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PubMed1992

Molecular cloning of the breakpoints of a complex Philadelphia chromosome translocation: identification of a repeated region on chromosome 17.

Complex translocations in chronic myelogenous leukemia involve various chromosomes, in addition to chromosomes 9 and 22, in a nonrandom fashion. We have analyzed the DNA from leukemia cells characterized by a complex translocation, t(9;22;10;17)(q34;q11;p13;q21), by using the techniques of Southern blot hybridization, in situ hybridization, and molecular cloning; one of the breakpoints is at 17q21, a band that is frequently involved in complex 9;22 translocations. All of the breakpoint junctions and the corresponding normal sequences from the four involved chromosomes have been molecularly cloned. Restriction mapping is consistent with a simple concerted exchange of chromosomal material among the four chromosomes, except that additional changes appeared to have occurred within the chromosome 17 sequences. The cloned sequences on chromosome 17 at band q21 were found to be repeated in normal cells. By fluorescence in situ hybridization, a strong signal is seen at 17q21, but a weaker signal is also present at 17q23. By comparison with other primate species, an inversion in chromosome 17 during evolution appears to be responsible for the splitting of the cluster of repeat units in normal human cells.

Animals↗

PubMed1987

Repeated sequence elements in the high molecular weight basic nuclear proteins from the winter flounder.

In maturing sperm of the winter flounder, histones are not replaced by protamines but instead joined by a group of high molecular weight basic nuclear proteins. Despite their large size and number of components, these proteins were reduced to a relatively simple set of peptides by a "limit" digestion with endoprotease Lys-C. Nine of these peptides, that together account for half of the mass of the digest, were purified by two rounds of chromatography on a C18 reverse-phase high pressure liquid chromatographic column and analysed by sequential Edman degradation. Their sequences can be divided into two homology groups. Seven of the peptides contain all or part of a dodecapeptide consensus sequence, NH2-Ser-Pro-Met-Arg-Ser-Arg-Ser-Pro-Ser-Arg-Ser-Lys-COOH, which appears to be tandemly repeated. This dodecapeptide contains a previously recognized consensus phosphorylation sequence, NH2-Arg-Ser-Arg-Ser-Pro-COOH, in which both serines are phosphorylated during the early stages of spermiogenesis. The other homology group has the sequence NH2-Arg-Arg-Val-X-X-Pro-Lys-COOH, where X-X is either Gln-Thr or Pro-Ser. The dodecapeptide and heptapeptide sequences form at least 35 and 11%, respectively, of the high molecular weight basic nuclear proteins and are, therefore, repeated many times over in these proteins. A search for identical or homologous sequences within the Protein Sequence Database indicated that they are unique. The closest matches were to protamines and some viral DNA-binding proteins.

Amino Acid Sequence↗

PubMed1981

Original domain for the serum albumin family arose from repeated sequences.

The characteristic three-domain structure has been conserved throughout mammalian evolution by serum albumin and its fetal counterpart, alpha-fetoprotein. Thus, one still detects 35.2% amino acid sequence homology between bovine serum albumin and murine alpha-fetoprotein. Yet, natural selection cannot be invoked as the major factor responsible for the observed conservation of these sequences, for the simple reason of their dispensability. Inherited analbuminemia is apparently a harmless trait in man and the rat. The conservation appears inherent in their repetitious origin. Each protein is made of triplicate copies of the ancestral domain. Furthermore, analysis of the published sequence data suggests that the original coding sequence for the ancestral domain arose as repeats of the 18-base-long primordial building block sequence TTC-ACA-GAG-GAG-CAG-CTG specifying Phe-Thr-Glu-Glu-Gln-Leu and its shorter subsidiary TTC-ATG-GAG-GAG specifying Phe-Met-Glu-Glu. Consequently, the homology between bovine serum albumin and alpha-fetoprotein is mostly confined to small segments still specified by recognizable descendants of these building block sequences. The point to be made here is that evolutionary conservation of coding sequences can be an inherent property; natural selection need not be invoked.

Amino Acid Sequence↗

PubMed1995

Complex behavior of simple repeats: the fragile X syndrome.

