On the physiological significance of alternative splicing events in higher plants

Protoplasma

Volumn 250, Issue 3, 2013, Pages 639-650

  Carvalho, Raquel F ^a   Feijão, Carolina V ^a   Duque, Paula ^a  

^a INSTITUTO GULBENKIAN DE CIÊNCIA   (Portugal)

Author keywords

Alternative splicing; Gene expression; Plant physiology and development; Precursor mRNA; Splice isoforms

Indexed keywords

ISOPROTEIN; MESSENGER RNA; PHYTOHORMONE; VEGETABLE PROTEIN;

ALTERNATIVE RNA SPLICING; ANIMAL; DISEASE RESISTANCE; GENETICS; HUMAN; IMMUNOLOGY; METABOLISM; PHYSIOLOGICAL STRESS; PLANT; PLANT DEVELOPMENT; REVIEW; SIGNAL TRANSDUCTION;

ALTERNATIVE SPLICING; ANIMALS; DISEASE RESISTANCE; HUMANS; PLANT DEVELOPMENT; PLANT GROWTH REGULATORS; PLANT PROTEINS; PLANTS; PROTEIN ISOFORMS; RNA, MESSENGER; SIGNAL TRANSDUCTION; STRESS, PHYSIOLOGICAL;

ARABIDOPSIS; EMBRYOPHYTA; EUKARYOTA; TRACHEOPHYTA;

EID: 84877945444     PISSN: 0033183X     EISSN: None     Source Type: Journal    
DOI: 10.1007/s00709-012-0448-9     Document Type: Review

References (107)

