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PubMed1979

New genes and promoters suggested by the DNA sequence near the end of the coliphage T7 early operon.

We have employed the dideoxynucleotide chain-terminating method to determine the nucleotide sequence of T7 DNA between the physical map positions 18.9% and 19.8%. The most striking features of this sequence are two perfect 21-basepair repeats, each of which appears to contain a promoter for late transcription. In each case the promoter sequence incorporates a putative translational terminator on its left (5'-side of the "sense" strand), and overlaps a potential ribosome-binding site on its right. The region probably lies immediately distal to the early operon, and may contain two short, hitherto unreported protein-coding sequences.

Base Sequence↗
PubMed2025

A transposable element insertion in AUX/IAA16 disrupts splicing and causes auxin resistance in Bassia scoparia.

A dicamba-resistant population of kochia (Bassia scoparia) identified in Colorado, USA in 2012 was used to generate a synthetic mapping population that segregated for dicamba resistance. Linkage mapping associating dicamba injury with genotype derived from restriction-site-associated DNA sequencing identified a single locus in the kochia genome associated with resistance on chromosome 4. A mutant version of Auxin/Indole-3-Acetic Acid 16 (AUX/IAA16; a gene previously implicated in dicamba resistance in kochia) was found near the middle of this locus in resistant plants. Long-read sequencing of dicamba-resistant plants identified a recently inserted long-terminal repeat (LTR) retrotransposon TRIM element near the beginning of the second exon of AUX/IAA16, leading to disruption of normal splicing and a mutated degron domain. Stable transgenic lines of Arabidopsis thaliana ectopically expressing the mutant and wild-type alleles of AUX/IAA16 were developed. Arabidopsis thaliana plants expressing the mutant AUX/IAA16 allele grew shorter roots on control media. However, transgenic root growth was less inhibited on media containing either dicamba (5 μM) or IAA (0.5 μM) when compared with non-transgenic plants or those expressing the wild-type allele of AUX/IAA16. In vitro assays indicate reduced binding affinity and more rapid dissociation of the mutant AUX/IAA16 with TIR1 in the presence of several auxins, and protein modeling suggests the substitution of the glycine residue in the degron domain of AUX/IAA16 is especially important for resistance. A fitness cost associated with the mutant allele of AUX/IAA16 has implications for resistance evolution and management of kochia populations with this resistance mechanism.

Indoleacetic Acids↗
PubMed1979

The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis.

In this study, we have located the sites of transcription initiation and termination on a cloned fragment of ribosomal DNA from X. laevis, and have sequenced the surrounding nucleotides. As reported previously (Reeder, Sollner-Webb and Wahn, 1977), about 25% of the 40S rRNA precursor molecules isolated from oocytes have polyphosphate 5' termini and are therefore presumed to represent primary transcripts. These ends hybridize specifically to the 221 bp DNA fragment and removed the overhanging DNA region with S1 nuclease. In the other, we hybridized 40S RNA to a 221 bp fragment of ribosomal DNA. The nucleotides encoding the 5' end of the 40S RNA were located more precisely by two methods. In one, we hybridized 40S RNA to the 221 bp DNA fragment and removed the overhanging DNA region with S1 nuclease. In the other, we hybridized 40S RNA to a smaller DNA fragment and extended the recessed 3' terminus of the DNA using reverse transcriptase. The resultant DNA fragments were sized on sequencing gels. Both determinations map the 5' end of 40S RNA at the same site in the rDNA, about 2250 bp upstream from the Eco RI site in the 18S rRNA coding sequence. At this site we find a DNA sequence beginning AGGGGAAGAC.... which agrees with partial sequence data from the 5' end of polyphosphorylated and bulk 40S rRNA. Features of this region of the ribosomal DNA will be discussed in this paper. A 227 nucleotide region surrounding the initiation site was also sequenced from an independently derived clone and found to differ in only one nucleotide. In addition, a sequence is found about 1100 nucleotides upstream from the 5' end of the gene that has 90% homology to the sequence from nucleotides minus 125 to +4 in the initiation region. At the termination region, X. laevis ribosomal DNA has a single recognition site for the restriction enzyme Hind III in each repeating unit. Using the S1 nuclease technique, the 3' termini of both the 40S precursor and mature 28S rRNA are seen to map within this recognition sequence. The sequence surrounding the Hind III site has striking homology to termination sites recognized by other RNA polymerase classes. Sequences with similar features are also found upstream from the initiation site.

