-title":"J. Immunol."},{"key":"e_1_3_1_24_2","unstructured":"Purified B cells (1.5 \u00d7 10 5 ) were cultured for 36 hours in 48-well dishes under conditions identical to the thymidine incorporation assays. Cells were then harvested stained with anti-B220\u2013FITC fixed in 50% ice-cold ethanol treated with ribonuclease and stained with propidium iodide. Aggregates and B220 \u2212 cells were excluded and the percentage of cells with sub-G1 DNA content was quantitated with CellQuest software. Similar results were observed when apoptosis was measured after 18 hours (10)."},{"key":"e_1_3_1_25_2","doi-asserted-by":"crossref","first-page":"2143","DOI":"10.4049\/jimmunol.156.6.2143","volume":"156","author":"Woodland R. T.","year":"1996","unstructured":"Woodland R. T., Schmidt M. R., Korsmeyer S. J., Gravel K. A., J. Immunol. 156, 2143 (1996);","journal-title":"J. Immunol."},{"key":"e_1_3_1_25_3","doi-asserted-by":"crossref","first-page":"10966","DOI":"10.1073\/pnas.93.20.10966","volume":"93","author":"Anderson J. S.","year":"1996","unstructured":"Anderson J. S., Teutsch M., Dong Z., Wortis H. H., Proc. Natl. Acad. Sci. U.S.A. 93, 10966 (1996) .","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_26_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.283.5400.390"},{"key":"e_1_3_1_27_2","doi-asserted-by":"crossref","first-page":"19704","DOI":"10.1016\/S0021-9258(17)45429-9","volume":"265","author":"Carpenter C. L.","year":"1990","unstructured":"Carpenter C. L., et al., J. Biol. Chem. 265, 19704 (1990).","journal-title":"J. Biol. Chem."},{"key":"e_1_3_1_28_2","unstructured":"We thank B. Sleckman S. Thomas G. Rathbun and C. Carpenter for suggestions K. Auger for anti-p110\u03b1 J. Lawitts for blastocyst injections D. Pollard for mouse husbandry and genotyping D. Kim for help with fibroblast culture and K. Lee-Fruman for a critical reading of the manuscript. Supported by grants from NIH (to L.C.C. F.W.A. and S.B.S.) a fellowship from the Damon Runyon\u2013Walter Winchell Cancer Research Fund (to D.A.F.) the Leukemia Society of America (to D.A.F.) and the Howard Hughes Medical Institute (to F.W.A.)."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.283.5400.393","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T03:34:27Z","timestamp":1705116867000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.283.5400.393"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1999,1,15]]},"references-count":34,"journal-issue":{"issue":"5400","published-print":{"date-parts":[[1999,1,15]]}},"alternative-id":["10.1126\/science.283.5400.393"],"URL":"https:\/\/doi.org\/10.1126\/science.283.5400.393","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1999,1,15]]}}}