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Cloning
Plastid
and
Mitochondrial
Genes - Cloning organellar genes presents rather a different prospect to nuclear genes.
Most mRNA molecules transcribed from organellar genes are non polyadenylated so conventional oligo dT-primed cDNA synthesis is not possible.
Since the genomes are small, cloning of the genes directly from purified organellar DNA is relatively straightforward compared to nuclear DNA.
The first problem is to prepare DNA which is free of contaminants.
Chloroplast DNA free from nuclear contamination has been reliably prepared from intact, purified chloroplasts treated with DNase prior to DNA isolation.
Other methods have also been used for chloroplast DNA.
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If it differs sufficiently in buoyant density from nuclear DNA, chloroplast DNA can be isolated by CsCl density gradient centrifugation.
However, this method is more applicable to algae as these differences in buoyant density are often too small in higher plants.
A number of chloroplast genes have now been cloned and it is apparent that some homology exists between species.
A method often used now is to take cloned sequences from one species and use them to probe for genes in other species. This homology greatly facilitates organellar gene cloning and analysis of the structure of the genome.
Mitochondrial DNA has been studied less extensively compared to chloroplast, DNA but interest has recently quickened.
Albeit the amount was small, mitochondrial DNA has been successfully prepared from purified, DNase treated mitochondria. As with chloroplasts, DNase treatment is necessary to remove nuclear contamination.
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The Ti plasmid transformation system has also been used to investigate light induced transcription of the small subunit of Rubisco.
Several different research groups have demonstrated that the small subunit promoter can function in a host cell.
In 1984, Broglie, together with colleagues from Rockefeller University and the Monsanto Company (Murphy and Thompson, 1988) inserted an entire pea small subunit gene into petunia protoplasts.
Four or five copies of the pea gene persisted in each cell of the callus that grew from the protoplasts. The pea gene was transcribed in the Petunia cells.
Several lines of evidence suggested that transcription was controlled by the pea genes own promoter.
For instance, the size of the transcript was the same regardless of the right handed or left handed orientation of the pea gene with respect to the other genes of the plasmid vector.
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Transcripts of the small subunit gene were at least 50 times more prevalent in light grown than in dark grown petunia callus cells.
Another transcript from the Ti plasmid, synthesized in the same cells but controlled by the nopaline synthetase promoter, was not affected by light, showing that the light effect on the small subunit gene was specific.
A different experiment, reported by Herrara-Estrella and colleagues, showed that the promoter from a different pea small subunit gene could be linked to a chloramphenicol acetyltransferase (cat) gene and inserted into tobacco protoplasts through a Ti system. Expression of the cat gene was stimulated 5- to 15-fold by light.
Apparently, the small subunit genes contain light-regulated promoters which maintain their properties even in foreign cells.
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The sequences that regulate small subunit gene expression can be located by modifying the genes used in transformation experiments.
The small subunit gene used in the Petunia cell experiments described above contained 1,052 base pairs upstream (51) from the origin of transcription.
Various segments of the 51-region could be deleted so that shorter lengths might be inserted into the host protoplasts.
When this experiment was performed, a Northern blot analysis of the RNA transcribed from the modified genes showed a pattern in dark and light that confirmed the promoter activity of the 51-sequences.
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Deletion of 700 bases, leaving 352 bases flanking the gene, cut the level of mRNA found in light-grown callus by more than a factor of five. Deletion of the 22 bases around the TATA box removed promoter activity entirely. We can conclude the following:
(a) Transcription is promoted by sequences around the TATA box, within 35 base pairs of the start of transcription;
(b) Such transcription is light-regulated. The actual base sequences within this region that confer the light response have not yet been reported, but there is a directly repeated sequence that looks interesting;
(c) In this gene the CAAT box sequences, though present, are not required for promoter activity. This does not mean, however, that they have no effect in their normal environment.
(d) Transcription is enhanced by sequences over 400 base pairs from the 5' -end of the gene.
Similar Ti transformation techniques are being used to study the properties of transit peptides that cause proteins to enter chloroplasts.
This is done by attaching segments of the small subunit gene to the gene of a protein that would not normally enter the plastids.
A fusion of the gene for the small subunit transit peptide (both with and without sequences that code for more of the small subunit) with the gene for neomycin phosphotransferase has been tested in tobacco protoplasts.
The transformed tobacco cells make a neomycin phosphotransferase with a transit peptide. Some of this protein is then transported into chloroplasts and processed just like the small subunit.
This experiment shows that the transit peptide by itself contains sufficient information to guide a protein into plastids.
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