Animals↗
PubMed1977

On the mechanism of oligonucleotide-primed RNA synthesis. II. Synthesis of specific primer-initiated RNA copies suitable for DNA sequence analysis.

The effect of temperature and primer concentration on oligonucleotide-primed transcription has been studied using the separated strands of a well-defined natural DNA as template. Results were similar to those obtained in the homopolymer-directed model systems. At high temperature and excess primer concentration mainly primer-initiated RNA copies are synthesized. Omission of one ribonucleoside triphosphate also makes the termination specific. The unique RNA fragments thus obtained have been used to determine the perfectly-repeated sequence of 68 base pairs in this DNA.

Base Sequence↗
PubMed1979

Comparison of the effect of linear gramicidin analogues on bacterial sporulation, membrane permeability, and ribonucleic acid polymerase.

Various analogues of linear gramicidin were tested for their biological activity in restoring the normal spore phenotype of gramicidin-negative mutants of Bacillus brevis and for their ability to increase cation conductivity of black lipid membranes and to inhibit bacterial RNA polymerase. Whereas many biologically active gramicidin analogues had no effect on membrane permeability, all biologically active peptides were able to inhibit ribonucleic acid (RNA) polymerase. These observations make it unlikely that membranes are the site of action of gramicidin during bacterial sporulation, but they are consistent with the notion that gramicidin functions to control RNA synthesis during the transition from vegetative growth to sporulation (Sarkar & Paulus, 1972). The relationship between peptide structure and the ability to restore normal sporulation and inhibit RNA polymerase showed that the eight amino-terminal residues have little influence on the function of gramicidin, whereas the highly nonpolar repeating sequence D-leucyl-L-tryptophan is essential for biological activity and may represent the site of interaction with RNA polymerase.

Bacillus↗
PubMed1979

Restriction cleavage map of SP01 DNA: general location of early, middle, and late genes.

A detailed restriction endonuclease map for the genome of Bacillus subtilis phage SP01 is presented. Sites of cleavage for the restriction enzymes BglII, EcoRI, HaeIII, and SalI were determined. This physical map showed that SP01 DNA was 140 kilobases in length and contained a repeated sequence of 12.4 kilobases at its termini. Combined with previously published information, we were also able to identify the general locations of genes expressed at early, middle, or late times in the phage lytic cycle. In particular, early genes were largely clustered in the terminal repeats, whereas a major cluster of late genes was located in the left-central portion of the genome.

Bacillus subtilis↗
PubMed1979

DNA of Epstein-Barr virus. V. Direct repeats of the ends of Epstein-Barr virus DNA.

