Amber Fragments Research Articles

Amber Beads from Slavic Early Medieval Sites: Analysis of the Source Base

In the article amber beads from Slavic sites of the early Middle Ages are presented. A catalogue was compiled, which included information on 21 archaeological monuments and complexes. These locations are divided into three regions of concentration. Such finds come from the hoards of Martynivka and Pastyrske circles and the Pastyrske hill-fort, inhumated burials (Mokhnach, Balaklia) and the hill-forts of the Kolochyn culture. According to the number of finds, the monuments and complexes can be divided into six groups (fig. 2): 1 — the number of finds is unknown; 2 — with the number of finds from one to eight beads; 3 — complexes with the number of finds of about one and a half dozen copies of amber beads; 4 — with about 20 beads; 5 — a burial from Balaklia, where 30 beads were found; 6 — a burial from Mokhnach with 117 items. At least 285 amber beads come from Slavic monuments. However, the number of finds in some of the complexes is unknown. Only 132 items are defined typologically. As a result of morphological analysis, seven types were identified based on general outlines, which were divided into variants based on the section (fig. 3; Table 2). All of these types were found in the hoards of Martynivka and Pastyrske circles, on the sites and in complexes associated with these two chronological groups. Only the materials of Demydivka settlement indicate the beginning of the existence of type 1 within the second half of the 5th — beginning / first half of the 6th centuries. Amber beads are not typical for the materials of previous Proto-Slavic archaeological cultures. They are known only on a few monuments of the Kyiv culture (Table. 3; fig. 11). Finds of amber beads are unknown for the materials of the Volyntseve, Romny and Raiky cultures. Amber beads appear only in Kyivan Rus period. The analysis and comparison of beads from two complexes — Khyttsi hoard and the collection of Kolomak region were carried out. An analysis of scientific publications, the geological situation of the region and direct research of artefacts by laboratory methods allows us to draw the following conclusions: - amber from all the monuments may be of local origin or imported from the deposits of the Right-Bank of the Dnipro River; - processing of amber was carried out by local craftsmen. To do so, they used a grinding stone (sandstone) and a polishing material (clay), which was applied to a cloth or felt. Holes were drilled mechanically using a metal flattened at the end of the needle. The shape of the beads generally depended on the primary morphology of the found amber fragments and did not differ in complexity. The most time-consuming, in our opinion, is a round disk-shaped one, since for its manufacture it was necessary to obtain a flat, even surface on both sides.

Five new species of Rohrthrips (Thysanoptera: Rohrthripidae) from Burmese amber, and the evolution of Tubulifera wings.

Rohrthripidae is a family of fossil thrips in the thysanopteran suborder Tubulifera. It includes a single genus, Rohrthrips, of which two species are previously known. Despite typical tubuliferan features, these thrips also show some terebrantian character states, particularly in the wings and antennae, and these are here interpreted as plesiomorphic. Five new species of these ancestral thrips are described from Burmese Cenomanian amber: Rohrthrips breviceps sp. n., Rohrthrips jiewenae sp. n., Rohrthrips maryae sp. n., Rohrthrips patrickmuelleri sp. n. and Rohrthrips schizovenatus sp. n.. Structures on the wings support the conclusion that the wings of Tubulifera are homologous with those of Terebrantia. Syninclusions in the amber fragments provide evidence of the diet and habitats of these ancestral thrips.