1. Ali GS, Reddy AS (2006) ATP, phosphorylation and transcription regulate the mobility of plant splicing factors. J Cell Sci 119: 3527-3538.
2. Ali GS, Reddy AS (2008) Regulation of alternative splicing of pre-mRNAs by stresses. Curr Top Microbiol Immunol 326: 257-275.
3. Ali GS, Golovkin M, Reddy AS (2003) Nuclear localization and in vivo dynamics of a plant-specific serine/arginine-rich protein. Plant J 36: 883-893.
4. Ali GS, Palusa SG, Golovkin M, Prasad J, Manley JL, Reddy AS (2007) Regulation of plant developmental processes by a novel splicing factor. PLoS One 2: e471.
5. Ali GS, Prasad KV, Hanumappa M, Reddy AS (2008) Analyses of in vivo interaction and mobility of two spliceosomal proteins using FRAP and BiFC. PLoS One 3: e1953.
6. Avanci NC, Luche DD, Goldman GH, Goldman MH (2010) Jasmonates are phytohormones with multiple functions, including plant defense and reproduction. Genet Mol Res 9: 484-505.
7. Barbazuk WB, Fu Y, McGinnis KM (2008) Genome-wide analyses of alternative splicing in plants: opportunities and challenges. Genome Res 18: 1381-1392.
8. Barta A, Kalyna M, Lorkovic ZJ (2008) Plant SR proteins and their functions. Curr Top Microbiol Immunol 326: 83-102.
9. Blencowe BJ (2006) Alternative splicing: new insights from global analyses. Cell 126: 37-47.
10. Braverman N, Dodt G, Gould SJ, Valle D (1998) An Isoform of Pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes. Hum Mol Genet 7: 1195-1205.
11. Brett D, Pospisil H, Valcarcel J, Reich J, Bork P (2002) Alternative splicing and genome complexity. Nat Genet 30: 29-30.
12. Brown L-A, Baker A (2008) Shuttles and cycles: transport of proteins into the peroxisome matrix (Review). Mol Membr Biol 25: 363-375.
13. Campbell MA, Haas BJ, Hamilton JP, Mount SM, Buell CR (2006) Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis. BMC Genomics 7: 327.
14. Carvalho RF, Carvalho SD, Duque P (2010) The plant-specific SR45 protein negatively regulates glucose and ABA signaling during early seedling development in Arabidopsis. Plant Physiol 154: 772-783.
15. Carvalho SD, Saraiva R, Maia TM, Abreu IA, Duque P (2012) XBAT35, a novel Arabidopsis RING E3 ligase exhibiting dual targeting of its splice isoforms, is involved in ethylene-mediated regulation of apical hook curvature. Mol Plant. doi: 10. 1093/mp/sss048.
16. Castillon A, Shen H, Huq E (2007) Phytochrome Interacting Factors: central players in phytochrome-mediated light signaling networks. Trends Plant Sci 12: 514-521.
17. Cheong JJ, Choi YD (2003) Methyl jasmonate as a vital substance in plants. Trends Genet 19: 409-413.
18. Chung HS, Howe GA (2009) A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis. Plant Cell 21: 131-145.
19. Chung HS, Niu Y, Browse J, Howe GA (2009) Top hits in contemporary JAZ: an update on jasmonate signaling. Phytochemistry 70: 1547-1559.
20. Chung HS, Cooke TF, Depew CL, Patel LC, Ogawa N, Kobayashi Y, Howe GA (2010) Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling. Plant J 63: 613-622.
21. Costanzo S, Jia Y (2009) Alternatively spliced transcripts of Pi-ta blast resistance gene in Oryza sativa. Plant Sci 177: 468-478.
22. Crafts-Brandner SJ, van De Loo FJ, Salvucci ME (1997) The two forms of ribulose-1, 5-bisphosphate carboxylase/oxygenase activase differ in sensitivity to elevated temperature. Plant Physiol 114: 439-444.
23. Deridder BP, Shybut ME, Dyle MC, Kremling KA, Shapiro MB (2012) Changes at the 3'-untranslated region stabilize Rubisco activase transcript levels during heat stress in Arabidopsis. Planta 236: 463-476.
24. Dinesh-Kumar SP, Baker BJ (2000) Alternatively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance. Proc Natl Acad Sci U S A 97: 1908-1913.
25. Duque P (2011) A role for SR proteins in plant stress responses. Plant Signal Behav 6: 49-54.
26. Feller U, Crafts-Brandner SJ, Salvucci ME (1998) Moderately high temperatures inhibit ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) Activase-Mediated Activation of Rubisco. Plant Physiol 116: 539-546.
27. Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW, Fox SE, Wong WK, Mockler TC (2010) Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res 20: 45-58.
28. Franklin KA, Lee SH, Patel D, Kumar SV, Spartz AK, Gu C, Ye S, Yu P, Breen G, Cohen JD, Wigge PA, Gray WM (2011) Phytochrome-interacting factor 4 (PIF4) regulates auxin biosynthesis at high temperature. Proc Natl Acad Sci U S A 108: 20231-20235.
