An Evaluation of the Critical Parameters for Abiotic Peptide Synthesis in Submarine Hydrothermal Systems

It has been proposed that oligopeptides may be formed in submarine hydrothermal systems (SHSs). Oligopeptides have been synthesized previously under simulated SHS conditions which are likely geochemically implausible. We have herein investigated the oligomerization of glycine under SHS–like conditions with respect to the limitations imposed by starting amino acid concentration, heating time, and temperature. When 10⁻¹ M glycine solutions were heated at 250°C for < 20 min glycine oligomers up to tetramers and diketopiperazine (DKP) were detectable. At 200°C, less oligomerization was noted. Peptides beyond glycylglycine (gly₂) and DKP were not detected below 150°C. At 10⁻² M initial glycine concentration and below, only gly₂, DKP, and gly₃ were detected, and then only above 200°C at < 20 min reaction time. Gly₃ was undetectable at longer reaction times. The major parameters limiting peptide synthesis in SHSs appear to be concentration, time, and temperature. Given the expected low concentrations of amino acids, the long residence times and range of temperatures in SHSs, it is unlikely that SHS environments were robust sources of even simple peptides. Possible unexplored solutions to the problems presented here are also discussed.     

Spontaneous and promoted association of linear oligoglycines

Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was observed for free oligoglycines H-(Gly)_m-OH (m = 3–5). At the same time, oligoglycylamides H-(Gly)_m-NH₂ (m = 3–5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an additional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolecular clustering of peptide chains, as expected, was observed. This means that the presence of several oligoglycine chains bound to each other in one center is not a necessary prerequisite for polyglycine II-type association.     

Diketopiperazine-mediated peptide formation in aqueous solution

Though diketopiperazines (DKP) are formed in most experiments concerning the prebiotic peptide formation, the molecules have not been paid attention in the studies of chemical evolution. We have found that triglycine, tetraglycine or pentaglycine are formed in aqueous solution of glycine anhydride (DKP) and glycine, diglycine or triglycine, respectively. A reaction of alanine with DKP resulted in the formation of glycylglycylalanine under the same conditions. These results indicate that the formation of the peptide bonds proceeds through the nucleophilic attack of an amino group of the amino acids or the oligoglycines on the DKP accompanied by the ring-opening.The formation of glycine anhydride, di-, tri- and tetraglycine was also observed in a mixed aqueous solution of urea and glycine in an open system to allow the evaporation of ammonia. A probable pathway is proposed for prebiotic peptide formation through diketopiperazine on the primitive Earth.     

Glycine-induced currents are insensitive to the glycine receptor α1 subunit-specific blocker, cyanotriphenylborate, in older circling mice

The pharmacologic characteristics of glycine receptors (GlyRs) in the lateral superior olive (LSO) of circling mice, animal model for inherited deafness, were investigated using a GlyR α₁ subunit-specific receptor blocker (cyanotriphenylborate [CTB]). There was a statistically significant age-dependent increase in the antagonistic effect of CTB in heterozygous (+/cir) mice. In postnatal (P)0–P3 heterozygous (+/cir) mice, glycine currents evoked by glycine puffs were reduced to 20.4 ± 2.6, 37.1 ± 3.1, and 63.9 ± 2.5% at 0.1, 1, and 10 μM CTB (n = 13) compared to controls, while the glycine currents were reduced to 22.3 ± 3.5, 52.9 ± 4.1, and 78.3 ± 3.5% at 0.1, 1, and 10 μM CTB (n = 7) in P8–P12 heterozygous (+/cir) mice. In contrast, the antagonistic effect of CTB was not strong and even less than that of younger animals in older homozygous (cir/cir) mice. In P0–P3 homozygous (cir/cir) mice, the extent of inhibition was 20.2 ± 3.7, 37.8 ± 4.3, and 66.8 ± 4.2% at 0.1, 1, and 10 μM CTB (n = 6) compared to controls, while the extent of inhibition was 18.7 ± 2.4, 28.1 ± 3.9, and 39.1 ± 8.2% (n = 6) in P8–P12 homozygous (cir/cir) mice. The age-dependent decrease in the antagonistic effect of CTB indicates the abnormal development of the α₁ subunit-containing GlyRs in homozygous (cir/cir) mice.     