Deuterium exchangeability in modern and fossil plant resins

Deuterium exchange experiments on modern and fossil plant resins (amber) were conducted to assess to what extent diagenetic alteration can overprint the stable hydrogen (δ2H) and carbon (δ13C) isotopic composition of these materials. Pairs of resins and amber fragments were placed together with deuterated water in sealed quartz-glass tubes to assure that all samples were exposed equally to the chosen experimental conditions. Experiments lasting up to one year were carried out at temperatures of 50 °C and 90 °C using water artificially enriched in deuterium (2H concentrations between 2861 and 8845 µg/g), which resulted in deuterium concentrations measured in samples well above natural concentrations (i.e., 2H ∼ 143 µg/g). Modern resins recorded a mean deuterium exchange with deuterium-enriched water of 2.0 ± 0.1%, compared to 1.3 ± 0.2% observed in their fossil counterparts. No significant changes were observed in the δ13C of modern and fossil resins during the experiments. Overall, these results indicate that the extent of 2H isotopic exchange in resins is minor and apparently occurs prior to polymerization. Consequently, the stable isotopic composition of fossil resins has the potential to serve as a useful proxy for paleoenvironmental reconstructions, given the widespread distribution of ambers in the Mesozoic and Cenozoic fossil record.

Намисто вельбарської культури з Хрінників

In 2016, a research of Khrinnyky 1 site in Rivne region was continued (fig. 1; 9). In particular, an object 27, determined as a dwelling of Wielbark culture (fig. 2), was investigated. The dwelling was dated by stages С2—С3 (230—330 CE) according to data of its ceramic complex (fig. 3, tab. 1). An interesting set of finds, carried out due to water flotation of the bottom filling of the object, was obtained. It could be a part of bead stringing or of another similar adornment. It includes a glass bead, three fish centra with through-holes and two small pieces of amber (fig. 4). A glass bead is close to type Alekseeva 137 and is dated back to the 2nd—3rd centuries CE. By its chemical composition (fig. 5, tab. 2) it can be characterized as low-quality product of local origin with the probable color of dark brown hues. Dating of the bead corresponds with the dating of the object’s ceramic complex (fig. 8). Fish vertebrae (fig. 6, tab. 3) belonged to the front tail part of the spine (vertebrae precaudales) of pike (Esox lucius) not less than 25 cm in length and with weight from 0.4 kg. The perforations could have been made with a thin point (an awl or a needle). The traces of polishing by soft abrasive were also traced on the surface that could also remain as a result of constant friction of beads on cloth. Probably the vertebrae played a role of seed beads. According to the results of IR-spectrometric and optical analyses, the amber could be of a local origin (fig. 7). The data of complex and context of the finds allow assuming the belonging of the amber fragments formerly to some product (fig. 10). On the basis of data set it was suggested the local origin of the decoration (fig. 11) and the possibility of its manufacture directly at the settlement of Khrinnyky 1.

Physico-Mechanical and Chemical Characteristics of Amber

Amber of amber-bearing deposits in Rivne-Volyn region of Ukraine has been analyzed. Relying upon instrumental techniques, physical and chemical as well as spectral analyses, and geological prospecting of the deposits, chemical composition and ultimate composition of amber occurring at the territory of Klesiv deposit (Ukraine, Rivne Region, Sarny District) have been identified. Klesiv amber contains the greatest part of inclusions; it contains 18 chemical elements. Basing upon the performed geological cross-section it has been determined that the amber occur in sandy soil and sandy-shale soil. The depth is insignificant – from 1 m to 10 – 15 m. Moreover, to determine the cost, experimental technique has been developed. The technique involves classification of the amber fragments according to their form, dimensions, and colour. Lithologic-and-facies sections of sites of Klesiv deposit have been obtained.

Amber in sediments of the Baltic Sea and the Curonian and Kaliningrad bays

In the Kaliningrad region, sediments of the Upper Eocene Prussian Formation accommodates the world’s largest explored amber deposits (up to 90% of world reserves). They are also partly tracked on the shelf of the southeastern Baltic region and subjected to bottom erosion, which is particularly intense during storm activity in the sea. Recent alongshore currents transport amber fragments over great distances, resulting in the formation of new (secondary) amber-bearing deposits in Holocene sediments in some places. The paper addresses formation conditions of such deposits. Catastrophic events, such as hydrospheric floods provoked by the regional glaciation, meteorite falls, and earthquakes (tsunamis), are of great significance.

