Heptanucleotide Sequence Research Articles

Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5′ end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5′ flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upsteam from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24–30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.

Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: Homology with Tn3

The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, γδ, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.

Promoter sequence for stringent control of bacterial ribonucleic acid synthesis.

In bacterial promoters subject to stringent control the heptanucleotide sequence spanning nucleotide positions -5 to +2 (defining the -10 region [D. Pribnow, J. Mol. Biol. 99:419-443, 1975] as positions -12 to -6) is highly conserved. This conserved region, for which the best consensus sequence is [Formula: see text] is absent from promoters which are not under stringent control.

DNA sequence of the gene for the outer membrane lipoprotein of E. coli: an extremely AT-rich promoter

The outer membrane lipoprotein is the most abundant protein in an E. coli cell. Its structural gene (Ipp) was cloned into a X phage vector and the nucleotide sequence of a DNA fragment of 814 bp encompassing the Ipp gene was determined. The promoter region of the gene was found to have the following features. First, a segment of 261 bp preceding the transcription initiation site (−1 to −261) has a very high AT content of 70%, in contrast to 53% for the mRNA region of 322 bp, 44% for a segment of 127 bp after the transcription termination site and 49% for the average AT content of the E. coli chromosome. Second, in particular, of the first 45 bp upstream from the transcription initiation site (−1 to −45), 36 bases (80%) are A or T. Third, there is a heptanucleotide sequence homologous to the “Pribnow box,” eight bases apart from the transcription initiation site. Fourth, a sequence homologous to the “RNA polymerase recognition site” exists on both strands between positions −27 and −39. Finally, there is a long dyad symmetry centered at the transcription initiation site.

Transposition of the Escherichia coli insertion element gamma generates a five-base-pair repeat.

We have determined DNA sequences surrounding the termini of the Escherichia coli insertion element gamma delta, both at its normal locus on the F (fertility) factor and at three different sites of insertion into the plasmid pBR322. After transposition, a five-base-pair pBR322 sequence is duplicated and appears in direct orientation adjacent to each end of the element. No such duplication flanks the ends of gamma delta in F, and there is no apparent homology between the sequences surrounding gamma delta in F and the five-base-pair duplications generated by insertion. These findings suggest that the duplications are not essential for transposition and that they do not act to direct gamma delta to a homologous site in the target chromosome. In addition, we find that the 35-base-pair inverted repeat that comprises the termini of gamma delta is strikingly similar in sequence to the ends of both the ampicillin-resistance transposon Tn3 and a 200-nucleotide-long sequence on the plasmid pSC101 which has been shown to mediate recombination with phage f1 replicative form. Within the terminal region, there is a specific heptanucleotide sequence common to each of the above elements and to bacteriophage Mu, all of which generate five-base-pair repeats upon insertion.

Clustering of Prm − mutations of bacteriophage λ in the region between 33 and 40 nucleotides from the cl transcription start point

Mutations affecting P RM-directed expression of the λ cI- rex operon have been isolated and characterized. Six (class I) mutations, including prm 116 ( K.-M. Yen and G. N. Gussin, 1973, Virology 56, 300–312), appear to be defective in the interaction between RNA polymerase and the P RM promoter. Five of the six mutations map in the RNA polymerase “recognition” region ( W. Gilbert, 1976, In “RNA Polymerase” (R. Losick and M. Chamberlin, eds.), pp. 193–205. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.) located approximately 35 nucleotides preceding the transcription start point, while one maps near the so-called Pribnow box, a heptanucleotide sequence common to some extent in all promoters sequenced thus far. The existence of mutations in two distinct regions of the promoter may provide the opportunity to distinguish between the functions of the two regions in transcription initiation. Three of the Prm − mutations (class II) are defective in repressor binding at O R. These mutations may be Prm − because repressor is unable to act as a positive regulator of P RM ( M. Ptashne, K. Backman, M. Z. Humayun, A. Jeffrey, R. Maurer, B. Meyer, and R. T. Sauer, 1976, Science 199, 156–161). A 10th mutant that exhibited some of the properties expected of a prm − mutant appears to be defective in the cI gene itself; most likely it is a cI missense mutant.

