Abstract
To better understand gene expression at very low levels, we have designed a method to eliminate cDNA clones representing abundant mRNAs. A cDNA library for drought-stressed hot pepper (Capsicum annuum) (Choi et al., 2002) underwent double-negative screening, once with probes made from a drought-stressed plant, the second time, with probes from a non-stressed plant. The cDNA clones that showed very weak or negative signals were isolated for further analysis, which resulted in 1399 cDNA clones from about 20,000 screened clones. When nucleotide sequences were determined, we obtained 1142 tentative unique genes, with a redundancy rate of 20.41%. An homology database search for the deduced amino acid sequences revealed that about 79% of the cDNA clones could not be matched for functioning with previously characterized sequences. However, when these uncategorized clones were subjected to classification based on functional domains, most could be cited. Notably, clones with possible functions in RNA transport, protein synthesis, and regulation of protein activity showed a dramatic increase in appearance while those coding for transposable elements, viral proteins, and plasmid proteins occupied a much smaller portion compared with those in theArabidopsis thaliana genome. In addition, those coding for proteins targeted to the endoplasmic reticulum were dramatically more abundant in our clones compared with theArabidopsis database.
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Literature Cited
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Capped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res25: 3389–3402
Arabidopsis Genome Initiative -- AGI (2000) Analysis of the genome sequence of the flowering plantArabidopsis thaliana. Nature408: 796–815
Asamizu E, Nakamura Y, Sato S, Tabata S (2000) A large scale analysis of cDNA inArabidopsis thaliana: Generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries. DNA Res7: 175–180
Caminci P, Shibata Y, Hayatsu N, Sugahara Y, Shibata K, Itoh M, Konno H, Okazaki Y, Muramatsu M, Hayashizaki Y (2000) Normalization and subtraction of captrapper selected cDNAs to prepare full-length cDNA libraries for high-rate new gene discovery. Genome Res10: 1617–1630
Choi KH, Hong CB, Kim WT (2002) Isolation and characterization of drought-induced cDNA clones from hot pepper. J Plant Biol45: 212–218
Cooke R, Raynal M, Laudie M, Grellet F, Delseny M, Morris PC, Guerrier D, Giraudat J, Quigley F, Clabault G, Li YF, Mache R, Krivitzky M, Gy lJ, Kreis M, Lecharny A, Parmentier Y, Marbach J, Fleck J, Clement B, Philipps G, Herve C, Bardet C, Tremousaygue D, Hofte H (1996) Further progress towards a catalogue of allArabidopsis genes: Analysis of a set of 5000 non-redundant ESTs. Plant J9: 101–124
Cushman JC, Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Curr Opin Plant Biol3: 117–124
Drawid A, Jansen R, Gerstein M (2000) Genome-wide analysis relating expression level with protein subcellular localization. Trends Genet16: 426–430
Fowler S, Thomashow MF (2002)Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell14: 1675–1690
Frishman D, Albermann K, Hani J, Heumann K, Metanomski A, Zollner A, Mewes HW (2001) Functional and structural genomics using PEDANT. Bioinformatics17: 44–57
Gong Z, Lee H, Xiong L, Jagendorf A, Stevenson B, Zhu JK (2002) RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance. Proc Natl Acad Sci USA99: 11507–11512
Greenbaum D, Jansen R, Gerstein M (2002) Analysis of mRNA expression and protein abundance data: An approach for the comparison of the enrichment of features in the cellular population of proteins and transcripts. Bioinformatics18: 585–596
Guo Y, Xiong L, Ishitani M, Zhu JK (2002) AnArabidopsis mutation in translation elongation factor 2 causes superinduction of CBF/DREB1 transcription factor genes but blocks the induction of their downstream targets under low temperature. Proc Natl Acad Sci USA99: 7786–7791
