Southern hybridization

The results from the dot blots and Northern hybridization experiments were further confirmed by Southern hybridization.Figure 4 shows the results of Southern hybridization of a Bam HI digest of plasmid pLI10 DNA with 32P-labeled bulk RNA，as described above.Plasmid pLI10 is a pBR322 derivative containing a trp promoter and having an insert of a 3.0-kb fragment of R.sphaeroides DNA containing the gene coding for RuBPCase Ⅱ［see Table 1，Figure 4（c）］.The results，employing different conditions of light and O2 are shown in Figure 4（a）；the right lane is an agarose gel on which the 4.9-kb vector and the 3.0-kb Bam HI insert were separated after electrophoresis.The bulk RNA was homologous only to the 3.0-kb Bam HI insert.The hybridization intensity，therefore，represents primarily the relative amount of mRNA coding for RuBPCase Ⅱ.Cells grown under high-intensity light had the highest concentration of mRNA for RuBPCase；these levels were greatly reduced when cells were grown under low-intensity light or incubated in the dark.There was no observable hybridization of RNA derived from aerobically grown cells.

Figure 4（b）demonstrates the changes in the levels of mRNA for RuBPCase Ⅱ when the cells were grown on different substrates.Butyrate-grown cells resulted in the highest mRNA level for RuBPCase when compared with the results obtained for cells grown on succinate and malate.The relative mRNA concentrations resulting from growth on the three substrates agreed well with the values determined from the dot hybridization experiments.

Figure 5 shows the Southern hybridization with plasmid pJW1 DNA and R.sphaeroides derived RNA.The pJW1 DNA was digested with SalI，KpnI，NruI，and Hine Ⅱ.Approximately 13 electrophoretic bands were obtained，as shown on the agarose gel on the right lanes of Figure 5（a）and（b）.A partial map of the restriction sites located on pJW1 is presented in Figure 5（c）.The 0.98-kb HincII-KpnI，1.06-kb KpnI-SalI，and 0.6-kb SalINruI fragments contain genes coding for LH-1，RC-L，and RC-M，respectively（P.Kiley and S.Kaplan，unpublished data）.The autoradiographs demonstrated that the LH-1 genes（0.98 kb）had the strongest hybridization signal.The 1.06-kb fragment had a rather strong hybridization signal，probably due to the fact that a small portion of the 3'end of the LH-1 genes extended into this fragment，which contains the entire RC-L gene.However，the 0.6-kb fragment，containing the RC-M gene and lacking any LH-1 fragment，had a relatively weak hybridization signal.Several other bands that were located on the 12.0-kb DNA insert of R.sphaeroides also hybridized with RNA to various degrees，suggesting that other genetic determinants were expressed under the conditions of this experiment.The Southern hybridization results verified the results obtained from the dot hybridization experiments，that is，that the genes for LH-1，RC-L，and RC-M are regulated by light and O2，but are not affected by the substrates used for cell growth to any great degree.(https://www.daowen.com)

Figure 4　Southern hydridization of pLI10 Bam HI digest with 32P-labeled RNA.The pLI10 DNA was disgested with BamHI，separated on a 1.0%agarose gel，transferred to Gene Screen，and hybridized with 32 P-labeled RNA from R.sphaeroides cells.The cells were grown on butyrate medium under different lighting conditions（a）or on different substrates（b）as described in the legend to Figure 1.The rightmost lanes of（a）and（b）are agarose gels，and the remaining lanes are autoradiograms of these gels hybridized with the indicated probes，where the 4.9-kb vector and 3.0-kb BamHI insert are indicated.Panel（c）shows the restriction map of pLI10 indicating the location of the gene coding for form Ⅱ RuBPCase