Transition step during assembly of HIV tat: P-TEFb transcription complexes and transfer to TAR RNA

Molecular and Cellular Biology

Volumn 32, Issue 23, 2012, Pages 4780-4793

  D'orso, Iván ^a   Jang, Gwendolyn M ^a   Pastuszak, Alexander W ^a   Faust, Tyler B ^a   Quezada, Elizabeth ^a   Booth, David S ^a,b   Frankel, Alan D ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIN DEPENDENT KINASE 9; RNA; RNA POLYMERASE II; TRANSACTIVATOR PROTEIN; TRANSCRIPTION ELONGATION FACTOR;

ACETYLATION; ARTICLE; CONTROLLED STUDY; GENETIC TRANSCRIPTION; HUMAN; HUMAN CELL; MOLECULAR RECOGNITION; MUTAGENESIS; PRIORITY JOURNAL; PROMOTER REGION; PROTEIN EXPRESSION; PROTEIN PHOSPHORYLATION; PROTEIN PROTEIN INTERACTION; PROTEIN RNA BINDING; PROTEIN STRUCTURE; RNA TRANSPORT; TRANSCRIPTION ELONGATION; TRANSCRIPTION INITIATION;

AMINO ACID SEQUENCE; CELL LINE; CONSERVED SEQUENCE; GENE EXPRESSION REGULATION, VIRAL; HIV; HIV INFECTIONS; HIV LONG TERMINAL REPEAT; HUMANS; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; MUTATION; POSITIVE TRANSCRIPTIONAL ELONGATION FACTOR B; RIBONUCLEOPROTEINS, SMALL NUCLEAR; RNA, VIRAL; RNA-BINDING PROTEINS; TAT GENE PRODUCTS, HUMAN IMMUNODEFICIENCY VIRUS; TRANSCRIPTIONAL ACTIVATION;

EUKARYOTA;

EID: 84871889026     PISSN: 02707306     EISSN: 10985549     Source Type: Journal    
DOI: 10.1128/MCB.00206-12     Document Type: Article

References (121)

