Effects of Light and Oxygen on the Levels of mRNAs...
The cellular levels of mRNA specifying the LH-Ⅰ，RC-L，and RC-M polypeptides in R.sphaeroides 2.4.1（pUI1A2）were significantly affected by light and oxygen as revealed by dot blot and Northern hybridization analysis.These results are similar to those reported earlier［10］for R.sphaeroides 2.4.1 lacking plasmid（pUI1A2）.Table 1 shows a quantitative estimate of the mRNA levels obtained by dot hybridization in cells grown under different physiological conditions.For these experiments an AluI-AluI（see Figure 1，fragment a，0.51 kb），PuuII-SalI（see Figure 1，fragment b，0.46 kb），or XhoI-NruI（see Figure 1，fragment c，0.37 kb）restriction fragment was used as a specific probe for the LH-Ⅰ，RC-L，and RC-M structural genes，respectively.Dark treatment of high light-grown cells for 1 h resulted in a 35%reduction in the level of mRNA specific for the LH-Ⅰ polypeptides and a 90%reduction in the level of pufL and pufM specific mRNA，suggesting a differential stability of these two transcripts.Steady-state photosynthetic growth at low light intensity（3 watts/m2）resulted in an increased level of the mRNA specifying these intracytoplasmic membrane proteins（3.5-fold for LH-Ⅰ；2.5-2.8-fold for RC-M and RC-L）when compared to growth at 10 watts/m2.These data parallel the whole cell specific Bchl content of these cells which was used to approximate the level of intracytoplasmic membrane（see Table 1）.Cells grown under steady-state aerobic conditions contained a detectable level of mRNA specific for LH-Ⅰ（31%of that found at high light photosynthetic growth），whereas the level of mRNA specific for pufL and pufM decreased nearly 20-fold relative to their steady-state levels in high light-grown cells.The levels of mRNA specifying lacZ from R.sphaeroides 2.4.1（pUI1A2）under different growth conditions were also measured using phage mpllw replicative form DNA as a probe for the lacZ gene.Under either high light or aerobic growth conditions R.sphaeroides 2.4.1（pUI1A2）had nearly identical growth rates，LacZ-specific mRNA，andβ-galactosidase activity（data not shown）.When the growth rate was reducedover 50%by growth in the presence of low light the Lac-specific mRNA level declined by a factor of 4，and the level ofβ-galactosidase activity was reduced by a factor of 3（data not shown）.
Table 1 Effects of light and O2 on bacteriochlorophyll content and mRNA levels specifying LH-Ⅰ，RC-L，and RC-M polypeptides of R.sphaeroides 2.4.1（pUI1A2）
ⓐHL，cells were photosynthetically grown on butyrate medium using high intensity light（10 watts/m2）.D，the HL-grown cells were shifted to darkness for 1 h.LL，cells were photosynthetically grown using low intensity light（3 watts/m2）.O2，cells were grown aerobically（25%O2）.
ⓑ The total bacteriochlorophyll（Bchl）content of cells was estimated by absorbance measurements at 775 nm of acetone/methanol（7∶2，v/v）extracts of whole cells assuming a millimolar extinction coefficient of 75［18.Protein concentrations were assayed by a modified Lowry procedure［19］.
ⓒ The relative amounts of mRNA were measured by dot hybridization and by scintillation counting of the filters and are expressed as the percentages of the mRNA level under high intensity light（HL）（set as 100）.Because different restriction fragment probes were employed，only the results within a column can be compared，not those between columns.
Figure 1 Restriction map of the puf operom.The genes coding for the β and a subunits of LH-I，RC-L，and RC-M polypeptides in R.sphaeroides lie in a single operon.Individual restriction sites used to generate the a（AluI），b（PvuII-SalII），c（XhoI-NruI），d（AccI），e（BstNI），and f（AccI-AluI）probes used for hybridization are indicated.The AccI，AluI，and BstNI sites depicted within the 5'-end of the puf operon are shown for the purposes of illustration；these do not represent the sole sites for these restriction endonucleases within this operon.bp，base pairs
As shown in Figure 2，the Northern hybridizations using the specific probes，a，b，and c referred to above，against RNA isolated from R.sphaeroides 2.4.1（pUI1A2）.Two major RNA species（2.6 and 0.5 kb）were observed when the AluI fragment was used as a specific probe for the LH-Ⅰ structural genes.The large puf operon transcript hybridized with the 32P-labeled probes specific for LH-Ⅰ，RC-L，and RC-M structural genes，whereas the small transcript only hybridized with the probe for the LH-Ⅰ structural genes.Therefore，the large transcript represents a polycistronic mRNA specifying the pufB，-A，-L and-M polypeptides，and the small transcript encodes only the pufB and-A polypeptides.The ratio of the small to the large transcript under either high or low light conditions shown in Figure 2 was approximately 10∶1.Both the experiments reported in this work and in a previous study［10］have documented the molar excess of the small puf operon transcript relative to the large mRNA with the range from numerous independent determinations on steady-state photosynthetic cells varying from approximately 7 to 18 to 1.The small puf operon transcript was even more abundant under nonphotosynthetic conditions，i.e.darkness（ratio＞50∶1）or oxygen（ratio 20∶1）.Besides these two major transcripts，several minor transcripts（2.0，1.4，1.1 kb）were observed only when hybridization was with the probes specific for either pufL and pufM.Therefore，this analysis suggests that these minor transcripts might represent specific degradation products of the large transcript（see Figure 2）.(https://www.daowen.com)
Figure 2 Northern hybridization of RNA from R.sphaeroides（pUI1A2）with probes specific for the LH-Ⅰ，RC-L，and RC-M polypeptides structural genes.RNA was isolated from R.sphaeroides cells grown photosynthetically on butyrate medium under the conditions described in the footnotes to Table 1.After glyoxylation，approximately 30 μg of bulk RNA was separated on a 1.0%agarose gel and transferred to GeneScreen.Hybridization with the specific probes for the LH-I，RC-L，and RC-M polypeptide structural genes listed in Figure 1 was performed as described under“Experimental Procedures.”The rightmost lane is an ethidium bromide-stained agarose gel showing the 14 and 16 S rRNA of R.sphaeroides［32］
