In Vitro Transcription of Chloroplast Genes
It has been known for some time［52，53；Haff and Bogorad，unpublished］that illumination of dark-grown maize seedlings quickly results in the production of more plastid RNAs as well as increased activity of the plastid DNA-dependent RNA polymerase.As noted earlier，some species of transcripts increase considerably in abundance during light-induced development.In the other developmental situation we have examined，ie，differentiation of mesophyll and bundle sheath cells in maize，as also described above，the rc-L gene is strikingly differently expressed in the two cell types［47，48］.In order to explore the mechanisms underlying such apparent transcriptional regulation of gene expression in plastids，a number of years ago we started to study details of the structure and properties of maize plastid DNA-depedent RNA polymerase［26，47，52，54-56］.
This enzyme was solubilized from thylakoids of sucrose gradient-purified plastids［54-56］.It has been shown to be different in properties and structure from the type Ⅱ nuclear DNAdependent RNA polymerase of maize［57-59］.Although purified preparations of nuclear polymerase Ⅱ and the maize plastid polymerase have some polypeptides of about the same size in common，these are，in fact，different［59］polypeptides as deduced from fingerprints［59］.Tewari and Goel［60］have recently found that purified preparations of pea plastid DNAdependent RNA polymerase contain polypeptides of the same sizes as purified preparations of the maize enzyme［55，58，61］.
When characterized cloned maize plastid sequences first became available［12，13，24，33，45，62，63］，it became possible to study their transcription by the plastid enzyme as a step toward developing an in vitro system capable of mimicking transcription of chloroplast genes as it occurs in vivo，ie，to experimentally test mechanisms of developmental regulation of gene transcription in maize plastids by synthesis of the system in vitro.
The first experiments toward this end［56］were directed at determining whether partially purified maize plastid polymerase can preferentially transcribe maize plastid DNA sequences in chimeric plasmids replicated in E.coli.Maize plastid RNA polymerase solubilized from the membranes of sucrose gradient-purified plastids was chromatographed on DEAEcellulose and eluted with a gradient of KCl.The bulk of the enzyme activity was eluted wich about 0.2 mol/L KCI.When tested with a chimeric plasmid template containing two cloned maize plastid DNA sequences—one of which included rRNA genes—the enzyme transcribed both the bacterial plasmid sequences and the cloned chloroplast sequences about equally，judging from Southern hybridization experiments.A fraction that eluted with about 0.5 mol/L KCl，when added to the polymerase activity elutcd with 0.2 mol/L KCl，stimulated the transcription of the plasmid DNA template，but the most striking effect was preferential transcription of some chloroplast sequences over others and over the bacterial vehicle DNA.
These results wre extended using as a template a relatively small fragment of plastid DNA cloned in the plasmid pMB9.The relative transcription of chloroplast and plasmid sequences was assessed quantitatively.The DEAE-cellulose-purifed RNA polymerase transcribed the chloroplast and vehicle sequences about equally.However，when the 27.5-kDa polypeptide（the S factor）that was purifed from the 0.5 mol/L KCl eluate of the DEAE column was included in the reaction mixture，transcription of the cloned chloroplast sequences was about an order of magnitude greater than transcription of the vehicle DNA.Another notable feature of this system is a requirement for a supercoiled DNA template.If the template was relaxed with E.coli topoisomerase most of the preferential transcription of plastid DNA sequences was lost.The latter observation also confirmed an assessment carried out by Southern hybridization on another fragment of chloroplast DNA.We concluded from these experiments that preferential transcription of chloroplast DNA sequences required the polymerase，the S factor polypeptide，and a supercoiled DNA template［57］.In the next set of experiments we showed that the 5'termini of in vivo and in vitro transcripts of the gene for tRNAHis are the same［26］.(https://www.daowen.com)
The earliest experiments［56］，as noted，indicated that not all chloroplast DNA sequences are transcribed equally in vitro by the maize polymerase plus S factor system.This question was examined quantitatively in a particularly attractive situation.The gene for the large subunit of ribulose bisphosphate carboxylase and a gene for a 2.2-kb transcript，later identified as cfl-BE that codes for the beta and epsilon subunits of the coupling factor CF1，are separated from one another by about 100，300 bp and are transcribed divergently.Maize plastid fragment Bam 9 contains all of rc-L and about 1.4 kbp of cfl-BE.This is an interesting pair to study because the two are differentially expressed in mesophyll and bundle sheath cells of maize［46，47］.
First，it was shown［47］using a BglⅡ subfragment of maize plastid DNA fragment Bam 9 cloned in the bacterial vector RSF1030 that even without the S factor chloroplast DNA sequences were transcribed almost nine times more actively than the RSF-1030 DNA but in the presence of the S factor hybridization to RSF-1030 was not detectable above the background level.When transcription of rc-L vs cf1-BE was studied，the ratio of transcription was about 1.1 in the absence of the S factor and 3.2 in its presence.Thus，the earlier observation regarding differential transcription of parts of chloroplast DNA cloned in E.coli［56］was confirmed（Another observation，related to the topography of the template，was made in the latter sets of experiments［47］.）.It was found that using two different sized fragments of linear chloroplast DNA as templates for RNA polymerase“run-off”experiments，RNAs of the expected sizes could not be found，leading us to conclude that supercoiled or circular forms of template might be required for initiation at specific sites.The latter has been confirmed in more recent experiments［L.D.Crossland and L.Bogorad，unpublished］.
In all，the experiments wich cloned maize chloroplast DNA sequences and maize chloroplast RNA polymerase in vitro encourage us to use this system to study developmentally regulated transcription.