1. Barboric M, Lenasi T. 2010. Kick-sTARting HIV-1 transcription elongation by 7SK snRNP deporTATion. Nat. Struct. Mol. Biol. 17:928-930.
2. Barboric M, et al. 2007. Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription. Nucleic Acids Res. 35: 2003-2012.
3. Barua B, Pamula MC, Hitchcock-DeGregori SE. 2011. Evolutionarily conserved surface residues constitute actin binding sites of tropomyosin. Proc. Natl. Acad. Sci. U. S. A. 108:10150-10155.
4. Berkhout B, Gatignol A, Rabson AB, Jeang KT. 1990. TARindependent activation of the HIV-1 LTR: evidence that tat requires specific regions of the promoter. Cell 62:757-767.
5. Bieniasz PD, Grdina TA, Bogerd HP, Cullen BR. 1999. Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription. Proc. Natl. Acad. Sci. U. S. A. 96:7791-7796.
6. Biglione S, et al. 2007. Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex. Retrovirology 4:47-58.
7. Blau J, et al. 1996. Three functional classes of transcriptional activation domain. Mol. Cell. Biol. 16:2044-2055.
8. Brown SA, Weirich CS, Newton EM, Kingston RE. 1998. Transcriptional activation domains stimulate initiation and elongation at different times and via different residues. EMBO J. 17:3146-3154.
9. Calabro V, Daugherty MD, Frankel AD. 2005. A single intermolecular contact mediates intramolecular stabilization of both RNA and protein. Proc. Natl. Acad. Sci. U. S. A. 102:6849-6854.
10. Carey M, Lin YS, Green MR, Ptashne M. 1990. A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives. Nature 345:361-364.
11. Chen D, Fong Y, Zhou Q. 1999. Specific interaction of Tat with the human but not rodent P-TEFb complex mediates the species-specific Tat activation of HIV-1 transcription. Proc. Natl. Acad. Sci. U. S. A. 96: 2728-2733.
12. Clement SL, Lykke-Andersen J. 2008. A tethering approach to study proteins that activate mRNA turnover in human cells. Methods Mol. Biol. 419:121-133.
13. Core LJ, Lis JT. 2008. Transcription regulation through promoterproximal pausing of RNA polymerase II. Science 319:1791-1792.
14. Cullen BR. 1990. The HIV-1 Tat protein: an RNA sequence-specific processivity factor? Cell 63:655-657.
15. Cunningham BC, Wells JA. 1989. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244:1081-1085.
16. Das A. 1992. How the phage lambda N gene product suppresses transcription termination: communication of RNA polymerase with regulatory proteins mediated by signals in nascent RNA. J. Bacteriol. 174:6711-6716.
17. Das C, Edgcomb SP, Peteranderl R, Chen L, Frankel AD. 2004. Evidence for conformational flexibility in the Tat-TAR recognition motif of cyclin T1. Virology 318:306-317.
18. de Hoon MJ, Imoto S, Nolan J, Miyano S. 2004. Open source clustering software. Bioinformatics 20:1453-1454.
19. Dorris DR, Struhl K. 2000. Artificial recruitment of TFIID, but not RNA polymerase II holoenzyme, activates transcription in mammalian cells. Mol. Cell. Biol. 20:4350-4358.
20. D'Orso I, Frankel AD. 2010. HIV-1 Tat: its dependence on host factors is crystal clear. Viruses 2:2226-2234.
21. D'Orso I, Frankel AD. 2010. RNA-mediated displacement of an inhibitory snRNP complex activates transcription elongation. Nat. Struct. Mol. Biol. 17:815-821.
22. D'Orso I, Frankel AD. 2009. Tat acetylation modulates assembly of a viral-host RNA-protein transcription complex. Proc. Natl. Acad. Sci. U. S. A. 106:3101-3106.
23. D'Orso I, Grunwell JR, Nakamura RL, Das C, Frankel AD. 2008. Targeting tat inhibitors in the assembly of human immunodeficiency virus type 1 transcription complexes. J. Virol. 82:9492-9504.
24. Dunker AK, Silman I, Uversky VN, Sussman JL. 2008. Function and structure of inherently disordered proteins. Curr. Opin. Struct. Biol. 18: 756-764.
25. Durney MA, D'Souza VM. 2010. Preformed protein-binding motifs in 7SK snRNA: structural and thermodynamic comparisons with retroviral TAR. J. Mol. Biol. 404:555-567.
26. Espinosa JM. 2010. The meaning of pausing. Mol. Cell 40:507-508.
27. Farrell S, Simkovich N, Wu Y, Barberis A, Ptashne M. 1996. Gene activation by recruitment of the RNA polymerase II holoenzyme. Genes Dev. 10:2359-2367.
28. Faust T, Frankel AD, D'Orso I. 2012. Transcription control by long non-coding RNAs. Transcription 3:78-86.
29. Feinberg MB, Baltimore D, Frankel AD. 1991. The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation. Proc. Natl. Acad. Sci. U. S. A. 88:4045-4049.
30. Fishburn J, Mohibullah N, Hahn S. 2005. Function of a eukaryotic transcription activator during the transcription cycle. Mol. Cell 18:369-378.
