Report of the Committee on Genetic Engineering

D. Report of the Committee on Genetic Engineering

Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, U.S.A.

This report reviews the work published within the last year or so on the molecular analysis of rice genes. A number of interesting papers appeared within this period and most of these are discussed in this report. The cloning of rice genes from genomic or cDNA libraries uses standard recombinant DNA techniques (Maniatis et al. 1982). DNA sequence analysis is usually by the dideoxynucleotide chain termination method. The DNA sequence comparisons of different sequences, and the deduction of the protein sequences from the DNA sequences, are made with the aid of computer programs (e.g. Queen and Korn 1984).

Hiratsuka et al. (1989) reported the complete rice (Oryza sativa) chloroplast genome sequence which consists of 134,525 base pairs (bp). This was a monumental task. The locations of genes were identified, along with open reading frames (ORFs), by comparing the sequences of rice chloroplast DNA with those of previously sequenced chloroplast genomes from tobacco and liverwort. A total of 91 genes, 3 pseudogenes and 36 ORFs were identified in the rice chloroplast genome. These include the genes coding for the 30 tRNAs and 4 rRNAs. Some of these genes are interrupted by introns. The length of the large inverted repeats in rice chloroplast DNA is 20,799 bp, about 4,500 bp shorter than those in tobacco.

Most ORFs that are extensively conserved between the chloroplast genomes of tobacco and liverwort are also conserved in rice. Apparently, the large single copy region of the rice chloroplast genome has undergone inversion which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes has been proposed (Hiratsuka et al. 1989). Prior to the publication of this work, about a dozen rice chloroplast genes, two pseudogenes and several ORFs were sequenced and reported by other investigators.

About 80% of the total rice seed proteins belong to the glutelin family.Okita et al. (1989) cloned and sequenced a near full-length cDNA and three genomis clones for rice glutelin. The three genomic clones (Gt1, Gt2 and Gt3) represent three Subfamilies of glutelin genes. A comparison of DNA sequences showed that Gt1 and Gt2 were closely related because the 5'-flanking and the coding sequences displayed 87% and 95% homology, respectively. In contrast, Gt3 showed almost no homology to Gt1 and Gt2 in the 5'-flanking sequences upstream of the putative CAAT boxes and exhibited significant divergence in all other portions of the gene. Expression studies indicated that glutelin mRNAs are differentially accumulated during endosperm development. Promoters from Gt2 and Gt3 were functional as shown by transient expression assays of chloramphenicol acetyl transferase activity in protoplasts of cultured tobacco and rice cells.

Takaiwa et al. (1989) cloned and sequenced another glutelin cDNA from rice. It gives a deduced amino acid sequence which encodes a 499 amino acid glutelin precursor with a signal peptide of 24 amino acids followed by a 279 amino acid acidic subunit and a 197 amino acid basic subunit. The sequence of this cDNA is significantly different from the two cDNA reported earlier by the same group and different from the three genomic glutelin clones of Okita et al. (1989).

About 5% of the total rice seed proteins belong to the prolamine family. Kim and Okita (1988) cloned and sequenced several cDNA clones encoding the rice seed storage protein prolamine. These clones can be divided into two homology classes. All clones encode a Putative prolamine precursor of 17.2 kda, with a predicted 14-amino acid signal peptide. The amino acid composition, deduced from the DNA sequence, is typical for prolamine with a high content of glutamine, alanine, proline, phenylalanine and tyrosine, and a low content of methionine and cysteine. In contrast to other cereal prolamines, the primary sequence of this rice prolamine is devoid of major tandem repetitive sequences.

Masumura et al. (1989) isolated a cDNA clone corresponding to a 10 kDa sulfur-rich prolamine. The deduced polypeptide sequence contains 134 amino acids including a 24 amino acid signal peptide predicted from Edman sequencing of the purified mature polypeptide and computer-assisted analysis of the putative membrane spanning region. This prolamine is very rich in sulfur containing amino acids, the mature polypeptide being composed of 20% of methionine and 10% of cysteine. The protein also contains a high percentage of glutamine and hydrophobic amino acids. Two repeats of amino acid sequence were found in the polypeptide.

ADP-glucose pyrophosphorylase is an important enzyme that controls the synthesis of starch in plants. Anderson et al. (1989) cloned an ADP-glucose pyrophosphorylase cDNA possessing about 1650 nucleotides which encodes a protein with 483 amino acids. The deduced primary polypeptide sequence includes a putative 28-amino acid leader peptide presumably required for transport of this nuclear encoded protein into the amyloplasts (starch containing plastids). The primary sequence of the putative mature subunit of the rice enzyme showed two regions displaying significant sequence identity to the E. coli enzyme. These two regions contain residues shown previously to be essential for the allosteric regulation and catalytic activity of the E. coli enzyme.