The fragile X syndrome of mental retardation is one of the most common genetic diseases. The mutation causing this disease was the first of a new class of mutations involving repeat sequences disturbing gene function. Fragile X mutations consist of an expansion of a CGG trinucleotide repeat in the FMR1 gene, which is inactivated as a result of this expansion. The lack of FMR1 protein is believed to be responsible for the mental retardation. The mechanism and the timing of the repeat amplification are still not known. Characterization of the repeat has clarified the genetics of fragile X syndrome, and has given tools to establish the diagnosis and to determine carrier status.

Animals↗

PubMed1987

A novel arrangement of sequence elements surrounding the rDNA promoter and its spacer duplications in tsetse species.

Variation in organization and sequence of the rDNA of six species of tsetse fly (Glossina) has been investigated. Several novel tsetse-specific features have been uncovered. Like many other species the spacer is composed of subrepeats, which in some species contain duplications of the true promoter at the spacer-ETS boundary. In tsetse, however, the first 90 base-pairs of the external transcribed spacer (ETS) (that is, +1 to +90 after transcription initiation) is the 3' end of the last subrepeat. The absence of a "unique" region between the last subrepeat and the ETS suggests that the tsetse rDNA unit may consist of multiple true promoters, that is there is no single ETS boundary. Furthermore, interspecific comparisons show that the 90 base-pair region is part of a conserved 202 base-pair region, consisting of 72 base-pairs upstream from the initiation site and a further 40 base-pairs downstream, which is shared by all promoters other than the last. In genera other than tsetse, subrepeat lengths between species are generally similar; in tsetse they differ due to (1) variation in copy-number of the subsubrepeat motif A9T6CAG, and (2) the presence of large regions flanked by direct simple repeats such as GA5 or TGGTCTC. Slippage-like mechanisms are probably responsible for (1), and recombination and subsequent excision involving the direct repeats for (2). Different structural and sequence variants are seen to be homogenized in the family and fixed in each species, reflecting continual unequal crossing-over. However, notwithstanding this process of differentiation, the available comparisons also reveal that there are two small conserved regions between Glossina and Drosophila: one is part of the promoter and the other is an ETS processing site. Such intergeneric and interspecific differences are discussed in relation to the problem of the maintenance of several essential functions within the rDNA repeating unit despite the continual differentiation of the unit into novel arrangements.

Animals↗

PubMed2006

Assessing radiation-associated mutational risk to the germline: repetitive DNA sequences as mutational targets and biomarkers.

This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

Animals↗

PubMed2003

Identification of novel p53-binding proteins by biomolecular interaction analysis combined with tandem mass spectrometry.

Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analysis (BIA) to develop a method of direct protein identification after real-time analysis of protein protein interactions. Using this method, called BIA-MS/MS, we detected multiple p53-interacting proteins in whole tissue extracts from human placenta and liver. Peptide sequencing revealed three proteins whose interaction with p53 had not been previously reported: a cyclin-dependent kinase inhibitor p57/Kip2, a serine/threonine protein phosphatase PP1C, and hemoglobin. Using our system, unambiguous sequence information can be obtained at the femto- to picomole level after repeating the recovery procedure five times. Furthermore, the association and dissociation constants are easily determined by kinetic analysis. This system provides a powerful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner.

Amino Acid Sequence↗

PubMed2005

Length-dependent energetics of (CTG)n and (CAG)n trinucleotide repeats.

Trinucleotide repeats are involved in a number of debilitating diseases such as myotonic dystrophy. Twelve to seventy-five base-long (CTG)n oligodeoxynucleotides were analysed using a combination of biophysical [UV-absorbance, circular dichroism and differential scanning calorimetry (DSC)] and biochemical methods (non-denaturing gel electrophoresis and enzymatic footprinting). All oligomers formed stable intramolecular structures under near physiological conditions with a melting temperature that was only weakly dependent on oligomer length. Thermodynamic analysis of the denaturation process by UV-melting and calorimetric experiments revealed an unprecedented length-dependent discrepancy between the enthalpy values deduced from model-dependent (UV-melting) and model-independent (calorimetry) experiments. Evidence for non-zero molar heat capacity changes was also derived from the analysis of the Arrhenius plots and DSC profiles. Such behaviour is analysed in the framework of an intramolecular 'branched-hairpin' model, in which long CTG oligomers do not fold into a simple long hairpin-stem intramolecular structure, but allow the formation of several independent folding units of unequal stability. We demonstrate that, for sequences ranging from 12 to 25 CTG repeats, an intramolecular structure with two loops is formed which we will call 'bis-hairpin'. Similar results were also found for CAG oligomers, suggesting that this observation may be extended to various trinucleotide repeats-containing sequences.