31. Fong YW, Zhou Q. 2000. Relief of two built-in autoinhibitory mechanisms in P-TEFb is required for assembly of a multicomponent transcription elongation complex at the human immunodeficiency virus type 1 promoter. Mol. Cell. Biol. 20:5897-5907.
32. Fong YW, Zhou Q. 2001. Stimulatory effect of splicing factors on transcriptional elongation. Nature 414:929-933.
33. Frankel AD, Kim PS. 1991. Modular structure of transcription factors: implications for gene regulation. Cell 65:717-719.
34. Frankel AD, Young JA. 1998. HIV-1: fifteen proteins and an RNA. Annu. Rev. Biochem. 67:1-25.
35. Fuda NJ, Ardehali MB, Lis JT. 2009. Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature 461:186-192.
36. Fujinaga K, et al. 1998. The ability of positive transcription elongation factor B to transactivate human immunodeficiency virus transcription depends on a functional kinase domain, cyclin T1, and Tat. J. Virol. 72:7154-7159.
37. Ganguly D, Chen J. 2009. Atomistic details of the disordered states of KID and pKID. Implications in coupled binding and folding. J. Am. Chem. Soc. 131:5214-5223.
38. Garber ME, et al. 2000. CDK9 autophosphorylation regulates highaffinity binding of the human immunodeficiency virus type 1 tat-P-TEFb complex to TAR RNA. Mol. Cell. Biol. 20:6958-6969.
39. Garber ME, Wei P, Jones KA. 1998. HIV-1 Tat interacts with cyclin T1 to direct the P-TEFb CTD kinase complex to TAR RNA. Cold Spring Harb. Symp. Quant. Biol. 63:371-380.
40. Garber ME, et al. 1998. The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein. Genes Dev. 12:3512-3527.
41. Gilchrist DA, et al. 2010. Pausing of RNA polymerase II disrupts DNAspecified nucleosome organization to enable precise gene regulation. Cell 143:540-551.
42. Gold MO, Yang X, Herrmann CH, Rice AP. 1998. PITALRE, the catalytic subunit of TAK, is required for human immunodeficiency virus Tat transactivation in vivo. J. Virol. 72:4448-4453.
43. Green M, Ishino M, Loewenstein PM. 1989. Mutational analysis of HIV-1 Tat minimal domain peptides: identification of trans-dominant mutants that suppress HIV-LTR-driven gene expression. Cell 58:215-223.
44. He N, et al. 2011. Human polymerase-associated factor complex (PAFc) connects the super elongation complex (SEC) to RNA polymerase II on chromatin. Proc. Natl. Acad. Sci. U. S. A. 108:E636-E645.
45. He N, et al. 2010. HIV-1 Tat and host AFF4 recruit two transcription elongation factors into a bifunctional complex for coordinated activation of HIV-1 transcription. Mol. Cell 38:428-438.
46. Hobson D, Uhlenbeck OC. 2006. Alanine scanning of MS2 coat protein reveals protein-phosphate contacts involved in thermodynamic hot spots. J. Mol. Biol. 356:613-624.
47. Janin J, Bahadur RP, Chakrabarti P. 2008. Protein-protein interaction and quaternary structure. Q. Rev. Biophys. 41:133-180.
48. Jeronimo C, et al. 2007. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme. Mol. Cell 27:262-274.
49. Kao SY, Calman AF, Luciw PA, Peterlin BM. 1987. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature 330:489-493.
50. Keaveney M, Struhl K. 1998. Activator-mediated recruitment of the RNA polymerase II machinery is the predominant mechanism for transcriptional activation in yeast. Mol. Cell 1:917-924.
51. Keen NJ, Gait MJ, Karn J. 1996. Human immunodeficiency virus type-1 Tat is an integral component of the activated transcription-elongation complex. Proc. Natl. Acad. Sci. U. S. A. 93:2505-2510.
52. Koh SS, Ansari AZ, Ptashne M, Young RA. 1998. An activator target in the RNA polymerase II holoenzyme. Mol. Cell 1:895-904.
53. Krueger BJ, et al. 2008. LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res. 36:2219-2229.
54. Krueger BJ, Varzavand K, Cooper JJ, Price DH. 2010. The mechanism of release of P-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activators includes a conformational change in 7SK. PLoS One 5:e12335. doi:10.1371/journal.pone.0012335.
55. Kurosu T, Peterlin BM. 2004. VP16 and ubiquitin; binding of P-TEFb via its activation domain and ubiquitin facilitates elongation of transcription of target genes. Curr. Biol. 14:1112-1116.
56. Laspia MF, Rice AP, Mathews MB. 1989. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell 59:283-292.
57. Li Q, Peterlin BM. 2009. Genetic analysis of P-TEFb function via heterologous nucleic acid tethering systems. Methods 48:375-380.
58. Li Q, et al. 2005. Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186. J. Biol. Chem. 280:28819-28826.
