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The repeat-induced point mutation (RIP) targets repeated sequences, such as transposable elements, in filamentous fungi. Host-transposable element coevolutionary dynamics have shaped taxonomically restricted eukaryotic defense systems, likely built on conserved ancestral mechanisms. Key questions surrounding homology recognition remain unresolved, and RIP offers a unique opportunity to answer them.
Bacillus subtilis-Bacillus globigii hybrids were made by integration of the B. globigii aromatic region (aroB to aroE) as an intergenote in the B. subtillis chromosome. Transformation of the heterologous intergenote by B. subtillis DNA (or vice versa) occurred at about 10% of the frequency of homologous transformation by hybrid donors into the same region. Heterologous intergenote crosses were unusually sensitive to shear fragmentations of donor DNA to sizes less than 30 X 10(6) to 40 X 10(6) daltons. In all cases, the entire intergenote was transferred en bloc. Homologous transformation of intergenote markers by B. globigii DNA was not unusually shear sensitive, and linkage was normal for markers in the intergenote. A model is proposed in which efficient heterologous intergenote transformation occurs by recognition and base pairing of homologous DNA sequences of both flanks of the intergenote.
Regulatory regions in DNA which have been sequenced have generally been found to contain one or more axes of two-fold rotational symmetry. If this symmetry is to be maintained in the helical sequence, the axis of rotation must be aligned with one of the two dyad axes of the helix. This is equivalent to saying that the rotational symmetry of the sequence can only be seen from certain viewing points in a circuit about the helix. More surprising is the fact that new symmetrical sequence arrangements can be seen at +/- 36 degrees, +/- 72 degrees, +/- 108 degrees, and +/- 144 degrees relative to the point at which the rotational symmetry is seen. This "amplification" of symmetry suggests a three-dimensional approach to sequence analysis. A specific reading frame, suggested by the geometry of the helix, is examined with regard to its elucidation of intra- and inter-sequence homologies. Two sequences are thus identified as being recurrent in a number of different regulatory sequences.
Genomic Xenopus borealis oocyte-specific 5S DNA (Xbo) contains clusters of 5S rRNA genes. The number of genes varies among clusters, and the distance between genes within a cluster is about 80 nucleotides. The spacer DNA between gene clusters is AT-rich and heterogeneous in length due in part to variable numbers of a tandemly repeated 21 nucleotide sequence. A cloned fragment of Xbo 5S DNA (Xbo1) containing three 5S rRNA genes has been sequenced. The sequences of Xbo1 genes 1 and 2 are very similar to the dominant 5S RNA sequence, whereas 15 of the 120 residues in the third gene are different. The sequence of gene 3 is as different from the dominant gene sequence as the X. laevis pseudogene is from the 5S RNA gene. Sequence analysis of genomic DNA shows that gene 3 is an abundant component of the multigene family. All three genes are transcribed when added to an extract of X. laevis oocyte nuclei, and a fragment of Xbo1 lacking the AT-rich spacer DNA and the 5' end of the first gene supports transcription of genes 2 and 3 in this in vitro system. Thus the 80 nucleotides preceding each 5S gene are sufficient for promoter function. Nucleic acid sequences preceding several eucaryotic genes that are transcribed by RNA polymerase III were analyzed and the following common features were found: a purine-rich region; at least one direct repeat; the absence of dyad symmetry; transcription beginning with a purine; a pyrimidine residue immediately preceding the first nucleotide of the gene; and the oligonucleotides AAAAG, AGAAG and GAC, located approximately 15, 25 and 35 nucleotides, respectively, before the start of transcription. The 10 base pair (bp) spacing between the homologous oligonucleotides is that expected for a recognition signal on one face of a DNA double helix. The extensive sequence differences between most of the spacers that precedes these genes make the three conserved oligonucleotides more striking. Parts of the 5' flanking regions of the three Xbo1 gene (-12 to -40), which include the conserved oligonucleotides, are identical. In contrast, 7 of the first 11 nucleotides that precede the third 5S RNA gene in Xbo1 differ from those that precede the first gene. The sequences following the X. borealis oocyte and somatic 5S genes are identical in 12 of the first 14 residues and contain two or more T clusters, as does the corresponding region of X. laevis oocyte 5S DNA. The 3' sequences of the Xenopus 5S rRNA genes and several other eucaryotic genes contain features in common with procaryotic transcription termination sites. The 3' end of the gene is GC-rich and contains a dyad symmetry. Termination occurs in an AT-rich region containing one or more T clusters on the noncoding strand.