Base Sequence↗

PubMed2005

Sequential behavior in the rat: a new model using food-reinforced instrumental behavior.

Sequential behavior, probably reflecting procedural learning, has intensively been investigated in humans. This work has mainly been done using so-called serial reaction time tasks. In such tasks, subjects have to respond rapidly to simple visual stimuli appearing at one of four locations by pressing a corresponding response key. Unknown to the subjects, these stimuli can follow a specific repeating sequence. Learning of such a sequence is typically inferred from faster reaction times to sequence as compared to random blocks of stimuli. In contrast to human subjects, the analysis of sequential behavior has received considerably less attention in rodents, possibly due to the lack of analogous animal models there. In order to establish such a model, a method was developed in rats to investigate serial reactions under conditions of random or sequential stimulus presentation. Operant testing chambers were used which consisted of four nose-poke holes with cue lights. These holes were arranged in a square fashion with a pellet receptacle in the center. The task of the rat was to rapidly respond to an illuminated hole by poking into it in order to obtain food. The stimulus locations varied permanently, and these changes pursued either a random or serial order. In three experiments with differing methodological details, responding under such conditions was analyzed with sequences consisting of 6, 12 or 13 positions. Evidence was obtained that rats can improve their performance under sequence as compared to random conditions, for example, with respect to the percentage of reinforcements obtained, or with respect to reaction times. Furthermore, methodological factors, like response requirements, were addressed which may critically affect experimental outcome. Together, this new kind of instrumental task might be useful to analyze sequential performance in the rat, and the brain mechanisms by which it is mediated.

Analysis of Variance↗

PubMed1998

Molecular evolution by staggered extension process (StEP) in vitro recombination.

We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65 degrees C is 50 times that of wild-type subtilisin E.

Bacillus subtilis↗

PubMed1992

Mapping genes in the mouse using single-strand conformation polymorphism analysis of recombinant inbred strains and interspecific crosses.

We have utilized a PCR-based analysis of single-strand conformation polymorphisms to identify polymorphisms that can be used for mapping cloned DNA sequences in the mouse. We have found that single-strand conformation polymorphism analysis of sequences that are potentially less subject to conservation (i.e., intron and 3' untranslated regions) is a relatively efficient means of detecting polymorphisms between inbred strains. Fifty percent of the tested primer pairs were polymorphic between inbred strains and 90% were polymorphic between mouse species, which is a frequency comparable to that found for microsatellite repeat sequences. We have found that this technique can be readily used to determine the strain distribution pattern in a recombinant inbred series and is a simple and rapid means to obtain a map position for cloned sequences. When this strategy was tested on a number of previously mapped cloned genes, the strain distribution patterns obtained were consistent with that to be expected on the basis of the known map position. We also tested the utility of this approach for characterizing genes that have not been previously mapped. Dvl, the mouse homolog of the putative Drosophila dishevelled gene, and Adfp, encoding an adipocyte differentiation-related protein, were found to map to chromosome 4. These results were confirmed using single-strand conformation polymorphism analysis of an interspecific backcross.

Animals↗

PubMed1997

Phylogenetic assessment of length variation at a microsatellite locus.

Sixty-six haplotypes at a locus containing a simple dinucleotide (CA)n microsatellite repeat were isolated by PCR-single-strand conformational polymorphism from populations of the horseshoe crab Limulus polyphemus. These haplotypes were sequenced to assess nucleotide variation directly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the microsatellite motif. In the repeat region itself, CA-number varied in integer values from 5 to 11 across alleles, except that a (CA)8 class was not observed. Differences among alleles were due also to polymorphisms at 22 sites in regions immediately flanking the microsatellite repeats. Nucleotide substitutions in these regions were used to estimate phylogenetic relationships among alleles, and the gene phylogeny was used to trace the evolution of length variation and CA repeat numbers. A low correlation between size variation and genealogical relationships among alleles suggests that absolute fragment size (as normally scored in microsatellite assays) is an unreliable indicator of historical affinities among alleles. This finding on the molecular fine structure of microsatellite variation suggests the need for caution in the use of repeat counts at microsatellite loci as secure indicators of allelic relationships.

