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PubMed2025

AVITI sequencing of a four-generation CEPH/Utah pedigree confirms low mutation rates at homopolymer loci despite their low sequence complexity.

BACKGROUND: Short tandem repeats (STRs) and homopolymers are among the most mutable loci in the human genome. Despite their presumed mutability owing to replication slippage, homopolymer loci exhibit lower mutation rates and minimal paternal age effects compared to other STRs. This paradox questions if technical limitations, rather than biological mechanisms, explain these observations. RESULTS: We used the Element Biosciences AVITI platform to sequence the genomes of a 48-member, four-generation CEPH/Utah pedigree. As the AVITI platform reduces error rates at repetitive sequences compared to Illumina, this design enabled accurate mutation discovery at 90% of assayed homopolymers and a 1.7-fold increase in discoverable mutations compared to Illumina. We identified a median of 35 de novo homopolymer mutations per trio and a mutation rate of 5.28 × 10-5 DNMs per locus per generation, confirming a lower rate than dinucleotides (1.94 × 10-4). Most DNMs were single base-pair expansions or contractions. Despite comprising <1% of homopolymer loci, G/C homopolymers showed 18-fold higher mutation rates than A/T homopolymers; in contrast, the high dinucleotide mutation rate is not driven by a particular motif class. Parent-of-origin analysis revealed 78% of homopolymer mutations are paternal in origin, but no significant paternal age effect was observed. CONCLUSIONS: This study confirms that homopolymers exhibit lower mutation rates and lack strong paternal age effects compared to other STRs, likely owing to the combination of a lower propensity to form slippage-causing secondary structures and more efficient mismatch repair. Our set of high-quality mutations suggest these phenomena are biological rather than technical in nature. Finally, we demonstrate that AVITI sequencing unlocks previously intractable regions of the genome and will be a powerful tool for continued investigation of repeat mutation.

AVITI↗

PubMed1987

Tailored removal of flanking homopolymer sequences from cDNA clones.

Terminal homopolymer sequences introduced during the synthesis and cloning of cDNA molecules often interfere with subsequent expression of the cloned cDNA. We describe a general method for the removal of homopolymer flanking sequences from cDNA inserts and subsequent tailoring of the resulting termini. The cDNA insert containing homopolymer tails is first subcloned into the multiple cloning site of an appropriate transcription vector. cDNA copies are then generated from in vitro-synthesized transcripts using oligonucleotide primers complementary to the nucleotide sequences adjacent to the homopolymer tails. The resulting double-stranded cDNA contains the homopolymer flanking sequences as 3'-terminal extensions that are conveniently removed by the 3'----5' exonuclease activity of T4 DNA polymerase. If the oligonucleotide primers also contain 5'-terminal noncomplementary sequences that specify potential restriction endonuclease sites, those sites are subsequently generated by the 5'----3' polymerase activity of the T4 DNA polymerase. Thus, in the same reaction, flanking homopolymer sequences are removed and the resulting termini are tailored to specify desired sequences.

Cloning, Molecular↗

PubMedDate not supplied

Frictional properties of dilute block-copolymer solutions and homopolymer solutions. Application to molecular weight determination.

An equation for the translational diffusion coefficient of block copolymers in dilute solution has been obtained by modifying Zimm's equation for homopolymers to take into account the existence of dissimilar segments in block copolymers. Illustrative calculations for homopolymers and block copolymers have been made and the results for homopolymers have been compared with experiments and with the calculations of Yamakawa and Fujii. A procedure has been proposed to determine the molecular weight of a block copolymer from measurements of its limiting viscosity number and its sedimentation coefficient or translational diffusion coefficient.

Mathematics↗

PubMed1992

Evaluation of polylactic acid homopolymers as carriers for bone morphogenetic protein.

Polylactic acid (PLA) homopolymers with molecular weights of 105,000; 21,000; 3300; 650; and 160d (PLA105000, PLA21000, PLA3300, PLA650, and PLA160, respectively) were prepared and evaluated as carriers for bone morphogenetic protein (BMP). Composites consisting of 4 mg of water-soluble, semipurified BMP and 100 mg of one of the PLA homopolymers were implanted into the dorsal muscles of mice. PLA105000/BMP, PLA21000/BMP, PLA3300/BMP, and PLA160/BMP composites failed to induce new bone formation; PLA105000, PLA21000, and PLA3300 elicited foreign-body reactions or chronic inflammation (or both), and PLA160 produced tissue necrosis. PLA650/BMP composites induced cartilage formation within one week and induced bone with hematopoietic marrow at three weeks postimplantation. PLA650/BMP composites were completely absorbed and replaced by new bone. The results suggest that within this group of PLA homopolymers, PLA650 may be the only type suitable for use as a BMP carrier.