29. Fu Y, Bannach O, Chen H, Teune JH, Schmitz A, Steger G, Xiong L, Barbazuk WB (2009) Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA. Genome Res 19: 913-921.
30. Gassmann W (2008) Alternative splicing in plant defense. Curr Top Microbiol Immunol 326: 219-233.
31. Gassmann W, Hinsch ME, Staskawicz BJ (1999) The Arabidopsis RPS4 bacterial-resistance gene is a member of the TIR-NBS-LRR family of disease-resistance genes. Plant J 20: 265-277.
32. Golovkin M, Reddy AS (1999) An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein. J Biol Chem 274: 36428-36438.
33. Haas BJ, Delcher AL, Mount SM, Wortman JR, Smith RK Jr, Hannick LI, Maiti R, Ronning CM, Rusch DB, Town CD, Salzberg SL, White O (2003) Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res 31: 5654-5666.
34. Hazen SP, Naef F, Quisel T, Gendron JM, Chen H, Ecker JR, Borevitz JO, Kay SA (2009) Exploring the transcriptional landscape of plant circadian rhythms using genome tiling arrays. Genome Biol 10: R17.
35. Hinsch M, Staskawicz B (1996) Identification of a new Arabidopsis disease resistance locus, RPs4, and cloning of the corresponding avirulence gene, avrRps4, from Pseudomonas syringae pv. pisi. Mol Plant Microbe Interact 9: 55-61.
36. Hu J, Baker A, Bartel B, Linka N, Mullen RT, Reumann S, Zolman BK (2012) Plant peroxisomes: biogenesis and function. Plant Cell 24: 2279-2303.
37. Hughes TA (2006) Regulation of gene expression by alternative untranslated regions. Trends in Genet 22: 119-122.
38. Iida K, Seki M, Sakurai T, Satou M, Akiyama K, Toyoda T, Konagaya A, Shinozaki K (2004) Genome-wide analysis of alternative pre-mRNA splicing in Arabidopsis thaliana based on full-length cDNA sequences. Nucleic Acids Res 32: 5096-5103.
39. Jordan T, Schornack S, Lahaye T (2002) Alternative splicing of transcripts encoding Toll-like plant resistance proteins-what's the functional relevance to innate immunity? Trends Plant Sci 7: 392-398.
40. Kalyna M, Simpson CG, Syed NH, Lewandowska D, Marquez Y, Kusenda B, Marshall J, Fuller J, Cardle L, McNicol J, Dinh HQ, Barta A, Brown JW (2012) Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis. Nucleic Acids Res 40: 2454-2469.
41. Kazan K (2003) Alternative splicing and proteome diversity in plants: the tip of the iceberg has just emerged. Trends Plant Sci 8: 468-471.
42. Kazan K, Manners JM (2008) Jasmonate signaling: toward an integrated view. Plant Physiol 146: 1459-1468.
43. Kikuchi S, Satoh K, Nagata T, Kawagashira N, Doi K, Kishimoto N, Yazaki J, Ishikawa M, Yamada H, Ooka H, Hotta I, Kojima K, Namiki T, Ohneda E, Yahagi W, Suzuki K, Li CJ, Ohtsuki K, Shishiki T, Otomo Y, Murakami K, Iida Y, Sugano S, Fujimura T, Suzuki Y, Tsunoda Y, Kurosaki T, Kodama T, Masuda H, Kobayashi M, Xie Q, Lu M, Narikawa R, Sugiyama A, Mizuno K, Yokomizo S, Niikura J, Ikeda R, Ishibiki J, Kawamata M, Yoshimura A, Miura J, Kusumegi T, Oka M, Ryu R, Ueda M, Matsubara K, Kawai J, Carninci P, Adachi J, Aizawa K, Arakawa T, Fukuda S, Hara A, Hashizume W, Hayatsu N, Imotani K, Ishii Y, Itoh M, Kagawa I, Kondo S, Konno H, Miyazaki A, Osato N, Ota Y, Saito R, Sasaki D, Sato K, Shibata K, Shinagawa A, Shiraki T, Yoshino M, Hayashizaki Y, Yasunishi A (2003) Collection, mapping, and annotation of over 28, 000 cDNA clones from japonica rice. Science 301: 376-379.
44. Kim E, Magen A, Ast G (2007) Different levels of alternative splicing among eukaryotes. Nucleic Acids Res 35: 125-131.
45. Kim E, Goren A, Ast G (2008) Alternative splicing: current perspectives. Bioessays 30: 38-47.
46. Kim SH, Kwon SI, Saha D, Anyanwu NC, Gassmann W (2009) Resistance to the Pseudomonas syringae effector HopA1 is governed by the TIR-NBS-LRR protein RPS6 and is enhanced by mutations in SRFR1. Plant Physiol 150: 1723-1732.
47. Kobza J, Edwards GE (1987) Influences of leaf temperature on photosynthetic carbon metabolism in wheat. Plant Physiol 83: 69-74.
48. Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, Franklin KA (2009) High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4. Curr Biol 19: 408-413.
49. Koncz C, Dejong F, Villacorta N, Szakonyi D, Koncz Z (2012) The spliceosome-activating complex: molecular mechanisms underlying the function of a pleiotropic regulator. Front Plant Sci 3: 9.
50. Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot 63: 1593-1608.
51. Law RD, Crafts-Brandner SJ (1999) Inhibition and acclimation of photosynthesis to heat stress is closely correlated with activation of ribulose-1, 5-bisphosphate Carboxylase/Oxygenase. Plant Physiol 120: 173-182.