In situ fabrication and electrochemical behavior of amino acid polyoxometalate nanoparticles-embedded microcapsules

Amino acid polyoxometalate nanoparticles-embedded microcapsules were in situ fabricated by layer-by-layer (LbL) self-assembly method [polyoxometalate, H₃PMo₁₂O₄₀·nH₂O (PMo₁₂); amino acid, glycine (Gly)]. The morphology of the obtained microcapsules was characterized by transmission electron microscopy and scanning electron microscopy. The electrochemical behavior of the amino acid polyoxometalate nanoparticles-embedded microcapsules was studied by cyclic voltammetry. The microcapsules show the pH-dependent properties, indicating that the pH of solution plays an important role in the electrochemical behavior of heteropolyanions.     

Transfer free energies of peptide backbone unit from water to aqueous electrolyte solutions at 298.15 K

Transfer free energies (ΔG_tr) of amino acids from water to aqueous electrolyte solutions have been determined from the solubility measurements, as a function of salt concentration at 298.15 K under atmospheric pressure. The investigated aqueous systems contain amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly₂), triglycine (Gly₃), and tetraglycine (Gly₄) and cyclic glycylglycine (c(GG)) with an electrolyte compound of potassium chloride (KCl), potassium bromide (KBr) or potassium acetate (KAc). The solubilities of glycine and diglycine in aqueous solution decrease with increasing the concentration of salts (salting-out effect), whereas those of triglycine and tetraglycine increase with increasing the concentration of salts (salting-in effect). Furthermore, salting-in effect was found in aqueous c(GG)/KBr system, while salting-out effect was observed in aqueous c(GG)/KCl or c(GG)/KAc system. The experimental results were used to estimate the transfer free energies (Δg_tr) of the peptide backbone unit (–CH₂CONH–) from water to the aqueous electrolyte solutions. We developed a new trail to determine the activity coefficients (γ) for aqueous and aqueous electrolyte solutions using an activity coefficient model, with which the total contribution of transfer free energy between solute and the solvent was calculated. We compared the difference between neglecting and using the activity coefficients term in predicting ΔG_tr. Since the transfer free energy contribution is negative, interactions between the ionic salts and the peptide backbone unit of zwitterionic glycine peptides are favorable and thus the ionic salts destabilize these amino acids. It was also found that KBr stabilizes c(GG), whereas KCl and KAc destabilize c(GG). These results provide evidence for the existence of interactions between the amide unit and ionic salts, in aqueous solution, which may be of importance in maintaining protein structure as well as in protein–solute and protein–solvent interactions.     

Crystal structures of two cyclic pseudopentapeptides containing ψ[CH2S] and ψ[CH2SO] backbone surrogates

The solid state conformations of cyclo[Gly–Proψ[CH₂S]Gly–D –Phe–Pro] and cyclo[Gly–Proψ[CH₂–(S)–SO]Gly–D –Phe–Pro] have been characterized by X-ray diffraction analysis. Crystals of the sulfide trihydrate are orthorhombic, P2₁2₁2₁, with a = 10.156(3) Å, b = 11.704(3) Å, c = 21.913(4) Å, and Z = 4. Crystals of the sulfoxide are monoclinic, P2₁, with a = 10.662(1) Å, b = 8.552(3) Å, c = 12.947(2) Å, β = 94.28(2), and Z = 2. Unlike their all-amide parent, which adopts an all-trans backbone conformation and a type II β-turn encompassing Gly-Pro-Gly-D -Phe, both of these peptides contain a cis Gly¹-Pro² bond and form a novel turn structure, i.e., a type II′ β-turn consisting of Gly–D –Phe–Pro–Gly. The turn structure in each of these peptides is stabilized by an intramolecular H bond between the carbonyl oxygen of Gly¹ and the amide proton of D -Phe⁴. In the cyclic sulfoxide, the sulfinyl group is not involved in H bonding despite its strong potential as a hydrogen-bond acceptor. The crystal structure made it possible to establish the absolute configuration of the sulfinyl group in this peptide. The two crystal structures also helped identify a type II′ β-turn in the DMSO-d₆ solution conformers of these peptides. © 1993 John Wiley & Sons, Inc.     

