Northern hybridization

Figure 2 shows a Northern hybridization of isolated RNA with DNA probes for the RuBPCase，LH-1，RC-L，and RC-M.The RNA species were extracted from R.sphaeroides cells grown on butyrate under different conditions of light and O2 as described above.After glyoxylation，the RNA was separated by agarose gel electrophoresis.Two strong bands of rRNA，16S and 14S，can be seen on the agarose gel（see Figure 2（a）（b），right lanes）.The ratio of 16S rRNA to 14S rRNA is roughly 2.1∶1，indicating that there has been little or no degradation of rRNA during isolation［20］.The RNA was transferred to Gene Screen and hybridized with 32P-labeled nick translated C-2-C DNA.Three transcripts（2.3，2.0，and 1.4 kb）were found when fragment C-2-C was used as the probe（see Figure 2（a））.Each of these hybridization complexes was very stable，resisting melting out up to a wash temperature of slightly over 90℃.The ratio of 2.3-，2.0-，and 1.4-kb transcripts is 1∶4∶2.5.Each of the three transcripts is coordinately light and oxygen regulated.Further，the amount of transcript apparent in low light-grown cells relative to high light-grown cells is precisely what one would expect from the dot blot results（see Figure 1）.When a 0.91-kb internal PstI fragment of the RuBPCase gene was used as a specific probe，the same three transcripts were obtained（data not shown）.No transcripts for RuBPCase were detected in aerobically grown cells.Even more interesting are the transcripts for the LH-1 polypeptides.The probe for LH-1 is the 1.2-kb KpnI-PstI fragment of pJW1 containing the LH-1 genes as well as a portion of the RC-L gene.Two major transcripts（2.6 and 0.8 kb）and two minor transcripts（2.0 and 1.45 kb）are seen in Figure 2（b），panel 1.Under high-intensity light with the KpnI-PstI probe，the ratios of the major transcripts（2.6 and 0.8 kb）to one another were approximately equal.However，after 1 h in the dark or in the presence of O2 the amount of larger transcript（2.6 kb）decreased，but the concentration of the small transcript（0.8 kb）changed only slightly；therefore，the ratio of the 0.8-kb transcript to the 2.6-kb transcript reached a value of 12，suggesting a greater rate of turn over of the 2.6-kb transcript relative to the 0.8-kb transcript.Under low-intensity light the ratio of the small transcript to the large transcript increased to a value of 1.9.

Figure 2　Northern hybridization of R.sphaeroides with cloned probes for RuBPCase，LH-1，RC-M，and RCL.Samples of glyoxylated RNA from R.sphaeroides cells，grown photosynthetically on the buryrate medium under the conditions described in the legend to Figure 1，were fractionated on 1.0%agarose gel（rightmost lanes of（a）and（b）），transferred to Gene Screen，and hybridized with32P-labeled probes for RuBPCase（a）and LH-1（b），panel 1，RC-L（b），panel 2，and RC-M（b），panel 3.The probes for RuBPCase and LH-1 are listed in Table 1.The cloned fragment of PvuII-SalI（0.46 kb）and XhoI-NruI（0.37 kb）were used as the probes specific for RC-L and RC-M，respectively.Both are the internal portions of their respective genes

Northern hybridizations were performed with probes specific for RC-L［see Figure 2（b），panel 2］and RC-M［see Figure 2（b），panel 3］，which indicated that these genes were expressed on the large transcript（2.6 kb），but were absent from the small transcript（0.8 kb）.Since these four structural genes coding for LH-1，RC-L，and RC-M are closely linked on the chromosome（［31］；P.Kiley and S.Kaplan，unpublished data），it seems reasonable to assume that the larger transcript represents a single polycistronic transcript specifying the four polypeptides：LH-1 a andβ，RC-L，and RC-M.The LH-1 polypeptides appear to be specified by the small transcript.Unlike the response observed when the C-2-C probe for RuBPCase was used，no significant changes were found in the relative abundance of the two major transcripts for LH-1 when the cells were grown on the different substrates（data not shown）.(https://www.daowen.com)

Because the KpnI-PstI probe contains，in addition to the LH-I genes，a portion of the distal RC-L gene，the observed ratio of the small transcript（0.8 kb）to the large transcript（2.6 kb）（1∶1 to 2∶1）is not an accurate measure of the true steady-state ratio of each of these transcripts，since the RC-L and RC-M coding regions reside only on the large transcript.Therefore，an AluI DNA fragment containing only the information for the LH-1 polypeptides as well as a small amount of proximal DNA was used for Northern hybridization analysis，and the relative levels of the small and large transcripts were determined（see Figure 3）.From the densitometer tracings of Figure 3，the steady-state ratio of the small transcript to the large transcript was 10∶1.From five independent determinations of this kind，the average ratio for the small to the large transcript was 11.2∶1，ranging from 7∶1to18∶1.

Figure 3　Northern hybridization of R.sphaeroides with a specific probe for LH-1.The hybridization conditions were the same as in Figure 1，except a 0.51-kb AluI-AluI fragment of pJW1 wa suse da sa specific probe for LH-1