Differential Hybridization of the puf Operon Trans...
The previously presented analysis of the 5'-ends of the small and large puf operon transcripts，their relative abundance and stabilities in vivo，strongly suggests that the small and large puf operon transcripts have different 5'termini upstream of the pufB structural gene（104 and 75 nucleotides 5'，respectively）.In order to test this hypothesis directly，two specific deoxyoligonucleotides were used as hybridization probes against bulk R.sphaeroides DNA.The LHI-4 deoxyoligonucleotide was synthesized to be complementary to a region within the mRNA coding for the puf B structural gene while the LHI-5 deoxyoligonucleotide was complementary to the mRNA from 78 to 96 nucleotides upstream of the pufB initiation codon.The results obtained when these deoxyoligonucleotides as well as an LH-Ⅰ structural gene probe were used in Northern blots against bulk R.sphaeroides 2.4.1 RNA from cells grown under different physiological conditions are shown in Figure 8.As expected，both the LH-I-specific and the LHI-4 probe hybridized to both the small and large puf operon transcripts under high stringency washing conditions（75℃for the LH-Ⅰ-specific probe，50℃for the LHI-4 probe）.In contrast，the LHI-5 probe hybridized to mRNA corresponding only to the small puf operon transcript under high stringency（65℃）washing conditions.Therefore，the results of this experiment when considered together with the previously presented data unambiguously show that the large puf operon transcript has a 5'terminus 75 nucleotides upstream of the start of the pufB structural gene with the transcript encompassing the four puf operon structural genes.In contrast，the small puf operon transcript contains a 5'terminus 104 nucleotides 5'of the pufB initiation codon and extends just beyond the puf B and A genes but does not include the L and M genes.The available information on the factors which may influence the origins of these two 5'-termini in vivo，the termination of the small puf operon transcript prior to the pu f L structural gene，and the ramifications of the differential use of two transcripts are summarized under“Discussion”.(https://www.daowen.com)