Thermodynamic Prediction of Glycine Polymerization as a Function of Temperature and pH Consistent with Experimentally Obtained Results

Prediction of the thermodynamic behaviors of biomolecules at high temperature and pressure is fundamental to understanding the role of hydrothermal systems in the origin and evolution of life on the primitive Earth. However, available thermodynamic dataset for amino acids, essential components for life, cannot represent experimentally observed polymerization behaviors of amino acids accurately under hydrothermal conditions. This report presents the thermodynamic data and the revised HKF parameters for the simplest amino acid “Gly” and its polymers (GlyGly, GlyGlyGly and DKP) based on experimental thermodynamic data from the literature. Values for the ionization states of Gly (Gly⁺ and Gly^?) and Gly peptides (GlyGly⁺, GlyGly^?, GlyGlyGly⁺, and GlyGlyGly^?) were also retrieved from reported experimental data by combining group additivity algorithms. The obtained dataset enables prediction of the polymerization behavior of Gly as a function of temperature and pH, consistent with experimentally obtained results in the literature. The revised thermodynamic data for zwitterionic Gly, GlyGly, and DKP were also used to estimate the energetics of amino acid polymerization into proteins. Results show that the Gibbs energy necessary to synthesize a mole of peptide bond is more than 10 kJ mol^?1 less than previously estimated over widely various temperatures (e.g., 28.3 kJ mol^?1 → 17.1 kJ mol^?1 at 25 °C and 1 bar). Protein synthesis under abiotic conditions might therefore be more feasible than earlier studies have shown.     

Studies on the conformations and interactions of elastin. Proton magnetic resonance of the repeating tetramer

Proton magnetic resonance studies at 220 MHz were carried out on synthetic polymers of the repeating tetrapeptide of elastin. Temperature dependence and solvent-mixture dependence of peptide proton chemical shifts were determined for both linear polymers, N-formyl-(Val-Pro-Gly₁-Gly₂)_n-Val OMe where n ? 8 and 40, and for cyclic polymers (Val-Pro-Gly₁-Gly₂)_n, where n = 3 and 4. The Gly₂ NH was found to be solvent shielded. In addition, by studying the polymers Boc-Val-Pro-Gly₁-Gly₂-OH, H-Pro-Gly₁-Gly₂-Val OMe, H(Pro-Gly₁-Gly₂-Val)₃OH, and others, it was demonstrated that the Val C–O immediately preceding the Gly₂ NH in the sequence was required for solvent shielding. Also the Gly₂ NH resonance is found at higher field than the Gly₁ NH resonance. This provides the basis for proposing a β turn in which the Pro and Gly₁ residues form the corners, i.e., residues i + 1 and i + 2, and in which the Gly₂ NH, residue i + 3 hydrogen bonds to the carbonyl of residue i, the Val residue. Studies on methanol–water solvent systems indicated retention of the β turn as a significant conformational feature. This suggests that the β turn occurs in the elastic fiber, which contains about 60% water but which utilizes association of hydrophobic groups as a primary force in fibrogenesis.     

A probable prebiotic peptide formation from glycineamide and related compounds in a neutral aqueous medium participation of nucleoside and 5′-mononucleotide

Summary Several glycine oligomers were formed up to a hexamer in neutral aqueous solution by using Gly-NH₂, an intermediate in the formation of glycine by the Strecker synthesis. The optimum pH was around 7 in order to produce (Gly)₂ in the presence of guanosine. The elongation of the glycine peptide was demonstrated by analyzing the n+1 mer of glycine from the reaction of the Gly-NH₂ and (Gly)_n system. (n=0, 2, 3). The peptide formation was promoted by a basic catalyst such as a nucleic base. Guanosine and GMP showed preferential catalytic effect in regard to peptide formation among nucleosides and 5′-mononucleotides, respectively. This is an example of a specific chemical interaction between an amino acid and nucleic base. Other peptides were formed by using Phac-Phe-NH₂ and β-asparagine. The aqueous mixture of phenylpyruvate, ammonium sulfate and glycine gave a peak corresponding to Phac-Phe-Gly in HPLC. The system including β-asparagine and glycine allowed for the formation of α-Asp-Gly and β-Asp-Gly in a ratio of about 1 to 4.     

Spontaneous and promoted association of linear oligoglycines

Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was observed for free oligoglycines H-(Gly)m-OH(m = 3-5). At the same time, oligoglycylamides H-(Gly)m-NH2 (m = 3-5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an additional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolecular clustering of peptide chains, as expected, was observed. This means that the presence of several oligoglycine chains bound to each other in one center is not a necessary prerequisite for polyglycine II-type association.     

