Cloning, mapping, and
            <i>in vitro</i>
            transcription-translation of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from spinach chloroplasts

Erion, Jack L.; Tarnowski, Joseph; Weissbach, Herbert; Brot, Nathan

doi:10.1073/pnas.78.6.3459

Cited by 27 publications

(12 citation statements)

References 34 publications

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“…procedure to study gene expression in a highly defined system. The use of different isoacceptor species now makes it possible to distinguish two different gene products that (21), a crude E. coli extract (22), and a highly defined system (7). The DNA sequence of the LS gene from corn and spinach is known (4,23), and, although the mature proteins from spinach and corn have alanine at the NH2 terminus (4,22), the DNA sequence indicates that the nascent proteins begin with fMet-Ser.…”

mentioning

confidence: 99%

Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

Cenatiempo¹,

Robakis²,

Meza-Basso³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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A simplified translation system coupled to DNA transcription that involves assaying the synthesis of the first dipeptide of a gene product has been described recently Procx NatL Acad. Sci USA 78,[4261][4262][4263][4264]. Using this dipeptide system, we have investigated the expression ofgenes carried on plasmids coding for (8-lactamase, ribosomal protein L12, and the chloroplast large subunit (LS) of ribulosebisphosphate carboxylase (RbuBPCase). Although all three nascent gene products begin with the sequence fMet-Ser, the formation of fMet-Ser can be used to distinguish between the synthesis of .8-lactamase and either L12 or the LS of RbuBPCase by using different serine isoacceptor tRNA species. In ,B-lactamase, the serine codon is AGU, which utilizes the serine isoacceptor species tRNASer; in L12 and the LS of RbuBPCase, the serine codewords are UCU and UCA, respectively, both of which are recognized by the serine isoacceptor species tRNA er. By using either pure tRNA1er or pure tRNA3er, the expression of each gene can be quantitated. In this system, guanosine-5'-diphosphate-3'-diphosphate inhibits the expression ofthe ,j-lactamase and L12 genes but stimulates the synthesis of the LS. In addition, the ratio of fMet-Ser/fMet-Ala (L12/L1O) synthesized was about 1 as compared with the ratio of 4 that has been obtained previously in vivo or in in vitro protein-synthesizing systems, in which the entire gene product was measured. A simplified DNA-directed coupled protein-synthesizing system has been developed to study the regulation ofthe synthesis of ribosomal protein L10 (1). In these initial experiments, plasmid pNF1337 DNA (2) was used as template, and the formation ofthe first dipeptide (fMet-Ala) ofprotein L10 was determined. An important advantage of this DNA-dependent system is that it can be constructed with only five highly purified proteins, i.e., RNA polymerase, initiation factor 1 (IF-1), IF-2, IF-3, and elongation factor Tu (EF-Tu). By using fMet-tRNA and the appropriate aminoacyl-tRNA species for the second amino acid, a specific dipeptide gene product can be selectively synthesized. Because dipeptide formation from a DNA template involves accurate transcription and proper initiation of translation, this system is ideally suited for studies on the regulation of gene expression.The ideal templates are plasmids or purified mRNAs that direct the synthesis of a limited number of products with different second amino acids. Plasmid pBR322 and recombinant plasmids derived from pBR322 contain the ,B-lactamase gene (3), which is efficiently expressed. Thus, the amino-terminal -fragment of ,B-lactamase (fMet-Ser) will be synthesized in the dipeptide system when fMet-tRNA and Ser-tRNA are present. A difficulty arises when a plasmid has another gene that also contains the serine codeword as the second amino acid. This is, in fact, the case with two recombinant plasmids derived from pBR322 that are described in this study. Plasmid pNF1337 contains, in addition to the fl-lactamase gene, the genes for LIO,...

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mentioning

confidence: 99%

Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

Cenatiempo¹,

Robakis²,

Meza-Basso³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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“…For higher plants, the most detailed information, including the nucleotide sequence, is available for the rbcL gene from tobacco (23,24), maize (5,10,11,17) and spinach (7,8,30,31). The results of these studies indicate that the gene is transcribed as a single 1.6 kilobase (Kb) mRNA species, colinear with the DNA sequence in all three plants (17,23,31).…”

Section: Introductionmentioning

confidence: 93%

“…Figure 1 shows the various constructs and the regions of the rbcL gene which they represent. The plasmids pZMe9019 and pZMel502 have been previously described (7). The plasmids pZH215 and pZH212 were constructed by ligating a 727 basepair (bp) Hha I fragment, isolated from pZMel502, into the Sma I site of pUC8 (29) and subsequently transformed into competent E. eoli K 12-JM83 cells (29).…”

Section: Plantsmentioning

confidence: 99%

Characterization of the mRNA transcripts of the maize, ribulose-1,5-bisphosphate carboxylase, large subunit gene

Erion¹

1985

Plant Mol Biol

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The analysis of RNA isolated from maize leaves indicates that there are two mRNA transcripts which are homologous to the chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The 5' end of the smaller transcript, 1.62 Kb in length, begins at a position which is 60 nucleotides upstream from the coding sequence of the gene, corresponding to the position mapped by earlier workers. The larger transcript, 1.86 Kb in length, has not been previously described and originates from a site on the gene which is 302 nucleotides upstream from the coding sequence. The increased size of the largerbcL mRNA transcript from maize, as compared to the transcripts from spinach and tobacco, results from the presence of a 130 nucleotide insert between the two maizerbcL gene mRNA start sites. The DNA sequence adjacent to the start site for the large mRNA transcript is shown to have greater than 90% homology to the DNA sequences adjacent to the mRNA start sites for the spinach and tobaccorbcL gene. Discounting the presence of the insert in the maizerbcL gene, the nucleotides upstream from the coding sequence in the maize, spinach and tobaccorbcL genes, all share approximately the same amount of homology to each other. This homology, along with other evidence, suggests that the large mRNA species are the primary transcripts and that the smaller RNA species are the result of post-transcriptional processing. The finding that spinach also contains two different mRNA transcripts for therbcL gene is consistent with this model.

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“…Additionally, the genes for the two RuBPCase subunits are located on different genomes; the large subunit (LS) gene on the chloroplast DNA and the small subunit gene encoded on nuclear DNA (1,11). The LS gene has been cloned from a variety of higher plants, algae and photosynthetic bacteria (6,7,9,13,15,21). Examination of the DNA sequence indicates a high degree of homology between the LS genes from different species (6,7,13,18,19,23).…”

Section: Introductionmentioning

confidence: 99%

Determination of the translation start site of the large subunit of ribulose-1,5-bisphosphate carboxylase from maize

et al. 1984

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Sequence studies of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from maize indicate the presence of two translation start sites, 18 bp apart. Each site is preceded by a suitable ribosome binding region. By using a simplified E. coli-based in vitro system which measures fromation of the first dipeptide of the gene product, we have determined that only the second methionine initiates translation.

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Cloning, mapping, and in vitro transcription-translation of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from spinach chloroplasts

Cited by 27 publications

References 34 publications

Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

Characterization of the mRNA transcripts of the maize, ribulose-1,5-bisphosphate carboxylase, large subunit gene

Determination of the translation start site of the large subunit of ribulose-1,5-bisphosphate carboxylase from maize

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