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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↗

PubMed1993

Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization.

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.

Bacteriological Techniques↗

PubMed1997

Low frequency of microsatellites in the avian genome.

A better insight into the occurrence of microsatellites in a range of taxa may help to understand the evolution of simple repeats. Previous studies have found the relative abundance of several repeat motifs to differ among mammals, invertebrates, and plants. Absolute numbers of microsatellites also tend to correlate positively with genome size. We analyzed the occurrence, frequency, and distribution of microsatellites in birds, a taxon with one of the smallest known genome sizes among vertebrates. Dot-blot hybridization revealed that about half of 22 different di-, tri-, and tetranucleotide repeat motifs were clearly more common in human than in three species of birds: chicken, woodpecker, and swallow. For the remaining motifs no clear difference was found. From searching avian database sequences we estimated there to be 30,000-70,000 microsatellites longer than 20 bp in the avian genome. The number of (CA) > or = 10 would be around 7000-9000 and the number of (CA) > or = 14 about 3000. The calculated density of avian microsatellites (total, one every 20-39 kb; (CA) > or = 10, one every 136-150 kb) is much lower than that estimated for the human genome (one every 6 and 30 kb, respectively). This may be explained by the fact that the avian genome contains relatively less noncoding DNA than most mammals and that avian SINE/LINE elements do not terminate in poly(A) tails, which are known to provide a resource for the evolution of simple repeats in mammals. We found no association between microsatellites and SINEs in birds. Primed in situ labeling suggested fairly even distribution of (CA)n repeats over chicken macrochromosomes and intermediate chromosomes, whereas the microchromosomes, a large part of the Z and W chromosomes, and most telomeres and centromeres had very low concentrations of (CA)n microsatellites. The scarcity of microsatellites on the microchromosomes is compatible to these regions likely being unusually rich in coding sequences. The low microsatellite density in the genome in general and on the microchromosomes in particular imposes an obstacle for the development of marker-rich genetic maps of chicken and other birds, and for the localization of quantitative trait genes.

Animals↗

PubMed1996

Northern blot analysis of simple repetitive sequence transcription in plants.

The presence of simple repetitive sequence motifs in RNA from various plant species was probed by Northern blot analysis. Hybridization of total, poly(A)(+)- and poly(A)(-)-RNA with microsatellite-complementary oligonucleotide probes revealed distinct bands with most but not all probe/species combinations, demonstrating the presence of di-, tri- and tetranucleotide repeat motifs in plant transcripts. Only trinucleotide repeat-derived hybridization signals were found to be enriched in the poly(A)(+)-fraction. The quality of Northern blot signals proved to be highly dependent on hybridization stringency. Thus, under the stringency conditions usually applied for oligonucleotide hybridization, some probes [(GT)8, (CAC)5, (TCC)5, and (CCTA)4] cross-hybridized to bands corresponding in size to 18S and/or 26S rRNA. Cross-hybridization to rRNA was significantly reduced at higher stringencies. These results stress the importance of carefully adjusting the hybridization conditions in Northern blot analysis of simple sequence transcripts.

Blotting, Northern↗

PubMed1994

Chromosomal homogeneity of Drosophila ribosomal DNA arrays suggests intrachromosomal exchanges drive concerted evolution.

BACKGROUND: The individual copies of tandemly repeated genes, such as ribosomal DNA (rDNA), evolve coordinately within a species. This phenomenon has been called concerted evolution, and is thought to be caused by sequence-homogenizing mechanisms, such as gene conversion or unequal crossing-over between individual copies of the gene family. As these processes would act between the arrays on homologous and non-homologous chromosomes, the whole family of repeats would be expected to undergo homogenization in a given interbreeding population. RESULTS: In order to study the homogenization process, we have examined polymorphisms within the internal transcribed spacer (ITS) of the rDNA in populations of Drosophila melanogaster at the sequence level, by DNA sequencing and temperature-gradient gel electrophoresis. Among 84 ITS clones sequenced from five different wild-type strains, we found three polymorphic sites that are apparently in the process of homogenization. However, these three sites, as well as combinations of them, occurred at different frequencies in the different strains. Moreover, temperature-gradient gel electrophoresis analysis of an ITS fragment including these three sites shows that single chromosomes from locally interbreeding populations can harbor rDNA arrays that are largely homogenized for different sequence variants. CONCLUSIONS: The presence of chromosomal arrays that are homogeneous for different variants in interbreeding populations of Drosophila melanogaster indicates that there is little recombination between the chromosomes while new mutations are being homogenized along the individual arrays. The most likely explanation for this finding is that intrachromosomal recombination events occur at much higher rates than recombination between homologous chromosomes. Thus, the first step of the homogenization process would occur mainly within chromosomal lines. Such behavior of tandem repeat arrays suggests a simple explanation of how selection can act on a multigene family, namely by acting on whole chromosomally confined repeat arrays rather than on individual repeat units.

Animals↗

PubMed1996

Motor stereotypy and diversity in songs of mimic thrushes.