Alleles↗

PubMed1988

Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules.

We have found abundant telomere-specific terminal transferase activity in crude macronuclear extracts from vegetatively growing cells of the hypotrichous ciliate Oxytricha nova. This activity adds two to seven tandem repeats of the sequence GGGGTTTT (the Oxytricha telomeric repeat) to the 3' end of oligonucleotide primers ending in repeats of G4T4 and always adds the repeats in the proper phase. The activity requires the presence of micromolar amounts of dGTP and dTTP as well as single-stranded oligomer primers ending 3' with repeats of the Oxytricha telomeric sequence. A nuclease activity is present in the extracts which is closely balanced with telomere terminal transferase activity. We propose a simple model for replication of the ends of linear DNA molecules based on the telomere terminal transferase.

Animals↗

PubMed1997

Effects of amino acid side-chain volume on chain packing in genetically engineered periodic polypeptides.

The fidelity of bacterial protein synthesis allows the production of architecturally well-defined polymeric materials through precise control of chain length, sequence, stereochemistry, and interchain interactions. In the present paper, we examine the relation between amino acid residue volume and crystalline unit cell dimensions, in a set of periodic protein polymers of repeating unit sequence -(AlaGly)3-X-Gly-, where X is Asn, Phe, Ser, Val, or Tyr. The proteins were overexpressed in Escherichia coli, purified by simple procedures based on acid/ethanol precipitation or insolubility in aqueous sodium dodecyl sulfate, and processed to form oriented crystalline mats by precipitation from formic acid under mechanical shear. X-ray diffraction analyses revealed that the basic structures of the -(AlaGly)3-X-Gly- polymers are identical to that previously reported for [(AlaGly)3-GluGly]36, [Krejchi, M.T., Atkins, E.D.T., Waddon, A.J., Fournier, M.J., Mason, T.L., and Tirrell, D.A. (1994) Science 265, 1427-1432], with the oligoalanylglycine segments forming antiparallel beta-sheets and the substituted amino acids occurring within three-residue folds at the lamellar surfaces. The X-ray diffraction signals for each member of the family index on an orthorhombic unit cell; the a-axis (hydrogen bond direction) and c-axis (chain direction) spacings remain invariant but the b-axis (sheet stacking direction) spacing increases with increasing volume of the substituted amino acid. The results obtained from a variant with alternating Glu and Lys substitution at the X position, together with the results previously reported for poly(L-alanylglycine) [Panitch, A., Matsuki, K., Cantor, E.J., Cooper, S.J., Atkins, E.D.T., Fournier, M.J., Mason, T.L., and Tirrell, D.A. (1997) Macromolecules 30, 42-49] are included for comparison. The average intersheet stacking distance (b/2) increases linearly with the volume of the amino acid inserted at position X. Because the chain-folded lamellar architecture adopted by these periodic polypeptides accommodates a wide range of residues differing in charge, steric bulk, and hydrophobicity, these results illustrate a new approach to the engineering of intermolecular interactions in polymeric solids.

Amino Acids↗

PubMed1997

Demonstration of apoptotic cells in tissue sections by in situ hybridization using digoxigenin-labeled poly(A) oligonucleotide probes to detect thymidine-rich DNA sequences.

The recognition of apoptotic cells by morphological appearance alone may be difficult. We have investigated the use of in situ hybridization (ISH) with digoxigenin-labeled poly(A) probes to detect apoptotic cells in tissue sections. This method was compared to conventional morphologic assessment and in situ end-labelling (ISEL) in a range of tissues in which apoptosis is known to occur. ISH with poly(A) probes detected apoptotic nuclei in all tissues in which there was evidence of apoptosis as judged by conventional histology. ISH and, to a lesser extent, ISEL preferentially labeled shrunken but still intact nuclei with margination of chromatin, presumably at an early stage of apoptosis. The poly(A) hybridization was abolished by pretreatment of tissue sections with DNAse. After denaturation of DNA, poly(A) hybridized to nuclei in all cells. No convincing hybridization signal was detected in alcohol-fixed or fresh-frozen sections. Both ISEL and ISH labeled some of the nuclei in ischemic tissues. ISH with poly(A) oligonucleotide probes offers a simple alternative to ISEL for detection of cells in early stages of apoptosis. These probes hybridize to thymidine-rich sequences of DNA in the highly repeated Alu sequences within the nuclear genome. These sequences are believed to become available for hybridization after formalin fixation and paraffin embedding as a result of the apoptosis-related increase in the susceptibility of nuclear DNA to denaturation.