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.
A cloned library of large, random embryonic human DNA fragments was constructed and screened for beta-globin sequences using the cloned human beta-globin cDNA plasmid pJW102 (Wilson et al., 1978) as a hybridization probe. Two independent clones were obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing. Each of the clones carries both the adult delta- and beta-globin genes. The two genes are separated by approximately 5.4 kilobases (kb) of DNA and their orientation with respect to the direction of transcription is 5'-delta--beta-3'. Both the delta- and beta-globin genes contain a large noncoding intervening sequence (950 and 900 bp, respectively) located between the codons for amino acids 104 (arginine) and 105 (leucine). Although the location of the large intervening sequence within the coding regions of the two genes is identical, the two noncoding sequences bear little sequence homology. A second, smaller intervening sequence similar to that found in other mammalian beta-globin genes was detected near the 5' end of the human beta-globin gene. The two independently isolated beta-globin clones differ from each other by the presence of a Pst I restriction enzyme cleavage site within the large intervening sequence of the delta-globin gene of one of the clones. This suggests that the human DNA carried in the two clones was derived from two homologous chromosomes which were heterozygous for the Pst I restriction enzyme recognition sequence.
Accurate detection of low-abundance single nucleotide polymorphisms (SNPs) against a large excess of homologous wild-type sequences requires both selective molecular recognition and effective transduction of small sequence differences into measurable signals. Here, we report a spatially confined electrochemical strategy that couples sequence-selective recognition with size-dependent mass-transport gating. DNA-hybridization-driven self-assembly of gold nanoparticles (AuNPs) forms a three-dimensional self-assembled electrode (3D-SAE) with a DNA-defined interparticle architecture. Competitive probes (SP/WP) convert single-base recognition into distinct molecular-size states: the SNP-associated pathway preferentially triggers a hybridization chain reaction (HCR), generating bulky AuNP-anchored HCR/methylene blue complexes (Au@HCR/MB) with reduced electrochemical accessibility through the porous 3D-SAE, whereas the wild-type pathway does not trigger HCR and maintains a high-current response from more readily accessible MB-containing species. Thus, sequence recognition is translated into a molecular-size difference and subsequently into an electrochemical signal through differential mass transport. Under buffer conditions, the platform achieved a statistically estimated detection limit of ∼0.47 fM and a quantitative range of 1 fM-100 pM. It discriminated a 0.1% mutant abundance in a fragmented genomic-DNA background. The downstream signal-transduction chemistry is enzyme-free and isothermal. This work establishes a mechanistical recognition-size-conversion-mass-transport-gating architecture for electrochemical nucleic acid analysis.
To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.
In many organisms, most notably Drosophila, homologous chromosomes in somatic cells associate with each other, a phenomenon known as somatic homolog pairing. Unlike in meiosis, where homology is read out at the level of DNA sequence complementarity, somatic homolog pairing takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, presumably mediated by different proteins that bind to these different regions. Here we consider an alternative model, which we term the "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. An important component of this model is that the buttons are non-uniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a non-homolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. We investigated several types of barcodes and examined their effect on pairing fidelity. We found that high fidelity homolog recognition can be achieved by arranging chromosome pairing buttons according to an actual industrial barcode used for warehouse sorting. By simulating randomly generated non-uniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.
