The nucleotide sequence of the DNA thought to contain the control region for the ilvGEDA operon in Escherichia coli has been determined by the Maxam-Gilbert procedure. The sequence includes a region that, upon transcription, would yield a leader transcript specifying a peptide 32 residues long. This putative peptide would contain four leucine, five isoleucine, and six valine residues. A model is proposed that correlates the multivalent control of the ilvGEDA operon with the extent to which this leader transcript is translated. In vitro transcription experiments yielded a transcript of about 183 nucelotides, compatible with the predictions of the model.
The DNA sequence of the intragenic region of the rat 45S ribosomal RNA precursor was determined. This sequence contains 2282 nucleotides and extends from the conserved EcoR I site near the 3' terminus of 18S rRNA to 69 nucleotides downstream of the 5' terminus of 28S rRNA. The sequences corresponding to 18S and 5.8S rRNA were identified by comparison with previously published data. The 5' terminus of rat 28S rRNA was identified by S1 nuclease protection and reverse transcriptase elongation assays. The internal transcribed spacers were found to be 1066 and 765 nucleotides long and had little homology with those of Xenopus and yeast. Regions of sequence homology between rat and Xenopus were found at the junctions of the internal transcribed spacers with 18S, 5.8S and 28S rRNA. These homologies suggest that these sequences may function as recognition sites for the processing of the ribosomal precursor RNA.
A strain of Escherichia coli K-12 containing a deletion extending from early in the ilvE gene toward the ilvG gene was shown to exhibit a higher expression of the downstream genes, ilvD and ilvA, than did an ilv+ strain. The elevated expression was under apparently normal ilv-specific control, however. The deletion was transferred to the ilv region of Ah80dilv and shown by restriction endonuclease and heteroduplex analysis to extend through the deoxyribonucleic acid (DNA) shown, in the preceding paper (C. S. Subrahmanyam, G. M. Mc-Corkle, and H. E. Umbarger, J. Bacteriol 142:547-555, 1980), to contain the ilvO determinant. The deletion was also transferred to an ilv-lac fusion strain and shown to cause an increase in ,B-galactosidase formation while allowing retention of ilv-specific control. Transducing phages excised from these fusion strains with and without the ilvO determinant were compared. The phage carrying the ilvO+
A low-copy-number plasmid was prepared that contained the entire ilv gene cluster of Escherichia coli. The introduction of an ilvO mutation allowed the ilvG gene of the plasmid to be expressed and imparted valine resistance to strains carrying it. Insertion of TnlO into the ilvG gene of the plasmid resulted in a strong polar effect on ilv genes E, D, and A. Replacement of a region of ilv deoxyribonucleic acid between two KpnI sites on the high-copy-number plasmid carrying the entire ilv gene cluster with a KpnI fragment carrying an ilv-lac fusion but not extending into the ilv-specific control region resulted in a plasmid expressing the lacZ gene under ilv control when the fusion had been inserted in its normal orientation but not when it had been inserted in the opposite orientation. These experiments indicate that ilv-specific control over ilvE, ilvD, and ilvA expression is dependent on these genes being continguous with deoxyribonucleic acid that lies upstream of ilvG. The results also add further support to the concept of an ilvGEDA operon in E. coli.The ilv gene cluster at 83 min on the Escherichia coli chromosome (Fig. 1) consists of five genes that specify enzymes responsible for isoleucine and valine biosynthesis. Among these, the ilvE, ilvD, and ilvA genes are expressed as a single transcriptional unit (23). The ilvC gene is a separate, inducible transcriptional unit (21). The ilvG gene has been located in this cluster more recently. It is expressed only when there is a mutation in the adjacent ilvO locus (8). The order of these loci has been established to be ilvG, ilvO, ilvEDA, and ilvC (1,18,22,23,25). All of these genes are transcribed in the same direction (4,17,23,25,27). Since ilvG is transcribed in the same direction as ilvEDA, a possibility arises that ilvG might be the first structural gene for an ilvGEDA operon. This idea was further supported by experiments showing that phages with ilv DNA extending through ilvO and into but not through ilvG did not exhibit ilv-specific gene control. In contrast, a phage carrying a deletion of ilvO and flanking portions of ilvE and ilvG but carrying DNA extending beyond ilvG exhibited ilv-specific gene control (9).The one observation contradictory to this idea came from several mutants in which valine-sensitive derivatives had been isolated from ilvO (valine-resistant) strains after Mu-i mutagenesis (cited but not documented in reference 22). The lesions were in ilvG and were presumed to have t Present address:
A plasmid carrying the 4,6-kilobase (kb) HindIII-derived fragment from an ilvO mutant derivative of lambda h80dilv imparted a valine-resistant phenotype on strains it carried. This fragment carries a small amount of the promoter-proximal end of ilvE, the ilvO determinant, and apparently the entire ilvG gene, which specifies the valine-insensitive acetohydroxy acid synthase. Comparable deoxyribonucleic acid (DNA) from the original lambda h80dilv did not carry the valine resistance marker. The valine-resistant phenotype was always correlated with the formation of the resistant enzymes. The ilvO determinant was shown to be carried within an approximately 600-based-pair region lying between the SalI and KpnI sites on the HindIII fragment and perhaps within the ilvG gene itself. Ribonucleic acid that hybridizes with the DNA corresponding to the ilvG gene is formed in wild-type K-12 cells. This fact, coupled with the fact that ilvG is transcribed from the same DNA strand as the ilvE, D, and A genes, led to the idea that transcription is normally initiated upstream from ilvG in both wild-type and ilvO strains. In wild-type strains either the formation or the translation of the transcript would be terminated with the ilvG gene, thus preventing expression of that gene.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.