Structural requirements essential for elastin coacervation: favorable spatial arrangements of valine ridges on the three‐dimensional structure of elastin‐derived polypeptide (VPGVG)n

The elastin precursor tropoelastin possesses a number of polymeric peptides with repeating 3–9 mer sequences. One of these is the pentapeptide Val‐Pro‐Gly‐Val‐Gly (VPGVG) present in almost all animal species, and its polymer (VPGVG)n coacervates just as does tropoelastin. In the present study, in order to explore the structural requirements essential for coacervation, (VPGVG)n and its shortened repeat analogs (VPGV)n, (VPG)n, and (PGVG)n were synthesized and their structural properties were investigated. In our turbidity measurements, (VPGVG)n demonstrated complete reversible coacervation in agreement with previous findings. The Gly⁵‐deleted polymer (VPGV)n also achieved self‐association, though the onset of self‐association occurred at a lower temperature. However, the dissociation of (VPGV)n upon temperature lowering was found to occur in a three‐step process; the Val_i⁴‐Val_i+1¹ structure arising in the VPGV polypeptide appeared to perturb the dissociation. No self‐association was observed for (VPG)n or (PGVG)n repeats. Spectroscopic measurements by CD, FT‐IR, and ¹H‐NMR showed that the (VPGV)n and (VPG)n both assumed ordered structures similar to that of (VPGVG)n. These results demonstrated that VPGVG is a structural element essential to achieving the β‐spiral structure required for self‐association followed by coacervation, probably due to the ideal spatial arrangement of the hydrophobic Val residues. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Energetics and possible mechanisms of ion-beam protein tritiation based on ab initio potential surfaces

Ab initio self-consistent field potential energy surfaces for the approach of T. T₂. T⁺. T⁺₃ and HeT⁺ to glycine in the gas phase have been determined and this data used to obtain insight into mechanisms of experimental ion-beam protein tritiation processes. Results of these calculations show that the ionic species T⁺, T⁺₃ and HeT⁺ can form stable adducts with glycine (Gly) and that each functions as a tritiation agent forming the complex GlyT⁺. Neutral T and T₂ experience a purely repulsive interaction with Gly and do not form an intermediate complex. These neutral species are expected to be less effective tritiation agents than the respective ions, in agreement with experimental observations. The fate of the stable GlyT⁺ complex is discussed and it is proposed that this species is neutralized by electron capture to give GlyT which spontaneously dissociates to either Gly+T or tritiated glycine (Gly^*)+H, with the latter reaction product channel favored statistically. The most likely site of exchange is predicted to be at the amine nitrogen although significance exchange is expected to occur at the α-carbon site by a somewhat more complex reaction mechanism.     

The formation of peptides from glycine thioesters

Summary The condensation products obtained from 0.01M S-glycyl-N-acetyl-cysteamine at different pH's were investigated. The highest yields of diketo-piperazine (approx. 50%) were observed in phosphate buffers between pH 7.5 and 8.5. The highest yields of diglycine (46%), triglycine (10%) and tetraglycine (2%) were observed in carbonate buffers at pH 9.5. At pH 8.0, over 90% of the glycyl residues of 0.15M S-glycyl-N-acetylcysteamine were incorporated into condensation products, mainly DKP (60–70%). The yields of products from the condensation of S-glycyl-ethanethiol under similar conditions closely re-sembled those obtained with S-glycyl-N-acetylcysteamine.Abbreviations Boc-gly N-tert-butyloxycarbonylglycine - Ac-cys N-acetylcysteine - csa cysteamine - Ac-csa N-acetylcysteamine - DKP diketopiperazine - (gly) ₂ diglycine - (gly) ₃ triglycine - (gly) ₄ tetraglycine - glySEt S-glycyl-ethanethiol - glyS-(Ac-cys) S-glycyl-N-acetylcysteine - glyS-(Ac-csa) S-glycyl-N-acetylcysteamine - Boc-glyS-(Ac-cys) S-(Boc-glycyl)-N-acetylcysteine - Boc-glyS-(Ac-csa) S-(Boc-glycyl)-N-acetylcysteamine - Boc-glySEt S-(Boc-glycyl)-ethanethiol - gly-bydrox glycine hydroxamate     