Roman amber identified as Siegburgite

A small amber fragment from the period of Roman occupation of Numantia (Garray, Spain) was characterised by Fourier transformed infrared spectroscopy and optical microscopy. The material was found to be a polystyrene-like material, which is a very rare fossil resin known as Siegburgite or class III amber. Until now, this material was found in Europe only at two sites, both localised in Germany, and it is the first time that this type of amber was identified in wrought archaeological artefacts. The discussion includes the applicability of the method to distinguish false from true and amber of different origins, its use and trading as well as a hypothesis for a transport route. The study highlights the need for a more systematic study of archaeological amber remains. The finding also proves the exploitation and trading of local, non-Baltic amber deposits throughout the Roman Empire.

Evidence that F-plasmid proteins TraV, TraK and TraB assemble into an envelope-spanning structure in Escherichia coli.

We have examined the role of the F-plasmid TraV outer membrane lipoprotein in the assembly of F-pili. Yeast two-hybrid analysis with a traV bait repeatedly identified traK, which is predicted to encode a periplasmic protein, among positive prey plasmids. A traK bait in turn identified traV and traB, which is predicted to encode an inner membrane protein. A traB bait exclusively identified traK preys. Several additional observations support the hypothesis that TraV, TraK and TraB form a complex in Escherichia coli that spans the cell envelope from the outer membrane (TraV) through the periplasm (TraK) to the inner membrane (TraB). First, two-hybrid analyses indicated that TraV and TraB bind to different TraK segments, as required if TraK bridges a ternary complex. Secondly, all three proteins fractionated with the E. coli outer membrane in tra+ cells. In contrast, TraB fractionated with the inner membrane in traV or traK mutant cells, and TraK appeared in the osmotic shock fluid from the traV mutant. These results are consistent with a TraV-TraK-TraB complex anchored to the outer membrane via the TraV lipoprotein. Further, in traK mutant cells, TraV failed to accumulate to a detectable level, and the TraB level was significantly reduced, suggesting that TraV and TraB must interact with TraK for either protein to accumulate to its normal level. Both TraK and TraV accumulated in traB2[Am] cells; however, the TraB2 amber fragment could be detected by Western blot, and sequence analysis indicated that the fragment retained the TraK-binding domain suggested by yeast two-hybrid analysis. We propose that TraV is the outer membrane anchor for a trans-envelope, Tra protein structure required for the assembly of F-pili and possibly for other events of conjugal DNA transfer.

Function in vivo of separate segments of the beta subunit of Escherichia coli RNA polymerase.

Transcription of genetic material is catalysed by the enzyme DNA-dependent, RNA polymerase. The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed beta and beta', are conserved throughout nature. The beta subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme. Several lines of evidence have suggested that the function of the beta subunit is not dependent upon the contiguity of the sequence blocks. In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the beta subunit of RNA polymerase. To this end, the beta structural gene rpoB was separated into four near-equal, non-overlapping segments (as well as 'half' genes) on the basis of 'split' genes in nature, known functional organization and sequence conservation. These segments were used to prepare sequence-specific antibodies against the four individual regions, as well as being expressed in vivo from a tight, lac-controlled high-copy number vector. Immunological probing of the holoenzyme in vitro suggested that the amino-terminal half of the beta polypeptide is buried within the enzyme complex. Of the four segments expressed in vivo, the extreme C-terminal segment was trans-dominant lethal (of the effect of large N-terminal amber fragments on cellular growth; Nene & Glass 1982) and this isolated region was shown to bind the translational elongation factor EF-Tu in vivo. These in vivo and in vitro studies, in conjunction with recent in vitro work (Severinov et al. 1995), unambiguously demonstrate that individual regions of beta may adopt structurally and functionally competent forms, and underline the possibility of in vivo investigation of separate regions of this massive polypeptide chain. A model is presented for the role of EF-Tu in stringent control.

Physical mapping of a collection of Mael-generating amber mutations in the beta gene of Escherichia coli RNA polymerase and the functional effect of internal deletions constructed through their manipulation.