Apoptosis↗

PubMed2001

The importance of time/space in diagnosing the causality of phylogenetic events: towards a "chronobiogeographical" paradigm?

A shift from a traditional biogeographical paradigm in cladistic biogeography to a chronobiogeographical paradigm is proposed. The chronobiogeographical paradigm aims to utilize temporal data in conjunction with spatial data in the detection of discrete historical events, such as vicariance and vicariant speciation, in cladograms. The concepts of primary and secondary congruency are introduced in relation to the distinction between repeated area relationships (primary congruency) and common extrinsic causality (secondary congruency). Simple hypothetical examples demonstrate that area cladograms cannot be safely interpreted purely as representing the sequence of area fragmentation; rather, they reflect recency of biotic interaction. Temporal data are shown to have a direct and potentially profound influence on the results of traditional cladistic biogeographical analyses, indicating the necessity of developing a chronobiogeographical approach. The implementation of the paradigm is considered first from a theoretical viewpoint and then in the context of the type of empirical data usually available. An as yet undevised "time/space algorithm" is deemed necessary for the latter, and guidelines are presented for the development of such an algorithm. Finally, we argue that the most rigorous and philosophically justified approach to the detection of phylogenetic causal events can be found only when temporal and spatial data are considered simultaneously. Consequently, the chronobiogeographical paradigm is seen as a logical elaboration of, not a replacement for, the biogeographical paradigm.

Algorithms↗

PubMed1990

[Polymerase chain reaction in clinical diagnosis].

The polymerase chain reaction (PCR) is a novel method that amplifies selective sequences of DNA or RNA molecules with unparalleled efficiency. The specificity of PCR amplification is based on two oligonucleotide primers which flank the DNA fragment to be multiplied and hybridize to opposite strands. Repeated cycles of thermal denaturation, primer annealing, and enzymatic extension by the heatstable Taq DNA polymerase result in exponential augmentation of the target DNA. PCR is simple, rapid, and highly sensitive. The nucleic acid content of a single cell is sufficient to detect a particular sequence. This method has an enormous impact on the detection of genetic mutations, sequence polymorphisms and infective agents. This review depicts the mechanism of PCR and explores its applications in clinical diagnostics.

DNA, Recombinant↗

PubMed1996

Modelling and design of cross-over trials.

There are many diseases and conditions that can be studied using a cross-over clinical trial, where the subjects receive sequences of treatments. The treatments are then compared using the repeated measurements taken 'within' subjects. The actual plan or design of the trial is usually obtained by consulting a published table of designs or by applying relatively simple rules such as using all possible permutations of the treatments. However, there is a danger is this approach because the model assumed for the data when the tables or rules were constructed may not be appropriate for the new trial being planned. Also, there may be restrictions in the new trial on the number of treatment sequences that can be used or on the number of periods of treatment particular subjects can be given. Such restrictions may mean that a published design of the ideal size cannot be found unless compromises are made. A better approach is to make the design satisfy the objectives of the trial rather than vice versa. In this paper we describe an approach to constructing such tailor-made designs which we hope will lead to ill-fitting 'off the peg' designs being a thing of the past. We use a computer algorithm to search for optimal designs and illustrate it using a number of examples. The criterion of optimality used in this paper is A-optimality but our approach is not restricted to one particular criterion. The model used in the search for the optimal design is chosen to suit the nature of the trial at hand and as an example a variety of models for three treatments are considered. We also illustrate the construction of designs for the comparison of two active treatments and a placebo where it can be assumed that the carry-over effects of the active treatments are similar. Finally, we illustrate an augmentation of a design that could arise when the objectives of a trial change.

Algorithms↗