Methyl groups placed on varphiXsB1 replicative form DNA by the Escherichia coli B modification enzyme are located in the overlap between fragments Mbo II-3 and Alu I-2, a 61-base-pair DNA segment. Mutations that led to loss of susceptibility to restriction by E. coli B occurred within this segment at three positions spanning 14 nucleotides. A sequence difference between varphiXsB1 and varphiXam3cs70, a varphiX174 strain not restricted by E. coli B, occurs at one of these positions. The site on simian virus 40 DNA methylated by the modification enzyme is located in the 115-base-pair overlap between fragments Hae III-I and Alu I-G. The sequences of these segments of varphiXsB1 and simian virus 40 DNA and two regions of phage f1 DNA recognized by the E. coli B restriction enzyme [Ravetch, J. V., Horiuchi, K. & Zinder, N. D. (1978) Proc. Natl. Acad. Sci. USA 75, 2266-2270] contain a homology of nine bases in the configuration:5'-T-G-A... 8N... T-G-C-T... 9N... T-N-N-T-3'. The sequence 5'-T-G-A... 8N... T-G-C-T-3' may constitute the restriction enzyme recognition site since it does not occur in varphiXam3cs70 DNA and occurs only once in simian virus 40 DNA, and since all observed mutations leading to loss of the site occur at one of the bases specified by this sequence. Analysis of the sequence of varphiXam3cs70 showed that if no other residues are recognized, all seven of these bases are essential for recognition and the interval between the two groups of specified bases must be precisely eight.
To determine the special feature of ribosomal RNA promoters that might account for the highly efficient and regulated synthesis of rRNA in E. coli, we have analyzed the beginnings of two ribosomal RNA operons, rrnD and rrnX. DNA sequences for 425 bp preceding those specifying mature 16s rRNA are reported. In vitro transcription of restriction endonuclease fragments containing this region from either operon reveals the presence of two promoters about 110 nucleotides apart; they are denoted P1 and P2. RNA synthesis from P1 is initiated with GTP at position -284 (relative to 16s sequences) in rrnD and with ATP at position -285 in rrnX. At P2, the RNA starts with CTP primarily at position-176 in both operons. The DNA sequences of the two operons are identical for 231 bp preceding the 16s rDNA (including a substantial region around P2); they then diverge almost completely, except for a notable 18 bp homology just preceding the transcription start site for P1. Certain sequences implicated in the recognition of promoters by E. coli RNA polymerase are clearly identifiable in both P1 and P2; other features include an extended region preceding P1 which is strikingly rich in AT base pairs. Possible mechansims by which these tandem promoters contribute to the high frequency of rRNA transcription and to the differential expression of the E. coli rrn operons are discussed.
Oligopeptides containing tyrosyl, lysyl, and alanyl residues bind to polynucleotides and nucleic acids as shown by proton magnetic resonance, fluorescence spectroscopy, and difference absorption spectroscopy. Proton magnetic resonance data indicate that stacking of tyrosyl residues with nucleic acid bases takes place only in single-stranded structures (such as poly(A) or denatured DNA). Stacking interactions lead to a quenching of tyrosine fluorescence. However, the tyrosyl fluorescence of the peptides is quenched in their complexes with both single-stranded and double-stranded nucleic acids. A comparison of the behavior of homologous peptides containing Tyr, methoxytyrosine, and Phe leads to the conclusion that hydrogen bonding of tyrosine with bases or phosphates is not involved in the investigated complexes. An energy transfer mechanism from tyrosine to nucleic acid bases is proposed to account for fluorescence quenching in oligopeptide complexes with double-stranded DNAs. Due to the specificity of its stacking interaction for single-stranded nucleic acid structures, tyrosine might be involved through such interactions in the selective recognition of single strands by proteins.
The outer membrane lipoprotein is the most abundant protein in an E. coli cell. Its structural gene (Ipp) was cloned into a lambda 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.