59. Lim SR, Hertel KJ. 2004. Commitment to splice site pairing coincides with A complex formation. Mol. Cell 15:477-483.
60. Lis J, Wu C. 1993. Protein traffic on the heat shock promoter: parking, stalling, and trucking along. Cell 74:1-4.
61. Lis JT, Mason P, Peng J, Price DH, Werner J. 2000. P-TEFb kinase recruitment and function at heat shock loci. Genes Dev. 14:792-803.
62. Majello B, Napolitano G, Lania L. 1998. Recruitment of the TATAbinding protein to the HIV-1 promoter is a limiting step for Tat transactivation. AIDS 12:1957-1964.
63. Malik S, Roeder RG. 2010. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation. Nat. Rev. Genet. 11:761-772.
64. Mancebo HS, et al. 1997. P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes Dev. 11:2633-2644.
65. Meng EC, Pettersen EF, Couch GS, Huang CC, Ferrin TE. 2006. Tools for integrated sequence-structure analysis with UCSF Chimera. BMC Bioinformatics 7:339-345.
66. Michels AA, et al. 2004. Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor. EMBO J. 23:2608-2619.
67. Milla ME, Brown BM, Sauer RT. 1994. Protein stability effects of a complete set of alanine substitutions in Arc repressor. Nat. Struct. Biol. 1:518-523.
68. Modesti N, Garcia J, Debouck C, Peterlin M, Gaynor R. 1991. Transdominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression. New Biol. 3:759-768.
69. Mondal T, Rasmussen M, Pandey GK, Isaksson A, Kanduri C. 2010. Characterization of the RNA content of chromatin. Genome Res. 20: 899-907.
70. Muniz L, Egloff S, Ughy B, Jady BE, Kiss T. 2010. Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat. PLoS Pathog. 6:e1001152. doi:10.1371/journal.ppat.1001152.
71. Naar AM, Lemon BD, Tjian R. 2001. Transcriptional coactivator complexes. Annu. Rev. Biochem. 70:475-501.
72. Nechaev S, Adelman K. 2011. Pol II waiting in the starting gates: regulating the transition from transcription initiation into productive elongation. Biochim. Biophys. Acta 1809:34-45.
73. Nevado J, Gaudreau L, Adam M, Ptashne M. 1999. Transcriptional activation by artificial recruitment in mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 96:2674-2677.
74. Nguyen VT, Kiss T, Michels AA, Bensaude O. 2001. 7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes. Nature 414:322-325.
75. Nilson KA, Price DH. 2011. The role of RNA polymerase II elongation control in HIV-1 gene expression, replication, and latency. Genet. Res. Int. 2011:726901. doi:10.4061/2011/726901.
76. Ott M, Geyer M, Zhou Q. 2011. The control of HIV transcription: keeping RNA polymerase II on track. Cell Host Microbe 10:426-435.
77. Perkins JR, Diboun I, Dessailly BH, Lees JG, Orengo C. 2010. Transient protein-protein interactions: structural, functional, and network properties. Structure 18:1233-1243.
78. Peterlin BM, Brogie JE, Price DH. 2011. 7SK snRNA: a noncoding RNA that plays a major role in regulating eukaryotic transcription. Wiley Interdiscip. Rev. RNA 3:92-103.
79. Peterlin BM, Price DH. 2006. Controlling the elongation phase of transcription with P-TEFb. Mol. Cell 23:297-305.
80. Ping YH, Rana TM. 1999. Tat-associated kinase (P-TEFb): a component of transcription preinitiation and elongation complexes. J. Biol. Chem. 274:7399-7404.
81. Price DH. 2000. P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol. Cell. Biol. 20:2629-2634.
82. Price DH. 2008. Poised polymerases: on your mark.get set.go! Mol. Cell 30:7-10.
83. Ptashne M, Gann A. 1997. Transcriptional activation by recruitment. Nature 386:569-577.
84. Purnell BA, Emanuel PA, Gilmour DS. 1994. TFIID sequence recognition of the initiator and sequences farther downstream in Drosophila class II genes. Genes Dev. 8:830-842.
85. Raha T, Cheng SW, Green MR. 2005. HIV-1 Tat stimulates transcription complex assembly through recruitment of TBP in the absence of TAFs. PLoS Biol. 3:e44. doi:10.1371/journal.pbio.0030044.
86. Rahl PB, et al. 2010. c-Myc regulates transcriptional pause release. Cell 141:432-445.
87. Rappaport J, Reinberg D, Zandomeni R, Weinmann R. 1987. Purification and functional characterization of transcription factor SII from calf thymus. Role in RNA polymerase II elongation. J. Biol. Chem. 262: 5227-5232.
88. Reines D, Chamberlin MJ, Kane CM. 1989. Transcription elongation factor SII (TFIIS) enablesRNApolymerase II to elongate through a block to transcription in a human gene in vitro. J. Biol. Chem. 264:10799-10809.