52. Lee JR, Jang HH, Park JH, Jung JH, Lee SS, Park SK, Chi YH, Moon JC, Lee YM, Kim SY, Kim J-Y, Yun D-J, Cho MJ, Lee KO, Lee SY (2006) Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis. Plant J 47: 457-466.
53. Long JC, Caceres JF (2009) The SR protein family of splicing factors: master regulators of gene expression. Biochem J 417: 15-27.
54. Lu T, Lu G, Fan D, Zhu C, Li W, Zhao Q, Feng Q, Zhao Y, Guo Y, Huang X, Han B (2010) Function annotation of the rice transcriptome at single-nucleotide resolution by RNA-seq. Genome Res 20: 1238-1249.
55. Macknight R, Bancroft I, Page T, Lister C, Schmidt R, Love K, Westphal L, Murphy G, Sherson S, Cobbett C, Dean C (1997) FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains. Cell 89: 737-745.
56. Macknight R, Duroux M, Laurie R, Dijkwel P, Simpson G, Dean C (2002) Functional significance of the alternative transcript processing of the Arabidopsis floral promoter FCA. Plant Cell 14: 877-888.
57. Mao DD, Tian LF, Li LG, Chen J, Deng PY, Li DP, Luan S (2008) AtMGT7: an Arabidopsis gene encoding a low-affinity magnesium transporter. J Integr Plant Biol 50: 1530-1538.
58. Marquez Y, Brown JW, Simpson C, Barta A, Kalyna M (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 22: 1184-1195.
59. Nagasaki H, Arita M, Nishizawa T, Suwa M, Gotoh O (2005) Species-specific variation of alternative splicing and transcriptional initiation in six eukaryotes. Gene 364: 53-62.
60. Ner-Gaon H, Fluhr R (2006) Whole-genome microarray in Arabidopsis facilitates global analysis of retained introns. DNA Res 13: 111-121.
61. Ner-Gaon H, Halachmi R, Savaldi-Goldstein S, Rubin E, Ophir R, Fluhr R (2004) Intron retention is a major phenomenon in alternative splicing in Arabidopsis. Plant J 39: 877-885.
62. Nicholson P, Yepiskoposyan H, Metze S, Zamudio Orozco R, Kleinschmidt N, Muhlemann O (2010) Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors. Cell Mol Life Sci 67: 677-700.
63. Nilsen TW, Graveley BR (2010) Expansion of the eukaryotic proteome by alternative splicing. Nature 463: 457-463.
64. Palusa SG, Ali GS, Reddy AS (2007) Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses. Plant J 49: 1091-1107.
65. Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40: 1413-1415.
66. Penfield S, Josse EM, Halliday KJ (2010) A role for an alternative splice variant of PIF6 in the control of Arabidopsis primary seed dormancy. Plant Mol Biol 73: 89-95.
67. Portis AR Jr (2003) Rubisco activase-Rubisco's catalytic chaperone. Photosynth Res 75: 11-27.
68. Quesada V, Macknight R, Dean C, Simpson GG (2003) Autoregulation of FCA pre-mRNA processing controls Arabidopsis flowering time. EMBO J 22: 3142-3152.
69. Reddy ASN (2001) Nuclear Pre-mRNA splicing in plants. Crit Rev Plant Sci 20: 523-571.
70. Reddy AS (2007) Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annu Rev Plant Biol 58: 267-294.
71. Robert-Seilaniantz A, Grant M, Jones JD (2011) Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. Annu Rev Phytopathol 49: 317-343.
72. Ru Y, Wang BB, Brendel V (2008) Spliceosomal proteins in plants. Curr Top Microbiol Immunol 326: 1-15.
73. Salvucci ME, Werneke JM, Ogren WL, Portis AR (1987) Purification and species distribution of rubisco activase. Plant Physiol 84: 930-936.
74. Salvucci ME, DeRidder BP, Portis AR Jr (2006) Effect of activase level and isoform on the thermotolerance of photosynthesis in Arabidopsis. J Exp Bot 57: 3793-3799.
75. Sanchez SE, Petrillo E, Beckwith EJ, Zhang X, Rugnone ML, Hernando CE, Cuevas JC, Godoy Herz MA, Depetris-Chauvin A, Simpson CG, Brown JW, Cerdan PD, Borevitz JO, Mas P, Ceriani MF, Kornblihtt AR, Yanovsky MJ (2010) A methyl transferase links the circadian clock to the regulation of alternative splicing. Nature 468: 112-116.
76. Sanchez SE, Petrillo E, Kornblihtt AR, Yanovsky MJ (2011) Alternative splicing at the right time. RNA Biol 8: 954-959.
77. Schmidt A, Baumann N, Schwarzkopf A, Frommer WB, Desimone M (2006) Comparative studies on Ureide Permeases in Arabidopsis thaliana and analysis of two alternative splice variants of AtUPS5. Planta 224: 1329-1340.
78. Schmucker D, Clemens JC, Shu H, Worby CA, Xiao J, Muda M, Dixon JE, Zipursky SL (2000) Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101: 671-684.
79. Seo PJ, Kim MJ, Ryu JY, Jeong EY, Park CM (2011) Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism. Nat Commun 2: 303.