We report the fine mapping of 55 of our 95 amber mutations in the beta gene of Escherichia coli RNA polymerase by virtue of the unique MaeI restriction sites created by this subset of nonsense mutations (i.e. CTAG, where the amber codon is underlined). [The full data are reported in Supplementary Publication SUP 50181 (12 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8-10.] The CTAG mutations, which have been positioned to within approx. 9-60 bp, are distributed almost along the entire length of the rpoB gene, the one exception being the interval 400-499. The lack of amber fragments for mutations within the 5' approx. 265 codons suggests lability of the extreme N-terminal region; further potential destabilizing 'signals' may be present in the non-conserved 'spacer' regions. The locations of four of the eleven rpoB amber mutations that are strongly polar on expression of the downstream rpoC gene have been determined through a combination of MaeI mapping, PCR amplification and DNA sequencing. Surprisingly, one such mutant carries two tandem CTAG sites but is viable with three of the nonsense suppressors tested. These polar amber sites define three different amino acids (Gln-31, Gln-83 and Trp-183) that fall within three sequence-conservation blocks in the N-terminal region. Six of the MaeI/Am (where MaeI/Am is an amber mutation generating a MaeI restriction site) rpoB alleles (Gln-83, Gln-276, Gln-327, Gln-618, Gln-649 and Trp-183) have been used to generate small in-frame deletions (31-100 codons) within conserved and non-conserved regions of the beta gene, and the properties of these deletion variants were assessed in vivo. The smallest deletion reported in this study removes 31 amino acids from the middle of a region common to the eubacterial/chloroplast subgroup of beta homologues, and our results strongly suggest beta(deltaQ618-Q649) is assembled into a holoenzyme form capable of transcriptional initiation in vivo.

Systematic mutation of bacteriophage T4 lysozyme

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid residue in response to the amber codon, tests the effects of multiple single amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).

Properties of monoclonal antibodies selected for probing the conformation of wild type and mutant forms of the P22 tailspike endorhamnosidase.

Eleven species of monoclonal antibodies directed against the trimeric P22 tailspike endorhamnosidase have been selected and characterized. Seven of these antibodies recognize the native tailspike, both isolated and assembled onto the virion, and prevent phage infection. Four antibodies react with denatured forms of the tailspike as well as with the plastic absorbed tailspike. Three of these latter prevent the tailspike from assembling onto the phage head. The antibodies have been tested against tailspike proteins carrying single amino acid substitutions at 15 different sites on the protein. Two of these mutations interfere with binding by a set of the monoclonals, indicating that they disrupt the epitopes for these antibodies. Since amino acid replacements corresponding to the temperature-sensitive folding mutations do not change the conformation of the native protein, these mutant proteins may be particularly useful for mapping epitopes. Amber fragments of the tailspike chain are recognized predominantly by the anti-denatured antibodies suggesting either that they are conformationally closer to folding intermediates than to the native tailspike or that the epitopes recognized by anti-native antibodies are carried by the C-terminal end of the native protein. Immunochemical detection by an anti-denatured antibody, after sucrose gradient sedimentation of a large 55-kDa amber fragment, indicates a monomeric rather than a trimeric state. This suggests that the missing C-terminal region is important for the trimerization reaction. Such N-terminal amber fragments may be useful models for studying with the monoclonal antibodies the nascent chain emerging from the ribosome.