The waxy locus of rice is responsible for the synthesis of amylose in the developing grain, and in the haploid pollen. The Wx gene encodes UDP-glucosyl transferase. The mutant gene, wx, lacks this enzyme so that the endosperm starch contains no amylose and 100% amylopectin. Okagaki and Wessler (1988) cloned a rice Wx gene from a genomic library. This gene encodes a 2.4 kb transcript that allows the in vitro synthesis of a 60 kDa pre-protein which is precipitated with maize waxy antisera. Southern blot analysis of 16 rice strains, ten containing waxy mutations, reveals that the waxy gene and flanking restriction fragments are virtually identical.

Rice lectin accumulates in a cell-type specific manner in various organs of developing embryos and young seedlings. Wilkins and Raikel (1989) isolated two cDNA clones encoding a rice lectin. The two cDNAs code for an identical 23 kDa pre-proprotein which is processed to yield a proprotein of 18 kDa. This proprotein is post-translationally cleaved to generate mature lectin polypeptides of 10 kDa and 8 kDa. RNA gel blot analysis showed that rice lectin is encoded by two mRNAs (0.9 and 1.1 kb). Both mRNAs are present in developing rice embryos but the mRNAs show different patterns of temporal expression.

Histones are small basic proteins that bind tightly to eucaryotic chromosomal DNA to form chromatin. Wu et al. (1989) cloned two histone 3 genes from a rice genomic library. The two genes share about 94% DNA sequence homology. Despite the high conservation in the coding region, the two clones exhibit very little homology in the flanking sequences except for several conserved sequence motifs in the 5' flanking regions. The deduced protein sequences of these two clones are identical to each other but differ by 3 amino acids (out of 135) from a previously published rice H3 gene, pRH3-2 (Peng and Wu 1986).

Using the cloned pRH3-2 histone 3 gene as a probe for in situ hybridization experiments, Raghavan (1989) found that the histone MRNA is differentially expressed during anther and pollen development in rice. In anther sections hybridized with a [³H]histone probe, gene expression was detected only in the endothecium of the premeiotic anther and in the bicellular pollen grains. During pollen development, expression of the histone gene appeared rather abruptly in the starch-filled bicellular pollen grains and it continued at a reduced level in the mature pollen grams.

Ethanol is produced immediately upon transfer of rice to an anaerobic environment. The activities of many enzymes increase under anaerobiosis, but only alcohol dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase show concomitant changes of isoenzyme profiles. Labeling experiments showed that both enzymes are synthesized de novo (Rivoal et al., 1989).

Two rice alcohol dehydrogenase genes, Adh1 and Adh2, were cloned (Xie and Wu, 1989). Both genes showed a 1128 bp coding region. Comparison of the DNA sequences of the Adh1 and Adh2 coding regions revealed 102 silent nucleotide changes and 56 nucleotide changes which alter the amino acid se- quences. Using the cloned gene as the probe to quantitate the mRNA level, we found that anaerobiosis resulted in a linear increase in the Adh mRNA level (about 10 fold) during the first 12 hours, indicating that the ADH enzyme synthesis is regulated at the transcriptional level. After returning the rice seedlings to aerobic conditions, the Adh mRNA level decreased and dropped to 50% level after approxiniately 10 hours.

For the transformation of rice protoplasts, it is important to have a reliable and convenient selectable marker to screen for the transformants. Dekeyser et al. (1989) tested six promoters, each joined separately to the neomycin phosphotransferase II gene (nptII) as the reporter. It was found that the 2' promoter of the octopine T DNA is 3-4 times more efficient than the CAMV 35S promoter and both are much stronger than 4 others tested in a transient assay using rice protoplasts.

The authors then tested six selective agents using suitable vectors for transformation. They found that rice callus growth is inhibited by low concentrations of methotrexate and phosphinothricin, and by moderate concentrations of G418 and hygromycin. The discrimination between transformed and untransformed rice callus tissue is optimal with 10 mg/L phosphinothricin or 100 mg/L G418. Southern blot hybridization analysis of the transformed calli demonstrated that 50% of the transformants contained a single vector copy and that nearly all integrated copies shared rearrangements. Thus, it is better to have several copies of the vector integrated per cell so that at least one copy is not rearranged (Dekeyser et al. 1989).