Previous data indicated that Epstein-Barr virus DNA is terminated at both ends by direct or inverted repeats of from 1 to 12 copies of a 3 X 10(5)-dalton sequence. Thus, restriction endonuclease fragments which include either terminus vary in size by 3 X 10(5)-dalton increments (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; S. D. Hayward and E. Kieff, J. Virol. 23:421--429, 1977). Furthermore, defined fragments containing either terminus hybridize to each other (Given and Kieff, J. Virol. 28:524--542, 1978). The 5' ends of the DNA are susceptible to lambda exonuclease digestion (Hayward and Kieff, J. Virol. 23:421--429, 1977). To determine whether the terminal DNA is a direct or inverted repeat, the structures formed after denaturation and reannealing of the DNA from one terminus and after annealing of lambda exonuclease-treated DNA were examined in the electron microscope. The data were as follows. (i) No inverted repeats were detected within the SalI D or EcoRI D terminal fragments of Epstein-Barr virus DNA. The absence of "hairpin- or pan-handle-like" structures in denatured and partially reannealed preparations of the SalI D or EcoRI D fragment and the absence of repetitive hairpin- or pan-handle-like structures in the free 5' tails of DNA treated with lambda exonuclease indicate that there is no inverted repeat within the 3 X 10(5)-dalton terminal reiteration. (ii) Denatured SalI D or EcoRI D fragments reanneal to form circles ranging in size from 3 X 10(5) to 2.5 X 1O(6) daltons, indicating the presence of multiple direct repeats within this terminus. (iii) Lambda exonuclease treatment of the DNA extracted from virus that had accumulated in the extracellular fluid resulted in asynchronous digestion of ends and extensive internal digestion, probably a consequence of nicks and gaps in the DNA. Most full-length molecules, after 5 min of lambda exonuclease digestion, annealed to form circles, indicating that there exists a direct repeat at both ends of the DNA. (iv) The finding of several circularized molecules with small, largely double-strand circles at the juncture of the ends indicates that the direct repeat at both ends is directly repeated within each end. Hybridization between the direct repeats at the termini is likely to be the mechanism by which Epstein-Barr virus DNA circularizes within infected cells (T. Lindahl, A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn, J. Mol. Biol. 102:511-530, 1976).

Animals↗
PubMed1975

Site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA.

It has been proposed that the nucleotide sequences of the 3' terminal extracistronic regions of phage RNA plus and minus strands have been strictly conserved during evolution because they are stringently required for recognition by the viral replicase. We have devised a method to generate point mutations at selected sites in the genome of phage Qbeta. The in vitro synthesis of Qbeta RAN with G leads to A transition in the 16th position from the 3' end, i.e., in the terminal extracistronic region of the genome, is outlined. When a mixture of about 60% wild-type and 40% mutant RNA was repeatedly replicated, the mutant RNA was enriched to 80%, showing that at least this point mutation in the terminal sequence of Qbeta RNA does not impair its in vitro replication, but in fact slightly accelerates it.

Base Sequence↗
PubMed1978

Characteristics of individual repetitive sequence families in the sea urchin genome studied with cloned repeats.

Cloned repetitive sequences from the S. purpuratus genome a few hundred to approximately 1000 nucleotides long were used to investigate the characteristics of individual repetitive sequence families. They wer terminally labeled by the kinase procedure and reacted with sheared S. purpuratus DNA. Repetition frequencies were measured for 26 individual families and were found to vary from a few to several thousand copies per genome. Estimates of sequence divergence were made for 18 cloned repeat families by measuring thermal stability of the heteroduplexes formed between the genomic DNA and the cloned fragments, compared with that of the renatured cloned fragments. The difference was less than 4 degrees C for three of the 18 families, and less than 10 degress C for 13 of the 18 families. These 13 repetitive sequence families lack any detectable highly divergent sequence relatives, and the results reported are shown not to change when the renaturation criterion is lowered below 55 degrees C in 0.18 M Na+. Five of the 18 cloned families displayed greater sequence divergence. The average sequence divergence of the total short repetitive sequence fraction of S. purpuratus DNA was found to match closely the average of the divergences of the cloned repeat sequences.

Base Sequence↗
PubMed1975

Anatomy of herpes simplex virus DNA. II. Size, composition, and arrangement of inverted terminal repetitions.

Electron microscope studies on self-annealed intact single strands and on partially denatured molecules show that herpes simplex virus 1 DNA consists of two unequal regions, each bounded by inverted redundant sequences. Thus the region L (70 percent of the contour length of the DNA) separates the left terminal region a1b from its inverted repeat b'a'1, each of which comprises 6 percent of the DNA. The region S (9.4 percent of DNA) separates the right terminal region cas (4.3 percent of the DNA) from its inverted repeat a'sc'. The regions of the two termini which are inverted and repeated itnernally differ in topology. Thus, cas is guanine plus cytosine rich, whereas only the terminal 1 percent of the a1b region, designated as subregion a1, is guanine plus cytosine rich.

