Instability of repetitive DNA sequences: The role of replication in multiple mechanisms

Rearrangements between tandem sequence homologies of various lengths are a major source of genomic change and can be deleterious to the organism. These rearrangements can result in either deletion or duplication of genetic material flanked by direct sequence repeats. Molecular genetic analysis of repetitive sequence instability in Escherichia coli has provided several clues to the underlying mechanisms of these rearrangements. We present evidence for three mechanisms of RecA-independent sequence rearrangements: simple replication slippage, sister-chromosome exchange-associated slippage, and single-strand annealing. We discuss the constraints of these mechanisms and contrast their properties with RecA-dependent homologous recombination. Replication plays a critical role in the two slipped misalignment mechanisms, and difficulties in replication appear to trigger rearrangements via all these mechanisms.

The consensus sequence of a major Alu subfamily contains a functional retinoic acid response element.

Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies. We show here that an Alu sequence encodes functional binding sites for retinoic acid receptors, which are members of the nuclear receptor family of transcription factors. The consensus sequences for the evolutionarily recent Alu subclasses contain three hexamer half sites, related to the consensus AGGTCA, arranged as direct repeats with a spacing of 2 bp, which is consistent with the binding specificities of retinoic acid receptors. An analysis was made of the DNA binding and transactivation potential of these sites from an Alu sequence that has been previously implicated in the regulation of the keratin K18 gene. These Alu double half sites are shown to bind bacterially synthesized retinoic acid receptors as assayed by electrophoretic mobility shift assays. These sites are further shown to function as a retinoic acid response element in transiently transfected CV-1 cells, increasing transcription of a reporter gene by a factor of approximately 35-fold. This transactivation requires cotransfection with vectors expressing retinoic acid receptors, as well as the presence of all-trans-retinoic acid, which is consistent with the known function of retinoic acid receptors as ligand-inducible transcription factors. The random insertion of potentially thousands of Alu repeats containing retinoic acid response elements throughout the primate genome is likely to have altered the expression of numerous genes, thereby contributing to evolutionary potential.

Conformation, energy, and folding ability of selected amino acid sequences.

Evolutionary selection of sequences is studied with a knowledge-based Hamiltonian to find the design principle for folding to a model protein structure. With sequences selected by naive energy minimization, the model structure tends to be unstable and the folding ability is low. Sequences with high folding ability have only the low-lying energy minimum but also an energy landscape which is similar to that found for the native sequence over a wide region of the conformation space. Though there is a large fluctuation in foldable sequences, the hydrophobicity pattern and the glycine locations are preserved among them. Implications of the design principle for the molecular mechanism of folding are discussed.

Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition.

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.

Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species

The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50°C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln). These are recurring themes, with all trends applying to 83–92% of the proteins for which complete sequences were available. Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias. We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement.

Identification of thyroid hormone response elements in the human fatty acid synthase promoter

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5′-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides −870 to −650) and TRE2 (nucleotides −272 to −40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter−luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides −716 to −731) represents TRE1 and that the sequence GGGTCC (nucleotides −117 to −112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.

SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor α (RARα) and PLZF-RARα oncoproteins associated with acute promyelocytic leukemia

Retinoic acid receptors (RARs) are hormone-regulated transcription factors that control key aspects of normal differentiation. Aberrant RAR activity may be a causal factor in neoplasia. Human acute promyelocytic leukemia, for example, is tightly linked to chromosomal translocations that fuse novel amino acid sequences (denoted PML, PLZF, and NPM) to the DNA-binding and hormone-binding domains of RARα. The resulting chimeric receptors have unique transcriptional properties that may contribute to leukemogenesis. Normal RARs repress gene transcription by associating with ancillary factors denoted corepressors (also referred to as SMRT, N-CoR, TRAC, or RIP13). We report here that the PML-RARα and PLZF-RARα oncoproteins retain the ability of RARα to associate with corepressors, and that this corepressor association correlates with certain aspects of the leukemic phenotype. Unexpectedly, the PLZF moiety itself can interact with SMRT corepressor. This interaction with corepressor is mediated, in part, by a POZ motif within PLZF. Given the presence of POZ motifs in a number of known transcriptional repressors, similar interactions with SMRT may play a role in transcriptional silencing by a variety of both receptor and nonreceptor transcription factors.