Nucleotide sequence and insertional inactivation of a Bacillus subtilis gene that affects cell division, sporulation, and temperature sensitivity

Located at 135 degrees on the Bacillus subtilis genetic map are several genes suspected to be involved in cell division and sporulation. Previously isolated mutations mapping at 135 degrees include the tms-12 mutation and mutations in the B. subtilis homologs of the Escherichia coli cell division genes ftsA and ftsZ. Previously, we cloned and sequenced the B. subtilis ftsA and ftsZ genes that are present on an 11-kilobase-pair EcoRI fragment and found that the gene products and organization of these two genes are conserved between the two bacterial species. We have since found that the mutation in the temperature-sensitive filamenting tms-12 mutant maps upstream of the ftsA gene on the same 11-kilobase-pair EcoRI fragment in a gene we designated dds. Sequence analysis of the dds gene and four other open reading frames upstream of ftsA revealed no significant homology to other known genes. It was found that the dds gene is not absolutely essential for viability since the dds gene could be insertionally inactivated. The dds null mutants grew slowly, were filamentous, and exhibited a reduced level of sporulation. Additionally, these mutants were extremely temperature sensitive and were unable to form colonies at 37 degrees C. Another insertion, which resulted in the elimination of 103 C-terminal residues, resulted in a temperature-sensitive phenotype less severe than that in the dds null mutant and similar to that in the known tms-12 mutant. The tms-12 mutation was cloned and sequenced, revealing a nonsense codon that was predicted to result in an amber fragment that was about 65% of the wild-type size (elimination of 93 C-terminal residues).

Conditionally lethal amber mutations in the leader peptidase gene of Escherichia coli

The lep gene of Escherichia coli encodes the leader peptidase which cleaves amino-terminal leader sequences of secreted proteins. To facilitate the study of structure-function relationships of the leader peptidase, 22 amber mutations in lep were isolated by localized mutagenesis. These amber mutants grew at 32 degrees C but not at 42 degrees C in the presence of a temperature-sensitive amber suppressor. Most of them were lethal under sup0 conditions. However, one amber mutant, the lep-9 mutant, exhibited temperature-sensitive growth in the sup0 strain, indicating that the amber fragment is active at 32 degrees C but not at 42 degrees C. Protein precursors of the maltose-binding protein and OmpA accumulate strikingly in the lep-9 mutant.

Assembly of amber fragments of the beta subunit of Escherichia coli RNA polymerase.

Immunoblotting of size-separated whole cell proteins permitted the study of protein-protein interaction. Briefly, proteins obtained from cleared cell lysates of Escherichia coli were separated by glycerol gradient centrifugation and analysed by blotting against a set of specific antibodies. We have applied this procedure to the assembly of 11 N-terminal amber fragments of the beta subunit of E. coli RNA polymerase ranging in size between 97% and 23% the length of the intact beta polypeptide (1342 amino acids). In this way, we have been able to define regions on the beta polypeptide involved in the assembly of RNA polymerase.

NusA amber mutation that causes temperature-sensitive growth of Escherichia coli

The nusA134 mutation was isolated from a sup0 strain as a temperature-sensitive mutant which grew at 32 degrees C but not at 42 degrees C. Immunoblot analysis showed that this mutant produced a 31,000-dalton nusA-encoded protein instead of the full-size 54,500-dalton product. Sequence and genetic analyses of the mutant nusA gene revealed a substitution of T for C at the PstI site (i.e., CTGCAG to CTGTAG), thereby creating a nonsense UAG codon. These results indicate that nusA134 is an amber mutation and that the 31,000-dalton amber fragment is active for Escherichia coli growth at 32 degrees C but not at 42 degrees C. Most lambda bacteriophage variants tested grew normally on the nusA134 mutant both at permissive and at nonpermissive temperatures. However, lambda r32, which carries an IS2 insertion beyond the tR1 terminator, was restricted at 42 degrees C. Defects in the transcriptional antitermination process, but not in transcription termination, were observed. A comparative study of nusA134 protein and a PstI-truncated protein suggests that truncation of the peptide chain at the PstI site by the amber mutation, rather than the loss of the glutamine residue, is primarily responsible for the defect in antitermination. The mode of the involvement of mutant nusA proteins in the N-mediated antitermination reaction is discussed.