Animals↗

PubMed1989

Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions.

A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.

Amino Acids↗

PubMed1986

OH-induced free radicals in purine nucleosides and their homopolymers: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane.

Free radicals produced by X-irradiation of N2O-saturated aqueous solutions of purine nucleosides (2'-deoxyadenosine, adenosine, 2'-deoxyguanosine, 3'-deoxyadenosine, guanosine and inosine) and the corresponding homopolymers (poly A and poly I) have been investigated by the technique of spin-trapping and e.s.r. spectroscopy. 2-Methyl-2-nitrosopropane was used as a spin-trap. For 2'-deoxyadenosine and 2'-deoxyguanosine, the resulting spin-adducts were separated by Bio-Gel P-2 column chromatography and analysed by e.s.r. spectroscopy. For homopolymers, e.s.r. spectra were recorded at 50 degrees C after enzymatic digestion to obtain signals with narrower line width. The e.s.r. signal consisting of only a primary triplet without further splittings, which is consistent with assignment to the trapping of an H-abstraction radical at the C4' position of the sugar moiety, was observed in all cases. For 2'-deoxyguanosine an e.s.r. signal consisting of a secondary triplet was observed. Examinations using other spin-trapping reagents such as PBN, 4-PyOBN and DMPO provided no positive evidence supporting the proposal that this was due to an alpha-nitrogen. The e.s.r. signal consisting of a secondary doublet which further splits into a doublet was observed for 2'-deoxyadenosine, adenosine, 3'-deoxyguanosine, 2'-deoxyguanosine, and inosine, and tentatively associated with a radical centered in the sugar moiety.

Adenosine↗

PubMed1976

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.

Cobalt↗

PubMed1976

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of Co+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fold higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3'-ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.

Cobalt↗

PubMed1990

The occurrence of alpha (1----2) linked N-acetylperosamine--homopolymer in lipopolysaccharides of non-O1 Vibrio cholerae possessing an antigenic factor in common with O1 V. cholerae.

Chemical analysis was carried out on lipopolysaccharides from Vibrio cholerae bio-serogroup Hakata 487-85. The O-specific chain of the phenol-soluble lipopolysaccharides was demonstrated by 13C-NMR spectroscopy and methylation analysis to contain a linear homopolymer of alpha(1----2) linked N-acetylperosamine (4-acetamido-4,6-dideoxy-D-mannopyranose), which was closely similar to but not identical to a linear alpha(1----2) linked N-3-deoxy-L-glycerotetronyl (S-2,4-dihydroxybutyryl) perosamine-homopolymer constituting that of O1 Vibrio cholerae lipopolysaccharides.

Amino Sugars↗

PubMed1989

Binding of globin mRNA, beta-globin mRNA segments and RNA homopolymers by immobilized protein of polysomal globin messenger ribonucleoprotein.

The binding of rabbit globin mRNA, in-vitro-generated beta-globin mRNA segments, and RNA homopolymers by proteins of rabbit reticulocyte polysomal messenger ribonucleoproteins (mRNP) after SDS gel electrophoresis and electroblotting was examined. The polysomal mRNP proteins have a higher affinity for mRNA than for rRNA and tRNA while having a higher affinity for polypurine than polypyrimidine homopolymers. Binding experiments with synthetic poly(A) and with segments of beta-globin mRNA transcribed from a cDNA in vitro revealed a set of polysomal mRNP proteins which preferentially bind the poly(A)-free beta-globin mRNA. A protein of Mr 90,000 binds specifically the 3'-nontranslated trailer of the poly(A)-free beta-globin mRNA and not the poly(A)-containing globin mRNA. Another set of proteins preferentially binds poly(A). The latter group of proteins contains a prominent species of Mr 72,000, which is most likely the rabbit poly(A)-binding protein. Three polysomal mRNP proteins which bound rabbit globin mRNA did not bind preferentially any of the other RNA probes used.

Animals↗

PubMed1982

Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. I. Cloning and characterization studies.