Fractionation of the chymotryptic precipitate of Bombyx mori silk fibroin

1. A solution of Bombyx mori silk fibroin was digested with chymotrypsin. Amino acid analyses of the chymotryptic precipitate showed in addition to the main constituents Gly, Ala, Ser and Tyr, very small amounts of Lys, His, Arg, Asp, Thr, Glu, Pro, Cys, Val, Met, Ile, Leu, Phe and Trp. 2. A stable solution of the chymotryptic precipitate in 6m-urea was obtained by dialysing a solution in 50% (w/v) lithium thiocyanate against 6m-urea. 3. The dinitrophenylated chymotryptic precipitate in 6m-urea was fractionated by gel filtration and by ion-exchange chromatography. On Dowex 1 (X2), a main fraction I_d and three further fractions with different amino acid compositions and molecular weights were obtained. 4. Specific rearrangement and fission of the bonds involving the serine nitrogen atoms of fraction I_d and fractionation of the resulting mixture by gel filtration yielded five fractions. Two of these fractions had the compositions DNP-Ser-(Gly₆,Ala₄,Ser) and DNP-Ser-(Gly₄,Ala₂ or Ala₃,Ser) and are presumably double repeating units according to the proposed formula of Lucas, Shaw & Smith (1957), namely [Ser-Gly-(Ala-Gly)_n]₂, for n values of 2 and 1 respectively.     

The Influence of Mineral Matrices on the Thermal Behavior of Glycine

On the Hadean–Early Archean Earth, the first islands must have provided hot and dry environments for abiotically formed organic molecules. The heat sources, mainly volcanism and meteorite impacts, were also available on Mars during the Noachian period. In recent work simulating this scenario, we have shown that neat glycine forms a black, sparingly water-soluble polymer (“thermomelanoid”) when dry-heated at 200 °C under pure nitrogen. The present study explores whether relevant minerals and mineral mixtures can change this thermal behavior. Most experiments were conducted at 200 or 250 °C for 2 or 7 days. The mineral matrices used were phyllosilicates (Ca-montmorillonites SAz-1 and STx-1, Na-montmorillonite SAz-1-Na, nontronite NAu-1, kaolinite KGa-1), salts (NaCl, NaCl-KCl, CaCl₂, artificial sea salt, gypsum, magnesite), picritic basalt, and three Martian regolith simulants (P-MRS, S-MRS, JSC Mars-1A). The main analytical method employed was high-performance liquid chromatography (HPLC). Glycine intercalated in SAz-1 and SAz-1-Na was well protected against thermomelanoid formation and sublimation at 200 °C: after 2 days, 95 and 79 %, respectively, had either survived unaltered or been transformed into the cyclic dipeptide (DKP) and linear peptides up to Gly₆. The glycine survival rate followed the order SAz-1?>?SAz-1-Na?>?STx-1?≈?NAu-1?>?KGa-1. Very good protection was also provided by artificial sea salt (84 % unaltered glycine after 200 °C for 7 days). P-MRS promoted the condensation up to Gly₆, consistent with its high phyllosilicate content. The remaining matrices were less effective in preserving glycine as such or as peptides.     

Genetic variation in piñon pine, Pinus edulis, associated with summer precipitation

Three previous reports of microgeographical variation of glycerate dehydrogenase (Gly) frequencies in piñon, Pinus edulis, established the hypothesis that Gly frequencies contribute to adaptation to heterogeneous environments, specifically to variation in soil moisture. In each of these studies, the frequency of the Gly‐3 allele or of Gly‐33 homozygotes was higher on dry sites than on nearby moist sites. Here we attempt to extend these observations by testing the hypothesis that Gly frequencies respond to soil moisture variation on a range‐wide scale. Gly frequencies were surveyed in 11 natural populations, and the frequency of the Gly‐3 allele varied from 0.27 to 0.65 among the sample sites. Elevation varied from 1650 to 3100 m, and summer precipitation, defined as precipitation from April to August, varied from 13.7 to 26.4 cm. The soil types at the collection sites were schist, quaternary volcanic or a mixture of shale and sandstone. Logistic regression revealed that Gly frequencies did not respond to either elevation or soil type, but were related to summer precipitation (P < 0.01). The correlation between summer precipitation and the frequency of the Gly‐3 allele was r = ?0.92 (P < 0.001). Thus, the patterns of differentiation on microgeographical scales are consistent with greater differentiation on a range‐wide scale.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. I. Synthesis of co-oligopeptides of tryptophan and glycine