Base Sequence↗
PubMed1978

Characterization of inverted repeated sequences in wheat nuclear DNA.

The properties of inverted repeated sequences in wheat nuclear DNA have been studied by HAP(1) chromatography, nuclease S1 digestion and electron microscopy. Inverted repeated sequences comprise 1.7% of wheat genome. The HAP studies show that the amount of "foldback HAP bound DNA" depends on DNA length. Inverted repeats appear to be clustered with an average intercluster distance of 25 kb. It is estimated that there are approximately 3 x 10(6) inverted repeats per haploid wheat genome. The sequences around inverted repeats involve all families of repetition frequencies. Inverted repeats are observed as hairpins in electron microscopy. 20% of hairpins are terminated by a single-stranded spacer ranging from 0.3 to 1.5 kb in length. Duplex regions of the inverted repeats range from 0.1 to 0.45 kb with number average values of 0.24 kb and 0.18 kb for unlooped and looped hairpin respectively. Thermal denaturations and nuclease S1 digestions have revealed a length of about 100 bases for duplex regions. The methods used to study inverted repeated sequences are compared and discussed.

Base Sequence↗
PubMed1979

The complete primary structure of a proline-rich phosphoprotein from human saliva.

The complete amino acid sequence of a calcium-binding "proline-rich phosphoprotein," named Protein A, from human saliva was determined by automated and manual Edman degradation of peptides obtained by enzymatic and chemical cleavage of the intact protein. The NH2-terminal pyrrolidone carboxylic acid was identified by means of NMR. The protein consists of 106 amino acids, including 24 residues of proline. The NH2-terminal 32 residues contain 13 of the 15 negatively charged residues including 2 phosphoserines, but only 1 proline. In spite of a high concentration of proline in the COOH-terminal part of the molecule, the longest oligoproline sequence is tetraproline. The protein contains a number of repeated sequences and there are also several sequences of 3 or 4 residues identical with known sequences of collagen, but the characteristic occurrence of glycine in every third position in collagen is not found in salivary Protein A.

Amino Acid Sequence↗
PubMedDate not supplied

Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9.

The transposable genetic element Tn9 consists of two direct repeats of the insertion sequence IS1 flanking a region of 1,102 base pairs which determines chloramphenicol resistance. Transposition of Tn9 leads to the duplication of a 9-base pair sequence which preexists at the site of insertion. One copy of this sequence is found at each end of the inserted element. The chloramphenicol resistance determined by Tn9, and by various other R plasmids, is due to the synthesis of the enzyme chloramphenicol acetyl transferase (CAT). This enzyme catalyses the formation of acetylated derivatives of chloramphenicol which are inactive as inhibitors of protein synthesis. By using the chain termination technique of DNA sequencing, we have now determined the nucleotide sequence of the 1,102 base pair region between the directly repeated IS1 sequence in the bacterial transposon Tn9 (encoding chloramphenicol resistance). The amino acid sequence of CAT predicted from the nucleotide sequence is identical to that determined by Shaw and coworkers. An analysis of the sequence suggests that the internal 1,102 base pair region is not directly involved in transposition.

Acetyltransferases↗
PubMed1979

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.

Base Sequence↗
PubMed1978

Structural analysis of the tRNA1Tyr gene of Escherichia coli. A 178 base pair sequence that is repeated 3.14 times.

The distal region of the tRNA1Tyr gene has been sequenced and found to have an unusual structure. It consists of a 178 base pair sequence that is repeated 3.14 times. The first repeat unit commences 19 base pairs before the end of the sequences encoding the mature tRNA, and these 19 base pairs are repeated faithfully at the beginning of each repeat unit. In the last fractional unit the repeated sequence extends only six base pairs beyond this 19 base pair sequence. Sequence information extends for 62 base pairs beyond the 3.14 repeating units, and no resemblance to the repeating sequence, or any other region of the tRNA1Tyr gene, is found. There are only 14 sites at which one of the repeats differs from the others; 11 of these are transitions, and the rest are transversions. The evolutionary implications of the differences are discussed. One of the differences, which occurs in the second repeat unit, corresponds to the location of the in vitro p-dependent transcription termination site. This is discussed along with other implications of the repeated structure.

