It has been previously reported that the 5' region of the rice actin 1 gene (Act1) promoted high-level expression of a beta-glucuronidase reporter gene (Gus) in transformed rice cells. In this paper we describe the construction of Act1-based expression vectors for use in monocot transformation. As part of the development of these vectors, we have evaluated the influence of the Act1 first intron, the Act1-Gus junction-encoded N-terminal amino acids, and the sequence context surrounding the Act1 and Gus translation initiation site on Act1-Gus gene expression in rice and maize cells. We have found that addition of Act1 intron 1 to the transcription unit of a Gus reporter gene under control of the cauliflower mosaic virus (CaMV) 35S promoter stimulated GUS activity more than 10-fold in transformed rice cells. Optimization of the sequence context around the Gus translation initiation site resulted in a 4-fold stimulation of Gus expression in transformed rice cells. By utilizing both the Act1 intron 1 and optimized Gus translation initiation site, a 40-fold stimulation in Gus expression from the CaMV 35S promoter has been achieved in transformed rice cells; very similar results were obtained in transformed maize cells. Taken together these results suggest that the Act1-based expression vectors described here should promote the expression of foreign genes in most, if not all, transformed monocot cells to levels that have not previously been attainable with alternative expression vectors.

Possible roles of untranslated sequences at the 3' ends of chloroplast genes, which include inverted repeat elements, were investigated in Chlamydomonas reinhardtii in vivo. Chlamydomonas chloroplast rbcL or psaB 3' flanking regions were coupled in various arrangements 3' to a chimeric gene consisting of a Chlamydomonas chloroplast atpB promoter sequence fused 5' to the Escherichia coli uidA (GUS) structural gene. These genes were introduced into the Chlamydomonas chloroplast genome at the same location by homologous recombination following microprojectile bombardment. Transformants harboring chimeric GUS genes fused to rbcL or psaB gene 3' inverted repeat sequences in their normal forward orientations accumulated GUS transcripts of a single size, whereas GUS transcripts of heterogenous sizes accumulated in transformants harboring the same gene lacking an inverted repeat sequence at its 3' end. Thus, the 3' flanking regions of the rbcL and psaB genes can define the location of the 3' terminus of a transcript in vivo. In chloroplast transformants harboring chimeric GUS genes fused to multiple inverted repeat sequences in their normal forward orientations, only GUS transcripts accumulated that were terminated by the first inverted repeat sequence. The latter data suggest that the 3' ends of these RNAs are the products of either transcription termination or endonucleolytic cleavage. Analyses of GUS transcripts in transformants harboring GUS genes terminated by rbcL or psaB gene 3' flanking regions in reversed orientations indicate that transcript 3' end formation in vivo requires nucleotide sequences located outside the inverted repeat elements. Inasmuch as decay rates of GUS transcripts were found to be independent of the presence of a 3' inverted repeat sequence, RNA stabilization does not appear to be a major in vivo function of these elements in the Chlamydomonas chloroplast transcripts studied.

Transcription from modified chloroplast genes has been studied in vitro, but only with the recently developed ability to stably introduce foreign DNA into Chlamydomonas reinhardtii chloroplast chromosomes in situ has it become possible to do so in vivo. Cloned chloroplast DNA sequences, into which had been inserted chimeric genes composed of the GUS coding sequence reporter under transcriptional control of chloroplast promoters for the C. reinhardtii atpA, atpB, and rbcL genes, were introduced into the cells on microprojectiles. These constructs become integrated into chloroplast chromosomes by homologous recombination. RNA gel blot analyses demonstrated that a single major 8-glucuronidase (GUS)-hybridizing transcript accumulates in each chloroplast transformant. We have found that: (1) Transcription of the chimeric gene begins at the same site as in the corresponding endogenous chloroplast gene; (2) the rates of transcription in vivo of the atpA:GUS and atpB:GUS genes relative to one another and to other genes are the same as those for the endogenous atpA and atpB genes, respectively, indicating that these promoters are fully functional despite being fused to a foreign gene and being at an alien location on the chloroplast chromosome; (3) in contrast to the atpA and atpB promoters, the rbcL promoter directs transcription of the rbcL:GUS gene at only 1% of the expected rate, suggesting that other features are required for optimal activity of this promoter; and (4) 22 base pairs upstream of the 5' end of the atpB:GUS transcript in the atpB promoter element is sufficient to confer wild-type levels of promoter activity.

As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam 10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correction mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.

As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam 10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correction mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.