The preparation of the co-oligopeptides of the series H-Gly-Trp-(Gly)_n-Trp-Gly-OH (n = 0, 1, and 2) and of a number of other unprotected co-oligopeptides of glycine and tryptophan is reported. The syntheses have been carried out by conventional methods, using, in general, N-hydroxysuccinimide esters for the coupling steps. All the oligopeptides were obtained after purfication as colorless and crystalline products, and gave only one spot on thin-layer chromatography. Specfic problems connected with the synthesis and purficiation of optically pure tryptophan-containing peptides are discussed.     

A high-resolution 13C-nmr study of collagenlike polypeptides and collagen fibrils in solid state studied by the cross-polarization–magic angle-spinning method. Manifestation of conformation-dependent 13C chemical shifts and application to conformational characterization

We have recorded high-resolution ¹³C-nmr spectra of collagen fibrils in the solid state by the cross-polarization–magic-angle-spinning(CP–MAS)method and analyzed the spectra with reference to those of collagenlike polypeptides. We used two kinds of model polypeptides to obtain reference ¹³C chemical shifts of major amino acid residues of collagen (Gly, Pro, Ala, and Hyp): the 3₁-helical polypeptides [(Gly)_nII, (Pro)_nII, (Hyp)_n, and (Ala? Gly? Gly)_nII], and the triple-helical polypeptides [(Pro? Gly? Pro)_n and (Pro? Ala? Gly)_n]. Examination of the ¹³C chemical shifts of these polypeptides, together with our previous data, showed that the ¹³C chemical shifts of individual amino acid residues are the same, within experimental error (±0.5 ppm), among different polypeptides with different primary sequences, if the conformations are the same. We found that the ¹³C chemical shifts of Ala residues of the 3₁-helical (Ala? Gly? Gly)_n and triple-helical (Pro? Ala? Gly)_n are significantly displaced, compared with those of the α-helix, β-sheet, and silk I form, and can be utilized as excellent probes to examine conformational features of collagen-like polypeptides. Further, the ¹³C chemical shifts of Gly and Pro residues in the triple-helical polypeptides are substantially displaced from those found in (Gly)_nII and (Pro)_nII of the 3₁-helix, reflecting further conformational change from the 3₁-helix to the supercoiled triple helix. In particular, the ¹³C chemical shifts of Gly C ? O carbons of the triple-helical polypeptides are substantially displaced upfield (4.1–5.1 ppm), with respect to those of the 3₁-helical polypeptides. These displacements are interpreted by that Gly C ? O of the former is not involved in NH …? O ? C hydrogen bonds, while this carbon of the latter is linked by these kinds of hydrogen bonds. On the basis of these ¹³C chemical shifts, as reference data for the collagenlike structure, we were able to assign the ¹³C-nmr peaks of Gly, Ala, Pro, and Hyp residues of collagen fibrils, which are in good agreement with the values expected from the model polypeptides mentioned above. We also discuss a plausible conformational change of collagen fibrils during denaturation.     

Recurrence of osteogenesis imperfecta because of paternal mosaicism: Gly862→Ser substitution in a type I collagen gene (COL1A1)

We determined that two siblings with type III osteogenesis imperfecta (OI) had the same single base substitution that converted the codon for glycine (Gly) 862 to a codon for serine (Ser) in exon 44 of the 1 chain of the type I ( 1(I)) collagen gene (COL1A1). The mutation was also detected in various paternal tissues; the mutant allele accounted for approximately 11% of the COL1A1 alleles in blood, 24% of those in fibroblasts, and 43% of those in sperm determined by allele-specific colony hybridization using amplified genomic sequences. These findings demonstrate that germ-line mosaicism in the phenotypically normal father is responsible for the recurrence. There is a cluster of serine substitutions for Gly (Gly⁸³², Gly⁸⁴⁴ and Gly⁹⁰¹) which is associated with nonlethal phenotypes and which is located between two lethal clusters. In the cases studied here, a Gly⁸⁶²Ser mutation was identified that is located inside the nonlethal cluster.