Primary structure and tissue distribution of two novel proline-rich γ-carboxyglutamic acid proteins

Two human cDNAs that encode novel vitamin K-dependent proteins have been cloned and sequenced. The predicted amino acid sequences suggest that both are single-pass transmembrane proteins with amino-terminal γ-carboxyglutamic acid-containing domains preceded by the typical propeptide sequences required for posttranslational γ-carboxylation of glutamic acid residues. The polypeptides, with deduced molecular masses of 23 and 17 kDa, are proline-rich within their putative cytoplasmic domains and contain several copies of the sequences PPXY and PXXP, motifs found in a variety of signaling and cytoskeletal proteins. Accordingly, these two proteins have been called proline-rich Gla proteins (PRGP1 and PRGP2). Unlike the γ-carboxyglutamic acid domain-containing proteins of the blood coagulation cascade, the two PRGPs are expressed in a variety of extrahepatic tissues, with PRGP1 and PRGP2 most abundantly expressed in the spinal cord and thyroid, respectively, among those tissues tested. Thus, these observations suggest a novel physiological role for these two new members of the vitamin K-dependent family of proteins.

The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a β-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

Detection of protein fold similarity based on correlation of amino acid properties

An increasing number of proteins with weak sequence similarity have been found to assume similar three-dimensional fold and often have similar or related biochemical or biophysical functions. We propose a method for detecting the fold similarity between two proteins with low sequence similarity based on their amino acid properties alone. The method, the proximity correlation matrix (PCM) method, is built on the observation that the physical properties of neighboring amino acid residues in sequence at structurally equivalent positions of two proteins of similar fold are often correlated even when amino acid sequences are different. The hydrophobicity is shown to be the most strongly correlated property for all protein fold classes. The PCM method was tested on 420 proteins belonging to 64 different known folds, each having at least three proteins with little sequence similarity. The method was able to detect fold similarities for 40% of the 420 sequences. Compared with sequence comparison and several fold-recognition methods, the method demonstrates good performance in detecting fold similarities among the proteins with low sequence identity. Applied to the complete genome of Methanococcus jannaschii, the method recognized the folds for 22 hypothetical proteins.

A very limited number of keywords (main patterns) describes all sequences of the human variable heavy (VH) and κ (Vκ) domains

Sequences of the variable heavy (VH) and κ (Vκ) domains of Ig structures were divided into 21 fragments that correspond to strands, loops, or parts of these structural units of the variable domains. Amino acid sequences of fragments (termed “words”) were collected from the 1,172 human heavy and 668 human κ chains available in the Kabat database. Statistical analysis of words of 17 fragments was performed (fragments that comprise the complementary determining regions′ fragments will not be discussed in this paper). The number of different words (those with different residues in at least one position) ranged, for various fragments, from 11 to 75 in the κ chains, and from 23 to 189 in the heavy chains. The main result of this study is that very few keywords, or main patterns of words, were necessary to describe over 90% of the sequences (no more than two keywords per fragment in the κ and no more than five per fragment in the heavy chains). No identical keywords were found for different fragments of the variable domains. Keywords of aligned fragments of the VH and Vκ domains were different in all but two instances. Thus, knowing the keywords, one can determine whether any given small part of a sequence belongs to a heavy or κ chain and predict its precise localization in the sequence. In addition, by using all of the keywords obtained through analysis of the Kabat database, it was possible to describe completely the sequences of the human VH and Vκ germ-line segments.

Protective DNA vaccination against organ-specific autoimmunity is highly specific and discriminates between single amino acid substitutions in the peptide autoantigen

DNA vaccines that encode encephalitogenic sequences in tandem can protect from subsequent experimental autoimmune encephalomyelitis induced with the corresponding peptide. The mechanism for this protection and, in particular, if it is specific for the amino acid sequence encoding the vaccine are not known. We show here that a single amino acid exchange in position 79 from serine (nonself) to threonine (self) in myelin basic protein peptide MBP68–85, which is a major encephalitogenic determinant for Lewis rats, dramatically alters the protection. Moreover, vaccines encoding the encephalitogenic sequence MBP68–85 do not protect against the second encephalitogenic sequence MBP89–101 in Lewis rats and vice versa. Thus, protective immunity conferred by DNA vaccination exquisitely discriminates between peptide target autoantigens. No bystander suppression was observed. The exact underlying mechanisms remain elusive because no simple correlation between impact on ex vivo responses and protection against disease were noted.