89. Rennell D, Bouvier SE, Hardy LW, Poteete AR. 1991. Systematic mutation of bacteriophage T4 lysozyme. J. Mol. Biol. 222:67-88.
90. Reynolds KA, McLaughlin RN, Ranganathan R. 2011. Hot spots for allosteric regulation on protein surfaces. Cell 147:1564-1575.
91. Rice AP, Carlotti F. 1990. Mutational analysis of the conserved cysteinerich region of the human immunodeficiency virus type 1 Tat protein. J. Virol. 64:1864-1868.
92. Rice AP, Carlotti F. 1990. Structural analysis of wild-type and mutant human immunodeficiency virus type 1 Tat proteins. J. Virol. 64:6018-6026.
93. Rice AP, Mathews MB. 1988. Transcriptional but not translational regulation of HIV-1 by the tat gene product. Nature 332:551-553.
94. Saunders A, Core LJ, Lis JT. 2006. Breaking barriers to transcription elongation. Nat. Rev. Mol. Cell Biol. 7:557-567.
95. Schulte A, et al. 2005. Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat. J. Biol. Chem. 280:24968-24977.
96. Sedore SC, et al. 2007. Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR. Nucleic Acids Res. 35:4347-4358.
97. Selby MJ, Peterlin BM. 1990. Trans-activation by HIV-1 Tat via a heterologous RNA binding protein. Cell 62:769-776.
98. Sikorski TW, Buratowski S. 2009. The basal initiation machinery: beyond the general transcription factors. Curr. Opin. Cell Biol. 21:344-351.
99. Smith CA, Calabro V, Frankel AD. 2000. An RNA-binding chameleon. Mol. Cell 6:1067-1076.
100. Smith E, Lin C, Shilatifard A. 2011. The super elongation complex (SEC) and MLL in development and disease. Genes Dev. 25:661-672.
101. Sobhian B, et al. 2010. HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP. Mol. Cell 38:439-451.
102. Southgate C, Zapp ML, Green MR. 1990. Activation of transcription by HIV-1 Tat protein tethered to nascent RNA through another protein. Nature 345:640-642.
103. Southgate CD, Green MR. 1991. The HIV-1 Tat protein activates transcription from an upstream DNA-binding site: implications for Tat function. Genes Dev. 5:2496-2507.
104. Stargell LA, Struhl K. 1996. A new class of activation-defective TATAbinding protein mutants: evidence for two steps of transcriptional activation in vivo. Mol. Cell. Biol. 16:4456-4464.
105. Strubin M, Struhl K. 1992. Yeast and human TFIID with altered DNAbinding specificity for TATA elements. Cell 68:721-730.
106. Suel GM, Lockless SW, Wall MA, Ranganathan R. 2003. Evolutionarily conserved networks of residues mediate allosteric communication in proteins. Nat. Struct. Biol. 10:59-69.
107. Tahirov TH, et al. 2010. Crystal structure of HIV-1 Tat complexed with human P-TEFb. Nature 465:747-751.
108. Tsiang M, et al. 1995. Functional mapping of the surface residues of human thrombin. J. Biol. Chem. 270:16854-16863.
109. Ujvari A, Luse DS. 2006. RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit. Nat. Struct. Mol. Biol. 13:49-54.
110. Wada T, Takagi T, Yamaguchi Y, Watanabe D, Handa H. 1998. Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro. EMBO J. 17:7395-7403.
111. Wei P, Garber ME, Fang SM, Fischer WH, Jones KA. 1998. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92: 451-462.
112. Xiao H, Lis JT, Jeang KT. 1997. Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment. Mol. Cell. Biol. 17: 6898-6905.
113. Yamaguchi Y, et al. 1999. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97:41-51.
114. Yang L, et al. 1997. Distinct transcriptional pathways of TAR-dependent and TAR-independent human immunodeficiency virus type-1 transactivation by Tat. Virology 235:48-64.
115. Yang XJ, Roberts JW. 1989. Gene Q antiterminator proteins of Escherichia coli phages 82 and lambda suppress pausing byRNApolymerase at a rho-dependent terminator and at other sites. Proc. Natl. Acad. Sci. U. S. A. 86:5301-5305.
116. Yang Z, Zhu Q, Luo K, Zhou Q. 2001. The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription. Nature 414: 317-322.
117. Yankulov K, Blau J, Purton T, Roberts S, Bentley DL. 1994. Transcriptional elongation by RNA polymerase II is stimulated by transactivators. Cell 77:749-759.
118. Zhang J, et al. 2000. HIV-1 TAR RNA enhances the interaction between Tat and cyclin T1. J. Biol. Chem. 275:34314-34319.
119. Zhou M, et al. 2000. Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription. Mol. Cell. Biol. 20:5077-5086.
120. Zhou Q, Yik JH. 2006. The Yin and yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation. Microbiol. Mol. Biol. Rev. 70:646-659.
121. Zhu Y, et al. 1997. Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. Genes Dev. 11:2622-2632.