Haas BJ, Volfovsky N, Town CD, Troukhan M, Alexandrov N, Feldmann KA, Flavell RB, White O, Salzberg SL (2002) Full-length messenger RNA sequences greatly improve genome annotation. Genome Biol3: 0029.1–0029.12
Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol51: 463–499
Huang X (1996) An improved sequence assembly program. Genomics33: 21–31
Jansen R, Gerstein M (2000) Analysis of the yeast transcriptome with structural and functional categories: Characterizing highly expressed proteins. Nucleic Acids Res28: 1481–1488
Knight H, Knight MR (2001) Abiotic stress signalling pathways: Specificity and cross-talk. Trends Plant Sci6: 262–267
Martin GB (1998) Gene discovery for crop improvement. Curr Opin Biotech9: 220–226
Mewes HW, Frishman D, Guldener U, Mannhaupt G, Mayer K, Mokrejs M, Morgenstern B, Munsterkotter M, Rudd S, Weil B (2002) MIPS: A database for genomes and protein sequences. Nucleic Acids Res30: 31–34
Mysore KS, Tuori RP, Martin GB (2001)Arabidopsis genome sequence as a tool for functional genomics in tomato. Genome Biol2: 1003.1–1003.4
Ohlrogge J, Benning C (2000) Unraveling plant metabolism by EST analysis. Curr Opin Plant Biol3: 224–228
Park JK, Hong CB (1991) A cDNA clone for red pepper (Capsicum annuum L.) actin. Mol Cells1: 301–308
Ribaut JM, Banziger M, Hoisington D (2002) Genetic dissection and plant improvement under abiotic stress conditions: Drought tolerance in maize as an example. JIRCAS Working Report 85–92
Rice P, Longden I, Bleasby A (2000) EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet16: 276–277
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. 2nd ed, Cold Spring Harbor Laboratory Press, New York
Schoof H, Zaccaria P, Gundlach H, Lemcke K, Rudd S, Kolesov G, Arnold R, Mewes HW, Mayer KF (2002) MIPSArahidopsis thaliana Database (MAtDB): An integrated biological knowledge resource based on the first complete plant genome. Nucleic Acids Res30: 91–93
Seki M, Narusaka M, Abe H, Kasuga M, Yamaguchi-Shinozaki K, Carninci P, Hayashizaki Y, Shinozaki K (2001) Monitoring the expression pattern of 1300Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray. Plant Cell13: 61–72
Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T, Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y, Shinozaki K (2002) Monitoring the expression profiles of 7000Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J31: 279–292
Soares MB (1997) Identification and cloning of differentially expressed genes. Curr Biol8: 542–546
van der Hoeven RV, Ronning C, Giovannoni JJ, Martin G, Tanksley SD (2002) Deductions about the number, organization and evolution of genes in the tomato genome based on analysis of a large EST collection and selective genomic sequencing. Plant Cell14: 1441–1456
White J, Todd J, Newman T, Focks N, Girke T, Martinez de llarduya OL, Jaworski J, Ohlrogge J, Benning C (2000) A new set ofArabidopsis expressed sequence tags from developing seeds. The metabolic pathway from carbohydrates to seed oil. Plant Physiol124: 1582–1594
Xing L, Brendel V (2000) MuSeqBox: A program for multiquery sequence BLAST output examination. Bioinformatics17: 744–745
Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 18 S165-S183
Yamaguchi-Shinozaki K, Shinozaki K (2002) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Novartis Found Symp236: 176–186
Zhang L, Ma XL, Zhang Q, Ma CL, Wang PR Sun YF, Zhao YX, Zhang H (2001) Expressed sequence tags from a NaCI-treatedSuaeda salsa cDNA library. Gene267: 193–200
Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol53: 247–73
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Cho, Y.B., Jeon, H.J. & Hong, C.B. Cloning and functional annotation of rare mRNA species from drought-stressed hot pepper (Capsicum annuum). J. Plant Biol. 46, 83–89 (2003). https://doi.org/10.1007/BF03030435
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DOI: https://doi.org/10.1007/BF03030435