UNLABELLED: Genome-wide association studies have identified genetic polymorphisms at 11p15 associated with Systemic Lupus Erythematosus (lupus). Statistical fine mapping prioritizes a highly prevalent coding haplotype within the IRF7 gene. Analysis of ancient DNA confirms that this haplotype has persisted at high frequencies in the global population for millennia. The IRF7 risk haplotype is sufficient to increase nuclear localization of IRF7 and transcriptional activity downstream of pattern recognition receptor pathways. This risk haplotype increases IRF7 DNA binding strength and alters IRF7 DNA sequence specificity, resulting in genotype-dependent increases in IFN-α production in numerous biological systems, including monocytes and airway epithelial cells. CRISPR engineering of a homologous risk variant in mouse Irf7 results in both enhanced innate control of virus infection and increased autoantibody titers in a model of autoimmunity. Altogether, we establish a persistent and prominent genetic IRF7 haplotype that amplifies IRF7 activity in a manner that has immunological risks and benefits. HIGHLIGHTS: Genetic analysis using modern and evolutionary datasets identifies a persistent and highly prevalent lupus-associated coding haplotype in IRF7 at 11p15 The IRF7 lupus risk haplotype increases IFN-α production by monocytes and airway epithelial cells The IRF7 lupus risk haplotype increases IRF7 DNA binding strength and alters DNA sequence specificity A homologous lupus risk variant in mouse Irf7 enhances control of vesicular stomatitis virus and exacerbates autoantibody production.
We describe a computer program designed to facilitate the analysis of nucleic acid sequences. The program can search several nucleic acid sequences for oligonucleotides common to all of them. It can examine a DNA or RNA sequence for two kinds of homologous regions--repetitions and dyad symmetries. The homologies need not be perfect: mismatches and "looping out" of nucleotides are allowed. The program also finds (A+T)- and (G+C)-rich regions, locates restriction enzyme recognition sites, determines the distribution of di- and trinucleotides, and performs various other functions. We include two representative applications of the program. All published prokaryotic transcription termination sequences (June 1977) were found to share the following features: (i) a string of at least five T residues, (ii) the sequence CGGGC or a close analog immediately preceding the T cluster, (iii) a region of strong dyad symmetry preceding the Ts and including the CGGGC sequence. A sequence of 221 nucleotides consisting of the Escherichia coli trp promoter, operator, and leader was found to contain two strong dyad symmetries. These homologies both occur at known regulatory sites; no comparable homologies occur in regions without regulatory significance.
A novel computer procedure has been used to search for homology among 17 known procaryotic promoter sequences. A model sequence, :formula: (see text), is compatible with the properties of all known promoter and operator mutations, predicts base positions for the initiation of RNA synthesis coinciding with those determined experimentally, is compatible with current models for the regulation of transcription, suggests that RNA polymerase could recognize the DNA double helix firstly in the B conformation then in the A.
Two sB mutations in the genome of bacteriophage fd were located by sequence analysis in the fd sequence at positions 971 and 6341. Base changes at or close to these positions in phage M13 and in phage fl am 124 also correlate with a loss of sensitivity to B restriction. From the sequence homology between the sequences at the two sB sites the recognition signal for the E. coli B restriction/modification enzzyme is predicted to be: 5' TGA---8N---TGCT 3' 3' ACT---8N---ACGA 5'.
The molecular structure of ribosome binding sites of ten phage genes and two messengers of Escherichia coli were compared concerning the signation parts which are presumably used by ribosomes for recognition and binding. With a simple calculation based on triplet patterns sofar unknown agreements between all of these sequences were found. In several cases it was shown that agreements between old sequences are easier recognizable if the purine- and pyrimidine bases are put into the triplets instead of the four A, G, C, and U (T) bases. In such cases "homologous" parts of sequences were recognized with more distinctness. This is true in our case for the double triplet (hexaplet) py-pu-pu-pu-pu-(pu) and the binding site triplet py-pu-pu, which are preceding the initiator. These triplets are in specific positions in all twelve sequences which were compared. The different course of the quaternary and the binary conformity curves (diagram 1) may show for the investigated area that the RNA phage gene-part is organized according to the well known quaternary triplet code. On the contrary the phage phi-gene-part seems to be organized according to a more simple, binary triplet sequence of purine and pyrimidine bases. The binary sequence seems to be the more original, the quaternary the derived one.