Using a transient gene expression system, Ingelbrecht et al. (1989) tested the functional role of the 3' end region on the level of expression of a chimeric reporter gene in tobacco cells. It was found that optimal expression of the reporter gene requires between 98 and 142 base pairs downstream from the most distal polyadenylation site of the octopine synthase gene. In the transient expression assay, all constructs directed similar neomycin phosphotransferase II activities using chimeric genes with 3' end sequences originating from different plant genes. However, in stably transformed tissues, these gene constructs gave characteristic expression levels which varied as much as 60-fold. The result suggests a role for 3' end sequences in mRNA processing or mRNA stability.

An efficient reporter gene constructed is expected to produce relatively stable mRNAs that can be efficiently translated to produce stable proteins. Gallie et al. (1989) tested the efficiency of expression of mRNA sequences with or without different upstream and downstream sequences by introducing the mRNA into protoplasts by electroporation. It was revealed that a translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5' end of the Beta- glucuronidase mRNA increased the translational efficiency 3- to 11-fold in maize and rice protoplasts. It was also found that a minimum length of 25 adenylate residues at the 3' end of the mRNA is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. The effect of the translational enhancer and a poly (A) tail function independently of each other, and the final enhancement is the multiplication of their individual effects.

This work is supported by research grant RF84066, Allocation No. 3, from the Rockefeller Foundation.

Anderson, J. M., J. Hnilo, R. Larson, T. W. Okita, M. Morell and J. Preiss, 1989. The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme. J. Biol. Chem. 264: 12238-12242.

Dekeyser, R., B. Claes, M. Marichal, M. Van Montagu and A. Caplan, 1989. Evaluation of selectable markers for rice transformation. Plant Physiol. 90: 217-223.

Gallie, D. R., W. J. Lucas and V. Walbot, 1989. Visualizing mRNA expression in plant protoplasts: Factors influencing efficient mRNA uptake and translation. Plant Cell 1: 301-311.

Hiratsuka, J., H. Shimada, R. Whittier, T. Ishibushi, M. Sakamoto, M. Mori, C. Kondo, Y. Honji, C. R. Sun, B. Y. Meng, Y. Q. Li, A. Kanno, Y. Nishizawa, A. Hirai, K. Shinozaki and M. Sugiura, 1989. The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol. Gen. Genet. 217: 185-194.

Ingelbrecht 1. L. W. L. M. F. Herman, R. A. Dekeyser, M. C. Van Mountagu and A. G. Depicker, 1989. Different 3' end regions strongly influence the level of gene expression in plant cells. Plant Cell 1:671-680.

Kim, W. T. and T. W. Okita, 1988. Structure, expression, and heterogeneity of the rice seed prolamines. Plant Physiol. 88: 649-655.

Maniatis, T., E. E. Fritsch and J. Sambrook, 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.

Masumura, T., D. Shibata, T. Hibino, T. Kato, K. Kawabe, G. Takeba, K. Tanaka and S. Fujii, 1989. cDNB cloning of an mRNA encoding a sulfur-rich 10kda prolamine polypeptide in rice seeds. Plant Mol. Biol. 12: 123-130.

Okagaki, R. J. and S. R. Wessler, 1988. Comparison of non-mutant and mutant waxv genes in rice and maize. Genetics 120: 1137-1143.

Okita, T. W., Y. S.Hwang, J. Hnilo, W. T. Kim, A. P. Aryan, R. Larson and H. B. Krishnan, 1989. Structure and expression of the rice glutelin multigene family. J. Biol. Chem. 264: 12573-12581.

Peng, Z. G. and R. Wu, 1986. A simple and rapid nucleotide sequencing strategy and its application in analyzing a rice histone 3 gene. Gene 45: 247-252.

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Raghavan, V.,1989. mRNAs and a cloned histone gene are differentially expressed during anther and pollen development in rice (Oryza sativa L.). J. Cell Science 92: 217-229.

Rivoal, J., B. Ricard and A. Pradet, 1989. Glycolytic and fermentative enzyme induction during anaerobiosis in rice seedlings. Plant Physiol. Biochem. 27: 43-52.

Takaiwa, F., S. Kikuchi and K. Oono, 1989. The complete nucleotide sequence of new type cDNA coding for rice storage protein glutelin. Nucleic Acids Res. 17: 3289.

Wilkins, T. A. and N. V. Raikhel, 1989. Expression of rice lectin is governed by two temporally and spatially regulated mRNAs in developing embryos. Plant Cell 1: 541-549.

Wu, S. C., Z. Vegh, X. M. Wang, C. C. Tan and D. Dudits, 1989. The nucleotide sequences of two rice histone H3 genes. Nucleic Acids Res. 17: 3297.

Xie, Y. and R. Wu, 1989. Rice alcohol dehydrogenase genes: Anaerobic induction, organ specific expression and characterization of CDNA clones. Plant Mol. Biol. 13: 53-68.