Localization of a DNA-binding determinant in the bacteriophage P22 Erf protein

Four amber fragments of the recombination-promoting P22 Erf protein were characterized. The intact Erf monomer contains 204 amino acids. The amber mutations produce fragments of 190, 149, 130 and 95 amino acid residues, all of which are inactive in vivo. The 190 residue fragment is more susceptible to proteolysis in cell extracts than is intact Erf. It breaks down to a stable remnant that is slightly larger than the 149 residue fragment. The 149 and 130 residue fragments are stable; electron microscopy of the purified fragments reveals that they have similar morphologies, retaining the ring-like oligomeric structure, but lacking the tooth-like protruding portions of intact Erf. Intact Erf and the 149 residue fragment have similar affinities for single-stranded DNA; the affinity of the 130 residue fragment is 40-fold lower in low salt at pH 6.0. The 95 residue fragment is unstable in vivo. These observations, combined with previous observations, are interpreted as suggesting that the boundary of the amino-terminal domain of the protein lies between residues 96 and 130, that certain residues between 131 and 149 form part of an interdomain DNA-binding segment of the protein, that the boundary of the carboxy-terminal domain lies to the C-terminal side of residue 149, and that the carboxy-terminal domain is not necessary for assembly of the ring oligomer, although it is essential for Erf activity in vivo.

Subunit assembly and metabolic stability of E. coli RNA polymerase.

Immunological cross-reaction was employed for identification of proteolytic fragments of E. coli RNA polymerase generated both in vitro and in vivo. Several species of partially denatured but assembled RNA polymerase were isolated, which were composed of fragments of the two large subunits, beta and beta', and the two small and intact subunits, alpha and sigma. Comparison of the rate and pathway of proteolytic cleavage in vitro of unassembled subunits, subassemblies, and intact enzymes indicated that the susceptibility of RNA polymerase subunits to proteolytic degradation was dependent on the assembly state. Using this method, degradation in vivo was found for some, but not all, of the amber fragments of beta subunit in merodiploid cells carrying both wild-type and mutant rpoB genes. Although the RNA polymerase is a metabolically stable component in exponentially growing cells of E. coli, degradation of the full-sized subunits was found in two cases, i.e., several temperature-sensitive E. coli mutants with a defect in the assembly of RNA polymerase and the stationary-phase cells of a wild-type E. coli. The in vivo degradation of RNA polymerase was indicated to be initiated by alteration of the enzyme structure.

Morphogenesis of the long tail fibers of bacteriophage T2 involves proteolytic processing of the polypeptide (gene product 37) constituting the distal part of the fiber

Gene 37 of phage T2 codes for a protein that, as a dimer, constitutes the most distal, receptor-recognizing part of its long tail fibers. It was found that, from a plasmid carrying a fragment of gene 37 that lacked a region of the gene encoding 87 CO 2H-terminal amino acid residues, a protein was expressed that was slightly larger than that present in the phage. This size difference could not be accounted for. The missing region of gene 37 and also gene 38 (which codes for the auxiliary protein required for dimerization of protein 37) were cloned. Plasmids were constructed with gene 37, or gene 37 together with gene 38, under inducible control. Independent of the presence of the latter gene, a protein was produced that had the same size as protein 37 in the phage. A pulse-chase experiment revealed that a precursor of protein 37 is synthesized and processed such that approximately 120 amino acid residues, most likely CO 2H-terminal, are removed. Therefore, the protein produced from the truncated gene was larger because it cannot be processed. This fact also solved an old puzzle: an amber fragment of protein 37 of phage T2 had been found to be larger than the mature protein. The amber codon could be located 24 codons away from the normal stop codon. Obviously, the fragment cannot be processed. The existence of this fragment demonstrates that processing occurs during phage maturation.

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. IX. The role of the carboxy-terminus in enzyme assembly.

The assembly of RNA polymerase was studied in Escherichia coli mutants encoding large N-terminal amber fragments of the beta subunit. Whereas the removal of up to 20% of the carboxy-terminus does not prevent the formation of premature core enzyme, the amber fragments seem to interfere with holoenzyme production. These studies permit, therefore, the localization of a region on the beta polypeptide involved in sigma binding.