Terminal-sequence analysis of bacterial ribosomal RNA. Correlation between the 3'-terminal-polypyrimidine sequence of 16-S RNA and translational specificity of the ribosome.

The 3'-terminal sequences of 16-S ribosomal RNA from a number of bacteria have been determined by a stepwise degradation and 3'-terminal labelling procedure. The sequences obtained were: Bacillus stearothermophilus, -G(Z)approximately 5 Y-U-C-C-U-U-U-C-U (A); B. subtilis, -G(Z)approximately 7 Y-C-U-U-U-C-U; Caulobacter crescentus, -G(Z)3 Y-U-C-C-U-U-U-C-U; Pseudomonas aerugionosa, -G-Z-Z-Y-C-U-C-U-C-C-U-U(A), where Z is any nucleotide other than G. Thus, as previously found in Escherichia coli, all bacterial 16-S rRNAs contain a pyrimidine-rich tract at the 3'-terminus. In B. stearothermophilus and Ps. aeruginosa this region shows substantial heterogeneity involving the 3'-terminal adenylic acid. A low level of 3'-terminal heterogeneity cannot be excluded for the other bacterial 16-S rRNAs examined. The 3'-termini of bacterial 16-S rRNA can be divided into two groups on the basis of sequence homology. The first group comprises E. coli and Ps. aeruginosa; the second, B. stearothermophilus, B. subtilis and C. crescentus. This division correlates with a previous separation of bacterial ribosomes into two categories based on ability to translate different mRNA preparations [Stallcup, Sharrock & Rabinowitz (1974) Biochem. Biophys. Res. Commun. 58, 92-98]. We have previously proposed that the precise base sequence at the 3'-terminus of 16-S rRNA determines the intrinsic capacity of bacterial ribosomes to translate a particular cistron [Shine & Dalgarno (1975) Nature (Lond.) 254, 34-38]. No difference was found in the 3'-terminal heptanucleotide sequence of 16-S rRNA from bacteriophage T7-infected E. coli, as compared to that in uninfected cells. Thus, the T7-induced alteration in translational specificity of E. coli ribosomes is probably not mediated by modification of the terminal seven nucleotides of the smaller rRNA. The 3'-terminal sequences of the 23-S rRNA species were also determined. The sequences obtained were: B stearothermophilus and B. subtilis, -Y-C; C. crescentus, -Y-C-U; Ps. aeruginosa, -Y-C-A; E. coli, -G-Y-U-U-A-A-C-C-U-U. No evidence for 3'-terminal heterogeneity was found. The results obtained are discussed in relation to possible base-pairing roles for the 3'-end of 16-S rRNA in bacterial protein synthesis.

The 3′-Terminal Heptanucleotide Sequence of the l Strand of T7 Deoxyribonucleic Acid

Abstract In a reaction catalyzed by the T4 DNA polymerase, the 3'-terminal deoxythymidylate of the l strand of T7 DNA can be exchanged for a [32P]deoxythymidylate without introducing any net covalent changes into the molecule. Degradation of the terminally labeled [32P]DNA to pyrimidine oligonucleotides using diphenylamine and formic acid revealed that almost all of the radioactivity was in a unique pyrimidine pentanucleotide. This fact indicates that the l strand has a 3'-terminal sequence of five pyrimidines. By 5'-terminal labeling and exonuclease digestion of the purified pentanucleotide, its structure was shown to be d-pTpCpCpCpT. This structure, together with data obtained earlier from nearest neighbor analyses (Englund, P. T. (1972) J. Mol. Biol. 66, 209–224), indicates that the 3'-terminal nucleotide sequence of the l strand of T7 DNA is ... pTpGpTpCpCpCpT.