Dot hybridization

Figure 1 presents the results obtained from dot hybridizations of DNA probes against bulk RNA isolated from cells grown under the conditions designated.The quantitative measurements obtained from scintillation counting of the filters are summarized in Tables 4 and Table 5.The quantitative results were similar when obtained by densitometric scans of the dot blots.The C-2-C clone contains a 1.0-kilobase（kb）SalI-BamHI fragment of pLIl0 cloned into M13 phage（P.Hallenback，personal communication）.This 1.0-kb fragment contains most of the gene for RuBPCase.The KpnI-PstI fragment of pJWl contains the LH-1 gene as well as a portion of RC-L，and the subclones pUI135 and pUI128 are the probes for LH-1 plus RC-L and for RC-M genes，respectively.It can be determined from the data in Figure 1（a）and Table 4 that the shift of cells from high light to dark for 1 h resulted in the degradation of greater than 70%of the mRNA for RuBPCase.Similarly，cells grown under steady-state low light intensity（3 W/m2）had a 20%reduction in the level of mRNA specifying RuBPCase when compared with cells growing at high light.Under aerobic growth conditions，no mRNA specifying RuBPCase was detectable.Although mRNA specifying LH-1，RC-L，and RC-M responded to light and O2，several major differences were observed when we compared these results with those observed for RuBPCase.First，the decreased light intensity used for growth from 10 to 3 W/m2 resulted in an increased level of mRNA specifying these membrane proteins；second，cells grown under partial aerobiosis still had a measurable level of mRNA（30%to 40%）.This is not unusual，since detectable levels of Bchl were present.However，under high p O2，LH-1 mRNAs were still present despite the absence of Bchl.On the other hand，a very different pattern of responses was observed when the same mRNA levels were measured in cells grown on different substrates（see Figure 1（b），Table 5）.The cells grown on butyrate synthesized substantially higher levels of mRNA for RuBPCase as compared with that synthesized when cells were grown on either malate（16-fold）or succinate（7-fold）.However，relatively small differences were observed in the levels of mRNA for the specific membrane proteins when cells were grown on the different carbon substrates.

Figure 1　Dot blot hybridization of R.sphaeroides RNA with cloned probes for RuBPCase，LH-1，RC-L，and RC-M.Cell cultures were grown and RNA was isolated as described in the text.Equal amounts of RNA（5 μg）after denaturation were spotted onto Gene Screen，hybridized with32P-labeled probes，and exposed to X-ray film.Each probe employed is listed above the figures and described in Table 1，and the culture conditions are listed to the left of the figures.Abbreviations：HL，high-intensity light；D，shift from highintensity light to dark for 1 h；LL，low-intensity light；O2，partially aerobic growth；B，butyrate；M，malate；S，succinate

Table 4　Effects of light and O2 on levels of mRNA coding for RuBPCase，LH-1，RC-L，and RC-M of R.sphaeroidesⓐ

ⓐThe relative amounts of mRNA were measured by dot hydridization，quantified by scintillation counting of the filters，and expressed as the percentages of mRNA level under high-intensity light（set as 100）.Only the results within a column can be compared，not those among columns.
ⓑ See footnote b of Table 2.

Table 5　Effects of carbon substrates on levels of mRNA coding for RuBPCase，LH-1，RC-L，and RC-M of R.sphaeroidesⓐ

ⓐ See footnote a of Table 4.