To assess the role of neighboring DNA sequences in gene regulation, poly(dA).poly(dT) and poly(dG).poly(dC) were cloned adjacent to promoters of the lactose control region. Recombinant plasmids were constructed which were suitable for large scale purification of restriction fragments containing these promoters, 95-base pair (bp) AluI fragments containing the lack operator and promoter for the lac wild type and for the catabolite gene activating the protein-independent mutant, lac UV5, were cloned into pBR322. Homopolymers of varying lengths were inserted into the -60 region of these promoters using recombinant DNA techniques. Six of the recombinant plasmids were chosen for detailed analysis: wild type (wt); wt-AT, containing 70 bp of poly(dA).poly(dT); wt-GC, containing 23 bp of poly(dG).poly(dC); UV5; UV5-AT, containing 70 bp of poly(dA).poly(dT) and finally UV5-GC, containing 43 bp of poly(dG).poly(dC). These plasmids were characterized by restriction mapping and DNA sequencing. The effects of the DNA homopolymers on the interaction of the Escherichia coli RNA polymerase with the promoters were studied using nitrocellulose filter binding. The results show that poly(dA).poly(dT) increases the level of RNA polymerase binding, whereas poly(dG).poly(dC) has no detectable effect.

Base Sequence↗

PubMed1975

Isolation of bacterial and phage proteins by homopolymer RNA-cellulose chromatography.

Nucleic acid-free extracts of Escherichia coli have been analyzed by chromatography on columns of cellulose, to which poly(A), poly(U), or poly(C) have been attached by ultraviolet irradiation. Proteins are released from the columns by stepwise elution with increasingly higher concentrations of salt, followed by washing with urea to remove very tightly bound molecules. The pattern of protein elution is reproducibly different for each of the homopolymer RNA-cellulose columns used: some proteins bind very tightly to one column, but poorly to others. Analysis by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, by immunological cross-reactivity in double diffusion tests, and by enzymological assays, has allowed the identification of a number of these proteins. The RNA polymerase core enzyme binds to poly(C)- and to poly(U)-cellulose columns, and can be purified to 20 to 30 percent homogeneity in a single step. Ribosomal protein S1 and the termination factor rho bind very tightly to poly(C)-cellulose, and both can be purified to homogeneity rapidly, in much higher yields than previously reported. Poly(A)-cellulose chromatography allows the isolation of large amounts of an 80,000 molecular weight protein having an as yet unassigned cellular function. The host factor required for RNA phage Qbeta RNA replication in vitro can also be obtained from poly(A)-cellulose, and chromatography of extracts of phage Qbeta-infected E. coli on RNA-cellulose columns results in very rapid isolation of the Qbeta replicase enzyme. Homopolymer RNA-cellulose chromatography thus appears to be a simple, general technique, useful for the efficient isolation of a variety of RNA-binding proteins.

Bacterial Proteins↗

PubMed1988

Enhancement of the sensitivity for in situ detection of alkaline phosphatase using a homopolymer of 2-acrylamide 2-methylpropanesulfonate.

In order to enhance the sensitivity of detection of alkaline phosphatase immobilized on nitrocellulose, a homopolymer of 2-acrylamide 2-methylpropanesulfonate (poly-AMPS) and agarose were added to the reaction mixture using 5-bromochloroindoxyl phosphate and nitroblue tetrazolium. By addition of the above ingredients, the amount of the reaction product deposited on the nitrocellulose filters on which alkaline phosphatase had been dot-blotted was increased, and darkening of the background (un-dot-blotted area) was prevented. Reflective densitometry revealed that the addition of poly-AMPS and agarose increased the sensitivity of the detection approximately three times higher than without the use of these compounds.

Acrylamides↗

PubMed1987

On the biodegradation of poly-beta-hydroxybutyrate (PHB) homopolymer and poly-beta-hydroxybutyrate-hydroxyvalerate copolymers.

The known biodegradability of PHB in certain biological environments has led to its proposed use as a 'biodegradable' implant material. Monofilaments of PHB homopolymer and two PHV-PHB copolymers have been studied in vivo and in vitro and assessed for changes in mechanical properties and topography. In vivo biodegradation was only observed with PHB when pre-degraded by 10.0 Mrad of gamma-irradiation before implantation. High temperature in vitro hydrolysis suggested that PHV copolymer additions retarded the rate of degradation of PHB. Hydration reactions had most effect on the ultimate tensile properties of the materials. In contrast, the elastic properties appeared to be relatively unaffected.