Mode matches and their locations in the hydrophobic free energy sequences of peptide ligands and their receptor eigenfunctions

Patterns in sequences of amino acid hydrophobic free energies predict secondary structures in proteins. In protein folding, matches in hydrophobic free energy statistical wavelengths appear to contribute to selective aggregation of secondary structures in “hydrophobic zippers.” In a similar setting, the use of Fourier analysis to characterize the dominant statistical wavelengths of peptide ligands’ and receptor proteins’ hydrophobic modes to predict such matches has been limited by the aliasing and end effects of short peptide lengths, as well as the broad-band, mode multiplicity of many of their frequency (power) spectra. In addition, the sequence locations of the matching modes are lost in this transformation. We make new use of three techniques to address these difficulties: (i) eigenfunction construction from the linear decomposition of the lagged covariance matrices of the ligands and receptors as hydrophobic free energy sequences; (ii) maximum entropy, complex poles power spectra, which select the dominant modes of the hydrophobic free energy sequences or their eigenfunctions; and (iii) discrete, best bases, trigonometric wavelet transformations, which confirm the dominant spectral frequencies of the eigenfunctions and locate them as (absolute valued) moduli in the peptide or receptor sequence. The leading eigenfunction of the covariance matrix of a transmembrane receptor sequence locates the same transmembrane segments seen in n-block-averaged hydropathy plots while leaving the remaining hydrophobic modes unsmoothed and available for further analyses as secondary eigenfunctions. In these receptor eigenfunctions, we find a set of statistical wavelength matches between peptide ligands and their G-protein and tyrosine kinase coupled receptors, ranging across examples from 13.10 amino acids in acid fibroblast growth factor to 2.18 residues in corticotropin releasing factor. We find that the wavelet-located receptor modes in the extracellular loops are compatible with studies of receptor chimeric exchanges and point mutations. A nonbinding corticotropin-releasing factor receptor mutant is shown to have lost the signatory mode common to the normal receptor and its ligand. Hydrophobic free energy eigenfunctions and their transformations offer new quantitative physical homologies in database searches for peptide-receptor matches.

Arrayed transposase-binding sequences on the ends of transposon Tn5090/Tn402

The transposon Tn5090/Tn402 encodes a 559 amino acid transposase, TniA, with a DDE motif. Gel mobility shifting and cleavage protection analysis with DNase I and hydroxyl radical probes revealed that TniA binds to multiple repeat sequences on either terminus of Tn5090/Tn402. Four of these TniA-binding 19mers occurred on the left-hand (t) end and two on the right-hand (i) end. Hydroxyl radical cleavage protection demonstrated the presence of 3–6 bp contact sequences on one face of the DNA helix. The binding pattern and organisation of repeats suggested parallels between Tn5090/Tn402 and Mu, which controls its transpositional activity in the assembly step of a higher order transpososome complex. The complex terminal structure and genes of transposase and nucleotide-binding proteins in tandem are hallmarks of the handful of Mu-like elements that are known to date.

Isolation of segments of homologous genes with only one conserved amino acid region via PCR

We present a method which allows the isolation of fragments from genes coding for homologous proteins via PCR when only one block of conserved amino acids is available. Sets of degenerated primers are defined by reverse translation of the conserved amino acids such that each set contains not more than 128 different sequences. The second primer binding site is provided by a special cassette that is designed such that it does not allow binding of the second primer prior to being copied by DNA synthesis. The cassette is ligated to partially-digested chromosomal DNA. The second primer is biotinylated to allow elimination of PCR products carrying degenerated primers on both sides via streptavidin binding. Fragments obtained after amplification and enrichment are cloned and sequenced. The feasibility of this method was demonstrated in a model experiment, where degenerated primers were deduced from six conserved amino acids within the family of homologs to the Escherichia coli Vsr protein.