Base Sequence↗
PubMed2026

Ribosome dynamics at the conserved PGP motif governs 2A peptide-bond-skipping efficiency.

Viral 2A oligopeptides drive an unusual ribosome recoding event in which peptide-bond formation fails at a conserved PG↓P motif, producing two discrete proteins without canonical termination. Despite decades of study, the molecular basis of 2A-mediated peptide-bond skipping remains poorly understood. Here, we combine quantitative 2A reporters with high-resolution ribosome profiling to interrogate ribosome dynamics at the core 2A sequences. We identify a pausing event at the terminal proline codon of the PGP motif that functions as a kinetic decision point: ribosome dwell time at this site inversely correlates with skipping efficiency. Increasing nascent chain flexibility by inserting linkers immediately upstream of the 2A sequence reduces ribosome occupancy at the terminal proline codon and enhances peptide-bond skipping. Strikingly, amino acid repeats positioned distally upstream also modulate 2A activity, indicating long-range coupling between nascent chain properties outside of the ribosome and the peptidyl transferase center inside the ribosome. In particular, hydrophobic residues potently suppress skipping, an effect that can be rescued by extending flexible segments within the peptide exit tunnel. Together, our findings support a model in which nascent chain features-beyond the core 2A motif-dynamically tune ribosomal recoding efficiency through co-translational feedback into the catalytic center.

Ribosomes↗
PubMed1978

Repeating covalent structure of streptococcal M protein.

We have attempted to identify the covalent structure of the M protein molecule of group A streptococci that is responsible for inducing type-specific, protective immunity. M protein was extracted from type 24 streptococci, purified, and cleaved with cyanogen bromide. Seven cyanogen bromide peptides were purified and further characterized. Together, the peptides account for the entire amino acid content of the M protein molecule. Each of the purified peptides possessed the type-specific determinant that inhibits opsonic antibodies for group A streptococci. The primary structures of the amino-terminal regions of each of the purified peptides was studied by automated Edman degradation. The partial sequences of two of the peptides were found to be identical to each other and to that of the uncleaved M protein molecule through at least the first 27 residues. The amino-terminal sequences of the remaining five peptides were identical to each other through the twentieth residue but completely different from the amino-terminal region of the other two peptides. However, the type-specific immunoreactivity and the incomplete analysis of the primary structure of the seven peptides suggest that the antiphagocytic determinant resides in a repeating amino acid sequence in the M protein molecule.

Amino Acid Sequence↗
PubMed2025

The chromosome-level genome of Stylosanthes guianensis provides insights into genome evolution and environmental adaptation.

Stylosanthes guianensis is a leguminous forage crop of significant economic importance, primarily distributed in tropical and subtropical regions. It exhibits strong adaptability to various stresses, yet the genetic basis underlying this trait remains unclear. In this study, we constructed the first chromosome-scale reference genome of S. guianensis using a combination of Nanopore and Hi-C sequencing technologies. The assembled genome size is 1254 Mb, with 10 pseudochromosomes. Using Nanopore full-length transcriptome data, we generated high-quality transcript-level gene annotations, identifying 36 585 gene models and 110 601 transcripts. The repetitive sequences in S. guianensis account for 79.16% of the genome, with the extensive expansion of Gypsy elements in long terminal repeats contributing to its genome size enlargement. Comparative genomic and transcriptomic analyses revealed that flavonoid metabolism plays a pivotal role in stress adaptation, providing new insights into the genetic basis of stress tolerance. Additionally, we generated whole-genome methylation profiles under cold treatment and control conditions, offering valuable data for future epigenomic research. These findings provide essential molecular resources for understanding stress resilience in S. guianensis and advancing its molecular breeding.

Genome, Plant↗