Animals↗

PubMed1989

On the structure of alpha-crystallin: construction of hybrid molecules and homopolymers.

The alpha A2 and alpha B2 subunits of bovine alpha-crystallin were purified by chromatofocussing in urea and assembled into homopolymers. Light-scattering measurements indicated their molecular masses were 360 and 420 kDa. The alpha A2 and alpha B2 polypeptides were also used to construct a series of hybrid molecules with alpha A/alpha B ratios ranging from 7:1 to 1:7. Sedimentation velocity analyses, isoelectric focussing under non-deaggregating conditions, circular dichroism spectroscopy and immunochemical analysis indicated that all of the subunits had copolymerized to alpha-crystallin-like aggregates with complete regeneration of the native structure. The polymers could be distinguished on the basis of their differing affinities for the antiserum. This was directly related to the proportion of alpha A2 subunits in each polymer. It was concluded that the alpha A2 and alpha B2 subunits are structurally equivalent and occupy equivalent site in the alpha-crystallin aggregates. It was also concluded that a micellar-like quaternary structure was consistent with most previous observations on the protein.

Animals↗

PubMed1968

The complexing of lysozyme with poly C and other homopolymers.

Lysozyme forms very large complexes with poly C, in acetate buffer solutions (pH 5.4), when the ratio of lysozyme to poly C concentration is 3/2. When this is less than 3/8 there is virtually no complexing, as evidenced by the low light-scattering power of such mixtures. At such relatively high poly C concentrations, the addition of pancreatic ribonuclease causes both the intensity and dissymmetry of scattering to rise to very high values after which time the intensity falls exponentially with time and with very little change in dissymmetry. Other homopolymers also form largest complexes with lysozyme at characteristic concentration ratios.

Adenine Nucleotides↗

PubMed1985

Interaction of DNA polymerase I of Escherichia coli with nucleotides. Antagonistic effects of single-stranded polynucleotide homopolymers.

Binding of deoxyribonucleoside 5'-triphosphates to DNA polymerase I of Escherichia coli was measured by using a microscale nonequilibrium dialysis method. It allowed rapid and economic measurement of dissociation constants, with negligible interfering side reactions. A stoichiometry of 1 mol of nucleoside 5'-triphosphate/mol of DNA polymerase was measured, and the occurrence of a single binding site was established, for which the nucleotides competed in the binary complex with the polymerase. Binding affinities decreased in the order dGTP greater than or equal to dATP greater than dCTP congruent to dTTP. These results are in agreement with previous findings [Englund, P. T., Huberman, J. A., Jovin, T. M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038-3044] except that, in a few cases, values of dissociation constants were smaller by factors of 2-3. The cations Mg2+ and Mn2+, as well as spermine, slightly enhanced complex stability at low levels and decreased it at high concentrations, while NaCl and Hg2+ had only destabilizing effects. Recognition between nucleoside 5'-triphosphates and nucleotide templates was studied by titration of the polymerase-[3H]dGTP complex with polynucleotide homopolymers. Complementary poly(dC) did not affect binding of dGTP, and non-complementary templates caused rejection of the nucleotide. Rejection of dGTP followed a saturation dependence with an equivalence of 110 +/- 10 monomer units of polynucleotides bound per molecule of DNA polymerase. The results favor a model by which recognition arises chiefly from the stereogeometrical fit of complementary template and nucleoside 5'-triphosphate into a rigid binding site.

DNA Polymerase I↗

PubMedDate not supplied

Chemical, physical, and toxicological characterization of fumes produced by heating tetrafluoroethene homopolymer and its copolymers with hexafluoropropene and perfluoro(propyl vinyl ether).

Submicrometer solid-particle (fume) aerosols with up to about 10(8) particles/cm3 are formed when dilute vapors of low molecular weight (MW) (ca. 5000-10,000) perfluoro polymers condense in air. The aerosol (e.g., 0.2 mg/m3) formed from these vapors is highly toxic to laboratory rats by inhalation (30-min exposure) when fresh but loses toxicity in minutes, apparently owing to coagulation. These fumes may result from (a) pyrolysis of tetrafluoroethene homopolymer or tetrafluoroethene-hexafluoropropene copolymer or (b) evaporation, at 200-320 degrees C, of isolated low MW polymer or low MW polymer present in perfluoro polymer resins. Some perfluoro polymer fume aerosols contain reactive functional groups, but others without detectable reactive groups are at least as toxic.

Animals↗