80. Shen JB, Orozco EM Jr, Ogren WL (1991) Expression of the two isoforms of spinach ribulose 1, 5-bisphosphate carboxylase activase and essentiality of the conserved lysine in the consensus nucleotide-binding domain. J Biol Chem 266: 8963-8968.
81. Shepard PJ, Hertel KJ (2009) The SR protein family. Genome Biol 10: 242.
82. Simpson GG (2004) The autonomous pathway: epigenetic and post-transcriptional gene regulation in the control of Arabidopsis flowering time. Curr Opin Plant Biol 7: 570-574.
83. Simpson GG, Dijkwel PP, Quesada V, Henderson I, Dean C (2003) FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition. Cell 113: 777-787.
84. Smalle J, Vierstra RD (2004) The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol 55: 555-590.
85. Staal J, Dixelius C (2008) RLM3, a potential adaptor between specific TIR-NB-LRR receptors and DZC proteins. Commun Integr Biol 1: 59-61.
86. Staal J, Kaliff M, Dewaele E, Persson M, Dixelius C (2008) RLM3, a TIR domain encoding gene involved in broad-range immunity of Arabidopsis to necrotrophic fungal pathogens. Plant J 55: 188-200.
87. Stamm S, Ben-Ari S, Rafalska I, Tang Y, Zhang Z, Toiber D, Thanaraj TA, Soreq H (2005) Function of alternative splicing. Gene 344: 1-20.
88. Stone SL, Hauksdottir H, Troy A, Herschleb J, Kraft E, Callis J (2005) Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol 137: 13-30.
89. Szymanski M, Barciszewska MZ, Erdmann VA, Barciszewski J (2003) 5S rRNA: structure and interactions. Biochem J 371: 641-651.
90. To KY, Suen DF, Chen SC (1999) Molecular characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase activase in rice leaves. Planta 209: 66-76.
91. Wahl MC, Will CL, Luhrmann R (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136: 701-718.
92. Wang BB, Brendel V (2006) Genomewide comparative analysis of alternative splicing in plants. Proc Natl Acad Sci U S A 103: 7175-7180.
93. Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456: 470-476.
94. Wang D, Lu Q, Li X, Jiang Q, Wu J, Jiang D (2009) Relationship between Rubisco activase isoform levels and photosynthetic rate in different leaf positions of rice plant. Photosynthetica 47: 621-629.
95. Wang D, Li XF, Zhou ZJ, Feng XP, Yang WJ, Jiang DA (2010) Two Rubisco activase isoforms may play different roles in photosynthetic heat acclimation in the rice plant. Physiol Plant 139: 55-67.
96. Ward AJ, Cooper TA (2010) The pathobiology of splicing. J Pathol 220: 152-163.
97. Weis E (1981) The temperature-sensitivity of dark-inactivation and light-activation of the ribulose-1, 5-bisphosphate car'òêylase in spinach chloroplasts. FEBS Lett 129: 197-200.
98. Werneke JM, Chatfield JM, Ogren WL (1989) Alternative mRNA splicing generates the two ribulosebisphosphate carboxylase/oxygenase activase polypeptides in spinach and Arabidopsis. Plant Cell 1: 815-825.
99. Whitham S, Dinesh-Kumar SP, Choi D, Hehl R, Corr C, Baker B (1994) The product of the tobacco mosaic virus resistance gene N: similarity to toll and the interleukin-1 receptor. Cell 78: 1101-1115.
100. Woodward AW, Bartel B (2005) The Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 Is necessary for peroxisome function and dependent on PEX5. Mol Biol Cell 16: 573-583.
101. Yamori W, Suzuki K, Noguchi K, Nakai M, Terashima I (2006) Effects of Rubisco kinetics and Rubisco activation state on the temperature dependence of the photosynthetic rate in spinach leaves from contrasting growth temperatures. Plant Cell Environ 29: 1659-1670.
102. Yan Y, Stolz S, Chetelat A, Reymond P, Pagni M, Dubugnon L, Farmer EE (2007) A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell 19: 2470-2483.
103. Zhang XC, Gassmann W (2003) RPS4-mediated disease resistance requires the combined presence of RPS4 transcripts with full-length and truncated open reading frames. Plant Cell 15: 2333-2342.
104. Zhang XC, Gassmann W (2007) Alternative splicing and mRNA levels of the disease resistance gene RPS4 are induced during defense responses. Plant Physiol 145: 1577-1587.
105. Zhang XN, Mount SM (2009) Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development. Plant Physiol 150: 1450-1458.
106. Zhang N, Portis AR Jr (1999) Mechanism of light regulation of Rubisco: a specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f. Proc Natl Acad Sci U S A 96: 9438-9443.
107. Zhang N, Kallis RP, Ewy RG, Portis AR Jr (2002) Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform. Proc Natl Acad Sci U S A 99: 3330-3334.