Discussion

The metabolic，morphological，and biophysical changes induced by the imposition of different environmental regimens on R.sphaeroides have been well studied11，16，17］；nevertheless，information specifying the molecular events governing such changes with respect to gene regulation is limited.The present investigation with carefully defined probes and methods provides substantial，detailed information about the regulation of mRNA coding for several photosynthetic membrane proteins as well as RuBPCase in cells subjected to a variety of cultural conditions.

Figure 5　Southern hybridization of pJW1 with 32 P-labeled RNA.The pJW1 DNA was digested with Hincll，KpnI，SalI，and NruI and blotted onto Gene Screen after fractionation on an agarose gel.The hybridization results employing different light and O2 treatments（a）and different substrates（b）are shown.The conditions were as described in the legend to Figure 1.The rightmost lanes are the agarose gels on which 1.06-，0.98-，and 0.6-kb fragments containing the genes for RC-L，LH-1，and RC-M，respectively，are indicated.Panel C shows the restriction map of pJW1

The hybridization experiments reported here demonstrate the importance of light as a trigger in the regulation of photosynthetic gene expression.Cells grown under low-light conditions revealed significantly reduced levels of mRNA for RuBPCase.However，under similar conditions the level of LH-1-specific mRNA was shown to be very high，as might be anticipated because more photosynthetic membrane is produced to compensate for the decreased light intensity and because there is a greater density of photosynthetic units per unit of membrane［32］.Based on the total radioactivity added for hybridization，the specific radioactivity of the RNA（3.6×107cpm/μg of RNA），and the amount of radioactivity shown to hybridize，we calculated that the mRNA specific for LH-1 was about 0.006%of total RNA，but 0.2%of the total mRNA.

Growth in the presence of O2 completely abolished the expression of the gene for RuBPCase，whereas a considerable level of mRNA specifying LH-1 was still synthesized in the presence of O2，although no Bchl was detected under high O2 partial pressure.On the other hand，the amounts of mRNA fro RC-L and RC-M significantly decreased（see Figure 2（b）），indicating that the mRNAs for RC-L and RC-M are very unstable.(https://www.daowen.com)

As might have been anticipated，the substrates used to support photosynthetic growth had a major influence on the mRNA levels for RuBPCase.Butyrate has been shown to be the best source of reducing power and shows a very high level of RuBPCase in R.sphaeroides［27］.The high level of RuBPCase can be accounted for by the increased transcriptional activity of the RuBPCase gene，which shows a 17-fold increase in cognate mRNA accompanied by a 20-fold increase in RuBPCase activity on butyrate as compared with that on malate.Although the data presented here cannot exclude the possibility of translational regulation of RuBPCase expression，the data are consistent with a transcriptional control mechanism.On the other hand，the substrates had relatively small effect on the expression of the genes for LH-1，RC-L，and RC-M.

The Northern hybridization experiments revealed that multiple transcripts may exist for both RuBPCase and LH-1.A single gene for the large subunit of RuBPCase from maize yields two differentially regulated mRNAs with sizes of 1.6 and 1.8 kb［10］.More complex patterns of RNA are seen for other photoregulated genes［3］.In this study，three R.sphaeroides transcripts for RuBPCase of 2.3，2.0，and 1.4 kb were found，with either C-2-C or an internal Pst fragment of the RuBPCase gene（data not shown）as a probe.The 1.4-kb transcript was the minimal size for the structural gene of RuBPCase.However，the 2.0-kb transcript was the major transcript observed.We also found that the levels of these three transcripts were regulated coordinately when the light and O2 pressure were changed.

Northern hybridization of the genes for LH-1，RC-L，and RC-M revealed that two major transcripts of 2.6 and 0.8 kb and two minor transcripts of 2.0 and 1.45 kb existed.The genes coding for RC-L［30］and RC-M［31］of R.sphaeroides have been sequenced.The LH-1 genes in R.capsulata have been sequenced and code for two polypeptides，B870α and B870β.In R.sphaeroides，the LH-1 genes have approximately 350 base pairs，with a spacer of about 150 base pairs，and are linked to the RC-L and RC-M genes（P.Kiley and S.Kaplan，unpublished data）.Although these four genes may have separate start codons，they probably constitute the same operon［33］.Since the 2.6-kb transcript is long enough to code for LH-1，RC-L，and RC-M，it probably gives rise to a polycistronic mRNA expressing at least these four genes.The 0.8-kb transcript is long enough to code for LH-1 and may be a single transcript of only the LH-1 genes.This assumption is confirmed by the results of the Northern hybridization of RNA with probes specific for RC-L and RC-M（see Figure 2（b））［2，3］，because only the 2.6-kb transcript，but not the 0.8-kb transcript，hybridized under these conditions.If we assume that each of these transcripts is initiated from the same site and ignore the mechanism by which the smaller as opposed to the larger is generated，then this may be the basis for the existence of a relatively fixed，15∶1 stoichiometry between LH-1 and the RC polypeptides［7］.The average ratio of the small transcript（0.8 kb）to the large transcript（2.6 kb）（11.2∶1）is within reasonable error identical to the fixed ratio observed for the gene products of the LH-1，RC-L，and RC-M genes.Therefore，the relative steady-state abundance of these two transcripts may be the sole basis for the stoichiometry of the gene products.The exact mechanism，initiation，termination，or selective degradation leading to the steady-state abundance of each of these transcripts in R.sphaeroides remains to be determined.In a paper that appeared recently，Belasco et al.［2］made essentially identical observations for the presence and relative abundance of two mRNA species for the LH-1，RC-L，and RC-M genes in R.capsulata.These authors concluded that selective degradation from the 3'end of the large transcript is responsible for the steady-state level of the small and large transcripts.The 2.6-kb transcript greatly diminished when the cells were shifted from light to dark or when cells were grown aerobically，whereas the concentration of the 0.8-kb transcript was maintained at almost the same levels under identical conditions（see Figure 2）.The different stability of two transcripts may be involved in the regulation of the LH-1，RC-L and RC-M genes in R.sphaeroides and，as suggested by Belasco et al.［2］，for R.capsulata.This result will be described in more detail in a separate paper.