The relationship between the motor and acoustic similarity of song was examined in brown thrashers (Toxostoma rufum) and grey catbirds (Dumetella carolinensis) (family Mimidae), which have very large song repertoires and sometimes mimic other species. Motor similarity was assessed by cross correlation of syringeal airflows and air sac pressures that accompany sound production. Although most syllables were sung only once in the song analyzed, some were repeated, either immediately forming a couplet, or after a period of intervening song, as a distant repetition. Both couplets and distant repetitions are produced by distinctive, stereotyped motor patterns. Their motor similarity does not decrease as the time interval between repetitions increases, suggesting that repeated syllables are stored in memory as fixed motor programs. The acoustic similarity between nonrepeated syllables, as indicated by correlation of their spectrograms, has a significant positive correlation with their motor similarity. This correlation is weak, however, suggesting that there is no simple linear relationship between motor action and acoustic output and that similar sounds may sometimes be produced by different motor mechanisms. When compared without regard to the sequence in which they are sung, syllables paired for maximum spectral similarity form a continuum with repeated syllables in terms of their acoustic and motor similarity. The prominence of couplets in the "syntax" of normal song is enhanced by the dissimilarity of successive nonrepeated syllables that make up the remainder of the song.

Animals↗

PubMed1993

Detection of somatic changes in human renal cell carcinomas with oligonucleotide probes specific for simple repeat motifs.

The purpose of our study was to detect somatic changes in renal cell carcinoma by multilocus fingerprinting. DNA fingerprints were generated from the DNA of normal and malignant renal tissue samples of 29 patients with nonhereditary kidney carcinoma by using oligonucleotide probes specific for simple repeat motifs such as (GTG)5, (CA)8, (GACA)4, or (TTAGGG)3. Each probe rendered a typical fingerprint pattern, because it is specific with respect to the target regions recognized in the genome. The restriction enzymes used were HinfI and HaeIII. Changed banding patterns were detected by using (GTG)5 in 20% of the tumors, in 20% for (CA)8 after HinfI digestion, and in 10% after HaeIII digestion. Even more informative probes were (GACA)4, showing 70% changes after HaeIII digestion, and (TTAGGG)3, with 80% changes after digestion with either enzyme. Since the simple repeat motifs recognized by (GACA)4 are localized on the short arms of the acrocentric chromosomes (13, 14, 15, 21, and 22), it is possible that sequences important for renal carcinogenesis are present in these regions. The observation of changes in regions to which (TTAGGG)3 hybridizes points to an involvement of DNA elements in telomeric sequence related regions in human kidney tumor formation.

Adult↗

PubMed1980

Characterization of two xenopus somatic 5S DNAs and one minor oocyte-specific 5S DNA.

The somatic 5S DNA from X. borealis (Xbs 5S DNA) and X. laevis (Xis 5S DNA) and a minor oocyte-specific 5S DNA from X. laevis (Xit 5S DNA) have been purified, and individual repeating units have been cloned and sequenced. The two somatic 5S DNAs differ from the major oocyte 5S DNAs in having GC-rich spacers, homogeneous repeat lengths and no "pseudogenes." The somatic 5S DNAs from the two species have similar spacer sequences with differences due to single base changes and insertions/deletions. The spacer of the minor oocyte-specific 5S DNA (Xit) has the AT-rich sequence characteristic of the major oocyte 5S DNAs from X. laevis and X. borealis, and contains one duplication that has diverged approximately 40%. Like the somatic 5S DNAs, Xit 5S DNA has a homogeneous length repeat and a unique nucleotide sequence in its spacer. The presence of variable-length spacer regions in a multigene family correlates with variables numbers of a simple sequence in the spacer regions.

Animals↗

PubMedDate not supplied

Triplet repeats, over-expanded in neuromuscular diseases, are under-represented in mammalian DNA: a survey of models.

Simple tandem repeats represent more than 1% of the human genome: occasionally they exhibit intergenerational expansibility and are associated with neuromuscular diseases. In transgenic mice the same sequences elicit similar symptoms, but do not expand. We have searched for di-, tri-, and tetra-repeats in the published DNA sequences of chromosomes 21 and 22 of Homo sapiens, as well as in more than five megabases of Mus musculus DNA. Human and murine DNA sequences show a shortage in frequency and base coverage of tri-repeats as compared to di- and tetra-repeats. In murine sequences the cumulative frequency of di-, tri-, and tetra-repeats and their overall base coverage are about threefold higher than in human. Models for both the shortage of tri-repeats found in man and mouse and for their dynamic expansions are discussed. We propose that some of the 10 possible tri-repeats may be more prone than others to assume unusual structures capable of interfering with DNA synthesis: hence the shortage of tri-repeats. If such repeats are located at the 3'end of a chain growing and thus approaching another chain annealed to the same template, as Okazaki fragments do during discontinuous and encumbered replication, a combination of strand displacement, template switch, and branch migration may produce structures resistant to removal, hence the expansion of tri-repeats.

Animals↗

PubMed1992

Genotyping Naegleria spp. and Naegleria fowleri isolates by interrepeat polymerase chain reaction.

