Construction of a Normalized cDNA Library for the Trypanosoma cruzi Genome Project
TURÁN P. ÜRMÉNYI
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, 21949-900, Brazil
Search for more papers by this authorMARIA F. BONALDO
Department of Pediatrics, The University of Iowa, Iowa City, Iowa
Search for more papers by this authorMARCELO B. SOARES
Department of Pediatrics, The University of Iowa, Iowa City, Iowa
Department of Physiology & Biophysics, The University of Iowa, Iowa City, Iowa, USA
Search for more papers by this authorCorresponding Author
EDSON RONDINELLI
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, 21949-900, Brazil
Corresponding Author: E. Rondinelli-Telephone number: 55 (21) 564–7364; FAX number: 55 (21) 280-8193; Email: edrondin@biof. ufrj.br.Search for more papers by this authorTURÁN P. ÜRMÉNYI
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, 21949-900, Brazil
Search for more papers by this authorMARIA F. BONALDO
Department of Pediatrics, The University of Iowa, Iowa City, Iowa
Search for more papers by this authorMARCELO B. SOARES
Department of Pediatrics, The University of Iowa, Iowa City, Iowa
Department of Physiology & Biophysics, The University of Iowa, Iowa City, Iowa, USA
Search for more papers by this authorCorresponding Author
EDSON RONDINELLI
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, 21949-900, Brazil
Corresponding Author: E. Rondinelli-Telephone number: 55 (21) 564–7364; FAX number: 55 (21) 280-8193; Email: edrondin@biof. ufrj.br.Search for more papers by this authorABSTRACT
Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.
References
- Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, J.M., Utterback, T. R., Nagle, J. W., Fields, C. & Craig Venter, J. 1992. Sequence identification of 2,375 human brain genes. Nature, 355: 632–634.
- Adams, M. D., Kelley, J. M., Gocayne, J. D., Dubnick, M., Polymer-opoulos, M. H., Xiao, H., Merril, C. R., Wu, A., Olde, B., Moreno, R. F., Kerlavage, A. R., McCombie, W. R. & Craig Venter, J. 1991. Complementary DNA sequencing: expressed sequence tags and human genome project. Science, 252: 1651–1656.
- Adams, M. D., Soares, M. B., Kerlavage, A. R., Fields, C. & Craig Venter, J. 1993. Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library. Nat. Genet., 4: 373–380.
- Berry, R., Stevens, T. J., Walter, N. A., Wilcox, A. S., Rubano, T., Hopkins, J. A., Weber, J., Goold, R., Soares, M. B. & Sikela, J. M. 1995. Gene-based sequence-tagged-sites (STSs) as the basis for a human gene map. Nature Genet., 10: 415–423.
- Boguski, M. S. 1995. The turning point in genome research. Trends Biochem. Sci., 20: 295–296.
- Bonaldo, M. F., Lennon, G. & Soares, M. B. 1996. Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res., 6: 791–806.
- Brando, A., Ürményi, T. P., Rondinelli, E., Gonzalez, A., Miranda, A. B. & Degrave, W. 1997. Identification of transcribed sequences (ESTs) in the Trypanosoma cruzi genome project. Mem. Inst. Os-waldo Cruz, 92: 863–866.
- Cano, M. I., Gruber, A., Vasquez, M., Cortés, A., Levin, M. J., Gonzáles, A., Degrave, W., Rondinelli, E., Zingales, B., Ramirez, J. L., Alonso, C., Requena, J. M. & Silveira, J. F. 1995. Molecular karyo-type of clone CL Brener chosen for the Trypanosoma cruzi genome project. Mol. Biochem. Parasitol., 71: 273–278.
- Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonucleasey, Biochemistry, 18: 5294–5299.
- The Trypanosoma cruzi Genome Consortium. 1997. The Trypanosoma cruzi genome initiative. Parasitol. Today, 13: 16–22.
- Han, J. H., Stratowa, C. & Rutter, W. J. 1987. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning. Biochemistry, 26: 1617–1625.
- Hillier, L. D., Lennon, G., Becker, M., Bonaldo, M. F., Chiapelli, B., Chissoe, S., Dietrich, N., DuBuque, T., Favello, A., Gish, W., Hawkins, M., Hultman, M., Kucaba, T., Lacy, M., Le, M., Le, N., Mardis, E., Moore, B., Morris, M., Parsons, J., Prange, C., Rifkin, L., Rohlfing, T., Schellenberg, K., Soares, M. B., Tan, F., Thierry-Meg, J., Trevaskis, E., Underwood, K., Wohldman, P., Waterston, R., Wilson, R. & Marra, M. 1996. Generation and analysis of 280,000 human expressed sequence tags. Genome Res., 6: 807–828.
- Okubo, K., Hori, N., Matoba, R., Niiyama, T., Fukushima, T., Kojima, Y. & Matsubara, K. 1992. Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nat. Genet., 2: 173–179.
-
Sambrook, J.,
Fritsch, E. F. &
Maniatis, T.
1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor
,
NY
.
10.1111/j.1095-8312.1996.tb01434.x Google Scholar
-
Soares, M. B.
1994. Construction of directionally cloned cDNA libraries in phagemid vectors, in: M. D. Adams,
C. Fields &
J. Craig Venter, (ed.), Automated DNA sequencing and analysis. Academic Press,
New York
. p.
110–114.
10.1016/B978-0-08-092639-1.50020-4 Google Scholar
- Soares, M. B., Bonaldo, M. F., Jelenc, P., Su, L., Lawton, L. & Efstratiadis, A. 1994. Construction and characterization of a normalized cDNA library. Proc. Natl. Acad. Sci. USA, 91: 9228–9232.
- Verdun, R. E., Di Paolo, N., Ürményi, T. P., Rondinelli, E., Frasch, C. C. & Sanchez, D. O. 1998. Gene discovery through expressed sequence tag sequencing in Trypanosoma cruzi. Infect. Immun., 66: 5393–5398.
