D. Report of the Committee on Genetic Engineering

(Molecular Analysis of Rice Genes)

Section of Biochemistry, Molecular & Cell Biology, Cornell University,

Ithaca, NY 14853, U.S.A.

Progress on the genetic engineering of rice continues to advance at a rapid pace. More and more biological scientists have become involved in rice biotechnology or in the molecular analysis of rice genes, as witnessed by the increasing number of publications within the last 12 months. For example, close to 100 abstracts were included in the proceedings of the Sixth Annual Meeting of the International Program on Rice Biotechnology (sponsored by the Rockefeller Foundation, NY) held in Thailand, in February 1993. In addition, approximately 200 papers were cited, and many of them were reviewed in the last four issues of the Rice Biotechnology Quarterly (edited by John E. Dille). Therefore, I will not review these papers in this report.

Regarding rice genome analysis, two noteworthy events have been initiated within the last year or two. First, a Rice Genome Research Program (RGP) was started in Japan in October 1991, and was close to full operation by mid 1992. The RGP covers all aspects of rice genome analysis, inlcuding the isolation and characterization of genes corresponding to traits which are agronomically, environmentally and economically desirable. The program includes large-scale sequencing of rice cDNA and genomic clones as well as the construction of RFLP and physical maps for all 12 rice chromosomes.

The first issue of Rice Genome, a semi-annual newsletter for rice genome analysis, was published in July 1992 by the Japanese RGP (J-RGP). This issue reported that about 1,000 clones were sequenced from a callus cDNA library, and among them 60 clones were found to encode known proteins as determined by data-base analysis. Independently, Uchimiya's group (1992) also sequenced about 1,000 clones from a cDNA library prepared from suspension cultured cells of rice. Results indicated that close to 100 cDNAs encode for known proteins, but less than 30% of these proteins are similar to those reported in the July 1992 issue of Rice Genome.

For physical mapping, the J-RGP scientists constructed two YAC libraries containing 4,000 and 8,000 clones, respectively. The average size of the rice DNA inserts was 350 kb. The second issue of Rice Genome reported the sequence analysis of clones from a root cDNA library. Among the 45 clones identified to match known protein sequences, 33 of them were new and different from those found in the callus cDNA library.

The third issue of Rice Genome was published in July 1993. Large-scale cDNA analysis by the J-RGP scientists revealed multigene families of certain genes, especially those which encode ribosomal proteins. The finding of multigene families among rice or plant genes was not new because other investigators had reported similar findings with other rice genes. Physical mapping using YAC libraries and cosmid libraries continued. Results showed that it is possible to construct two contigs spanning across 3 to 5 centimorgans (cM). Information on RFLP

RAY WU, Convener

linkage mapping and database systems of the RGP was also included in both the 1992 and 1993 issues of Rice Genome. However, these subjects are beyond the scope of this report. One policy of the J-RGP is that it intends to share its cDNA clones and RFLP probes with anyone who requests them. This is an excellent policy because other investigators can then benefit from the RGP without having to isolate these clones or probes themselves.

Second, a Chinese Rice Genome Program (C-RGP) was intiated in August 1992. This program also covers all aspects of genome analysis, which include RFLP mapping, isolating, sequencing and characterizing rice genes of biological importance. cDNA libraries and genomic libraries have been constructed. A progress report on the C-RGP is not yet available.

I now include several brief comments on the J-RGP's recent analysis of rice cDNA clones. Scientists of the J-RGP have made excellent progress by sequencing over 1,000 cDNA clones each from a callus cDNA library and a root cDNA library. After the nucleotide sequence data were translated to amino acid sequences, approximately 100 proteins were represented and only around 25% of them were common in both libraries. This is probably related to the fact that the number of clones in the two cDNA libraries is small (e.g., below 5,000). Thus, it is likely that only highly expressed genes are represented in these libraries. It has been estimated by different investigators that rice contains approximately 50,000 expressed genes; thus 50,000 different cDNA clones need to be analyzed. It is likely that certain genes are expressed at very low levels, especially those involved in the regulation of gene expression (e.g., transcription factors). If particular regulatory gene produces a level of mRNA that amounts to 5% of the average level of all rice genes, then one needs a cDNA library with one million clones to include this particular cDNA. Even if one allows only a three-fold (instead of five-fold) redundancy in order to include most cDNA clones, the cDNA library still needs to include three million clones. If one starts with a cDNA library of this size, most clones will be represented 60 times on the average. Thus, for further analysis, scientists of any RGP need to use all of the 1,000 cDNA (or more) clones that have been sequenced already as probes to identify identical clones from larger, more complete cDNA libraries. Then, after sequencing every X number of clones, one again needs to use them as probes to eliminate from consideration identical clones from the remaining clones in the clones from a cDNA library with several million clones have been sequenced. Once the final job is completed, or even before it is complete, the DNA sequence of all or most clones from one or several cDNA libraries will be known. This information is very valuable because it will allow scientists to go one or two steps forward in eventually understanding the nature, regulation and expression of all the genes in rice.

References

Rice Genome (Minobe, Y., ed.) Vol. 1, No. 1, July 1992; No. 2, December 1992; Vol. 2, No. 1, July 1993. Tsukuba, 305, Japan.

Uchimiya, H., S. Kidou, T. Shimazaki, S. Aotsuka, S. Takamatsu, R. Nishi, H. Hashimoto, Y. Matsubayashi, N. Kidou, M. Umeda, and A. Kato 1992. Random sequencing of cDNA libraries reveals a versatility of expressed genes in cultured cells of rice (Oryza sativa). Plant Journal 2: 1005-1009.