All six Naegleria species recognized to date were studied by interrepeat polymerase chain reaction (PCR). Priming at repeat sequences, which are known to be variable among eukaryotes, yielded electrophoretic DNA banding patterns that were specific for any single species. With a single PCR and simple gel electrophoresis, species determination could be performed in less than 1 day. Unambiguous discrimination between the pathogen N. fowleri and nonpathogenic Naegleria species appeared to be possible. Analysis of DNAs obtained from 20 separate isolates of N. fowleri revealed that geographic variation of the genetic fingerprints rarely occurs. All but 3 of 20 isolates of N. fowleri which were investigated showed identical banding patterns; for two isolates from New Zealand and one from Australia, a limited number of additional bands was detected, independent of the PCR primers used. These data corroborate previous findings on the genetic stability of pathogenic N. fowleri.

Animals↗

PubMed2001

Genotyping Encephalitozoon cuniculi by multilocus analyses of genes with repetitive sequences.

Encephalitozoon cuniculi infects a wide range of mammalian hosts. Three genotypes based on the number of GTTT repeats in the internal transcribed spacer (ITS) of the rRNA have been described, of which genotypes I and III have been identified in humans. In this study, the genetic diversity of E. cuniculi was examined at the polar tube protein (PTP) and spore wall protein I (SWP-1) loci. Nucleotide sequence analysis of the PTP gene divided 11 E. cuniculi isolates into three genotypes in congruence with the result of analysis of the ITS of the rRNA gene. The three PTP genotypes differed from one another by the copy number of the 78-bp central repeat as well as point mutations. These E. cuniculi isolates also differed from one another in the number of 15- and 36-bp repeats in the SWP-1 gene. In addition, some E. cuniculi isolates had heterogeneous copies of the SWP-1 gene with various numbers of repeats. Intragenotypic variation was also observed at the SWP-1 locus. Based on the length polymorphism and sequence diversities of the PTP and SWP-1 genes, two simple PCR tests were developed to differentiate E. cuniculi in clinical samples.

Animals↗

PubMed1995

[Direct detection of loci with pathologic trinucleotide repeats in diseases with anticipation].

We describe a simple method for the identification of pathologically expanded (CCG)n and (CTG)n three nucleotides repeat arrays in the human genome and for the recovery of flanking sequences. We were able to detect the presence of novel high-molecular-weight alleles in at least two of three subjects known to have expanded (CCG)n tracts at the FRAXA locus. The above method may be used for testing of small families or even single affected individuals with disease thought to display clinical evidence of anticipation. The (CCG)n > 200 and (CTG)n > 250 probes may also be useful for individual "DNA fingerprint" identifications.

Alleles↗

PubMed1995

The complete primary structure of human nebulin and its correlation to muscle structure.

Nebulin is a giant filamentous protein specific for vertebrate skeletal muscles. The correlation of its size to thin filament lengths in vertebrates suggests that nebulin may function as a molecular ruler to determine thin filament length. We have isolated a full-length cDNA of 20.8 kb encoding human nebulin and determined its sequence. The cDNA's predicted peptide has a molecular weight of 773 kDa, and 97% of its mass consists of 185 copies of -35-residue module. Within the molecule, different sub-families of modules can be distinguished, and their arrangement is correlated to the structure of the thin filament. The central 154 copies are grouped into 22 seven-module super repeats corresponding to 38.5 nm thin filament repeats. In the thin filament ruler region, multiple isoforms are generated by alternative exon usage which is likely to explain the developmental and tissue-specific size variations of nebulins previously found in vertebrate skeletal muscles. We propose that different types of nebulin molecular rulers are expressed in the different types of skeletal muscles by differential splicing. Outside the super repeat region, the presence of distinct module arrangements implies functional diversity of the nebulin module family. A novel "simple repeat" family together with an SH3 domain at the C-terminus appear to anchor the nebulin filament system in the Z-disc. Nebulin's SH3 domain is highly related in sequence to the SH3 domains in yeast actin binding protein ABP-1 and to the src substrate p80/85 in chicken, both proteins which are involved in regulating actin assembly of the cytoskeleton in non-muscle cells. Study of nebulins terminal sequences is likely to reveal how integration of the nebulin filament into the sarcomere is regulated.

Alternative Splicing↗

PubMed1979

Subunit structure of alpha-satellite DNA containing chromatin from African green monkey cells.

alpha-Satellite DNA containing chromatin from African green monkey cells (CV-1 cells) has been used to study the question whether or not nucleosomes are arranged in phase with the 172 bp repeat unit of the satellite DNA. Digestion experiments with DNAase II led us to exclude a simple phase relationship between the nucleosomal and the satellite DNA repeats. Digestion of CV-1 nuclei with micrococcal nuclease and endogenous nuclease (s) produced a series of sharp bands in the satellite DNA register over a background of heterogeneous length fragments. This observation is explained by a preferential cleavage of certain nucleotide sequences by these nucleases and is not in contradiction to our conclusion that a simple phase relationship does not exist.

Animals↗